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What is targeted by Pexidartinib?

Pexidartinib is a selective tyrosine kinase inhibitor against CSF1R.

Background Pexidartinib, a novel, orally administered small-molecule tyrosine kinase inhibitor, has strong selectivity against colony-stimulating factor 1 receptor. This phase I, nonrandomized, open-label multiple-dose study evaluated pexidartinib safety and efficacy in Asian patients with symptomatic, advanced solid tumors. Materials and Methods Patients received pexidartinib: cohort 1, 600 mg/d; cohort 2, 1000 mg/d for 2 weeks, then 800 mg/d. Primary objectives assessed pexidartinib safety and tolerability, and determined the recommended phase 2 dose; secondary objectives evaluated efficacy and pharmacokinetic profile. Results All 11 patients (6 males, 5 females; median age 64, range 23-82; cohort 1 n = 3; cohort 2 n = 8) experienced at least one treatment-emergent adverse event; 5 experienced at least one grade ≥ 3 adverse event, most commonly (18%) for each of the following: increased aspartate aminotransferase, blood alkaline phosphatase, gamma-glutamyl transferase, and anemia. Recommended phase 2 dose was 1000 mg/d for 2 weeks and 800 mg/d thereafter. Pexidartinib exposure, area under the plasma concentration-time curve from zero to 8 h (AUC0-8h), and maximum observed plasma concentration (Cmax) increased on days 1 and 15 with increasing pexidartinib doses, and time at Cmax (Tmax) was consistent throughout all doses. Pexidartinib exposure and plasma levels of adiponectin and colony-stimulating factor 1 increased following multiple daily pexidartinib administrations. One patient (13%) with tenosynovial giant cell tumor showed objective tumor response. Conclusions This was the first study to evaluate pexidartinib in Asian patients with advanced solid tumors. Pexidartinib was safe and tolerable in this population at the recommended phase 2 dose previously determined for Western patients (funded by Daiichi Sankyo; clinicaltrials.gov number, NCT02734433). LESSONS LEARNED: The combination of pexidartinib and binimetinib was safe and tolerable and demonstrated encouraging signs of efficacy in two patients with advanced gastrointestinal stromal tumor (GIST) refractory to tyrosine kinase inhibitors (TKIs).Molecular profiling of GISTs at diagnosis and upon progression may provide insight into the mechanisms of response or resistance to targeted therapies.Additional trials are needed to further explore combined KIT and MEK inhibition in treatment-naïve and TKI-refractory patients with advanced GIST. BACKGROUND: Nearly all patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Dual inhibition of KIT and MAPK pathways has synergistic antitumor activity in preclinical GIST models. METHODS: This was an investigator-initiated, phase I, dose escalation study of the MEK inhibitor binimetinib combined with pexidartinib, a potent inhibitor of CSF1R, KIT, and FLT3, in patients with advanced or metastatic GIST who progressed on imatinib. The primary endpoint was phase II dose determination; secondary endpoints included safety, tolerability, and efficacy. An expansion cohort to further evaluate safety and efficacy was planned. RESULTS: Two patients were treated at dose level one (binimetinib 30 mg b.i.d. and pexidartinib 400 mg every morning and 200 mg every evening), after which the study was terminated by the manufacturer. No dose-limiting toxicities (DLTs) were reported, and treatment was well tolerated. The only grade ≥3 treatment-emergent adverse event (TEAE) was asymptomatic elevated creatine phosphokinase (CPK). Both patients had a best response of stable disease (SD) by RECIST. Progression-free survival (PFS) and overall survival (OS) were 6.1 and 14.6 months, respectively, in one patient with five prior lines of therapy. The second patient with NF1-mutant GIST had a 27% decrease in tumor burden by RECIST and remains on study after 19 months of treatment. CONCLUSION: Pexidartinib combined with binimetinib was tolerable, and meaningful clinical activity was observed in two imatinib-refractory patients. BACKGROUND: Tenosynovial giant cell tumour (TGCT), a rare, locally aggressive neoplasm, overexpresses colony-stimulating factor 1 (CSF1). Surgery is standard with no approved systemic therapy. We aimed to evaluate pexidartinib, a CSF1 receptor inhibitor, in patients with TGCT to provide them with a viable systemic treatment option, especially in cases that are not amenable to surgical resection. METHODS: This phase 3 randomised trial had two parts. Part one was a double-blind study in which patients with symptomatic, advanced TGCT for whom surgery was not recommended were randomly assigned via an integrated web response system (1:1) to the pexidartinib or placebo group. Individuals in the pexidartinib group received a loading dose of 1000 mg pexidartinib per day orally (400 mg morning; 600 mg evening) for the first 2 weeks, followed by 800 mg per day (400 mg twice a day) for 22 weeks. Part two was an open-label study of pexidartinib for all patients. The primary endpoint, assessed in all intention-to-treat patients, was overall response at week 25, and was centrally reviewed by RECIST, version 1.1. Safety was analysed in all patients who received at least one dose of the study drug. This study is registered with ClinicalTrials.gov, number NCT02371369. FINDINGS: Between May 11, 2015, and Sept 30, 2016, of 174 patients assessed for eligibility, 120 patients were randomly assigned to, and received, pexidartinib (n=61) or placebo (n=59). There were 11 dropouts in the placebo group and nine in the pexidartinib group. Emergence of mixed or cholestatic hepatotoxicity caused the data monitoring committee to stop enrolment six patients short of target. The proportion of patients who achieved overall response was higher for pexidartinib than placebo at week 25 by RECIST (24 [39%] of 61 vs none of 59; absolute difference 39% [95% CI 27-53]; p<0·0001). Serious adverse events occurred in eight (13%) of 61 patients in the pexidartinib group and one (2%) of 59 patients in the placebo group. Hair colour changes (67%), fatigue (54%), aspartate aminotransferase increase (39%), nausea (38%), alanine aminotransferase increase (28%), and dysgeusia (25%) were the most frequent pexidartinib-associated adverse events. Three patients given pexidartinib had aminotransferase elevations three or more times the upper limit of normal with total bilirubin and alkaline phosphatase two or more times the upper limit of normal indicative of mixed or cholestatic hepatotoxicity, one lasting 7 months and confirmed by biopsy. INTERPRETATION: Pexidartinib is the first systemic therapy to show a robust tumour response in TGCT with improved patient symptoms and functional outcomes; mixed or cholestatic hepatotoxicity is an identified risk. Pexidartinib could be considered as a potential treatment for TGCT associated with severe morbidity or functional limitations in cases not amenable to improvement with surgery. FUNDING: Daiichi Sankyo. Pexidartinib (PLX3397) is a small molecule tyrosine kinase and colony-stimulating factor-1 inhibitor with FDA breakthrough therapy designation for tenosynovial giant-cell tumor, and currently under study in several other tumor types, including breast cancer, non-Hodgkin's lymphoma, and glioblastoma. Here, we report a case of severe drug-induced liver injury requiring liver transplantation due to vanishing bile duct syndrome (VBDS) after exposure to pexidartinib in the I-SPY 2 Trial, a phase 2 multicenter randomized neoadjuvant chemotherapy trial in patients with Stage II-III breast cancer. We also review the current literature on this rare, idiosyncratic, and potentially life-threatening entity. PURPOSE: To evaluate the safety, recommended phase II dose (RP2D) and efficacy of pexidartinib, a colony stimulating factor receptor 1 (CSF-1R) inhibitor, in combination with weekly paclitaxel in patients with advanced solid tumors. PATIENTS AND METHODS: In part 1 of this phase Ib study, 24 patients with advanced solid tumors received escalating doses of pexidartinib with weekly paclitaxel (80 mg/m2). Pexidartinib was administered at 600 mg/day in cohort 1. For subsequent cohorts, the dose was increased by ⩽50% using a standard 3+3 design. In part 2, 30 patients with metastatic solid tumors were enrolled to examine safety, tolerability and efficacy of the RP2D. Pharmacokinetics and biomarkers were also assessed. RESULTS: A total of 51 patients reported ≥1 adverse event(s) (AEs) that were at least possibly related to either study drug. Grade 3-4 AEs, including anemia (26%), neutropenia (22%), lymphopenia (19%), fatigue (15%), and hypertension (11%), were recorded in 38 patients (70%). In part 1, no maximum tolerated dose was achieved and 1600 mg/day was determined to be the RP2D. Of 38 patients evaluable for efficacy, 1 (3%) had complete response, 5 (13%) partial response, 13 (34%) stable disease, and 17 (45%) progressive disease. No drug-drug interactions were found. Plasma CSF-1 levels increased 1.6- to 53-fold, and CD14dim/CD16+ monocyte levels decreased by 57-100%. CONCLUSIONS: The combination of pexidartinib and paclitaxel was generally well tolerated. RP2D for pexidartinib was 1600 mg/day. Pexidartinib blocked CSF-1R signaling, indicating potential for mitigating macrophage tumor infiltration. Pexidartinib (TURALIO™) is an orally administered small molecule tyrosine kinase inhibitor with selective activity against the colony-stimulating factor 1 (CSF1) receptor, KIT proto-oncogene receptor tyrosine kinase (KIT) and FMS-like tyrosine kinase 3 harboring an internal tandem duplication mutation (FLT3-ITD). In August 2019, the US FDA approved pexidartinib capsules for the treatment of adult patients with symptomatic tenosynovial giant cell tumor (TGCT) associated with severe morbidity or functional limitations and not amenable to improvement with surgery. This approval was based on positive results from the phase III ENLIVEN trial. Pexidartinib is being investigated in various maligcies as monotherapy or combination therapy. This article summarizes the milestones in the development of pexidartinib leading to its first approval for TGCT. Because genetic alterations including mutations, overexpression, translocations, and dysregulation of protein kinases are involved in the pathogenesis of many illnesses, this enzyme family is currently the subject of many drug discovery programs in the pharmaceutical industry. The US FDA approved four small molecule protein kinase antagonists in 2019; these include entrectinib, erdafitinib, pexidartinib, and fedratinib. Entrectinib binds to TRKA/B/C and ROS1 and is prescribed for the treatment of solid tumors with NTRK fusion proteins and for ROS1-postive non-small cell lung cancers. Erdafitinib inhibits fibroblast growth factor receptors 1-4 and is used in the treatment of urothelial bladder cancers. Pexidartinib is a CSF1R antagonist that is prescribed for the treatment of tenosynovial giant cell tumors. Fedratinib blocks JAK2 and is used in the treatment of myelofibrosis. Overall, the US FDA has approved 52 small molecule protein kinase inhibitors, nearly all of which are orally effective with the exceptions of temsirolimus (which is given intravenously) and netarsudil (an eye drop). Of the 52 approved drugs, eleven inhibit protein-serine/threonine protein kinases, two are directed against dual specificity protein kinases, eleven target non-receptor protein-tyrosine kinases, and 28 block receptor protein-tyrosine kinases. The data indicate that 46 of these drugs are used in the treatment of neoplastic diseases (eight against non-solid tumors such as leukemias and 41 against solid tumors including breast and lung cancers; some drugs are used against both tumor types). Eight drugs are employed in the treatment of non-maligcies: fedratinib, myelofibrosis; ruxolitinib, myelofibrosis and polycythemia vera; fostamatinib, chronic immune thrombocytopenia; baricitinib, rheumatoid arthritis; sirolimus, renal graft vs. host disease; nintedanib, idiopathic pulmonary fibrosis; netarsudil, glaucoma; and tofacitinib, rheumatoid arthritis, Crohn disease, and ulcerative colitis. Moreover, sirolimus and ibrutinib are used for the treatment of both neoplastic and non-neoplastic diseases. Entrectinib and larotrectinib are tissue-agnostic anti-cancer small molecule protein kinase inhibitors. These drugs are prescribed for the treatment of any solid cancer harboring NTRK1/2/3 fusion proteins regardless of the organ, tissue, anatomical location, or histology type. Of the 52 approved drugs, seventeen are used in the treatment of more than one disease. Imatinib, for example, is approved for the treatment of eight disparate disorders. The most common drug targets of the approved pharmaceuticals include BCR-Abl, B-Raf, vascular endothelial growth factor receptors (VEGFR), epidermal growth factor receptors (EGFR), and ALK. Most of the approved small molecule protein kinase antagonists (49) bind to the protein kinase domain and six of them bind covalently. In contrast, everolimus, temsirolimus, and sirolimus are larger molecules (MW ≈ 1000) that bind to FK506 binding protein-12 (FKBP-12) to generate a complex that inhibits the mammalian target of rapamycin (mTOR) protein kinase complex. This review presents the physicochemical properties of all of the FDA-approved small molecule protein kinase inhibitors. Twenty-two of the 52 drugs have molecular weights greater than 500, exceeding a Lipinski rule of five criterion. Excluding the macrolides (everolimus, sirolimus, temsirolimus), the average molecular weight of the approved drugs is 480 with a range of 306 (ruxolitinib) to 615 (trametinib). More than half of the antagonists (29) have lipophilic efficiency values of less than five while the recommended optima range from 5 to 10. One of the troublesome problems with both targeted and cytotoxic drugs in the treatment of maligt diseases is the near universal development of resistance to every therapeutic modality. INTRODUCTION: Tenosynovial giant cell tumor (TGCT) is a benign clonal neoplastic proliferation arising from the synovium often causing pain, swelling, joint stiffness, and reduced quality of life. The optimal treatment strategy in patients with diffuse-type TGCT (dt-TGCT) is evolving. Surgery is the main treatment, with a high recurrence rate and surgery-related morbidity. Radiotherapy is associated with important side effects. TGCT cells overexpress colony-stimulating factor 1 (CSF1). Pexidartinib (Turalio™) is a selective CSF1 R inhibitor, which was recently approved by the FDA for the treatment of TGCT. AREAS COVERED: This article reviews the pharmacological properties, clinical efficacy, and safety of pexidartinib. EXPERT OPINION: Pexidartinib was effective with an acceptable safety profile for advanced TGCT in phase I-III studies. The phase III trial (ENLIVEN) in unresectable TGCT met its primary endpoints of overall response rate. These results led to FDA approval for this TGCT population. Mixed or cholestatic hepatotoxicity was observed in rare cases. For this reason, pexidartinib is currently available only through a Risk Evaluation and Mitigation Strategy (REMS) Program in the USA. TGCT significantly impairs patients' quality of life. The approval of pexidartinib has changed the therapeutic armamentarium for this condition. However, strict monitoring of liver function is warranted. PURPOSE: Pexidartinib (PLX3397) is a colony-stimulating factor-1 receptor (CSF-1R) inhibitor under clinical evaluation for potential CNS tumor treatment. This study aims to evaluate plasma pharmacokinetic parameters and estimate CNS penetrance of pexidartinib in a non-human primate (NHP) cerebrospinal fluid (CSF) reservoir model. METHODS: Five male rhesus macaques, each with a previously implanted subcutaneous CSF ventricular reservoir and central venous lines, were used. NHPs received a single dose of 40 mg/kg pexidartinib (human equivalent dose of 800 mg/m2), administered orally as 200 mg tablets. Serial paired samples of blood and CSF were collected at 0-8, 24, 48, and 72 h. Pexidartinib concentrations were assayed by Integrated Analytical Solutions, Inc. (Berkeley, CA, USA) using HPLC/MS/MS. Pharmacokinetic (PK) analysis was performed using noncompartmental methods. RESULTS: Samples from four NHPs were evaluable. Average (± SD) plasma PK parameters were as follows: Cmax = 16.50 (± 6.67) μg/mL; Tmax = 5.00 (± 2.58) h; AUClast = 250.25 (± 103.76) h*μg/mL; CL = 0.18 (± 0.10) L/h/kg. In CSF, pexidartinib was either quantifiable (n = 2), with Cmax values of 16.1 and 10.1 ng/mL achieved 2-4 h after plasma Tmax, or undetected at all time points (n = 2, LLOQCSF = 5 ng/mL). CONCLUSION: Pexidartinib was well-tolerated in NHPs, with no Grade 3 or Grade 4 toxicities. The CSF penetration of pexidartinib after single-dose oral administration to NHPs was limited. Tenosynovial giant cell tumor (TGCT) is a rare benign tumor that involves the synovium, bursa, and tendon sheath, resulting in reduced mobility of the affected joint or limb. The current standard of care for TGCT is surgical resection. However, some patients have tumor recurrence, present with unresectable tumors, or have tumors that are in locations where resection could result in amputations or significant debility. Therefore, the development of systemic agents with activity against TGCT to expand treatment options is a highly unmet medical need. Pathologically, TGCT is characterized by the overexpression of colony-stimulating factor 1 (CSF-1), which leads to the recruitment of colony-stimulating factor-1 receptor (CSF-1R) expressing macrophages that make up the primary cell type within these giant cell tumors. The binding of CSF-1 and CSF-1R controls cell survival and proliferation of monocytes and the switch from a monocytic to macrophage phenotype contributing to the growth and inflammation within these tumors. Therefore, molecules that target CSF-1/CSF-1R have emerged as potential systemic agents for the treatment of TGCT. Given the role of macrophages in regulating tumorigenesis, CSF1/CSF1R-targeting agents have emerged as attractive therapeutic targets for solid tumors. Pexidartinib is an orally bioavailable and potent inhibitor of CSF-1R which is one of the most clinically used agents. In this review, we discuss the biology of TGCT and review the pre-clinical and clinical development of pexidartinib which ultimately led to the FDA approval of this agent for the treatment of TGCT as well as ongoing clinical studies utilizing pexidartinib in the setting of cancer. PURPOSE: Simultaneously targeting the tumor and tumor microenvironment may hold promise in treating children with refractory solid tumors. Pexidartinib, an oral inhibitor of tyrosine kinases including colony stimulating factor 1 receptor (CSF-1R), KIT, and FLT3, is FDA approved in adults with tenosynovial giant cell tumor. A phase I trial was conducted in pediatric and young adult patients with refractory leukemias or solid tumors including neurofibromatosis type 1-related plexiform neurofibromas. PATIENTS AND METHODS: A rolling six design with dose levels (DL) of 400 mg/m2, 600 mg/m2, and 800 mg/m2 once daily for 28-day cycles (C) was used. Response was assessed at regular intervals. Pharmacokinetics and population pharmacokinetics were analyzed during C1. RESULTS: Twelve patients (4 per DL, 9 evaluable) enrolled on the dose-escalation phase and 4 patients enrolled in the expansion cohort: median (lower, upper quartile) age 16 (14, 16.5) years. No dose-limiting toxicities were observed. Pharmacokinetics appeared linear over three DLs. Pharmacokinetic modeling and simulation determined a weight-based recommended phase II dose (RP2D). Two patients had stable disease and 1 patient with peritoneal mesothelioma (C49+) had a sustained partial response (67% RECIST reduction). Pharmacodynamic markers included a rise in plasma macrophage CSF (MCSF) levels and a decrease in absolute monocyte count. CONCLUSIONS: Pexidartinib in pediatric patients was well tolerated at all DL tested, achieved target inhibition, and resulted in a weight-based RPD2 dose.

What is the microgenderome?

The sexually dimorphic microbiome has been termed the 'microgenderome'.

The 'microgenderome' provides a paradigm shift that highlights the role of sex differences in the host-microbiota interaction relevant for autoimmune and neuro-immune conditions. Analysis of cross-sectional self-report and faecal microbial data from 274 patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) suggests that commensal gut microorganisms may play both protective and deleterious roles in symptom expression. Results revealed significant sex-specific interactions between Firmicutes (Clostridium, Streptococcus, Lactobacillus and Enterococcus) and ME/CFS symptoms (including neurological, immune and mood symptoms), regardless of compositional similarity in microbial levels across the sexes. Extending animal studies, we provide support for the microgenderome in a human clinical population. Applied and mechanistic research needs to consider sex-interactions when examining the composition and function of human microbiota. The microgenderome defines the interaction between microbiota, sex hormones and the immune system. Our recent research inferred support for the microgenderome by showing sex differences in microbiota-symptom associations in a clinical sample of patients with myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). This addendum expands upon the sex-specific pattern of associations that were observed. Interpretations are hypothesized in relation to genera versus species-level analyses and D-lactate theory. Evidence of sex-differences invites future research to consider sex comparisons in microbial function even when microbial abundance is statistically similar. Pairing assessment of clinical symptoms with microbial culture, DNA sequencing and metabolomics methods will help advance our current understandings of the role of the microbiome in health and disease. Sex differences in immunity are well described in the literature and thought to be mainly driven by sex hormones and sex-linked immune response genes. The gastrointestinal tract (GIT) is one of the largest immune organs in the body and contains multiple immune cells in the GIT-associated lymphoid tissue, Peyer's patches and elsewhere, which together have profound effects on local and systemic inflammation. The GIT is colonised with microbial communities composed of bacteria, fungi and viruses, collectively known as the GIT microbiota. The GIT microbiota drives multiple interactions locally with immune cells that regulate the homeostatic environment and systemically in diverse tissues. It is becoming evident that the microbiota differs between the sexes, both in animal models and in humans, and these sex differences often lead to sex-dependent changes in local GIT inflammation, systemic immunity and susceptibility to a range of inflammatory diseases. The sexually dimorphic microbiome has been termed the 'microgenderome'. Herein, we review the evidence for the microgenderome and contemplate the role it plays in driving sex differences in immunity and disease susceptibility. We further consider the impact that biological sex might play in the response to treatments aimed at manipulating the GIT microbiota, such as prebiotics, live biotherapeutics, (probiotics, synbiotics and bacteriotherapies) and faecal microbial transplant. These alternative therapies hold potential in the treatment of both psychological (e.g., anxiety, depression) and physiological (e.g., irritable bowel disease) disorders differentially affecting males and females.

What causes yellowing of the skin and eyes, also known as jaundice, in patients with liver failure?

Jaundice refers to yellow coloration of the skin and the sclera (white of the eyes) of newborn babies that result from the accumulation of bilirubin in the skin and mucous membranes.

Although liver test abnormalities are frequently identified in patients with acquired immunodeficiency syndrome (AIDS), the causes, evaluation, and outcome of jaundice in these patients have not been systematically evaluated. From August 1, 1990 through September 1, 1994, all human immunodeficiency virus (HIV)-infected patients with liver test abnormalities seen by the gastroenterology service at a large, inner-city hospital were prospectively identified. Jaundice was defined as a serum bilirubin concentration > or = 3 mg/dL. The etiology of jaundice was determined by the pattern of liver biochemistry test abnormalities, radiographic studies, liver biopsy, clinical follow-up, and autopsy. During the study period, 541 HIV-infected patients (511 with AIDS) were evaluated for liver disease by our service; 36 of these patients had jaundice (7 percent). The most common causes of jaundice were drug-induced hepatitis, occurring in 11 patients (31 percent), and alcoholic liver disease, occurring in 5 (13 percent). Opportunistic infections or neoplasms were identified as the cause of jaundice in 11 patients (30 percent), with 4 having intrahepatic disease and 7 having extrahepatic disease. Multiple potential causes were seen in 3 patients. Abdominal ultrasonography (US) and computed tomography (CT) were helpful in suggesting the underlying cause of disease. The short-term mortality was high, with 9 patients dying during the hospitalization (25 percent) and 7 patients dying within 6 months of evaluation. Liver disease was the cause of death in 7 of these patients. In conclusion, jaundice is uncommon in AIDS and may result from a variety of both opportunistic and non-opportunistic etiologies. Drug-induced hepatitis is the most common cause and may be fatal. Long-term survival was poor. Jaundice in an adult patient can be caused by a wide variety of benign or life-threatening disorders. Organizing the differential diagnosis by prehepatic, intrahepatic, and posthepatic causes may help make the work-up more manageable. Prehepatic causes of jaundice include hemolysis and hematoma resorption, which lead to elevated levels of unconjugated (indirect) bilirubin. Intrahepatic disorders can lead to unconjugated or conjugated hyperbilirubinemia. The conjugated (direct) bilirubin level is often elevated by alcohol, infectious hepatitis, drug reactions, and autoimmune disorders. Posthepatic disorders also can cause conjugated hyperbilirubinemia. Gallstone formation is the most common and benign posthepatic process that causes jaundice; however, the differential diagnosis also includes serious conditions such as biliary tract infection, pancreatitis, and maligcies. The laboratory work-up should begin with a urine test for bilirubin, which indicates that conjugated hyperbilirubinemia is present. If the complete blood count and initial tests for liver function and infectious hepatitis are unrevealing, the work-up typically proceeds to abdominal imaging by ultrasonography or computed tomographic scanning. In a few instances, more invasive procedures such as cholangiography or liver biopsy may be needed to arrive at a diagnosis. Jaundice is a yellowish pigmentation of skin and mucous membranes caused by hyperbilirubinemia, which itself has various causes. Jaundice related to maligt tumors is classified as obstructive jaundice. This disease proceeds from biliary tract obstruction and liver failure by progression of intrahepatic tumors, including metastases from other maligcies. Biliary tract cancer, pancreatic head cancer, or lymph nodes metastases from other sites of cancer are mainly responsible for the obstruction of the bile duct. In patients with obstructive jaundice, biliary drainage is often required in order to give treatments such as chemotherapy. In patients with biliary drainage, various complications arise, such as cholangitis due to obstruction ofa biliary stent, and bleeding from the ulcer due to a dislodged stent to the duodenum. It is crucial to manage those complications as oncologic emergencies. Jaundice of liver failure due to hepatic metastases is often observed in patients with gastrointestinal maligcies such as gastric cancer or colorectal cancer. Although chemotherapy is the usual application for those patients, useful anti-cancer agents are limited. It is crucial to diagnose and decide the best treatments as soon as possible for patients with very advanced hepatic metastases. Hepatitis viruses are infectious agents that can infect liver and cause inflammation. The infection triggers immune response against infected cells that leads to the destruction of hepatic cells. This destruction has two consequences: leaking ALT and AST liver enzymes which increases during the course of disease and accumulation of bilirubin- a red pigmented compound released from dead red cells- which causes the yellow coloration of eyes and skin. These viruses transmit through diverse routes i.e. blood transfusion, sexual contacts and consuming water or food contaminated by feces. Enteric hepatitis viruses use the latter route for transmission; hence their outbreaks are more common in underdeveloped countries. There are currently two distinguished enteric hepatitis viruses, hepatitis A and hepatitis E. These viruses belong to different family of viruses and their epidemiological characteristics are different. These infections can be diagnosed by an ELISA for IgM antibody. A vaccine has been developed in last decade of twentieth century for hepatitis A virus, which is administered mostly in the developed world i.e. U.S and Japan. Treatment for these infections is mostly supportive; however, in the case of fulmit hepatitis the liver transplantation might be necessary. BACKGROUND: Neonatal jaundice refers to yellow coloration of the skin and the sclera (whites of the eyes) of newborn babies that result from the accumulation of bilirubin in the skin and mucous membranes. Because bilirubin is potentially toxic to the central nervous system. Genetic disorders of bilirubin conjugation, particularly the common Gilbert's syndrome, can also contribute to neonatal hyperbilirubinemia. OBJECTIVE: The aim of this study was to evaluate the lipid per-oxidation and antioxidant enzyme activities in patients with neonatal jaundice before and after phototherapy. MATERIALS AND METHODS: The study includes 50 neonatal jaundice patients with average age 2-15 days. All patients of neonatal jaundice receiving phototherapy except feeding, cleaning. Subjects selected were from the patients attending Pediatrics Department. Plasma malondialdehyde (MDA), erythrocyte glutathione peroxidase (GPX), superoxide dismutase and catalase (CAT) to monitor the bilirubin level. RESULTS: The results show increased levels of bilirubin compared with controls (P < 0.001) shows the level of plasma MDA in control, before and after phototherapy. Represents the level of GPX was significantly increased in after the phototherapy group when compared with before phototherapy and control SPSS soft ware: (P < 0.001). Shows the reduced glutathione (GSH) level in plasma was significantly decreased in the after phototherapy group when compared with before phototherapy and control (P < 0.001). And finally with ascorbic acid and CAT. CONCLUSION: It is evident from the study that increased oxidative stress in neonatal jaundice babies leads to decrease in the levels of antioxidants like GSH and ascorbic acid and disturb their metabolism, that weaken their ability to fight the growing stress. Intense oxidative stress and decreased antioxidants may contribute to neural cell death and alter the erythrocytomembrane structure processing in neonatal jaundice. BACKGROUND: Febrile jaundice results clinically in generalized yellow coloration of the teguments and mucous membranes due to excess plasma bilirubin, accompanied by fever. Two types are found: conjugated and unconjugated bilirubin jaundice. Jaundice is a sign in several diseases due to viruses (viral hepatitis and arbovirus), parasites (malaria) and bacteria (leptospirosis). In the Central African Republic (CAR), only yellow fever is included on the list of diseases for surveillance. The aim of this study was to identify the other pathogens that can cause febrile jaundice, for better management of patients. METHODS: Between 2008 and 2010, 198 sera negative for yellow fever IgM were randomly selected from 2177 samples collected during yellow fever surveillance. Laboratory analyses targeted four groups of pathogens: hepatitis B, C, delta and E viruses; dengue, chikungunya, Zika, Crimean-Congo haemorrhagic fever, West Nile and Rift Valley arboviruses; malaria parasites; and bacteria (leptospirosis). RESULTS: Overall, 30.9% sera were positive for hepatitis B, 20.2% for hepatitis E, 12.3% for hepatitis C and 8.2% for malaria. The majority of positive sera (40.4%) were from people aged 16-30 years. Co-infection with at least two of these pathogens was also found. CONCLUSION: These findings suggest that a systematic investigation should be undertaken of infectious agents that cause febrile jaundice in the CAR. The presence of abnormal amounts of bilirubin in the blood stream and skin, usually referred to as hyperbilirubinemia, is associated with a wide range of pathologies that can pose considerable risks for human health. The early and effective screening of the severity degrees of this medical condition can play an important role on the selection of the appropriate treatment for the associated pathologies. This, in turn, can minimize the need for more aggressive and costly therapeutic interventions which can themselves pose considerable risks for morbidity and mortality. The current noninvasive protocols used to differentiate these severity degrees, however, are hindered by the relatively limited knowledge about the impact of different amounts of extravascular bilirubin on skin spectral responses and on the onset of jaundice, the resulting yellow-tinted skin appearance. In this paper, we address this open problem through controlled in silico experiments supported by measured data provided in the related literature. Our experimental findings bring biophysically-based insights to bear on the clarification of this biomedical entanglement, and unveil optical features that can potentially lead to more effective screening protocols for the noninvasive differentiation and monitoring of variable degrees of hyperbilirubinemia severity.

Is Tcf3 associated with the Wnt pathway?

Tcf3 is a component of the Wnt/β-catenin and Notch signaling pathways.

Menin is a tumor suppressor protein mutated in patients with multiple endocrine neoplasia type 1. We show that menin is essential for canonical Wnt/beta-catenin signaling in cultured rodent islet tumor cells. In these cells, overexpression of menin significantly enhances TCF gene assay reporter activity in response to beta-catenin activation. Contrastingly, inhibition of menin expression with Men1 siRNA decreases TCF reporter gene activity. Likewise, multiple endocrine neoplasia type 1 disease associated missense mutations of menin abrogate the ability to increase TCF reporter gene activity. We show that menin physically interacts with proteins involved in the canonical Wnt signaling pathway, including beta-catenin, TCF3 (TCFL1), and weakly with TCF4 (TCFL2). Menin overexpression increases expression of the Wnt/beta-catenin downstream target gene Axin2, which is associated with increased H3K4 trimethylation of the Axin2 gene promoter. Moreover, inhibition of menin expression by siRNA abrogates H3K4 trimethylation and Axin2 gene expression. Based on these studies, we hypothesized that Wnt signaling could inhibit islet cell proliferation because loss of menin function is thought to increase endocrine tumor cell proliferation. TGP61 rodent islet tumor cells treated with a glycogen synthase kinase 3beta inhibitor that increases Wnt pathway signaling had decreased cell proliferation compared with vehicle-treated cells. Collectively, these data suggest that menin has an essential role in Wnt/beta-catenin signaling through a mechanism that eventually affects histone trimethylation of the downstream target gene Axin2, and activation of Wnt/beta-catenin signaling inhibits islet tumor cell proliferation. Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and domit negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. The canonical Wnt/β-catenin signaling pathway classically functions through the activation of target genes by Tcf/Lef-β-catenin complexes. In contrast to β-catenin-dependent functions described for Tcf1, Tcf4 and Lef1, the known embryonic functions for Tcf3 in mice, frogs and fish are consistent with β-catenin-independent repressor activity. In this study, we genetically define Tcf3-β-catenin functions in mice by generating a Tcf3ΔN knock-in mutation that specifically ablates Tcf3-β-catenin. Mouse embryos homozygous for the knock-in mutation (Tcf3(ΔN/ΔN)) progress through gastrulation without apparent defects, thus genetically proving that Tcf3 function during gastrulation is independent of β-catenin interaction. Tcf3(ΔN/ΔN) mice were not viable, and several post-gastrulation defects revealed the first in vivo functions of Tcf3-β-catenin interaction affecting limb development, vascular integrity, neural tube closure and eyelid closure. Interestingly, the etiology of defects indicated an indirect role for Tcf3-β-catenin in the activation of target genes. Tcf3 directly represses transcription of Lef1, which is stimulated by Wnt/β-catenin activity. These genetic data indicate that Tcf3-β-catenin is not necessary to activate target genes directly. Instead, our findings support the existence of a regulatory circuit whereby Wnt/β-catenin counteracts Tcf3 repression of Lef1, which subsequently activates target gene expression via Lef1-β-catenin complexes. We propose that the Tcf/Lef circuit model provides a mechanism downstream of β-catenin stability for controlling the strength of Wnt signaling activity during embryonic development. Regulatory factors controlling stem cell identity and self-renewal are often active in aggressive cancers and are thought to promote their growth and progression. TCF3 (also known as TCF7L1) is a member of the TCF/LEF transcription factor family that is central in regulating epidermal and embryonic stem cell identity. We found that TCF3 is highly expressed in poorly differentiated human breast cancers, preferentially of the basal-like subtype. This suggested that TCF3 is involved in the regulation of breast cancer cell differentiation state and tumorigenicity. Silencing of TCF3 dramatically decreased the ability of breast cancer cells to initiate tumor formation, and led to decreased tumor growth rates. In culture, TCF3 promotes the sphere formation capacity of breast cancer cells and their self-renewal. We found that in contrast to ES cells, where it represses Wnt-pathway target genes, TCF3 promotes the expression of a subset of Wnt-responsive genes in breast cancer cells while repressing another distinct target subset. In the normal mouse mammary gland, Tcf3 is highly expressed in terminal end buds, structures that lead duct development. Primary mammary cells are dependent on Tcf3 for mammosphere formation, and its overexpression in the developing gland disrupts ductal growth. Our results identify TCF3 as a central regulator of tumor growth and initiation, and a novel link between stem cells and cancer. Wnt signaling pathways are a highly conserved pathway, which plays an important role from the embryonic development to bone formation. The effect of Wnt pathway on osteogenesis relies on their cellular environment and the expression of target genes. However, the molecular mechanism of that remains unclear. On the basis of the preliminary results, we observed the contrary effect of canonical Wnt signaling on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the different culture environment. Furthermore, we found that the expression level of miR-17 was also varied with the change in the culture environment. Therefore, we hypothesized that miR-17 and canonical Wnt signaling may have potential interactions, particularly the inner regulation relationship in different microenvironments. In this paper, we observed that canonical Wnt signaling promoted osteogenesis of PDLSCs in the fully culture medium, while inhibited it in the osteogenic differentiation medium. Interestingly, alteration in the expression level of endogenous miR-17 could partially reverse the different effect of canonical Wnt signaling. Furthermore, the role of miR-17 was because of its target gene TCF3 (transcription factor 3), a key transcription factor of canonical Wnt pathway. Overexpression of TCF3 attenuated the effect of miR-17 on modulating canonical Wnt signaling. Finally, we elucidated that TCF3 enhanced osteogenesis both in vitro and in vivo. In brief, the different level of miR-17 was the main cause of the different effect of canonical Wnt signaling, and TCF3 was the crucial node of miR-17-canonial Wnt signaling regulation loop. This understanding of microRNAs regulating signaling pathways in different microenvironments may pave the way for fine-tuning the process of osteogenesis in bone-related disorders. Canonical Wnt signaling supports the pluripotency of embryonic stem cells (ESCs) but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Furthermore, canonical Wnt signaling upregulates the activity of the Pou5f1 distal enhancer via the Sox motif in ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor essential for ESC culture suggests MEK/ERK signaling and canonical Wnt signaling combine to promote ESC differentiation. Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells. Embryonic stem cells (ESCs) have both the ability to self-renew and to differentiate into various cell lineages. Retinoic acid (RA), a metabolite of Vitamin A, has a critical function in initiating lineage differentiation of ESCs through binding to the retinoic acid receptors. Additionally, the Wnt signaling pathway plays a role in pluripotency and differentiation, depending on the activation status of the canonical and noncanonical pathways. The activation of the canonical Wnt signaling pathway, which requires the nuclear accumulation of β-catenin and its interaction with Tcf1/Lef at Wnt response elements, is involved in ESC stemness maintece. The noncanonical Wnt signaling pathway, through actions of Tcf3, can antagonize the canonical pathway. We show that RA activates the noncanonical Wnt signaling pathway, while concomitantly inhibiting the canonical pathway. RA increases the expression of ligands and receptors of the noncanonical Wnt pathway (Wnt 5a, 7a, Fzd2 and Fzd6), downstream signaling, and Tcf3 expression. RA reduces the phosphorylated β-catenin levels by fourfold, although total β-catenin levels do not change. We show that RA signaling increases the dissociation of Tcf1 and the association of Tcf3 at promoters of genes that regulate stemness (e.g., NR5A2, Lrh-1) or differentiation (e.g. Cyr61, Zic5). Knockdown of Tcf3 increases Lrh-1 transcript levels in mESCs and prevents the RA-associated, fourfold increase in Zic5, indicating that RA requires Tcf3 to effect changes in Zic5 levels. We demonstrate a novel role for RA in altering the activation of these two Wnt signaling pathways and show that Tcf3 mediates some actions of RA during differentiation. During mouse neocortical development, the Wnt-β-catenin signaling pathway plays essential roles in various phenomena including neuronal differentiation and proliferation of neural precursor cells (NPCs). Production of the appropriate number of neurons without depletion of the NPC population requires precise regulation of the balance between differentiation and maintece of NPCs. However, the mechanism that suppresses Wnt signaling to prevent premature neuronal differentiation of NPCs is poorly understood. We now show that the HMG box transcription factor Tcf3 (also known as Tcf7l1) contributes to this mechanism. Tcf3 is highly expressed in undifferentiated NPCs in the mouse neocortex, and its expression is reduced in intermediate neuronal progenitors (INPs) committed to the neuronal fate. We found Tcf3 to be a repressor of Wnt signaling in neocortical NPCs in a reporter gene assay. Tcf3 bound to the promoter of the proneural bHLH gene Neurogenin1 (Neurog1) and repressed its expression. Consistent with this, Tcf3 repressed neuronal differentiation and increased the self-renewal activity of NPCs. We also found that Wnt signal stimulation reduces the level of Tcf3, and increases those of Tcf1 (also known as Tcf7) and Lef1, positive mediators of Wnt signaling, in NPCs. Together, these results suggest that Tcf3 antagonizes Wnt signaling in NPCs, thereby maintaining their undifferentiated state in the neocortex and that Wnt signaling promotes the transition from Tcf3-mediated repression to Tcf1/Lef1-mediated enhancement of Wnt signaling, constituting a positive feedback loop that facilitates neuronal differentiation. BACKGROUND AND OBJECTIVES: Transcription factor 3 (TCF3) implicates Wnt signaling pathway and regulates E-cadherin expression, which is involved in aggressiveness of tumors. This study aims to investigate the role of TCF3 in predicting prognosis of patients with stage II and III colorectal cancer (CRC). METHODS: Real-Time quantitative PCR was performed in 64 fresh CRC tissues and 6 cell lines to examine TCF3 mRNA expression. TCF3 protein expression dynamics were detected by immunohistochemistry of 118 paraffin-embedded specimens, and the clinical significance of TCF3 was assessed by clinical correlation and Kaplan-Meier analyses. Aberrant hypomethylation of TCF3 promoter was also investigated using bisulfite sequencing and methylation specific PCR. RESULTS: The up-regulation of TCF3 mRNA was frequently detected both in CRC tissues with recurrence and metastasis-derived cell lines. The expression level of TCF3 protein was significantly correlated with histological type (P = 0.038) and disease-free survival time (P = 0.002). Higher TCF3 expression indicated poor prognostic outcomes (P<0.05, log-rank test). Multivariate analysis also showed strong TCF3 protein expression and perineural invasion were independent adverse prognosticators in CRC (P = 0.010, 0.000). Moreover, it was showed that promoter hypomethylation of TCF3 is associated with its up-expression. CONCLUSIONS: This study highlighted the prognostic value of TCF3 in stage II and III CRC. The up-regulation of TCF3, which is mainly caused by promoter hypomethylation, is one of the molecular mechanisms involved in the development and progression of CRC. The rat whisker hair follicle (HF) is a model for studying the reconstruction of the HF or dermal papilla (DP), and involves the Wnt/β-catenin signaling pathway, which is a key pathway in HF development and HF cycling after birth. It has been reported that Wnt/catenin signaling plays an indispensable role in human or rat pelages development and postnatal growth. However, the distribution of some Wnt/β-catenin signaling pathway factors and their relationship with the epithelial stem cell markers in whisker follicles has not been characterized. In this study, we investigated the immunolocalization of Wnt/catenin signaling pathway members, including Wnt10b, Wnt10a, Wnt5a, β-catenin, and downstream lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 3 (TCF3), as well as, HF stem-cell markers CD34, CK15 and proliferating cell nuclear antigen (PCNA) protein, in rat anagen phase whisker follicles. β-catenin, Wnt5a, Wnt10b, Wnt10a, LEF1, and TCF3 were expressed in the outer root sheath (ORS), inner root sheath, matrix and hair shaft of anagen follicles. β-catenin, Wnt10b, LEF1, and TCF3 were highly expressed and Wnt5a and Wnt10a weakly expressed in DP and dermal sheath (DS) regions. The expression of α-smooth muscle actin was strong in the lower DS and it was also detected in some DP cells. CD34, CK15 and PCNA were all expressed in the ORS; and CD34 and PCNA were also detected in the matrix, however CD34 was extensively expressed in DP and DS regions. Our studies located the position of Wnts, downstream LEF1 and TCF3 and stem cell marker proteins, which provide new information in understanding the role of the Wnt singaling pathway in whisker follicles' growth. Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors. B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with TCF3-PBX1 fusion gene expression has constitutively elevated levels of Wnt16b and ROR1 (receptor tyrosine kinase-like orphan receptor), a ligand and a receptor from the Wnt signaling pathway, respectively. Although survival rate is usually high after the initial chemotherapy, many TCF3-PBX1 BCP-ALL patients relapse and subsequently develop treatment resistance, resulting in poor prognosis. Here, we aimed to investigate the molecular signaling associated with Wnt16b and ROR1 overexpression in TCF3-PBX1 cell lines and primary samples, and to identify effective treatment options via ROR1 targeting. We detected higher ROR1 expression on TCF3-PBX1 leukemic cells even at a later stage of patient relapse, providing a strong rationale for the use of ROR1-targeted therapy. We found that Wnt5a-ROR1 signaling enhances proliferation of TCF3-PBX1 cells via RhoA/Rac1 GTPases activation and STAT3 upregulation. Wnt16b also activated the RhoA/Rac1 signaling cascade suggesting the activation of a non-canonical Wnt pathway in TCF3-PBX1 cells. Wnt16 could interact with ROR1 but not in TCF3-PBX1 cells, suggesting that Wnt5a is the ligand signaling via ROR1 in TCF3-PBX1 cells. By high throughput drug-sensitivity testing of TCF3-PBX1 cells before and after ROR1 knockdown we found that targeting ROR1 significantly improves the therapeutic efficacy of Bcl-2 family inhibitors venetoclax and navitoclax, and this synergism was confirmed ex vivo using a drug-resistant primary sample from a relapsed TCF3-PBX1 patient. Our work underlines a new type of targeted combination therapy that could be clinically advantageous for patients with TCF3-PBX1 BCP-ALL.

Have the rotavirus vaccines changed the predominant rotavirus genotypes?

The increased diversity and differences in genotype dominance observed in states using RotaTeq (G12P[8]), and in states and territories using Rotarix (equine-like G3P[8] and G2P[4]), suggest that these vaccines exert different immunological pressures that influence the diversity of rotavirus strains.

BACKGROUND: Introduction of rotavirus vaccines into national immunization programs (NIPs) could result in strain selection due to vaccine-induced selective pressure. This study describes the distribution and diversity of rotavirus genotypes before and after rotavirus vaccine introduction into the Australian NIP. State-based vaccine selection facilitated a unique comparison of diversity in RotaTeq and Rotarix vaccine states. METHODS: From 1995 to 2015, the Australian Rotavirus Surveillance Program conducted genotypic analysis on 13051 rotavirus-positive samples from children <5 years of age, hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined using serological and heminested multiplex reverse-transcription polymerase chain reaction assays. RESULTS: G1P[8] was the domit genotype nationally in the prevaccine era (1995-2006). Following vaccine introduction (2007-2015), greater genotype diversity was observed with fluctuating genotype domice. Genotype distribution varied based on the vaccine implemented, with G12P[8] domit in states using RotaTeq, and equine-like G3P[8] and G2P[4] domit in states and territories using Rotarix. CONCLUSIONS: The increased diversity and differences in genotype domice observed in states using RotaTeq (G12P[8]), and in states and territories using Rotarix (equine-like G3P[8] and G2P[4]), suggest that these vaccines exert different immunological pressures that influence the diversity of rotavirus strains circulating in Australia.

Which syndrome is caused by dysfunction of the ciliary ARMC9/TOGARAM1 protein?

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome.

Intellectual disability (ID) refers to deficits in mental abilities, social behavior, and motor skills to perform activities of daily living as compared to peers. Numerous genetic and environmental factors may be responsible for ID. We report on elucidation of molecular basis for syndromic ID associated with ptosis, polydactyly, and MRI features suggestive of Joubert syndrome using homozygosity mapping followed by exome sequencing. The analysis revealed a novel synonymous variation p.T293T (c.879G>A) which leads to a splicing defect in ARMC9 gene. The variant is present in conserved region of ARM domain of ARMC9 protein, which is predicted to form a platform for protein interaction. This domain is likely to be altered in patient due to splicing defect caused by this synonymous variation. Our report of variant in ARMC9 Leading to Joubert syndrome phenotype (JS30), elucidates the genetic heterogeneity of Joubert syndrome, and expands the gene list for ciliopathies. Author information: (1)Department of Human Genetics and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands. (2)Department of Pediatrics, University of Washington, Seattle, Washington, USA. (3)Institute of Medical Genetics, and. (4)Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland. (5)Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. (6)Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. (7)Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, United Kingdom. (8)Department of Paediatric Neurology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom. (9)Department of Human Genetics, Medical Research Institute, Alexandria University, Alexandria, Egypt. (10)The University of Washington Center for Mendelian Genomics is detailed in Supplemental Acknowledgments. (11)University of Washington Center for Mendelian Genomics, Seattle, Washington, USA. (12)Department of Genome Sciences, University of Washington, Seattle, Washington, USA. (13)Medical Proteome Center, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany. (14)Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia. (15)Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, Washington, USA.

Does head ct increase brain tumor risk?

Yes, there appears to be a small but higher than expected lifetime risk of secondary brain tumors in persons who underwent CT scans during childhood.

OBJECTIVE: We aimed to estimate the risks of radiation exposure from a single head CT scan to children of different ages. MATERIALS AND METHODS: We constructed a multistate time-dependent Markov model to simulate the course of children exposed to a head CT. The relevant literature was reviewed for probabilities, which were used to calculate tumor types, latencies after exposure and outcomes in the model. Where multiple approximations of the same probability had been reported, meta-analytic techniques were employed to compute pooled estimates. The model was then used to calculate the effect of the radiation exposure on life expectancy and quality of life for children following head CT at different ages. RESULTS: The tumors likely to be induced by low-level cranial irradiation include thyroid carcinoma (47%), meningioma (34%) and glioma (19%). According to the model, a single head CT is likely to cause one of these tumors in 0.22% of 1-year-olds, 30% of whom will consequently die. The exposure will shorten the life expectancy of all exposed 1-year-olds by an average of 0.04 years and their expected quality of life by 0.02 quality-adjusted life years. The risks of radiation exposure diminish for older children. CONCLUSIONS: The model predicts that the effective radiation dose from a single head CT is capable of inducing a thyroid or brain tumor in an infant or child. These tumors can severely impact both quality of life and life expectancy. Care should be taken before ordering CT scans in children, particularly in infants and toddlers. A complete assessment of late effects of X-ray diagnostics should take into account that radiation sensitivity varies considerably for the different ages at exposure and, furthermore, that not only maligt diseases but also benign neoplasms are induced which also may lead to severe detriment of the patient. Risk estimates are derived for paediatric head CTs as well as for brain tumours in adults. Dose-effect relationships for tumours of the brain, skin, thyroid, and other sites of the head region, leukaemia, and cataracts are taken from the literature. On the basis of estimates for Germany about the number of head scans, the annual rate of radiation-induced diseases is calculated. 1,000 annual paediatric CT investigations of the skull will lead to about 3 excess neoplasms in the head region, i.e., the probability of an induced late effect must be suspected in the range of some thousandths. Additionally, a relevant increase of cataracts must be considered. The radiation-induced occurrence of meningiomas and other brain tumours most probably contributes to the continuously increasing incidence of these diseases which is observed in several industrial nations, as well as the exposure of the bone marrow by CT to the increase of childhood leukaemia. This study aims to investigate the contribution of diagnostic exposures to the rising rates of brain tumours and other neoplasms which are observed in several industrial nations. Included are benign tumours in the head and neck region and cataracts which are neglected in usual risk estimates by international and national radiation protection committees. Dose-effect relationships for tumours of the brain, skin, thyroid and other sites of the head region, leukaemia and cataracts are taken from the literature. Risk estimates are derived for paediatric head computed tomographies (CTs) as well as for brain tumours in adults. On the basis of estimates for Germany about the number of head scans, the annual rate of radiation-induced diseases is calculated. About 1000 annual paediatric CT investigations of the skull will lead to about three excess neoplasms in the head region, i.e. the probability of an induced late effect must be suspected in the range of some thousands. Additionally, a relevant increase of cataracts must be considered. The radiation-induced occurrence of meningiomas and other brain tumours most probably contributes to the continuously increasing incidence of these diseases which is observed in several industrial nations, as well as the exposure of the bone marrow by CT to the increase of childhood leukaemia. BACKGROUND AND PURPOSE: Neurointerventions in children have dramatically improved the clinical outlook for patients with previously intractable cerebrovascular conditions, such as vein of Galen malformations and complex arteriovenous fistulas. However, these complex and sometimes lengthy procedures are performed under fluoroscopic guidance and thus unavoidably expose vulnerable pediatric patients to the effects of ionizing radiation. Recent epidemiologic evidence from a national registry of children who underwent CT scans suggests a higher-than-expected incidence of secondary tumors. We sought to calculate the predicted risk of secondary tumors in a large cohort of pediatric neurointerventional patients. MATERIALS AND METHODS: We reviewed our cohort of pediatric neurointerventions, tabulated radiation dose delivered to the skin, and calculated the range of likely brain-absorbed doses by use of previously developed mathematical models. The predicted risk of secondary tumor development as a function of brain-absorbed dose in this cohort was then generated by use of the head CT registry findings. RESULTS: Maximal skin dose and brain-absorbed doses in our cohort were substantially lower than have been previously described. However, we found 1) a statistically significant correlation between radiation dose and age at procedure, as well as number and type of procedures, and 2) a substantial increase in lifetime predicted risk of tumor above baseline in the cohort of young children who undergo neurointerventions. CONCLUSIONS: Although neurointerventional procedures have dramatically improved the prognosis of children facing serious cerebrovascular conditions, the predicted risk of secondary tumors, particularly in the youngest patients and those undergoing multiple procedures, is sobering. BACKGROUND: To evaluate the possible association between paediatric head computed tomography (CT) examination and increased subsequent risk of maligcy and benign brain tumour. METHODS: In the exposed cohort, 24 418 participants under 18 years of age, who underwent head CT examination between 1998 and 2006, were identified from the Taiwan National Health Insurance Research Database (NHIRD). Patients were followed up until a diagnosis of maligt disease or benign brain tumour, withdrawal from the National Health Insurance (NHI) system, or at the end of 2008. RESULTS: The overall risk was not significantly different in the two cohorts (incidence rate=36.72 per 100 000 person-years in the exposed cohort, 28.48 per 100 000 person-years in the unexposed cohort, hazard ratio (HR)=1.29, 95% confidence interval (CI)=0.90-1.85). The risk of benign brain tumour was significantly higher in the exposed cohort than in the unexposed cohort (HR=2.97, 95% CI=1.49-5.93). The frequency of CT examination showed strong correlation with the subsequent overall risk of maligcy and benign brain tumour. CONCLUSIONS: We found that paediatric head CT examination was associated with an increased incidence of benign brain tumour. A large-scale study with longer follow-up is necessary to confirm this result. OBJECTIVE: To perform a systematic review to evaluate the risk of maligcy associated with computed tomography (CT) of the head and/or neck in infants, children, and adolescents. DATA SOURCES: Pubmed, EMBASE, and the Cochrane Library were assessed from the date of their inception to January 2014. Additionally, manual searches of bibliographies were performed and topic experts were contacted. REVIEW METHODS: Data were obtained from studies measuring or estimating the risks of maligcy associated with radiation from head and/or neck CT in pediatric populations according to an a priori protocol. Two independent evaluators corroborated the extracted data. RESULTS: There were 16 criterion-meeting studies that included data from n = 858,815 patients. The radiation-related risk of maligcy was estimated using primary patient data for both the exposure and outcome in a minority of studies, with most analyses utilizing mathematical modeling techniques. The data regarding otolaryngology-specific studies were limited and suggested a borderline significant increase in the risk of all combined cancers after facial CT (incidence rate ratio [IRR] = 1.14; 95% CI, 1.01-1.28) and neck/spine CT (IRR = 1.13; 95% CI, 1.00-1.28). Cohort data suggest that 1 excess brain maligcy occurred after 4000 brain CTs (40 mSv per scan) and that the estimated risk in the 10 years following CT exposure was 1 brain tumor per 10,000 patients exposed to a 10 mGy scan at less than 10 years of age. CONCLUSION: Detailed understanding of any potential maligcy risk associated with pediatric imaging of the head and neck furthers our ability to engage in rational, shared, informed decision making with families considering CT scan. BACKGROUND: Computed tomography (CT), a strong diagnostic tool, delivers higher radiation doses than most imaging modalities. As CT use has increased rapidly, radiation protection is important, particularly among children. We evaluate leukemia and brain tumor risk following exposure to low-dose ionizing radiation from CT scans in childhood. METHODS: For a nationwide retrospective cohort of 168 394 children who received one or more CT scans in a Dutch hospital between 1979 and 2012 who were younger than age 18 years, we obtained cancer incidence, vital status, and confounder information by record linkage with external registries. Standardized incidence ratios were calculated using cancer incidence rates from the general Dutch population. Excess relative risks (ERRs) per 100 mGy organ dose were calculated with Poisson regression. All statistical tests were two-sided. RESULTS: Standardized incidence ratios were elevated for all cancer sites. Mean cumulative bone marrow doses were 9.5 mGy at the end of follow-up, and leukemia risk (excluding myelodysplastic syndrome) was not associated with cumulative bone marrow dose (44 cases). Cumulative brain dose was on average 38.5 mGy and was statistically significantly associated with risk for maligt and nonmaligt brain tumors combined (ERR/100 mGy: 0.86, 95% confidence interval = 0.20 to 2.22, P = .002, 84 cases). Excluding tuberous sclerosis complex patients did not substantially change the risk. CONCLUSIONS: We found evidence that CT-related radiation exposure increases brain tumor risk. No association was observed for leukemia. Compared with the general population, incidence of brain tumors was higher in the cohort of children with CT scans, requiring cautious interpretation of the findings. BACKGROUND: Advanced diagnostic imaging has provided tremendous benefits; however, increased use of ionizing radiation modalities such as cranial computed tomography (CT) may be associated with an increased risk of developing central nervous system tumors. METHODS: A literature review identified studies published for more than the last 50 years from 1968 to 2018 that explored the association between head CT scans and developing central nervous system tumors in pediatrics. We reviewed seven studies that described and analyzed the risk of brain tumors. RESULTS: A positive correlation between exposure to CT scans and developing central nervous system tumors was evident in all cohorts. The strength of the association varied across the studies. Exclusion of patients with predisposing factors to central nervous system tumors was examined in four studies with a decreased risk to develop central nervous system tumors noted in three studies. Two studies reported nonsignificant reduction in the excess relative risk per milliGray of brain dose after adjusting for predisposing factors, whereas the reduction was significant in one study. The frequency of CT exposure was proportional to the risk of developing tumors in two studies although not significantly maintained in two other studies. Gender had no significant effect on the central nervous system tumor risk. The calendar year at the time of imaging showed decreasing risk in those exposed to CT in more recent years compared with prior decades. CONCLUSIONS: Prospective epidemiologic studies are needed to examine the precise carcinogenic effect of exposure to ionizing radiation and help tailor further preventive measures.

Which main viral protein is targeted by the drug remdesivir?

Viral Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.

Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.

What can be isolated from Pleurotus mutilus?

Pleuromutilins are antibiotics, isolated from the fungus, Pleurotus mutilus, that selectively inhibit bacterial translation and are semisynthetic derivatives of the naturally occurring tricyclic diterpenoid pleuromutilins.

Pleuromutilins are antibiotics that selectively inhibit bacterial translation and are semisynthetic derivatives of the naturally occurring tricyclic diterpenoid pleuromutilin, which received its name from the pleuromutilin-producing fungus Pleurotus mutilus Tiamulin and valnemulin are two established derivatives in veterinary medicine for oral and intramuscular administration. As these early pleuromutilin drugs were developed at a time when companies focused on major antibacterial classes, such as the β-lactams, and resistance was not regarded as an issue, interest in antibiotic research including pleuromutilins was limited. Over the last decade or so, there has been a resurgence in interest to develop this class for human use. This has resulted in a topical derivative, retapamulin, and additional derivatives in clinical development. The most advanced compound is lefamulin, which is in late-stage development for the intravenous and oral treatment of community-acquired bacterial pneumonia and acute bacterial skin infections. Overall, pleuromutilins and, in particular, lefamulin are characterized by potent activity against Gram-positive and fastidious Gram-negative pathogens as well as against mycoplasmas and intracellular organisms, such as Chlamydia spp. and Legionella pneumophila Pleuromutilins are unaffected by resistance to other major antibiotic classes, such as macrolides, fluoroquinolones, tetracyclines, β-lactam antibiotics, and others. Furthermore, pleuromutilins display very low spontaneous mutation frequencies and slow, stepwise resistance development at sub-MIC in vitro. The potential for resistance development in clinic is predicted to be slow as confirmed by extremely low resistance rates to this class despite the use of pleuromutilins in veterinary medicine for >30 years. Although rare, resistant strains have been identified in human- and livestock-associated environments and as with any antibiotic class, require close monitoring as well as prudent use in veterinary medicine. This review focuses on the structural characteristics, mode of action, antibacterial activity, and resistance development of this potent and novel antibacterial class for systemic use in humans. BACKGROUND Pleuromutilin is a natural tricyclic, derived from the fungus, Pleurotus mutilus. This study aimed to investigate the effects of pleuromutilin on migration and proliferation of A2780 and Caov-3 human ovarian carcinoma cells and the growth of A2780 tumor xenografts in mice and the molecular mechanisms involved. MATERIAL AND METHODS A2780 and Caov-3 human ovarian carcinoma cells were cultured with and without 40, 160, and 200 μM of pleuromutilin. The Edu fluorescence assay, the wound-healing assay, and Matrigel were used to measure A2780 and Caov-3 cell proliferation, migration, invasion, and adhesion in vitro, respectively. Western blot measured protein levels of FAK, p-FAK, MMP-2, and MMP-9. A2780 cells were injected subcutaneously into mice to determine the effects of pleuromutilin on the growth of tumor xenografts. RESULTS Pleuromutilin significantly reduced A2780 and Caov-3 cell proliferation at 48 h in a dose-dependent manner (P<0.05), and at 200 μM, pleuromutilin reduced cell proliferation by 21.43% and 23.65%, respectively. Treatment of A2780 cells with pleuromutilin significantly reduced cell migration, invasion, and adhesion and the expression of p-FAK, MMP-2, and MMP-9 compared with untreated controls. In the mouse tumor xenograft model, treatment with pleuromutilin significantly reduced tumor size compared with the untreated group and inhibited tumor metastasis to the intestine, spleen, and peritoneal cavity. CONCLUSIONS In A2780 and Caov-3 human ovarian carcinoma cells, pleuromutilin inhibited cell proliferation, migration, invasion, and adhesion in a dose-dependent manner, and reduced tumor growth and metastases in a mouse A2780 cell tumor xenograft model.

Which type of pluripotency is Otx2 associated with?

transcription factor Otx2 acts as a negative switch in the regulation of transition from naive to primed pluripotency. Otx2 and Oct4 drive early activation during embryonic stem cell transition from naive pluripotency.

Sall1 is a multi-zinc finger transcription factor that regulates kidney organogenesis. It is considered to be a transcriptional repressor, preferentially localized on heterochromatin. Mutations or deletions of the human SALL1 gene are associated with the Townes-Brocks syndrome. Despite its high expression, no function was yet assigned for Sall1 in embryonic stem (ES) cells. In the present study, we show that Sall1 is expressed in a differentiation-dependent manner and physically interacts with Nanog and Sox2, two components of the core pluripotency network. Genome-wide mapping of Sall1-binding loci has identified 591 genes, 80% of which are also targeted by Nanog. A large proportion of these genes are related to self-renewal and differentiation. Sall1 positively regulates and synergizes with Nanog for gene transcriptional regulation. In addition, our data show that Sall1 suppresses the ectodermal and mesodermal differentiation. Specifically, the induction of the gastrulation markers T brachyury, Goosecoid, and Dkk1 and the neuroectodermal markers Otx2 and Hand1 was inhibited by Sall1 overexpression during embryoid body differentiation. These data demonstrate a novel role for Sall1 as a member of the transcriptional network that regulates stem cell pluripotency. Mouse embryonic stem cells (ESCs) represent the naïve ground state of the preimplantation epiblast and epiblast stem cells (EpiSCs) represent the primed state of the postimplantation epiblast. Studies have revealed that the ESC state is maintained by a dynamic mechanism characterized by cell-to-cell spontaneous and reversible differences in sensitivity to self-renewal and susceptibility to differentiation. This metastable condition ensures indefinite self-renewal and, at the same time, predisposes ESCs for differentiation to EpiSCs. Despite considerable advances, the molecular mechanism controlling the ESC state and pluripotency transition from ESCs to EpiSCs have not been fully elucidated. Here we show that Otx2, a transcription factor essential for brain development, plays a crucial role in ESCs and EpiSCs. Otx2 is required to maintain the ESC metastable state by antagonizing ground state pluripotency and promoting commitment to differentiation. Furthermore, Otx2 is required for ESC transition into EpiSCs and, subsequently, to stabilize the EpiSC state by suppressing, in pluripotent cells, the mesendoderm-to-neural fate switch in cooperation with BMP4 and Fgf2. However, according to its central role in neural development and differentiation, Otx2 is crucially required for the specification of ESC-derived neural precursors fated to generate telencephalic and mesencephalic neurons. We propose that Otx2 is a novel intrinsic determit controlling the functional integrity of ESCs and EpiSCs. Reported pig induced pluripotent stem cells (piPSCs) have shown either a bFGF-dependent state with human embryonic stem cell (ESC) and mouse epiblast stem cell (EpiSC) morphology and molecular features or piPSCs exist in a LIF-dependent state and resemble fully reprogrammed mouse iPSCs. The features of authentic piPSCs and molecular events during the reprogramming are largely unknown. In this study, we assessed the transcriptome profile of multiple piPSC lines derived from different laboratories worldwide and compared to mouse and human iPSCs to determine the molecular signaling pathways that might play a central role in authentic piPSCs. The results demonstrated that the up-regulation of endogenous epithelial cells adhesion molecule (EpCAM) was correlated with the pluripotent state of pig pluripotent cells, which could be utilized as a marker for evaluating pig cell reprogramming. Comparison of key signaling pathways JAK-STAT, NOTCH, TGFB1, WNT and VEGF in pig, mouse and human iPSCs showed that the core transcriptional network to maintain pluripotency and self-renewal in pig were different from that in mouse, but had significant similarities to human. Pig iPSCs, which lacked expression of specific naïve state markers KLF2/4/5 and TBX3, but expressed the primed state markers of Otx2 and Fabp7, share defining features with human ESCs and mouse EpiSCs. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) were silenced in piPSCs as previously seen in mouse iPSCs that have limited ability to contribute to chimaeras. These key differences in naïve state gene and imprinting gene expression suggests that so far known piPSC lines may be more similar to primed state cells. The primed state of these cells may potentially explain the rare ability of piPSCS to generate chimeras and cloned offspring. Naive and primed pluripotency is characterized by distinct signaling requirements, transcriptomes, and developmental properties, but both cellular states share key transcriptional regulators: Oct4, Sox2, and Nanog. Here, we demonstrate that transition between these two pluripotent states is associated with widespread Oct4 relocalization, mirrored by global rearrangement of enhancer chromatin landscapes. Our genomic and biochemical analyses identified candidate mediators of primed state-specific Oct4 binding, including Otx2 and Zic2/3. Even when differentiation cues are blocked, premature Otx2 overexpression is sufficient to exit the naive state, induce transcription of a substantial subset of primed pluripotency-associated genes, and redirect Oct4 to previously inaccessible enhancer sites. However, the ability of Otx2 to engage new enhancer regions is determined by its levels, cis-encoded properties of the sites, and the signaling environment. Our results illuminate regulatory mechanisms underlying pluripotency and suggest that the capacity of transcription factors such as Otx2 and Oct4 to pioneer new enhancer sites is highly context dependent. Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency. Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that the reactivation of anterior neural transcription factor OTX2, eye field transcription factor RX1 and SIX3 in iPSCs is a feature of pluripotency acquisition and predetermines the potential of RPE differentiation. Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development. The enhancer landscape is dramatically restructured as naive preimplantation epiblasts transition to the post-implantation state of primed pluripotency. A key factor in this process is Otx2, which is upregulated during the early stages of this transition and ultimately recruits Oct4 to a different set of enhancers. In this study, we discover that the acetylation status of Oct4 regulates the induction of the primed pluripotency gene network. Maintece of the naive state requires the NAD-dependent deacetylase, SirT1, which deacetylates Oct4. The activity of SirT1 is reduced during the naive-to-primed transition; Oct4 becomes hyper-acetylated and binds to an Otx2 enhancer to induce Otx2 expression. Induction of Otx2 causes the reorganization of acetylated Oct4 and results in the induction of the primed pluripotency gene network. Regulation of Oct4 by SirT1 may link stem cell development to environmental conditions, and it may provide strategies to manipulate epiblast cell state. Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of their expression and the mechanisms underlying their functions are still not definitively understood. Here we address the above-mentioned issues in the first steps of mouse embryonic stem cell (ESC) differentiation. We observed that the expression of Lin28 genes is transiently induced soon after the exit of ESCs from the naive ground state and that this induction is due to the Hmga2-dependent engagement of Otx2 with enhancers present at both Lin28 gene loci. These mechanisms are crucial for Lin28 regulation, as demonstrated by the abolishment of the Lin28 accumulation in Otx2- or Hmga2-knockout cells compared to the control cells. We have also found that Lin28 controls Hmga2 expression levels during ESC differentiation through a let-7-independent mechanism. Indeed, we found that Lin28 proteins bind a highly conserved element in the 3' UTR of Hmga2 mRNA, and this provokes a down-regulation of its translation. This mechanism prevents the inappropriate accumulation of Hmga2 that would modify the proliferation and physiological apoptosis of differentiating ESCs. In summary, we demonstrated that during ESC differentiation, Lin28 transient induction is dependent on Otx2 and Hmga2 and prevents an inappropriate excessive rise of Hmga2 levels.-Parisi, S., Passaro, F., Russo, L., Musto, A., Navarra, A., Romano, S., Petrosino, G., Russo, T. Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events. The transcription factor Otx2 acts as a negative switch in the regulation of transition from naive to primed pluripotency in mouse pluripotent stem cells. However, the molecular features and function of porcine OTX2 have not been well elucidated in porcine-induced pluripotent stem cells (piPSCs). By studying high-throughput transcriptome sequencing and interfering endogenous OTX2 expression, we demonstrate that OTX2 is able to downgrade the self-renewal of piPSCs. OTX2 is highly expressed in porcine brain, reproductive tissues, and preimplantation embryos, but is undetectable in fibroblasts and most somatic tissues. However, the known piPSC lines reported previously produced different levels of OTX2 depending on the induction procedures and culture conditions. Overexpression of porcine OTX2 can reduce the percentage of alkaline phosphatase-positive colonies and downregulate NANOG and OCT4 expression. In contrast, knockdown of OTX2 can significantly increase endogenous expressions of NANOG, OCT4, and ESRRB, and stabilize the pluripotent state of piPSCs. On the other hand, NANOG can directly bind to the OTX2 promoter as shown in ChIP-seq data and repress OTX2 promoter activity in a dose-dependent manner. These observations indicate that OTX2 and NANOG can form a negative feedback circuitry to regulate the pluripotency of porcine iPS cells. Embryonic stem cells (ESCs) cultured in leukemia inhibitory factor (LIF) plus fetal bovine serum (FBS) exhibit heterogeneity in the expression of naive and primed transcription factors. This heterogeneity reflects the dynamic condition of ESCs and their versatility to promptly respond to signaling effectors promoting naive or primed pluripotency. Here, we report that ESCs lacking Nanog or overexpressing Otx2 exhibit an early primed identity in LIF + FBS and fail to convert into 2i-induced naive state. Conversely, Otx2-null ESCs possess naive identity features in LIF + FBS similar to Nanog-overexpressing ESCs and convert poorly into FGF-induced early primed state. When both Nanog and Otx2 are inactivated, ESCs cultured in LIF + FBS exhibit primed identity and weakened ability to convert into naive state. These data suggest that, through mutual antagonism, NANOG and OTX2 specify the heterogeneous identity of ESCs cultured in LIF + FBS and individually predispose them for optimal response to naive or primed inducing factors. Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. Here, we use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regulators including a zinc finger protein, ZNF207, that plays diverse roles during development. In hESCs, ZNF207 partners with master pluripotency TFs to govern self-renewal and pluripotency while simultaneously controlling commitment of cells towards ectoderm through direct regulation of neuronal TFs, including OTX2. The distinct roles of ZNF207 during differentiation occur via isoform switching. Thus, a distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm. The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predomit attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis. Porcine OTX2 was found to be highly activated in porcine iPS cells (piPSCs) that were reported by different laboratories worldwide. To reveal the regulatory function of OTX2 in porcine reprogrammed cells, we screened porcine miRNA-seq databases and found two miRNAs, miR-1343 and miR-545, that could specifically bind to 3'UTR of OTX2 and suppress endogenous OTX2 expression in piPSCs. Knockdown of OTX2 by miR-1343 and miR-545 could significantly increase the expression of SOX2 and ESRRB, but did not alter the expressions of OCT4 and KLF4, and improve the pluripotency of piPSCs. The promoter-based assays showed that OTX2 potentially bound to the promoter region of SOX2 and ESRRB and suppressed their expression. On the other hand, SOX2 could interact with OTX2 promoter. Ectopic expression of SOX2 could significantly decrease OTX2 promoter activity, showing that there is a negative feedback loop between SOX2 and OTX2. Additionally, SOX2 and ESRRB significantly stimulated miR-1343 expression in piPSCs, but OTX2 down regulated the expression of miR-1343 in either direct or indirect manners. In summary, this study demonstrates that there is a regulatory network mediated by miR-1343, in which downregulation of OTX2 by miR-1343 can elevate the expression of pluripotent genes that were then sustain the pluripotency of piPSCs.

Can AGY be used as antidiuretic replacement therapy?

No, AGY is an oral egg yolk anti-gliadin antibody used to neutralize gluten. It is used in patients with celiac disease.

What is caused by de novo sox6 variants?

SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. De novo variants of the SOX6 gene have been identified in a large family with a complex phenotype variably associating attention-deficit/hyperactivity disorder (ADHD) with craniosynostosis, hearing impairment, developmental delay and carpal and tarsal fusions.

Author information: (1)Department of Surgery, Division of Orthopaedic Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. (2)Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI 48201, USA. (3)Division of Medical Genetics, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. (4)Division of Medical Genetics, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA. (5)Division of Medical Genetics, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. (6)Department of Clinical Genetics, Leiden University Medical Centre, 2300 LC Leiden, the Netherlands. (7)Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, ON M5G 1X8, Canada; Institute of Medical Sciences, University of Toronto, Toronto, ON M5G 1X8, Canada. (8)Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany. (9)Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; Institute für Humangenetik, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany. (10)Neurovascular Unit and Cognitive Disorders (EA-3808 NEUVACOD), Université de Poitiers, 86073 Poitiers, France; Service de Génétique Clinique, Centre Hospitalier Universitaire de Poitiers, 86021 Poitiers, France. (11)Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, Département de Génétique, 75013 Paris, France. (12)Service de Génétique, Centre Hospitalier Universitaire de Nice Hôpital de l'Archet 2,151 route Saint Antoine de la Ginestière, 062002 Nice, France. (13)Center for Human Genetics, University Hospitals Leuven, 3000 Leuven, Belgium. (14)Laboratory for Molecular Diagnosis, Center for Human Genetics, University Hospitals Leuven, 3000 Leuven, Belgium. (15)University of Pittsburgh Medical Center, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA. (16)Roberts Individualized Medical Genetics Center, Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. (17)Division of Genomic Diagnostics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. (18)MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK. (19)MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK; Clinical Genetics Service, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham NG5 1PB, UK. (20)Clinical Genetics Service, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham NG5 1PB, UK. (21)Wessex Clinical Genetics Services, University Hospital Southampton NHS Foundation Trust, Southampton SO16 5YA, UK. (22)GeneDx, Gaithersburg, MD 20877, USA. (23)Division of Medical Genetics, Department of Pediatrics, Duke University, Durham, NC 27707, USA. (24)Service de Génétique, Génomique, et Procréation, Centre Hospitalier Universitaire Grenoble Alpes, 38700 La Tronche, France; INSERM 1209, CNRS UMR 5309, Institute for Advanced Biosciences, Université Grenoble Alpes, 38706 Grenoble, France. (25)Section of Genetics, Department of Pediatrics, University of Colorado School of Medicine and Children's Hospital Colorado, Aurora, CO 80045, USA. (26)Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, 1000 Ljubljana, Slovenia. (27)Centre Hospitalier Universitaire Nantes, Service de Génétique Médicale, 44000 Nantes, France. (28)Centre Hospitalier Universitaire Nantes, Service de Génétique Médicale, 44000 Nantes, France; Université de Nantes, CNRS, INSERM, L'Institut du Thorax, 44000 Nantes, France. (29)Service de Cytogénétique, Centre Hospitalier Universitaire de Le Mans, 72037 Le Mans, France. (30)Department of Surgery, Division of Orthopaedic Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Electronic address: [email protected]. (31)Centre Hospitalier Universitaire Nantes, Service de Génétique Médicale, 44000 Nantes, France; Centre Hospitalier Universitaire Toulouse, Service de Génétique Médicale, 31000 Toulouse, France. Electronic address: [email protected].

Can secondary glioblastoma be caused by brain irradiation?

Yes, brain irradiation can cause secondary glioblastoma.

A cerebellar glioblastoma was discovered in a 28 year old woman, 5 years after a focal 50 grays brain irradiation for meningioma of the clivus. This case fulfilled the accepted criteria for radiation-induced neoplasms of the central nervous system, namely a second histologically-proven tumor different from the first lesion, a location within the irradiated area and a long latent period. The growing incidences of secondary maligcies in long-term survivors of childhood leukemia following allogeneic bone marrow transplantation (alloBMT) are increasingly being reported. Among the late complications of conventional myeloablative alloBMT, the occurrence of secondary maligt solid tumors is of major concern. Secondary maligt and benign brain tumors such as astrocytoma, meningioma and glioblastoma have been described in long-term survivors of conventional myeloablative alloBMT. Here we report a case of secondary anaplastic oligoastrocytoma that developed 7 years after matched unrelated alloBMT for relapsing childhood acute myeloid leukemia (AML) with CNS involvement. Although isolated CNS relapse of primary leukemia following alloBMT is not uncommon, it is important to identify and define potential risk factors that may lead to the development of secondary brain tumors in children who received high-dose chemotherapy and irradiation prior to alloBMT presenting with progressive neurological symptoms and to differentiate them from leukemia relapse with CNS involvement. BACKGROUND: Secondary maligt neoplasms (SMN) in CNS tumor survivors has become problem of increasing concern over the last 20 years. These tumors usually occur in a different site from the primary brain tumor several years after treatment. CASE REPORT: We report secondary cranial maligt neoplasms in three patients who were treated with irradiation and chemotherapy for their primary brain tumors. The first case is a male survivor of an orbital rhabdomyosarcoma who developed a meningioma 8 years later. The other two cases are female survivors of ependymomas who were irradiated at the age of 3 and developed secondary gliomas 8 and 17 years after therapy respectively. CONCLUSION: Patients carry a risk of developing SMNs many years later since irradiation is still an important part of the treatment. An SMN may have a benign course, as in meningioma, or be a dilemma for the patient, as in glioblastoma. Gliomas of astrocytic, oligodendroglial and ependymal origin account for more than 70% of all brain tumors. The most frequent (65%) and most maligt histological type is the glioblastoma. Since the introduction of computerized tomography and magnetic resoce imaging, the incidence rates of brain tumors have been rather stable, with a tendency of higher rates in highly developed, industrialized countries. Some reports indicate that Caucasians have higher incidence than black or Asian populations, but to some extent, this may reflect socio-economic differences and under-ascertainment in some regions, rather than a significant difference in genetic susceptibility. With the exception of pilocytic astrocytomas, the prognosis of glioma patients is still poor. Less than 3% of glioblastoma patients are still alive at 5 years after diagnosis, higher age being the most significant predictor of poor outcome. Brain tumors are a component of several inherited tumor syndromes, but the prevalence of these syndromes is very low. Several occupations, environmental carcinogens, and diet (N-nitroso compounds) have been reported to be associated with an elevated glioma risk, but the only environmental factor unequivocally associated with an increased risk of brain tumors, including gliomas, is therapeutic X-irradiation. In particular, children treated with X-irradiation for acute lymphoblastic leukemia show a significantly elevated risk of developing gliomas and primitive neuroectodermal tumor (PNET), often within 10 years after therapy. TP53 mutations are frequent in low-grade gliomas and secondary glioblastomas derived therefrom. Approximately 60% of mutations are located in the hot spot codons 248 and 273, and the majority of these are G:C-->A:T transitions at CpG sites. TP53 mutations are significantly more frequent in low-grade astrocytomas with promoter methylation of the O(6)-methylguanine-DNA methyltransferase repair gene, suggesting that, in addition to deamination of 5-methylcytosine, exogenous or endogenous alkylation in the O(6) position of guanine may contribute to the formation of these mutations. A 22 year-old-man with acute lymphoblastic leukaemia had received prophylactic cranial irradiation and intrathecal chemotherapy. Eighteen years later a cerebellar glioblastoma multiforme was diagnosed. The authors speculate about the possibility that this tumor may have been radiation and/or chemotherapy induced. Improvement in neuroimaging techniques, in particular magnetic resoce imaging, has helped characterize Central Nervous System abnormalities, namely secondary brain tumours. The most common secondary neoplasms which occur following cranial radiation therapy are sarcoma and meningioma. The occurrence of glioblastoma multiforme following radiation and chemotherapy in acute lymphocytic leukaemia (ALL) is rare. We report 3 cases of glioblastoma multiforme in children developing 11-72 months following completion of chemotherapy/radiotherapy for ALL. The exact cause for the development of glioblastoma multiforme following therapy for ALL is not clear. A genetic predisposition may be essential for the occurrence of such a highly maligt primary brain tumour in leukaemia patients, irrespective of radiation and/or chemotherapy. The pathogenesis and surgical management are discussed, and the literature on the subject is reviewed. Secondary brain tumors rarely arise after cranial irradiation; among them, meningiomas and glioblastomas are the most common and secondary oligodendroglial tumors the most rare. We present a 48-year-old man who developed an oligodendroglial tumor 38 years after receiving 50 Gy of cranial irradiation to a pineal tumor. He underwent gross total removal of a calcified, ring-enhanced mass in the right temporal lobe. The tumor was histologically diagnosed as anaplastic oligodendroglioma. Our review of previously reported secondary oligodendroglial tumors that developed after cranial irradiation revealed that these rare tumors arose after low-dose cranial irradiation or at the margin of a field irradiated with a high dose. We suggest that secondary oligodendroglial tumors arising after cranial irradiation are more aggressive than primary oligodendrogliomas. Radiation therapy has been widely applied for cancer treatment. Childhood acute lymphocytic leukemia (ALL), characterized by frequent central nervous system involvement, is a well documented disease for the effect of prophylactic cranio-spinal irradiation. Irradiation, however, acts as an oncogenic factor as a delayed effect and it is rare that glioblastoma multiforme develops during the remission period of ALL. We experienced a pediatric radiation-induced GBM patient which developed during the remission period of ALL, who were primarily treated with chemotherapeutic agents and brain radiation therapy for the prevention of central nervous system (CNS) relapse. Additionally, we reviewed the related literature regarding on the effects of brain irradiation in childhood and on the prognosis of radiation induced GBM. Secondary glioblastoma multiforme (sGBM) can occur after a long latency period following radiation treatment of various diseases including brain tumors, leukemia, and more benign disorders like tinea capitis. Outcomes of radiation-induced sGBM remain poor in both children and adults. We report a case of a 16-year-old girl with a history of disseminated juvenile pilocytic astrocytoma treated with chemotherapy and craniospinal radiation 9 years prior who developed sGBM in the absence of a tumor predisposition syndrome. She presented with a several-week history of headaches and no acute findings on computed tomography compared to baseline neuroimaging 3 months prior. Repeat computed tomography performed just 3 weeks later for worsening headaches revealed a new large posterior fossa tumor where pathology confirmed the diagnosis of sGBM. In spite of maximal surgical resection, reirradiation, and adjuvant chemotherapy, she died 1 year postdiagnosis. Our case highlights the potential late effects of high-dose cranial radiation, how symptomatology may precede neuroimaging findings, and the rapid formation of sGBM that mirrors that of de novo Glioblastoma Multiforme. Stereotactic radiosurgery (SRS) using the Gamma Knife (GK) is now being increasingly utilized for the treatment of brain metastases. However, there are a few reported cases of SRS-induced brain neoplasms. We herein report the case of a Japanese woman with metastatic non-small cell lung cancer (NSCLC) harboring epidermal growth factor (EGFR)-mutations who was treated four times with a GK for brain metastases. She developed glioblastoma 5.7 years after the initial GK surgery. Radiation-induced secondary neoplasms generally appear after a latency period of several years. Advances in cancer therapy have improved the survival of patients with NSCLC, providing enough time for secondary neoplasms to appear after SRS. BACKGROUND: Glioblastoma multiforme (GBM), the most common maligt brain tumor, mainly manifests as a primary de novo and less frequently as a secondary glial neoplasm. GBM has been demonstrated to overexpress the NK-1 receptor and substance P can be used as a ligand for targeted therapy. Alpha emitters, e.g. 213Bi, that deposit their high energy within a short range allow the selective irradiation of tumor cells while sparing adjacent neuronal structures. MATERIAL AND METHODS: Among 50 glioma patients of different subtypes that have to date been treated with targeted alpha therapy at the Medical University Warsaw, we report here the data on nine patients with secondary GBM. Following surgery, chemo- and radiotherapy, recurrent GBM was treated by intracavitary injection of 1-6 doses of 0.9-2.3 GBq 213Bi- DOTA-[Thi8,Met(O2)11]-substance P (213Bi-DOTA-SP) in 2-month intervals. 68Ga-DOTA-[Thi8,Met(O2)11]-substance P (68Ga-DOTA-SP) was co-injected with the therapeutic doses to assess biodistribution using PET/CT. Therapeutic response was monitored with MRI. RESULTS: Treatment with activities ranging from 1.4 to 9.7 (median 5.8) GBq 213Bi- DOTA-SP was well tolerated with only mild transient adverse reactions, mainly headaches due to a transient perfocal edema reaction. The median progression free survival and overall survival time following the initiation of alpha therapy was 5.8 and 16.4 months, respectively. The median overall survival time from the first diagnosis was 52.3 months. Two out of nine patients are still alive 39 and 51 months, respectively, after the initiation of the therapy. CONCLUSIONS: Targeted alpha therapy of secondary GBM with 213Bi-DOTA-SP is safe and well tolerated and may evolve as a promising novel therapeutic option for secondary GBM. Glioblastomas are lethal brain tumors that are treated with conventional radiation (X-rays and gamma rays) or particle radiation (protons and carbon ions). Paradoxically, radiation is also a risk factor for GBM development, raising the possibility that radiotherapy of brain tumors could promote tumor recurrence or trigger secondary gliomas. In this study, we determined whether tumor suppressor losses commonly displayed by patients with GBM confer susceptibility to radiation-induced glioma. Mice with Nestin-Cre-driven deletions of Trp53 and Pten alleles were intracranially irradiated with X-rays or charged particles of increasing atomic number and linear energy transfer (LET). Mice with loss of one allele each of Trp53 and Pten did not develop spontaneous gliomas, but were highly susceptible to radiation-induced gliomagenesis. Tumor development frequency after exposure to high-LET particle radiation was significantly higher compared with X-rays, in accordance with the irreparability of DNA double-strand breaks (DSB) induced by high-LET radiation. All resultant gliomas, regardless of radiation quality, presented histopathologic features of grade IV lesions and harbored populations of cancer stem-like cells with tumor-propagating properties. Furthermore, all tumors displayed concomitant loss of heterozygosity of Trp53 and Pten along with frequent amplification of the Met receptor tyrosine kinase, which conferred a stem cell phenotype to tumor cells. Our results demonstrate that radiation-induced DSBs cooperate with preexisting tumor suppressor losses to generate high-grade gliomas. Moreover, our mouse model can be used for studies on radiation-induced development of GBM and therapeutic strategies. SIGNIFICANCE: This study uncovers mechanisms by which ionizing radiation, especially particle radiation, promote GBM development or recurrence. Publisher: Медуллобластома — наиболее часто встречающаяся злокачественная опухоль центральной нервной системы у детей, возникающая в задней черепной ямке (мозжечок, ствол, IV желудочек) и имеющая высокий риск метастазирования по ликворным путям. Существенный прогресс в понимании развития этой опухоли и поиске соответствующих оптимальных схем ее лечения связан с выделением различных молекулярных категорий первичных медуллобластом на основании изучения факторов цитогенетической и транскрипционной пролиферации. Современные подходы к лечению для пациентов старше трех лет включают в себя максимально возможную хирургическую резекцию, краниоспинальное облучение с «бустом» на послеоперационное ложе с последующей химиотерапией платиносодержащими препаратами. Применение радиотерапии в конвенциональном режиме, в том числе краниоспинальное облучение, часто приводит к возникновению осложнений, увеличение количества которых можно обнаружить по мере наблюдения за пациентами. При этом особого внимания заслуживают вторичные опухоли, в том числе глиобластомы, так как их возникновение предопределяет фатальный исход. Отчасти этим можно объяснить тот факт, что проведение и изменение схемы химиотерапии без повторной морфологической верификации в различных вариантах не всегда приводит к контролю роста опухоли при «рецидивах» медуллобластом. В работе рассмотрены случаи возникновения радиоиндуцированных глиобластом, «вторичных» по отношению к первично выявленным медуллобластомам, у пациентов, получивших ранее краниоспинальное облучение в качестве компонента комбинированного лечения после удаления опухоли. Выяснилось, что частота подобного явления довольно значительная и составляет около 10% среди пациентов с рецидивами медуллобластом, что существенно выше предполагаемого и отмеченного ранее уровня. Прослежены закономерности возникновения радиоиндуцированных глиобластом в зависимости от молекулярно-генетической группы, к которой отнесены эти пациенты после первичной операции, и их клинических характеристик, приведены результаты лечения. Сделан вывод о необходимости морфологической верификации после выявления отдаленного во времени рецидива после комбинированного лечения медуллобластом для проведения адекватного лечения. Intracranial germinomas are considered one of the most radiosensitive tumors and are curable by radiotherapy alone. Although patients can expect long-term survival, the adverse effects of radiotherapy and late sequelae in survivors are a major concern. Radiation-induced secondary neoplasms are one of those sequelae and are a serious concern because they are often connected directly with life prognosis. We describe a case of radiation-induced glioblastoma after radiotherapy for germinoma. An 11-year-old boy with basal ganglia germinoma was successfully treated with postoperative cranial irradiation. At the age of 40 years, he was admitted to our hospital for aphasia and memory disturbance. CT and MRI revealed a tumor in the left parietal lobe with dissemination. The tumor of the parietal lobe was removed surgically, and pathohistologically, it was diagnosed as glioblastoma. Long-term survivors who receive radiotherapy for germinomas in childhood are at risk for late complications, including radiation-induced neoplasms. Therefore, careful follow-up neurological examinations are recommended in these patients, even 20-30 years after radiotherapy.

Is aggrephagy a variant of autophagy?

Yes, the selective branch of autophagy that deals with identification, capture and degradation of protein aggregates is called aggrephagy.

Many neurodegenerative disorders feature the presence of misfolded polypeptide-containing intracellular inclusion bodies biochemically and morphologically analogous to cellular aggresomes. However, it is largely unknown how misfolded polypeptides form aggresomes and are eventually cleared by the aggresome-macroautophagy/autophagy pathway, so-called aggrephagy. Our recent study revealed that when the ubiquitin-proteasome system is impaired, the accumulated misfolded polypeptides are selectively recognized and transported to the aggresome by a CED complex. This complex is composed of CTIF, originally identified as a specific factor for nuclear cap-binding protein complex (a heterodimer of NCBP1/CBP80 and NCBP2/CBP20)-dependent translation (CT), and its associated factors EEF1A1 and DCTN1. Aggresomal targeting of a misfolded polypeptide via the CED complex is accompanied by CTIF release from the CT complex and thereby inhibits CT efficiency. Therefore, our study provides new mechanistic insights into the crosstalk between translational inhibition and aggresome formation under the influence of a misfolded polypeptide. Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson's disease (PD), XCT 790 exerted neuroprotective effects in the dopaminergic neurons of nigra by inducing autophagy to clear toxic protein aggregates and, in addition, ameliorated motor co-ordination deficits. Using a chemical biology approach, we unrevealed the role of ERRα in regulating autophagy and can be therapeutic target for neurodegeneration. A proteostasis view of neurodegeneration (ND) identifies protein aggregation as a leading causative reason for damage seen at the cellular and organ levels. While investigative therapies that aim at dissolving aggregates have failed, and the promises of silencing expression of ND associated pathogenic proteins or the deployment of engineered induced pluripotent stem cells (iPSCs) are still in the horizon, emerging literature suggests degrading aggregates through autophagy-related mechanisms hold the current potential for a possible cure. Macroautophagy (hereafter autophagy) is an intracellular degradative pathway where superfluous or unwanted cellular cargoes (such as peroxisomes, mitochondria, ribosomes, intracellular bacteria and misfolded protein aggregates) are wrapped in double membrane vesicles called autophagosomes that eventually fuses with lysosomes for their degradation. The selective branch of autophagy that deals with identification, capture and degradation of protein aggregates is called aggrephagy. Here, we cover the workings of aggrephagy detailing its selectivity towards aggregates. The diverse cellular adaptors that bridge the aggregates with the core autophagy machinery in terms of autophagosome formation are discussed. In ND, essential protein quality control mechanisms fail as the constituent components also find themselves trapped in the aggregates. Thus, although cellular aggrephagy has the potential to be upregulated, its dysfunction further aggravates the pathogenesis. This phenomenonwhen combined with the fact that neurons can neither dilute out the aggregates by cell division nor the dead neurons can be replaced due to low neurogenesis, makes a compelling case for aggrephagy pathway as a potential therapeutic option.

List the blood group antigens, associated with blood type

ABO antigens are highly abundant in many human cell types, including platelets, vascular endotheliums, and red blood cells.

Using monoclonal antibodies (MoABs) against blood group determits and related carbohydrate sequences, it is now possible to clarify their carcinoma-associated modulation at a molecular level. In the present study a panel of MoABs against different type 1 chain derived blood group antigens, comprising A, B, H type 1, Le(a), sialyl-Le(a) (CA 19-9), sialyl type 1 structure (CA 50), and Le(b) was used to investigate their immunoreactivity in 38 medullary carcinomas of the thyroid (MTC) and in normal thyroid tissue. The antigens were not expressed in normal follicular or C-cells but were expressed to a various extent in MTC. The studies revealed some characteristic anomalies in the frequency and patterns of tumor-associated antigen expression. The MoAB C 50 stained 32 of the 38 tumors, H type 1 (Le(d)) was demonstrated in 21 and the Le(b) antigen in 27. The Le(a)- and the A antigen were detected in 10 and 12 tumors and the B antigen in one. From the results some rules about the pathways for tumor-associated re-expression of these antigens can be deduced. Le(a) antigen expression was significantly correlated with the CA 50 and Le(b) antigens. The significant relation observed between A-, H1-, and Le(b) antigen formation in MTC suggests the existence of a carcinoma-associated fucosyltransferase committing the type 1 precursor chain along the H1-antigen pathway, and by further glycosylation to an A-, B-, or a Le(b) antigen. Comparative studies of tumor-associated H type 1 and H type 2 antigen expression revealed that H type 2 antigen synthesis was significantly related to a blood type 0 in the host. On the other hand, H1 antigen reactivity was independent of the AB0 blood type of the hosts and was also detected in H type 2 antigen-negative tumors. These findings support the proposal that even in tumor tissue, H antigen expression is still determined by the interaction of at least two different genes. Despite the occurrence of the precursor substance (CA 50) and the formation of the Le(a)- and Le(b) antigens, indicating the presence of a alpha 1,4-fucosyl-transferase (Lewis-enzyme), only two tumors showed the formation of CA 19-9. In conclusion, the investigations demonstrated the domit re-expression of three type 1 chain-derived structures in MTC, namely H type 1, Le(b), and CA 50. These findings support the general concept demonstrated in other carcinomas, that fucosyl- and sialyltransferases are preferentially activated in MTC.(ABSTRACT TRUNCATED AT 400 WORDS) Blood group A, B, H, Le, Leb, Lex, and Ley antigenicity as well as the expression of CA 19-9 were examined in pancreatic cancer specimens from 30 patients, using monoclonal antibodies to the respective antigen and immunohistochemical techniques, and the findings were correlated with the blood group types (ABO and Lewis) of the individuals. Compatible antigen expression was found in 82, 75, and 50% of tumors from patients with A, B, and O blood group types, respectively. Deletion of the compatible antigen was found in 10 (33%) of the cases, predomitly in patients of blood group O type, and incompatible expression (of B antigen only) in 4 (13%). Lea was detected in 87%, Leb in 90%, Lex in 30%, and Ley in 43% of the specimens, regardless of ABH and Lewis phenotype of the patients. Coexpression of Lea and Leb was found in 87%, of Lex and Ley in 13%, of Lea and Lex in 23%, and of Leb and Ley in 40% of the cases. CA 19-9 was expressed in 80% of the tumors; it was present in the tumor tissue of 21 of 22 patients from Lea-b+, in all 4 individuals from Lea+b-, but in none of the 4 patients from Lea-b- phenotype (P less than 0.01). Heterogeneity in the expression of each of the antigens was found. The overall results indicate that blood group antigen expression in pancreatic tumor differs from that of other gastrointestinal cancers and that the Lewis antigen expression in pancreatic cancer cells is independent of the blood group phenotype of the patients and thus may be useful as a tumor marker. In many tissues, carcinogenesis mimics embryonic development. This is true for the pancreas. The first alteration seen during pancreatic tumor induction in the hamster model is proliferation of poorly differentiated ductular (tubular) structures intermingled with endocrine cells, a pattern consistent with findings in the embryonic and fetal pancreas. However, unlike the fetal tissue, various cell types of intestinal epithelium appear in the advance stages of pancreatic carcinogenesis. Moreover, contrary to the situation in the fetal and adult hamster pancreas, the induced pancreatic lesion expresses antigens with human blood group type specificities, including A, B, H, Leb, Lex, and Ley, antigens that are expressed, however, by fetal and adult duodenal epithelium. Considering the origin of the pancreas from the duodenal mucosa, the overall findings indicate that during pancreatic carcinogenesis some genes, acquired from the progenitor (duodenal) cells, which are inactive in embryonic and normal pancreatic cells, are activated, possibly as a function of some oncogenes. Comparative studies in human tissue lead to the same conclusion. The immunohistological distribution of blood group (BG)-related antigens including A, B, H type 2, and sialylated Lex in lung adenocarcinomas was examined using monoclonal antibodies. BG-A, B, and H type 2 compatible with the ABO status in tumor cells were expressed in 60% of the cases. Accumulation of H type 2, associated with loss of BG-A and B, was observed in tumor cells of patients with BG status other than 0. Tumor-associated antigens, Lex and sialylated Lex were detected in 36.0% and 72.0%, respectively. Modification of carbohydrate antigens in cancer may be associated with incomplete synthesis; accumulation of precursor antigen; and activated sialylation. To clarify the clinico-pathological characteristic of poorly differentiated adenocarcinoma of gastric cancer with medullary growth pattern, the immunohistochemical study was performed using antibodies against blood group related antigens (Lewis(a), Lewis(b), Lewis(x), Lewis(y)) and gastrointestinal tissue related antigens (CEA, AFP and NSE). The following results were obtained. 1. Foveolar epithelium of normal gastric mucosa has type 1 blood group associated antigen (Lewis(a), Lewis(b)) and deep gland has type 2 antigen (Lewis(x), Lewis(y)) respectively, as the differentiation antigens. 2. Expression of Lewis(a) and Lewis(b) are more often observed in poorly differentiated adenocarcinoma with medullary and scirrhous growth pattern than in well and moderately differentiated adenocarcinoma. Poorly differentiated adenocarcinomas postulated to have closer relation with normal gastric deep gland in terms of antigen expression. 3. Poorly differentiated adenocarcinoma with medullary growth pattern is characterized as more differentiated type cancer than scirrhous type with respect to expression of blood group related antigen. 4. Poorly differentiated adenocarcinoma with medullary growth pattern may be divided into 3 subgroups by each antigen expression of CEA, NSE, and AFP. The abnormal expression of blood group related antigens has been reported in many maligt tumours; however, such expression in cholangiocarcinoma has not been examined systematically. The expression of blood group-related antigens (A, B, H, Lewis(a), Lewis(b), Lewis(x), Lewis(y), carbohydrate antigen 19-9 and carcinoembryonic antigen) was investigated immunohistochemically in 75 cases of cholangiocarcinoma (31 peripheral type and 44 hilar type). In non-neoplastic bile ducts, A, B, and H antigens were expressed in large bile ducts, while Lewis(a,b,y) and carbohydrate antigen 19-9 were variably expressed in both large and small bile ducts. Lewis(x) and carcinoembryonic antigen was not found in non-neoplastic bile ducts. In cholangiocarcinomas, A, B, and H, antigens were more frequent in the hilar type than in the peripheral type, although the difference was not significant. The expression of the blood-group related antigens, particularly A, Lewis(a,b,y), carcinoembryonic antigen, and carbohydrate antigen 19-9, was frequent in the tumour cells in well differentiated adenocarcinomas, while their immunoreactivity was less frequent in poorly differentiated adenocarcinomas. The superanuclear and luminal expression of these antigens in carcinoma cells was frequent in well differentiated adenocarcinomas, and the diffuse, cell membranous and stromal expression of these antigens was relatively frequent in poorly differentiated adenocarcinomas and adenosquamous carcinoma. The A, B, and H immunoreactivity of both non-neoplastic bile ducts and cholangiocarcinomas was consistent with the host blood group type. These findings suggest that both the expression and intracellular distribution of blood group-related antigens in cholangiocarcinoma are related to the differentiation of cholangiocarcinoma and, possibly, to the parent structure. Associations between blood type and disease have been studied since the early 1900s when researchers determined that antibodies and antigens are inherited. In the 1950s, the chemical identification of the carbohydrate structure of surface antigens led to the understanding of biosynthetic pathways. The blood type is defined by oligosaccharide structures, which are specific to the antigens, thus, blood group antigens are secondary gene products, while the primary gene products are various glycosyltransferase enzymes that attach the sugar molecules to the oligosaccharide chain. Blood group antigens are found on red blood cells, platelets, leukocytes, plasma proteins, certain tissues, and various cell surface enzymes, and also exist in soluble form in body secretions such as breast milk, seminal fluid, saliva, sweat, gastric secretions, urine, and amniotic fluid. Recent advances in technology, biochemistry, and genetics have clarified the functional classifications of human blood group antigens, the structure of the A, B, H, and Lewis determits and the enzymes that produce them, and the association of blood group antigens with disease risks. Further research to identify differences in the biochemical composition of blood group antigens, and the relationship to risks for disease, can be important for the identification of targets for the development of nutritional intervention strategies, or the identification of druggable targets. WIREs Syst Biol Med 2016, 8:517-535. doi: 10.1002/wsbm.1355 For further resources related to this article, please visit the WIREs website. Portable, on-site blood typing methods will help provide life-saving blood transfusions to patients during an emergency or natural calamity, such as significant earthquakes. We have previously developed waveguide-mode (WM) sensors for forward ABO and Rh(D) blood typing and detection of antibodies against hepatitis B virus and hepatitis C virus. In this study, we evaluated a WM-sensor for reverse ABO blood typing. Since reverse ABO blood typing is a method for detection of antibodies against type A and type B oligosaccharide antigens on the surface of red blood cells (RBCs), we fixed a synthetic type A or type B trisaccharide antigen on the sensor chip of the WM sensor. We obtained significant changes in the reflectance spectra from a WM sensor on type A antigen with type B plasma and type O plasma and on type B antigen with type A plasma and type O plasma, and no spectrum changes on type A antigen or type B antigen with type AB plasma. Signal enhancement with the addition of a peroxidase reaction failed to increase the sensitivity for detection on oligosaccharide chips. By utilizing hemagglutination detection using regent type A and type B RBCs, we successfully determined reverse ABO blood groups with higher sensitivity compared to a method using oligosaccharide antigens. Thus, functionality of a portable device utilizing a WM sensor can be expanded to include reverse ABO blood typing and, in combination with forward ABO typing and antivirus antibody detection, may be useful for on-site blood testing in emergency settings. Blood group systems based on red blood cell antigens are genetically determined and can identify patients at risk. Type non-O of the ABO blood group system has been associated with coronary artery disease, thrombosis, and a worse prognosis. The present study evaluated the distribution of blood group types in patients with heart failure (HF) and the impact on clinical outcome. We evaluated the ABO and Rhesus D antigen (RhD) blood types in a large cohort of chronic HF patients (n = 3,815). ABO blood type distribution in the HF population was not significantly different to that reported in the general national population (A 40%, B 20%, AB 8%, and O 33%). The percentage of Rh-negative per blood type was also similar (A 10%, B 9%, AB 10%, and O 7%). Patients with type O were more likely to be hypertensive compared with non-O type. Mean follow-up was 4.2 years. Overall survival during follow-up was 55%. Cox regression analysis after adjustment for significant predictors demonstrated that RhD-negative was associated with a worse prognosis in patients with ischemic cardiomyopathy (n = 2,881, 76%): hazard ratio 1.26, 95% confidence interval 1.04 to 1.53, p = 0.02. Type non-O was also independently associated with a worse prognosis compared with type O in patients with non-ischemic cardiomyopathy: hazard ratio 1.32, 95% confidence interval 1.04 to 1.67, p = 0.02. In conclusion, blood group type distribution in HF patients are similar to the general population. RhD-negative is associated with a worse prognosis in patients with ischemic cardiomyopathy. PURPOSE: The aim of our study was to investigate the impact of the ABO blood groups and blood-based biomarkers on the growth kinetics of renal angiomyolipoma (AML). METHODS: A total of 124 patients with AML who were followed-up between 2010 and 2018 were retrospectively reviewed. The patients' characteristics were recorded, including age, body mass index (BMI), blood pressure, smoking history, and ABO blood group. Baseline laboratory test results, including serum creatinine, AST, ALT, platelet, neutrophil and lymphocyte count, were used to calculate the estimated glomerular filtration rate (eGFR), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and De Ritis ratio. The Cox regression analysis was used to evaluate the relationship between variables and tumor growth. RESULTS: The study population comprised 71 women and 44 men with a median age of 47.3 (28-65) years. Among patients classified according to the blood groups, no significant differences were observed regarding age, BMI, smoking history, co-morbidities, NLR, PLR, De Ritis ratio, eGFR, or tumor size and localisation. The mean growth rate from baseline to the last scan was 0.36 ± 0.27 cm, 0.21 ± 0.21 cm, 0.14 ± 0.11 cm, and 0.19 ± 0.17 cm for blood type O, A, B, and AB, respectively. In multivariate analysis, eGFR < 60 (p = 0.044), central tumor localisation (p = 0.030), presence of blood group-0 (p = 0.038), and De Ritis ratio ≥ 1.24 (p = 0.047) were statistically associated with tumor growth. CONCLUSION: Our study demonstrates that both the ABO blood groups and the De Ritis ratio might represent independent predictors of tumor growth rate in patients with renal AML. INTRODUCTION: Pemphigus is a chronic inflammatory and autoimmune disease which can cause blisters and mucocutaneous erosions. ABO secretor refers to those who secrete ABO blood group antigens based on their blood type in body fluids such as saliva, sweat, tears, semen, and serum. Previous studies showed that nonsecretor people are more prone to certain autoimmune diseases. AIM: The aim of this study was to determine the ABO secretor status in the saliva of patients with pemphigus vulgaris. MATERIALS AND METHODS: This case-control study was conducted on 35 patients with pemphigus vulgaris and 35 healthy controls. The two groups were matched for age and gender. Pemphigus vulgaris diagnosis was confirmed by histopathology and direct immunofluorescence microscopy. ABO blood grouping was done, and 5 ml of unstimulated saliva was collected to determine secretor status. Secretors were recognized from nonsecretors by the Wiener agglutination inhibition test. Results were extracted by using statistical chi-square and Fisher's exact tests. RESULTS: 16 male and 19 female patients aged 49.43 ± .12.37 years were compared with 16 male and 19 female controls aged 46.43 ± 11.88 years. The most frequent blood group among case and control groups was O (54.3% and 60%, respectively). There was no significant difference in blood groups (P=0.73). 90% of the samples were ABO secretors. The patient group included 31 (88.6%) and the control group included 32 (91.4%) ABO secretors; there was no significant difference between the two groups (P=1.000). CONCLUSION: In this study, we observed that the people with nonsecretor status in comparison with the people with secretor status are not more susceptible to develop pemphigus vulgaris.

How many DNaseI hypersensitive sites (DHS) mark the murine beta globin locus region?

The expression of genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR) Targeted deletion of 5'HS1 and 5’HS4 of the beta-globin locus Control region reveals additive activity of the sites . The LCR is composed of a series of 5 DNase . sites (5'HSs), that form in the nucleus of erythroid precursors .

In addition to local sequence elements the regulation of the high-level, development- and tissue-specific expression of the human beta globin gene cluster appears to require distant regulatory sequences which have been termed locus control region. In the chromatin of erythroid cells the locus control region is characterized by four DNaseI hypersensitive sites that are located 6-18 kb 5' of the epsilon globin gene. The definition of the sequences minimally required for locus control region activity is likely to further the understanding of its physiology and will be of interest for the development of somatic gene therapy strategies of the hemoglobinopathies. We present here the analysis of a family with a 3,030-bp deletion of sequences upstream of the epsilon globin gene including the most 3' locus control region element and cosegregating beta(0) thalassemia. The deletion is linked in cis to a structurally and functionally normal beta globin gene. The proximal element of the locus control region does not therefore appear to be necessary for beta globin gene activity in vivo. The human beta-globin domit control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus. The human beta-globin locus control region (LCR) consists of four erythroid-specific DNaseI hypersensitive sites (HSs) at the 5' end of the beta-globin cluster. The LCR functions over a long distance on chromosome 11 to regulate transcription and replication of the beta-globin genes. To determine whether the HSs function independently or as an integrated unit, we analyzed the requirements for long-range transcriptional activation. If the HSs function independently, individual HSs would be expected to have long-range activity. In contrast, if long-range activity requires multiple HSs, individual HSs would have a limited functional distance. HS2, HS3, and a miniLCR containing multiple HSs, were separated from a gamma-globin promoter by fragments of phage lambda DNA. After stable transfection into K562 cells, HS2 had strong enhancer activity, but only when positioned close to the promoter. HS3 also had strong enhancer activity, although it was weaker than HS2 and more sensitive to the spacer DNA. The miniLCR had the strongest enhancer activity and functioned even at a distance of 7.3 kb. A model is proposed in which synergistic interactions between HSs confer long-range activation by creating a stable LCR nucleoprotein structure, which is competent for recruiting chromatin-modifying enzymes. These enzymes would mediate the well-characterized activity of the LCR to modulate chromatin structure. The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3. Mammalian beta-globin loci are composed of multiple orthologous genes whose expression is erythroid specific and developmentally regulated. The expression of these genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR). The LCR encompasses 5 major HSs, each of which is highly homologous among humans, mice, and other mammals. To analyze the function of individual HSs in the endogenous murine beta-globin LCR, we have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In this report, we demonstrate that deletion of the conserved region of 5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion site but has no influence on the formation of the remaining HSs in the LCR. Therefore, in the endogenous murine locus, there is no domit or initiating site whose formation must precede the formation of the other HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of beta-globin regulation. The mammalian beta-globin locus is a multigenic, developmentally regulated, tissue-specific locus from which gene expression is regulated by a distal regulatory region, the locus control region (LCR). The functional mechanism by which the beta-globin LCR stimulates transcription of the linked beta-like globin genes remains unknown. The LCR is composed of a series of 5 DNaseI hypersensitive sites (5'HSs) that form in the nucleus of erythroid precursors. These HSs are conserved among mammals, bind transcription factors that also bind to other parts of the locus, and compose the functional components of the LCR. To test the hypothesis that individual HSs have unique properties, homologous recombination was used to construct 5 lines of mice with individual deletions of each of the 5'HSs of the endogenous murine beta-globin LCR. Here it is reported that deletion of 5'HS1 reduces expression of the linked genes by up to 24%, while deletion of 5'HS4 leads to reductions of up to 27%. These deletions do not perturb the normal stage-specific expression of genes from this multigenic locus. In conjunction with previous studies of deletions of the other HSs and studies of deletion of the entire LCR, it is concluded that (1) none of the 5'HSs is essential for nearly normal expression; (2) none of the HSs is required for proper developmental expression; and (3) the HSs do not appear to synergize either structurally or functionally, but rather form independently and appear to contribute additively to the overall expression from the locus. The distal locus control region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the beta-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1-5) mark the LCR. Sequence conservation and direct experimental evidence have implicated sequences within and between the HS cores in function of the LCR. In this report we confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences. The human beta-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional beta-like globin genes, epsilon, (G)gamma, (A)gamma, delta, and beta, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNaseI-hypersensitive sites located 6-22 Kb upstream of the epsilon -globin gene. During ontogeny two switches occur in beta-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic epsilon - to fetal gamma-globin occurs at six weeks of gestation. The second switch from gamma- to adult delta- and beta-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching. The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp "flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However, human transgenes in which only individual HS core regions were deleted showed drastic loss of expression accompanied by changes in chromatin structure. To address these disparate results, we have deleted the core region of 5'HS2 from the endogenous murine beta-LCR. The phenotype was similar to that of the larger 5'HS2 deletion, with no apparent disruption of chromatin structure. These results demonstrate that the greater severity of HS core deletions in comparison to full HS deletions is not a general property of the beta-LCR.

What is the outcome of COVID-19 patients treated with tocilizumab?

Preliminary clinical results have indicated that tocilizumab can improve the outcomes of patients with severe or critical COVID-19 while maintaining a good safety profile.

Which loss-of-function ABCC8 mutation is associated with Pulmonary Arterial Hypertension (PAH)?

A de novo novel heterozygous predicted deleterious missense variant c.G2873A (p.R958H) in ABCC8 in a child with idiopathic PAH.

Author information: (1)Department of Pharmacology, College of Physicians and Surgeons (M.S.B., K.J.S., R.S.K.), Columbia University, New York, NY. (2)Department of Pediatrics, College of Physicians and Surgeons (L.M., N.Z., U.K., E.B.R., W.K.C.), Columbia University, New York, NY. (3)Department of Systems Biology (N.Z., H.Q., Y.S.), Columbia University, New York, NY. (4)Department of Applied Physics and Applied Mathematics (H.Q., Y.S.), Columbia University, New York, NY. (5)Department of Cell Biology and Physiology (C.M., C.G.N.) and Center for the Investigation of Membrane Excitability Diseases (C.M., C.G.N.), Washington University School of Medicine, Washington University in St. Louis, MO. (6)Regeneron Genetics Center, Regeneron Pharmaceuticals, Inc, Tarrytown, NY (C.G.-J., F.E.D., J.D.O., J.G.R., A.R.S., A.B.). (7)Department of Medicine (M.B., C.H., M.H., J.M.M., M.T., C.M.T., K.Y., S.G., N.W.M.), University of Cambridge, United Kingdom. (8)VU University Medical Center, Amsterdam, the Netherlands (H.J.B., A.C.H., A.V.N.). (9)Golden Jubilee National Hospital, Glasgow, Scotland (C.C., A.J.P.). (10)Royal Free Hospital, London, England (G.C.). (11)Newcastle University (P.A.C.) and Newcastle upon Tyne Hospitals National Health Service Foundation Trust (P.A.C.), United Kingdom. (12)Dépat de Génétique, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris (M.E., F.S.) and UMR_S 1166-ICAN, INSERM (Institut National de la Santé et de la Recherche Médicale) (M.E., F.S.), UPMC (Pierre and Marie Curie University) Sorbonne Universités, France. (13)National Heart and Lung Institute, Imperial College London, United Kingdom (J.S.R.G., S.J.W.). (14)AP-HP (Assistance Publique - Hôpitaux de Paris), Centre de référence de l'hypertension pulmonaire sévère, INSERM UMR_S 999, Hôpital Bicêtre, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France (B.G., M.H., C.G., D.M.). (15)Sheffield Clinical Research Facility, Royal Hallamshire, Sheffield, United Kingdom (D.G.K.). (16)Department of Infection, Immunity, and Cardiovascular Disease, University of Sheffield, Sheffield, United Kingdom (A.L.). (17)Royal United Bath Hospitals, Bath, United Kingdom (R.V.M.R., J.S.). (18)Papworth Hospital, Cambridge, United Kingdom (J.P.-Z., M.T.). (19)Division of Genetics and Molecular Medicine, King's College London, London, England (R.C.T.). (20)Department of Medicine, Imperial College London, Hammersmith Campus, London, United Kingdom (J.W., M.R.W.). (21)Royal Brompton Hospital, London, United Kingdom (S.J.W.). (22)Department of Hematology (S.G.), Addenbrookes Hospital, University of Cambridge, United Kingdom.

Which disease is rated using the Fahn-Tolosa-Marin scale?

The Fahn-Tolosa-Marin clinical tremor rating scale is used for essential tremor.

We sought to determine whether mirtazapine is safe and well-tolerated as a treatment for essential tremor (ET). We studied mirtazapine in a randomized, double-blind, placebo-controlled, crossover study of 17 ET patients. Patients were started with 15 mg per day of either mirtazapine or placebo for 1 week and the dose was escalated weekly until the targeted dose of 45 mg per day was achieved. This dose was maintained for 2 weeks. Tremor was assessed at baseline and after 14 days of 45 mg of mirtazapine or placebo. There was a minimum washout period of 14 days between the two arms of the study. Tremor assessments included global improvement, Fahn Tolosa Marin Tremor Rating Scale, Beck Depression Inventory and the Parkinson's Disease Questionnaire-39. Patient global improvement ratings indicated that in the placebo condition 12 patients were unchanged and 1 patient was mildly improved. In the mirtazapine condition, 10 patients were unchanged, 2 were moderately improved and 1 was markedly improved. There was no significant improvement with mirtazapine or placebo compared to baseline as measured by the Tremor Rating Scale. Adverse effects were more common in the mirtazapine group and included drowsiness, confusion, dry mouth, weight gain, polyuria, itching, nausea, gait and balance problems, blurred vision, and bad taste. We conclude that the majority of the ET patients do not benefit from mirtazapine. Mirtazapine has significant adverse effects and should be used cautiously in ET patients. The purpose of this study was to evaluate interrater and intrarater reliability of the Fahn-Tolosa-Marin Tremor Rating Scale (TRS) in essential tremor (ET). Proper treatment of ET is contingent upon correct assessment of the severity, loss of function, and disability related to tremor. Videotape recordings of 17 subjects with ET evaluated with the TRS were produced and sent to 59 raters. Once the raters returned the videotape and completed the score sheet, they were mailed a second tape with the same recordings presented in a different order. In the interrater reliability evaluation, modified Kappa statistics for seven tremor type composites ranged from 0.10 to 0.65 in the first videotape and 0.17 to 0.62 in the second videotape. Interrater reliabilities were greater for Part A items (magnitude of tremor in different body parts) than for Part B items (tremor in writing and drawings) of the TRS. The average Spearman correlation was 0.87, indicating very good consistency between the two videotapes, but correlations for Part A were somewhat better than for Part B. It is best when the same rater performs repeated measures of tremor on a patient, particularly when judging tremor in handwriting and drawings. Training of raters on use of the TRS would help standardize judgement. BACKGROUND: Gamma knife thalamotomy (GKT) has been used as a therapeutic option for patients with disabling tremor refractory to medications. Impressive improvement of tremor has been reported in the neurosurgical literature, but the reliability of such data has been questioned. OBJECTIVE: To prospectively evaluate clinical outcomes after GKT for disabling tremor with blinded assessments. DESIGN: Prospective study with blinded independent neurologic evaluations. SETTING: University hospital. PATIENTS: Consecutive patients who underwent unilateral GKT for essential tremor and Parkinson disease tremor at our center. These patients were unwilling or deemed unsuitable candidates for deep brain stimulation or other surgical procedures. INTERVENTIONS: Unilateral GKT and regular follow-up evaluations for up to 30 months, with blinded video evaluations by a movement disorders neurologist. MAIN OUTCOME MEASURES: Clinical outcomes, as measured by the Fahn-Tolosa-Marin Tremor Rating Scale and activities of daily living scores, and incidence of adverse events. RESULTS: From September 1, 2006, to November 30, 2008, 18 patients underwent unilateral GKT for essential tremor and Parkinson disease tremor at our center. Videos for 14 patients (11 with essential tremor, 3 with Parkinson disease tremor) with at least 6 months' postoperative follow-up were available for analysis (mean [SD] follow-up duration, 19.2 [7.3] months; range, 7-30 months). The Fahn-Tolosa-Marin Tremor Rating Scale activities of daily living scores improved significantly after GKT (P = .03; median and mean change scores, 2.5 and 2.7 points, respectively [range of scale was 0-27]), but there was no significant improvement in other Fahn-Tolosa-Marin Tremor Rating Scale items (P = .53 for resting tremor, P = .24 for postural tremor, P = .62 for action tremor, P = .40 for drawing, P > .99 for pouring water, P = .89 for head tremor). Handwriting and Unified Parkinson's Disease Rating Scale activities of daily living scores tended to improve (P = .07 and .11, respectively). Three patients developed delayed neurologic adverse events. CONCLUSIONS: Overall, we found that GKT provided only modest antitremor efficacy. Of the 2 patients with essential tremor who experienced marked improvement in tremor, 1 subsequently experienced a serious adverse event. Further prospective studies with careful neurologic evaluation of outcomes are necessary before GKT can be recommended for disabling tremor on a routine clinical basis. The Movement Disorder Society established a task force to review rating scales for the assessment of tremor. Screening instruments used in identifying patients with tremor were also reviewed. Seven tremor severity scales, six activities of daily living (ADL)/disability scales, four quality-of-life scales, and five screening instruments were identified by searching PubMed.gov. The availability, use, acceptability, reliability, validity, and sensitivity to change were reviewed for each scale; and each scale was classified as recommended, suggested or listed based on whether 3, 2, or 1 of the following criteria were met: (1) used in the assessment of tremor (yes/no), (2) used in published studies by people other than the developers (yes/no), and (3) successful clinimetric testing (yes/no). Five tremor severity scales (the Fahn-Tolosa-Marin Tremor Rating Scale, the Bain and Findley Clinical Tremor Rating Scale, the Bain and Findley Spirography Scale, the Washington Heights-Inwood Genetic Study of Essential Tremor Rating Scale, and the Tremor Research Group Essential Tremor Rating Assessment Scale), one ADL/disability scale (the Bain and Findley Tremor ADL Scale), one quality-of-life scale (the Quality of Life in Essential Tremor Questionnaire), and one screening instrument (the Washington Heights-Inwood Genetic Study of Essential Tremor Rating Scale, version 1) are recommended using these criteria. However, all scales need a more comprehensive analysis of sensitivity to change in order to judge their utility in clinical trials and individual patient assessments. The task force recommends that further work with existing recommended scales be performed as opposed to the development of new tremor scales. The aim of this study was to compare the efficacy of the branded and a generic extended-release ropinirole formulation in the treatment of advanced Parkinson's disease (PD). Of 22 enrolled patients 21 completed the study. A rater blinded to treatment evaluated Unified Parkinson's Disease Rating Scale, Fahn-Tolosa-Marin Tremor Rating Scale, Nonmotor Symptoms Assessment Scale, and a structured questionnaire on ropinirole side effects. Besides, the patients self-administered EQ-5D, Parkinson's Disease Sleep Scale (PDSS-2), and Beck Depression Inventories. Branded and generic ropinirole treatment achieved similar scores on all tests measuring severity of motor symptoms (primary endpoint, UPDRS-III: 27.0 versus 28.0 points, P = 0.505). Based on patient diaries, the lengths of "good time periods" were comparable (10.5 and 10.0 hours for branded and generic ropinirole, resp., P = 0.670). However, generic ropinirole therapy achieved almost 3.0 hours shorter on time without dyskinesia (6.5 versus. 9.5 hours, P < 0.05) and 2.5 hours longer on time with slight dyskinesia (3.5 versus. 1.0 hours, P < 0.05) than the branded ropinirole did. Except for gastrointestinal problems, nonmotor symptoms were similarly controlled. Patients did not prefer either formulation. Although this study has to be interpreted with limitations, it demonstrated that both generic and branded ropinirole administration can achieve similar control on most symptoms of PD. There is a growing amount of evidence to suggest that besides motor features, patients with essential tremor (ET) may exhibit significant nonmotor features, such as mild cognitive deficits, fatigue, neuropsychiatric symptoms, and sleep disturbances. The goal of this study was to examine nonmotor features in young patients with ET and their impact on quality of life. 45 patients (24.55 ± 7.16 years old) with ET and 35 controls were evaluated using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale, Beck Depression Inventory, Beck Anxiety Scale, Fatigue Severity Scale, and Short Form-36. Cognitive functions were evaluated using the Turkish version of the Montreal Cognitive Assessment Battery (MoCA). We ruled out other possible causes of the tremor. The tremor rate was evaluated using the Fahn-Tolosa-Marin Tremor Rating Scale. Poor sleep quality, fatigue, anxiety, and depressive symptoms were more common, and MoCA total scores were lower in the patient group. Fatigue, depressive symptoms, and higher anxiety levels were seen to have a negative effect on physical and mental health. Excessive daytime sleepiness had a negative effect on physical health. There is an emerging interest in nonmotor features of ET. This study showed that even young patients have nonmotor features that decrease their quality of life. This might tell us that nonmotor symptoms could be a part of the disease in the early stages. OBJECTIVES: Ventralis intermedius deep brain stimulation is an established intervention for medication-refractory essential tremor. Newer constant current stimulation technology offers theoretical advantage over the traditional constant voltage systems in terms of delivering a more biologically stable therapy. There are no previous reports on the outcomes of constant current deep brain stimulation in the treatment of essential tremor. This study aimed to evaluate the long-term efficacy of ventralis intermedius constant current deep brain stimulation in patients diagnosed with essential tremor. MATERIALS AND METHODS: Essential tremor patients implanted with constant current deep brain stimulation for a minimum of three years were evaluated. Clinical outcomes were assessed using the Fahn-Tolosa-Marin tremor rating scale at baseline and postoperatively at the time of evaluation. The quality of life in the patients was assessed using the Quality of Life in Essential Tremor questionnaire. RESULTS: Ten patients were evaluated with a median age at evaluation of 74 years (range 66-79) and a mean follow up time of 49.7 (range 36-78) months since starting stimulation. Constant current ventralis intermedius deep brain stimulation was well tolerated and effective in all patients with a mean score improvement from 50.7 ± 5.9 to 17.4 ± 5.7 (p = 0.0020) in the total Fahn-Tolosa-Marin rating scale score (65.6%). Furthermore, the total combined mean Quality of Life in Essential Tremor score was improved from 56.2 ± 4.9 to 16.8 ± 3.5 (p value = 0.0059) (70.1%). CONCLUSION: This report shows that long-term constant current ventralis intermedius deep brain stimulation is a safe and effective intervention for essential tremor patients. OBJECTIVE Unilateral Gamma Knife thalamotomy (GKT) is a well-established treatment for patients with medically refractory tremor who are not eligible for invasive procedures due to increased risk of compications. The purpose of this study was to evaluate whether staged bilateral GKT provides benefit with acceptable risk to patients suffering from disabling medically refractory bilateral tremor. METHODS Eleven patients underwent staged bilateral GKT during a 17-year period (1999-2016). Eight patients had essential tremor (ET), 2 had Parkinson's disease (PD)-related tremor, and 1 had multiple-sclerosis (MS)-related tremor. For the first GKT, a median maximum dose of 140 Gy was delivered to the posterior-inferior region of the nucleus ventralis intermedius (VIM) through a single isocenter with 4-mm collimators. Patients who benefitted from unilateral GKT were eligible for a contralateral GKT 1-2 years later (median 22 months). For the second GKT, a median maximum dose of 130 Gy was delivered to the opposite VIM nucleus to a single 4-mm isocenter. The Fahn-Tolosa-Marin (FTM) clinical tremor rating scale was used to score tremor, drawing, and drinking before and after each GKT. The FTM writing score was assessed only for the domit hand before and after the first GKT. The Karnofsky Performance Status (KPS) was used to assess quality of life and activities of daily living before and after the first and second GKT. RESULTS The median time to last follow-up after the first GKT was 35 months (range 11-70 months). All patients had improvement in at least 1 FTM score after the first GKT. Three patients (27.3%) had tremor arrest and complete restoration of function (noted via FTM tremor, writing, drawing, and drinking scores equaling zero). No patient had tremor recurrence or diminished tremor relief after the first GKT. One patient experienced new temporary neurological deficit (contralateral lower-extremity hemiparesis) from the first GKT. The median time to last follow-up after the second GKT was 12 months (range 2-70 months). Nine patients had improvement in at least 1 FTM score after the second GKT. Two patients had tremor arrest and complete restoration of function. No patient experienced tremor recurrence or diminished tremor relief after the second GKT. No patient experienced new neurological or radiological adverse effect from the second GKT. Statistically significant improvements were noted in the KPS score following the first and second GKT. CONCLUSIONS Staged bilateral GKT provided effective relief for medically refractory, disabling, bilateral tremor without increased risk of neurological complications. It is an appropriate strategy for carefully selected patients with medically refractory bilateral tremor who are not eligible for deep brain stimulation. OBJECTIVE Multiple sclerosis (MS) is a neurodegenerative disease that can lead to severe intention tremor in some patients. In several case reports, conventional radiotherapy has been reported to possibly exacerbate MS. Radiosurgery dramatically limits normal tissue irradiation to potentially avoid such a problem. Gamma Knife thalamotomy (GKT) has been established as a minimally invasive technique that is effective in treating essential tremor and Parkinson's disease-related tremor. The goal in this study was to analyze the outcomes of GKT in patients suffering from medically refractory MS-related tremor. METHODS The authors retrospectively studied the outcomes of 15 patients (mean age 46.5 years) who had undergone GKT over a 15-year period (1998-2012). Fourteen patients underwent GKT at a median maximum dose of 140 Gy (range 130-150 Gy) using a single 4-mm isocenter. One patient underwent GKT at a dose of 140 Gy delivered via two 4-mm isocenters (3 mm apart). The posteroinferior region of the nucleus ventralis intermedius (VIM) was the target for all GKTs. The Fahn-Tolosa-Marin clinical tremor rating scale was used to evaluate tremor, handwriting, drawing, and drinking. The median time to the last follow-up was 39 months. RESULTS After GKT, 13 patients experienced tremor improvement on the side contralateral to surgery. Four patients noted tremor arrest at a median of 4.5 months post-GKT. Seven patients had excellent tremor improvement and 6 had good tremor improvement. Four patients noted excellent functional improvement, 8 noted good functional improvement, and 1 noted satisfactory functional improvement. Three patients experienced diminished tremor relief at a median of 18 months after radiosurgery. Two patients experienced temporary adverse radiation effects. Another patient developed a large thalamic cyst 60 months after GKT, which was successfully managed with Ommaya reservoir placement. CONCLUSIONS Gamma Knife thalamotomy was found to be a minimally invasive and beneficial procedure for medically refractory MS tremor. Although essential tremor is the most common movement disorder, there is little knowledge about the pathophysiological mechanisms of this disease. Therefore, we explored brain connectivity based on slow spontaneous fluctuations of blood oxygenation level dependent (BOLD) signal in patients with essential tremor (ET). A cohort of 19 ET patients and 23 healthy individuals were scanned in resting condition using functional magnetic resoce imaging (fMRI). General connectivity was assessed by eigenvector centrality (EC) mapping. Selective connectivity was analyzed by correlations of the BOLD signal between the preselected seed regions and all the other brain areas. These measures were then correlated with the tremor severity evaluated by the Fahn-Tolosa-Marin Tremor Rating Scale (FTMTS). Compared to healthy subjects, ET patients were found to have lower EC in the cerebellar hemispheres and higher EC in the anterior cingulate and in the primary motor cortices bilaterally. In patients, the FTMTS score correlated positively with the EC in the putamen. In addition, the FTMTS score correlated positively with selective connectivity between the thalamus and other structures (putamen, pre-supplementary motor area (pre-SMA), parietal cortex), and between the pre-SMA and the putamen. We observed a selective coupling between a number of areas in the sensorimotor network including the basal ganglia and the ventral intermediate nucleus of thalamus, which is widely used as neurosurgical target for tremor treatment. Finally, ET was marked by suppression of general connectivity in the cerebellum, which is in agreement with the concept of ET as a disorder with cerebellar damage. BACKGROUND: There is a significant need for a targeted therapy for essential tremor (ET), as medications have not been developed specifically for ET, and the ones prescribed are often not well-tolerated, so that many patients remain untreated. Recent work has shown that, unlike previous experience, kinematically guided individualized botulinum toxin type A (BoNT-A) injections provide benefit along with minimal weakness. Ours is the first long-term (96-week) safety and efficacy study of BoNT-A as monotherapy for ET using kinematically driven injection parameters. METHODS: Ten ET patients were administered six serial BoNT-A treatments every 16 weeks and were assessed at 6 weeks following treatment. During each study visit, the Fahn-Tolosa-Marin (FTM) scale, the Unified Parkinson's Disease Rating Scale, and the Quality of Life for Essential Tremor Questionnaire (QUEST) were administered along with kinematic assessment of the treated limb. Participants performed scripted tasks with motion sensors placed over each arm joint. Dosing patterns were determined using the movement disorder neurologist's interpretation of muscles contributing to the kinematically analyzed upper limb tremor biomechanics. RESULTS: There was a 33.8% (p<0.05) functional improvement (FTM part C) and a 39.8% (p<0.0005) improvement in QUEST score at week 96 compared to pretreatment scores at week 0. Although there was a 44.6% (p<0.0005) non-dose-dependent reduction in maximal grip strength, only 2 participants complained of mild weakness. Following the fourth serial treatment, mean action tremor score was reduced by 62.9% (p=0.001) in the treated and by 44.4% (p=0.03) in the untreated arm at week 96 compared to week 48. CONCLUSIONS: Individualized BoNT-A dosing patterns to each individual's tremor biomechanics provided an effective monotherapy for ET as function improved without functionally limiting muscle weakness. Essential tremor is a neurological syndrome of heterogeneous pathology and aetiology that is characterized by tremor primarily in the upper extremities. This tremor is commonly hypothesized to be driven by a single or multiple neural oscillator(s) within the cerebello-thalamo-cortical pathway. Several studies have found an association of blood-oxygen level-dependent (BOLD) signal in the cerebello-thalamo-cortical pathway with essential tremor, but there is behavioural evidence that also points to the possibility that the severity of tremor could be influenced by visual feedback. Here, we directly manipulated visual feedback during a functional MRI grip force task in patients with essential tremor and control participants, and hypothesized that an increase in visual feedback would exacerbate tremor in the 4-12 Hz range in essential tremor patients. Further, we hypothesized that this exacerbation of tremor would be associated with dysfunctional changes in BOLD signal and entropy within, and beyond, the cerebello-thalamo-cortical pathway. We found that increases in visual feedback increased tremor in the 4-12 Hz range in essential tremor patients, and this increase in tremor was associated with abnormal changes in BOLD amplitude and entropy in regions within the cerebello-thalamo-motor cortical pathway, and extended to visual and parietal areas. To determine if the tremor severity was associated with single or multiple brain region(s), we conducted a birectional stepwise multiple regression analysis, and found that a widespread functional network extending beyond the cerebello-thalamo-motor cortical pathway was associated with changes in tremor severity measured during the imaging protocol. Further, this same network was associated with clinical tremor severity measured with the Fahn, Tolosa, Marin Tremor Rating Scale, suggesting this network is clinically relevant. Since increased visual feedback also reduced force error, this network was evaluated in relation to force error but the model was not significant, indicating it is associated with force tremor but not force error. This study therefore provides new evidence that a widespread functional network is associated with the severity of tremor in patients with essential tremor measured simultaneously at the hand during functional imaging, and is also associated with the clinical severity of tremor. These findings support the idea that the severity of tremor is exacerbated by increased visual feedback, suggesting that designers of new computing technologies should consider using lower visual feedback levels to reduce tremor in essential tremor. OBJECTIVES: We investigated the effects of deep brain stimulation (DBS) or lesions of the ventral intermediate nucleus (Vim) of the thalamus for spinocerebellar ataxia (SCA) and examined the pathophysiological role of neuronal activity of the Vim underlying ataxia. METHODS: Five patients with SCA with cortical atrophy (ages 60-69 years; 2 sporadic and three familial SCA) and five patients with essential tremor (ET) (ages 57-71 years) were treated with Vim surgery. Intraoperatively, we recorded neuronal activity from single neurons in the Vim thalamus while patients were at rest and compared the physiological properties of those neurons between patients with SCA and those with ET. RESULTS: Postsurgery mean scores for the Fahn-Tolosa-Marin Tremor Scale were improved from 78 to 44 in SCA patients and from 54 to 21 in ET patients. Stronger stimulation was necessary to optimize outcomes in SCA as compared to ET patients. We analyzed 68 Vim neurons in SCA and 60 Vim neurons in ET. Mean discharge rates, burst characteristics, and oscillatory activity were similar for both patient groups, however, we observed that the ratio of cells responding to passive manipulation was significantly smaller (P = 0.0001) in SCA (22%) than in ET (71%). INTERPRETATION: Thalamic surgery led to a significant improvement in tremor in SCA patients. One potential mechanism underlying ataxia in SCA may be disruption of cerebellar sensory feedback, which modulates motor commands in the cerebello-thalamo-cortical network. BACKGROUND: Besides fluctuations, therapy refractory tremor is one of the main indications of deep brain stimulation (DBS) in patients with idiopathic Parkinson syndrome (IPS). Although thalamic DBS (ventral intermediate nucleus [Vim] of thalamus) has been shown to reduce tremor in 85-95% of patients, bradykinesia and rigidity often are not well controlled. The dentato-rubro-thalamic tract (DRT) that can directly be targeted with special diffusion tensor magnetic resoce imaging sequences has been shown as an efficient target for thalamic DBS. The subthalamic nucleus (STN) is typically chosen in younger patients as the target for dopamine-responsive motor symptoms. This study investigates a one-path thalamic (Vim/DRT) and subthalamic implantation of DBS electrodes and possibly a combined stimulation strategy for both target regions. OBJECTIVE: This study investigates a one path thalamic (Vim/DRT) and subthalamic implantation of DBS electrodes and a possibly combined stimulation strategy for both target regions. METHODS: This is a randomized, active-controlled, double-blinded (patient- and observer-blinded), monocentric trial with three treatments, three periods and six treatment sequences allocated according to a Williams design. Eighteen patients will undergo one-path thalamic (Vim/DRT) and STN implantation of DBS electrodes. After one month, a double-blinded and randomly-assigned stimulation of the thalamic target (Vim/DRT), the STN and a combined stimulation of both target regions will be performed for a period of three months each. The primary objective is to assess the quality of life obtained by the Parkinson's Disease Questionnaire (39 items) for each stimulation modality. Secondary objectives include tremor reduction (obtained by the Fahn-Tolosa-Marin tremor rating scale, video recordings, the Unified Parkinson's disease rating scale, and by tremor analysis), psychiatric assessment of patients, and to assess the safety of intervention. RESULTS: At the moment, the recruitment is stopped and 12 patients have been randomized and treated. A futility analysis is being carried out by means of a conditional power analysis. CONCLUSIONS: The approach of the OPINION trial planned to make, for the first time, a direct comparison of the different stimulation conditions (Vim/DRT, compared to STN, compared to Vim/DRT+STN) in a homogeneous patient population and, furthermore, will allow for intraindividual comparison of each condition with the "quality of life" outcome parameter. We hypothesize that the combined stimulation of the STN and the thalamic (Vim/DRT) target will be superior with respect to the patients' quality of life as compared to the singular stimulation of the individual target regions. If this holds true, this work might change the standardized treatment described in the previous section. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02288468; https://clinicaltrials.gov/ct2/show/NCT02288468 (Archived by WebCite at http://www.webcitation.org/6wlKnt2pJ); and German Clinical Trials Register: DRKS00007526; https://www.drks.de/drks_ web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00007526 (Archived by WebCite at http://www.webcitation.org/6wlKyXZZL). OBJECTIVE Transcranial magnetic resoce-guided focused ultrasound surgery (tcMRgFUS) is one of the emerging noninvasive technologies for the treatment of neurological disorders such as essential tremor (ET), idiopathic asymmetrical tremor-domit Parkinson's disease (PD), and neuropathic pain. In this clinical series the authors present the preliminary results achieved with the world's first tcMRgFUS system integrated with a 1.5-T MRI unit. METHODS The authors describe the results of tcMRgFUS in a sample of patients with ET and with PD who underwent the procedure during the period from January 2015 to September 2017. A monolateral ventralis intermedius nucleus (VIM) thalamic ablation was performed in both ET and PD patients. In all the tcMRgFUS treatments, a 1.5-T MRI scanner was used for both planning and monitoring the procedure. RESULTS During the study period, a total of 26 patients underwent tcMRgFUS thalamic ablation for different movement disorders. Among these patients, 18 were diagnosed with ET and 4 were affected by PD. All patients with PD were treated using tcMRgFUS thalamic ablation and all completed the procedure. Among the 18 patients with ET, 13 successfully underwent tcMRgFUS, 4 aborted the procedure during ultrasound delivery, and 1 did not undergo the tcMRgFUS procedure after stereotactic frame placement. Two patients with ET were not included in the results because of the short follow-up duration at the time of this study. A monolateral VIM thalamic ablation in both ET and PD patients was performed. All the enrolled patients were evaluated before the treatment and 2 days after, with a clinical control of the treatment effectiveness using the graphic items of the Fahn-Tolosa-Marin tremor rating scale. A global reevaluation was performed 3 months (17/22 patients) and 6 months (11/22 patients) after the treatment; the reevaluation consisted of clinical questionnaires, neurological tests, and video recordings of the tests. All the ET and PD treated patients who completed the procedure showed an immediate amelioration of tremor severity, with no intra- or posttreatment severe permanent side effects. CONCLUSIONS Although this study reports on a small number of patients with a short follow-up duration, the tcMRgFUS procedure using a 1.5-T MRI unit resulted in a safe and effective treatment option for motor symptoms in patients with ET and PD. To the best of the authors' knowledge, this is the first clinical series in which thalamotomy was performed using tcMRgFUS integrated with a 1.5-T magnet. INTRODUCTION: Movement disorders are not rare in demyelinating diseases but there are few studies comparing their frequency between multiple sclerosis and neuromyelitis optica spectrum disorder. Our aim was to determine the frequency and the related features of movement disorders in a cohort of patients with multiple sclerosis and neuromyelitis optica spectrum disorder. METHODS: It is a cross-sectional study of patients with multiple sclerosis and neuromyelitis optica spectrum disorder. Patients were evaluated by a movement disorder specialist. Data from a personal interview and neurological examination were collected. Fahn-Tolosa-Marin tremor rating scale was used for tremor evaluation. Health-related quality of life was assessed using EuroQol instrument. RESULTS: Two hundred fifty-three patients were included (mean [SD] age, 40 [12] years; 74.3% female; median [IQR] EDSS score 2.5 [1.0-6.0]); 26% presented with movement disorders. Paroxysmal dystonia (n = 32) and tremor (n = 27) were the most common movement disorders. Patients with multiple sclerosis and low Expanded Disability Status Scale score (below 4.0) have fewer movement disorders than patients with neuromyelitis optica spectrum disorder. The diagnosis of neuromyelitis optica spectrum disorder was strongly associated with paroxysmal dystonia (OR = 22.07, 95% CI = 2.56-189.78; p = 0.005). Patients with multiple sclerosis and patients without movement disorders have a slightly better quality of life. CONCLUSIONS: Paroxysmal dystonia was the most common movement disorder in demyelinating diseases and strongly associated with neuromyelitis optica spectrum disorder. INTRODUCTION: Tremor is the most frequent and disabling neurological side effect under Calcineurin inhibitor-induced immunosuppression, but no studies have defined its phenomenology, severity, distribution, the impact on quality of life, as well as of other neurological symptoms associated. METHODS: 126 consecutive kidney-transplanted patients, under treatment with Cyclosporin A, Tacrolimus and non-Calcineurin inhibitors, within therapeutic range, were enrolled. Participants underwent a deep neurological examination by two blinded to the treatment raters, and a blood sampling to assess plasmatic immunosuppressant level and nephrological function tests. Tremor and cerebellar signs were scored according to the Fahn-Tolosa-Marin and the SARA scale. Parkinsonism was excluded applying the UPDRS (part III). RESULTS: Tremor was more common and severe in the Tacrolimus group, similar to impairment in ADL. Regardless of treatment, tremor involved both upper and lower limbs and was activated by action, but in about 50% of cases presented in action and rest condition. Plasmatic level of Tacrolimus was higher in patients with tremor than in those without, while cholesterol was significantly lower. Cerebellar and neuropathic signs were overall mild and were not significantly different across the three groups comparing patients with and without tremor. CONCLUSIONS: Non-Calcineurin inhibitors such as Sirolimus have the lowest propensity to induce tremor and with a milder severity, while Calcineurin inhibitors, especially Tacrolimus, the highest, and regardless of the formulation. Plasmatic concentration of Tacrolimus was higher in tremulous patients; further research needs to validate the role of cholesterol plasmatic concentration in predicting the occurrence of tremor in patients on Tacrolimus. INTRODUCTION: To evaluate the safety and efficacy of incobotulinumtoxinA (IncoA) injection for treatment of essential hand tremor. In essential tremor and Parkinson's disease tremor, administration of onabotulinumtoxinA via a fixed injection approach improves the tremor but a high percentage of patients (30-70%) develop moderate to severe hand weakness which has limited its use in clinical practice. METHODS: This study was performed from July 2013 to July 2016 on 33 subjects. This is a double-blind, placebo-controlled, crossover trial injecting 80-120 units of IncoA into 8-14 hand and forearm muscles using a customized approach. The subjects were followed for 28 weeks. The treatment efficacy was evaluated by the Fahn Tolosa Marin tremor rating score and NIH genetic criteria for tremor severity at 4 and 8 weeks after each of the two sets of treatments. Hand strength was assessed by an ergometer. RESULTS: There was statistically significant improvement in clinical rating score of tremor at 4 and 8 weeks following the IncoA injection. CONCLUSION: In this study, injection of IncoA treatment via a customized approach improved essential tremor on the clinical scales and patient's perception with a low occurrence of significant hand weakness. OBJECTIVE: To investigate the effect of directional current steering and short pulse stimulation in the ventral intermediate thalamic nucleus (VIM) on stimulation-induced side effects in patients with essential tremor. METHODS: We recruited 8 patients with essential tremor in a stable postoperative condition (>3 months after electrode implantation of deep brain stimulation [DBS] electrodes) with segmented contacts implanted in the VIM. Tremor severity on acute stimulation was assessed by the Fahn-Tolosa-Marin Tremor Rating Scale. Cerebellar impairment was evaluated with the International Cooperative Ataxia Rating Scale. Patients rated paresthesia intensity with a visual analog scale. RESULTS: In all patients, tremor was reduced to the same extent by VIM stimulation regardless of pulse width using energy dose-equivalent amplitudes. Short pulse stimulation diminished stimulation-induced ataxia of the upper extremities and paresthesia compared with conventional parameters. Directional steering with monopolar stimulation of single segments successfully suppressed tremor but also induced ataxia. No differences in adverse effects were found between single-segment stimulation conditions. CONCLUSION: These proof-of-principle findings provide evidence that acute short pulse stimulation is superior to directional steering in the subthalamic area to decrease stimulation-induced side effects while preserving tremor suppression effects in patients with tremor. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that for patients with tremor with thalamic DBS, acute short pulse stimulation reduces adverse effects, while directional steering does not provide a generalizable benefit regarding adverse effects. BACKGROUND: The Fahn-Tolosa-Marin Clinical Rating Scale for Tremor (FTM) has been used in large trials for essential tremor (ET), but its anchors for ratings from 0 to 4 of upper limb tremor are probably too low for patients with severe tremor (tremor amplitude >4 cm; grade 4). The Essential Tremor Rating Assessment Scale (TETRAS) is a validated clinical scale designed specifically for the assessment of ET severity. TETRAS has anchors that span a larger range of tremor amplitudes (>20 cm = grade 4), making it more suitable for assessing patients with severe ET. However, there is no direct comparison of these scales in any clinical trial. METHODS: Upper limb postural and kinetic tremor items from both scales were compared using blinded, video-recorded examinations of patients with moderate-to-severe ET who participated in a trial of focused ultrasound thalamotomy. RESULTS: FTM ratings of postural and kinetic tremor correlated strongly with those of TETRAS. However, FTM exhibited a ceiling effect for severe tremor. Rest tremor, exclusive to FTM, correlated poorly with postural and kinetic tremor and had very poor test-retest reliability. In contrast, wing-beating postural tremor, exclusive to TETRAS, exhibited excellent test-retest reliability and a strong correlation with kinetic and limbs-extended-forward postural tremor. Test-retest reliabilities of the other TETRAS and FTM ratings were excellent, and both scales had good sensitivity to treatment effect. CONCLUSIONS: TETRAS has 2 main advantages over FTM in the assessment of tremor severity: (1) the absence of a ceiling effect in patients with severe ET, and (2) the inclusion of wing-beating tremor. Despite being considered as a benign, genetic and monosymptomatic disorder, ET is a poorly understood entity with etiological and pathological heterogeneity. The aim of the present study was to examine the relation between proximal and distal muscle strength and upper limb functionality and tremor severity in patients with essential tremor (ET). The study enrolled 25 tremor patients followed at the neurology clinic of a university hospital and 19 healthy controls. Demographic data, risk factors, disease duration and domit hand of the participants were recorded. Back and leg strength was assessed using a back and leg dynamometer and hand dynamometer and pinchmeter were used to determine hand strength. Functional ability of the participants was evaluated using the Minnesota Manual Dexterity Test (MMDT), Perdue Pegboard (PPBT) and Nine Hole Peg Test (NHPT). Tremor severity was assessed using the Fahn-Tolosa-Marin Tremor Rating Scale (FTMTRS) and the Lower Extremity Clinical Tremor Assessment Scale (LECTAS). A significant difference was found in the average back and leg muscle strength between ET patients and healthy controls (p < 0.05). The mean values for right/left hand muscle strength were not significantly different between the two groups (p > 0.05). Among the upper limb functional ability tests, significant differences were found between the two groups in the mean time to complete NHPT, Minnesota placing subtest and PPBT test (p < 0.05). While gender and risk factors were not significantly different between the two groups (p > 0.05), there was a significant difference with respect to the educational level (p < 0.01). No significant difference was found between back and leg muscle strength and FTMTRS and LECTAS (p > 0.05). A negative correlation and a significant association were found between average strength measurements obtained with the left hand dynamometer and FTMTRS in the ET group (p = 0.030, r = - 0.434). A positive correlation and a significant association were found between left hand strength and mean turning time in the MMDT in the control group (p = 0.041, r = 0.473). ET patients experience loss of proximal muscle strength and functional disability. Further studies are planned to investigate the effects of physical therapy modalities targeting increased proximal muscle strength on tremor severity and functional ability in ET patients. Tremor and parkinsonism are recognized side effects of valproate; however, the relationship between rest tremor and other signs of parkinsonism has not been addressed in patients taking valproate. We studied a cohort of 125 consecutive patients treated with valproate due to epilepsy or migraine, evaluated with the Fahn-Tolosa-Marin Tremor Rating Scale (FTM-TRS). A total of 14 (11.2%) patients had rest tremor (bilateral n = 10, unilateral n = 4). Patients with rest tremor had significant higher scores in the FTM-TRS (P < 0.001), but only one was diagnosed with parkinsonism. Patients may have valproate-induced parkinsonism or exacerbated motor features of Parkinson's disease by valproate. The frequency of parkinsonism was 1.6% in this cohort and of 3% in the pooled data of 717 patients from previous reports. Rest tremor is observed in 11.2% of patients treated with valproate and is related to the burden of valproate-induced tremor, rather than the presence of parkinsonism. BACKGROUND: Several scales are available for rating the severity of tremor at present. However, the sensitivity to change of these instruments has remained to be clarified. OBJECTIVE: To compare the sensitivity of the Fahn-Tolosa-Marin Tremor Rating Scale, the Part III of the Movement Disorder Society-sponsored Unified Parkinson's Disease Rating Scale (MDS-UPDRS) and the MDS-UPDRS Tremor Scale to the effects of various antitremor treatments. METHODS: Enrolling subjects with parkinsonism associated with tremor, we analyzed two scenarios: (1) tremor changes associated with acute levodopa challenge (n = 287) and (2) a 12-month outcome of different treatment options (n = 512) including deep brain stimulation (n = 146), levodopa/carbidopa intestinal gel infusion (n = 30), and initiating (n = 63) or adjusting oral antiparkinsonian medication (n = 273). Changes in tremor scales were assessed by effect size values (Cohen's d and eta-square). RESULTS: Part B of the Fahn-Tolosa-Marin Tremor Rating Scale was the most sensitive to acute levodopa challenge (Cohen's d = -1.04, η2 = 0.12). However, Part A of the Fahn-Tolosa-Marin Tremor Rating Scale showed the highest effect size, which was a small one (Cohen's d = -0.33, η2 = 0.03), for detecting a treatment-related change in the severity of tremor during long-term follow-up. CONCLUSIONS: The Fahn-Tolosa-Marin Tremor Rating Scale has a better ability to capture changes due to levodopa challenge or antiparkinsonian treatment than MDS-UPDRS Part III or MDS-UPDRS Tremor Scale.

List the core SNARE complex proteins.

VAMP2 Syntaxin SNAP25

SNAP-25 is a protein involved in regulated membrane fusion and part of the SNARE complex. It exists as two splicing variants, SNAP-25a and SNAP-25b, which differ in 9 out of 206 amino acids. SNAP-25 together with Syntaxin 1 and VAMP-2 forms the ternary SNARE complex essential for mediating activity-dependent release of hormones and neurotransmitters. The functional difference between SNAP-25a and SNAP-25b is poorly understood as both can participate in SNARE complexes and mediate membrane fusion. However, we recently demonstrated that SNAP-25b-deficiency results in metabolic disease and increased insulin secretion. Here we investigated if SNAP-25a and SNAP-25b differently affect interactions with other SNAREs and SNARE-interacting proteins in mouse hippocampus. Adult mice almost exclusively express the SNAP-25b protein in hippocampus whereas SNAP-25b-deficient mice only express SNAP-25a. Immunoprecipitation studies showed no significant differences in amount of Syntaxin 1 and VAMP-2 co-precipitated with the different SNAP-25 isoforms. In contrast, Munc18-1, that preferentially interacts with SNAP-25 via Syntaxin 1 and/or the trimeric SNARE complex, demonstrated an increased ability to bind protein-complexes containing SNAP-25b. Moreover, we found that both SNAP-25 isoforms co-precipitated the Gβγ subunits of the heterotrimeric G proteins, an interaction known to play a role in presynaptic inhibition. We have identified Gβ1 and Gβ2 as the interacting partners of both SNAP-25 isoforms in mouse hippocampus, but Gβ2 was less efficiently captured by SNAP-25a. These results implicate that the two SNAP-25 isoforms could differently mediate protein interactions outside the ternary SNARE core complex and thereby contribute to modulate neurotransmission.

What is septicemia?

Septicemia occurs when a bacterial infection elsewhere in the body, such as the lungs or skin, enters the bloodstream.

BACKGROUND AND PURPOSE: Rapid identification of microbes in the bloodstream is crucial in managing septicemia because of its high disease severity, and direct identification from positive blood culture bottles through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can shorten the turnaround time. Therefore, we developed a simple method for rapid microbiological identification from positive blood cultures by using MALDI-TOF MS. METHODS: We modified previously developed methods to propose a faster, simpler and more economical method, which includes centrifugation and hemolysis. Specifically, our method comprises two-stage centrifugation with gravitational acceleration (g) at 600g and 3000g, followed by the addition of a lysis buffer and another 3000g centrifugation. RESULTS: In total, 324 monomicrobial bacterial cultures were identified. The success rate of species identification was 81.8%, which is comparable with other complex methods. The identification success rate was the highest for Gram-negative aerobes (85%), followed by Gram-positive aerobes (78.2%) and anaerobes (67%). The proposed method requires less than 10 min, costs less than US$0.2 per usage, and facilitates batch processing. CONCLUSION: We conclude that this method is feasible for clinical use in microbiology laboratories, and can serve as a reference for treatments or further complementary diagnostic testing.

Which is the master oncogenic transcription factor in T-cell acute lymphoblastic leukemia?

The oncogenic transcription factor TAL1/SCL induces an aberrant transcriptional program in T-cell Acute lymphoblastic Leukemia (T-ALL) cells.

The Tal1 oncogene is a class II basic helix-loop-helix (bHLH) transcription factor, overexpressed in as much as 60% of T cell acute lymphoblastic leukemia cases. Like other class II bHLH proteins, Tal1 can heterodimerize with the class I bHLH proteins, such as E47, and bind to a DNA recognition sequence termed E box. Therefore, it is believed that the oncogenic capacity of Tal1 lies in its ability, as a heterodimer with E47, to activate aberrantly a set of "leukemogenic" genes in T cells. However, compared with E47 homodimers, Tal1/E47 heterodimers are very poor transactivators. Thus the effect of Tal1 is actually to inhibit E47 homodimer activity. Here we propose that the transforming properties of Tal1 are the result of its ability to inhibit E47 activity. We address the mechanism of Tal1 inhibition and demonstrate that Tal1/E47 heterodimers cannot activate transcription because their respective activation domains are incompatible. Furthermore, we present data showing that Tal1 can inhibit E47-mediated activation of the CIP1 gene. Finally, we demonstrate that Tal1 inhibits E47 activity in leukemic T cells. Aberrant expression of transcription factor oncogenes such as HOX11, HOX11L2, TAL1/SCL, LYL1, LMO1, and LMO2 can be detected in lymphoblasts from up to 80% of patients with acute T-cell lymphoblastic leukemia (T-ALL). Transcriptional activation of these oncogenes in leukemic cells typically results from chromosomal rearrangements that place them next to highly active cis-acting transcriptional regulatory elements. However, biallelic activation of TAL1 in some T-ALL cases has been previously proposed. We have used allele-specific mRNA analysis to show that trans-acting mechanisms leading to biallelic overexpression of TAL1 are involved in 10 (42%) of 24 TAL1+ informative T-ALL cases, 2 (17%) of 12 HOX11+ informative cases, and 7 (64%) of 11 LMO2+ informative cases. We propose that aberrant expression of oncogenic transcription factors in a significant fraction of T-ALLs may result from loss of the upstream transcriptional mechanisms that normally down-regulate the expression of these oncogenes during T-cell development. The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells. T-cell acute lymphoblastic leukemia (T-ALL) is caused by the cooperation of multiple oncogenic lesions. We used exome sequencing on 67 T-ALLs to gain insight into the mutational spectrum in these leukemias. We detected protein-altering mutations in 508 genes, with an average of 8.2 mutations in pediatric and 21.0 mutations in adult T-ALL. Using stringent filtering, we predict seven new oncogenic driver genes in T-ALL. We identify CNOT3 as a tumor suppressor mutated in 7 of 89 (7.9%) adult T-ALLs, and its knockdown causes tumors in a sensitized Drosophila melanogaster model. In addition, we identify mutations affecting the ribosomal proteins RPL5 and RPL10 in 12 of 122 (9.8%) pediatric T-ALLs, with recurrent alterations of Arg98 in RPL10. Yeast and lymphoid cells expressing the RPL10 Arg98Ser mutant showed a ribosome biogenesis defect. Our data provide insights into the mutational landscape of pediatric versus adult T-ALL and identify the ribosome as a potential oncogenic factor. Previous results indicated that miR-146b-5p is downregulated by TAL1, a transcription factor critical for early hematopoiesis that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL) where it has an oncogenic role. Here, we confirmed that miR-146b-5p expression is lower in TAL1-positive patient samples than in other T-ALL cases. Furthermore, leukemia T-cells display decreased levels of miR-146b-5p as compared to normal T-cells, thymocytes and other hematopoietic progenitors. MiR-146b-5p silencing enhances the in vitro migration and invasion of T-ALL cells, associated with increased levels of filamentous actin and chemokinesis. In vivo, miR-146b overexpression in a TAL1-positive cell line extends mouse survival in a xenotransplant model of human T-ALL. In contrast, knockdown of miR-146b-5p results in leukemia acceleration and decreased mouse overall survival, paralleled by faster tumor infiltration of the central nervous system. Our results suggest that miR-146b-5p is a functionally relevant microRNA gene in the context of T-ALL, whose negative regulation by TAL1 and possibly other oncogenes contributes to disease progression by modulating leukemia cell motility and disease aggressiveness. In hematopoietic cell development, the transcriptional program is strictly regulated in a lineage- and stage-specific manner that requires a number of transcription factors to work in a cascade or in a loop, in addition to interactions with nonhematopoietic cells in the microenvironment. Disruption of the transcriptional program alters the cellular state and may predispose cells to the acquisition of genetic abnormalities. Early studies have shown that proteins that promote cell differentiation often serve as tumor suppressors, whereas inhibitors of those proteins act as oncogenes in the context of acute leukemia. A prime example is T-cell acute lymphoblastic leukemia (T-ALL), a maligt disorder characterized by clonal proliferation of immature stage thymocytes. Although a relatively small number of genetic abnormalities are observed in T-ALL, these abnormalities are crucial for leukemogenesis. Many oncogenes and tumor suppressors in T-ALL are transcription factors that are required for normal hematopoiesis. The transformation process in T-ALL is efficient and orchestrated; the oncogene disrupts the transcriptional program directing T-cell differentiation and also uses its native ability as a master transcription factor in hematopoiesis. This imbalance in the transcriptional program is a primary determit underlying the molecular pathogenesis of T-ALL. In this review, we focus on the oncogenic transcription factor TAL1 and the tumor-suppressor E-proteins and discuss the maligt cell state, the transcriptional circuit, and the consequence of molecular abnormalities in T-ALL.

List as many European influenza vaccines as possible.

Three split-virion vaccines (Vaxigrip, Begrivac, and Influsplit/Fluarix) and three subunit vaccines containing only viral surface glycoproteins (Influvac, Agrippal, and Fluvirin).

Three split-virion vaccines (Vaxigrip, Begrivac, and Influsplit/Fluarix) and three subunit vaccines containing only viral surface glycoproteins (Influvac, Agrippal, and Fluvirin) available for the 1994-95 season were analysed by biological, molecular, and biochemical methods. Although all vaccines are required by health authorities to contain 15 micrograms haemagglutinin per dose of each virus strain, there were significant differences in haemagglutination titres among the examined vaccines of both types. The enzymatic activity of neuraminidase was present in all vaccines except Fluvirin. Total protein content was lower for subunit vaccines. Viral nucleoprotein was detected in all split vaccines but to varying levels according to SDS-PAGE and Western blot analyses. The ovalbumin content was low in general but was about tenfold higher for Influvac than for the other vaccines analysed. This protein may induce hypersensitive reactions among persons with severe egg allergy. All three split-virion vaccines were found to contain the matrix protein; however, it was not detected in the subunit vaccines. Differences in influenza antigen variety in currently available vaccines may affect efficacy, whereas differences in concentrations of nonviral compounds such as ovalbumin and endotoxin may lead to different postvaccination reactogenicity profiles.

What is canSAR?

canSAR (http://cansar.icr.ac.uk) is a public integrative cancer-focused knowledgebase for the support of cancer translational research and drug discovery. Through the integration of biological, pharmacological, chemical, structural biology and protein network data, it provides a single information portal to answer complex multidisciplinary questions including--among many others--and what is known about a protein, in which cancers is it expressed or mutated, and what chemical tools and cell line models can be used to experimentally probe its activity.

canSAR (http://cansar.icr.ac.uk) is a public integrative cancer-focused knowledgebase for the support of cancer translational research and drug discovery. Through the integration of biological, pharmacological, chemical, structural biology and protein network data, it provides a single information portal to answer complex multidisciplinary questions including--among many others--what is known about a protein, in which cancers is it expressed or mutated, and what chemical tools and cell line models can be used to experimentally probe its activity? What is known about a drug, its cellular sensitivity profile and what proteins is it known to bind that may explain unusual bioactivity? Here we describe major enhancements to canSAR including new data, improved search and browsing capabilities and new target, cancer cell line, protein family and 3D structure summaries and tools. canSAR (http://cansar.icr.ac.uk) is a publicly available, multidisciplinary, cancer-focused knowledgebase developed to support cancer translational research and drug discovery. canSAR integrates genomic, protein, pharmacological, drug and chemical data with structural biology, protein networks and druggability data. canSAR is widely used to rapidly access information and help interpret experimental data in a translational and drug discovery context. Here we describe major enhancements to canSAR including new data, improved search and browsing capabilities, new disease and cancer cell line summaries and new and enhanced batch analysis tools. canSAR (http://cansar.icr.ac.uk) is a public, freely available, integrative translational research and drug discovery knowlegebase. canSAR informs researchers to help solve key bottlenecks in cancer translation and drug discovery. It integrates genomic, protein, pharmacological, drug and chemical data with structural biology, protein networks and unique, comprehensive and orthogonal 'druggability' assessments. canSAR is widely used internationally by academia and industry. Here we describe major enhancements to canSAR including new and expanded data. We also describe the first components of canSARblack-an advanced, responsive, multi-device compatible redesign of canSAR with a question-led interface.

What is the mechanism of action of magrolimab?

Magrolimab is an anti-CD47 antibody with promising results for myelodysplastic syndromes and acute myeloid leukemia.

In recent years, immunotherapies have been clinically investigated in AML and other myeloid maligcies. While most of these are focused on stimulating the adaptive immune system (including T cell checkpoint inhibitors), several key approaches targeting the innate immune system have been identified. Macrophages are a key cell type in the innate immune response with CD47 being identified as a domit macrophage checkpoint. CD47 is a "do not eat me" signal, overexpressed in myeloid maligcies that leads to tumor evasion of phagocytosis by macrophages. Blockade of CD47 leads to engulfment of leukemic cells and therapeutic elimination. Pre-clinical data has demonstrated robust anti-cancer activity in multiple hematologic maligcies including AML and myelodysplastic syndrome (MDS). In addition, clinical studies have been underway with CD47 targeting agents in both AML and MDS as monotherapy and in combination. This review will describe the role of CD47 in myeloid maligcies and pre-clinical data supporting CD47 targeting. In addition, initial clinical data of CD47 targeting in AML/MDS will be reviewed, and including the first-in-class anti-CD47 antibody magrolimab.

List types of mutations.

point mutations deletions insertions inversions translocations

With the advancement and improvement of new sequencing technology, next-generation sequencing (NGS) has been applied increasingly in cancer genomics research fields. More recently, NGS has been adopted in clinical oncology to advance personalized treatment of cancer. NGS is utilized to novel diagnostic and rare cancer mutations, detection of translocations, inversions, insertions and deletions, detection of copy number variants, detect familial cancer mutation carriers, provide the molecular rationale for appropriate targeted, therapeutic and prognostic. NGS holds many advantages, such as the ability to fully sequence all types of mutations for a large number of genes (hundreds to thousands) and the sensitivity, speed in a single test at a relatively low cost compared to be other sequencing modalities. Here we described the technology, methods and applications that can be immediately considered and some of the challenges that lie ahead. Alpha-thalassemia (α-thal) is probably the most prevalent monogenic condition in the world. Deletions are the most common types of mutations in α-thal, followed by point mutations and small insertion/deletion. In the context of national screening program for prevention of thalassemia and hemoglobinopathies in Iran, α-thal carriers have come to more attention. Therefore, the frequency and distribution of α-globin mutations in various regions of the country have been studied in recent years. A comprehensive search was performed in PubMed, Scopus, and national databases for finding reports on mutation detection in α-thal carriers and HbH disease with Iranian origin. The mutation data of 10849 α-thal carriers showed that -α3.7 and α-5NT were the most common deletional and nondeletional mutations, respectively. In HbH disease cases, the -α3.7/--MED was the most prevalent genotype. Overall, 42 different mutations have been identified in α-globin cluster reflecting the high heterogeneity of the mutations in Iranian populations.

HER-2 belongs to what family of proteins?

Her-2 belongs to the family of the human epidermal growth factor receptors (EGFRs).

The family of protein kinases includes many oncogenes and growth factor receptors, many of which have been linked to the pathogenesis and progression of cancer. Protein tyrosine kinases such as HER-2/c-erbB-2 and the epidermal growth factor receptor (EGFR) have been linked specifically to breast cancer, and perturbations of HER-2 affect response to chemotherapy. We have reviewed the biology of protein kinases in human breast cancer, as well as their translational applications to breast cancer patients. We have studied the spectrum of protein kinases expressed in human breast cancer cells and have identified four protein kinases with potentially important functions in breast cancer: rak (src-related), TK5 (which we now designate JAK3), the focal adhesion kinase (FAK), and STK1 (human M015/CAK). We describe the potential significance of these genes in breast cancer, as well as our methodology for identifying and characterizing novel genes in breast cancer. The product of the HER-2/neu proto-oncogene, HER2, is the second member of the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors and has been suggested to be a ligand orphan receptor. Ligand-dependent heterodimerization between HER2 and another HER family member, HER1, HER3 or HER4, activates the HER2 signaling pathway. The intracellular signaling pathway of HER2 is thought to involve ras-MAPK, MAPK-independent S6 kinase and phospholipase C-gamma signaling pathways. However, the biological consequences of the activation of these pathways are not yet completely known. Amplification of the HER2 gene and overexpression of the HER2 protein induces cell transformation and has been demonstrated in 10% to 40% of human breast cancer. HER2 overexpression has been suggested to associate with tumor aggressiveness, prognosis and responsiveness to hormonal and cytotoxic agents in breast cancer patients. These findings indicate that HER2 is an appropriate target for tumor-specific therapies. A number of approaches have been investigated: (1) a humanized monoclonal antibody against HER2, rhuMAbHER2 (trastuzumab), which is already approved for clinical use in the treatment of patients with metastatic breast cancer; (2) tyrosine kinase inhibitors, such as emodin, which block HER2 phosphorylation and its intracellullar signaling; (3) active immunotherapy, such as vaccination; and (4) heat shock protein (Hsp) 90-associated signal inhibitors, such as radicicol derivatives, which induce degradation of tyrosine kinase receptors, such as HER2. Human epidermal growth factor receptor-2 (HER2/erbB-2) belongs to a family of four transmembrane receptors involved in signal transduction pathways that regulate cell growth and differentiation. Overexpression/amplification of HER2 is associated with maligcy and a poor prognosis in breast cancer. HER2 acts as a networking receptor that mediates signaling to cancer cells, causing them to proliferate. HER receptors exist as monomers but dimerize on ligand binding. HER ligands are bivalent growth factor molecules whose low-affinity site binds to HER2. No HER2-specific ligand has been identified but HER2 is the preferred heterodimerization partner for other HER receptors. HER2-containing heterodimers are relatively long-lived and potent. HER3 has no inherent activity and is the major and most potent dimerization partner of HER2. HER2 overexpression biases the formation of HER2-containing heterodimers, leading to enhanced responsiveness to stromal growth factors and oncogenic transformation. Removal of HER2 from the cell surface or inhibition of its intrinsic enzymatic activity may reduce oncogenicity. Our research suggests that the antitumor efficacy of HER2-specific antibodies such as Herceptin relates to their ability to direct HER2 to a Cbl- dependent endocytosis and degradation pathway. The reported clinical therapeutic efficacy of anti-HER2 monoclonal antibodies in breast cancer highlights the importance of understanding the biology of HER2. The EGF family of receptors belongs to the tyrosine kinase receptor (TKR) family and plays an important role during embryonic and postnatal development and also in the progression of tumors. Her-2/neu/c-erbB-2, a member of the epidermal growth factor receptor family, can be cleaved into a soluble extra cellular domain (ECD) and a membrane-bound stub fragment. Her-2 ECD from a breast cancer cell line SKBR3 was immunopurified and analyzed with matrix-assisted laser desorption ionization (MALDI) and carboxyl terminal amino acid sequencing. A sequence within the juxtamembrane region (only 11 amino acid residues) PAEQR ASP was identified most likely as a primary site of cleavage, PA EQRASP as a minor site, that generate the ECD. The sites of cleavage are within the signature motif P/GX(5-7)P/G highly conserved in the EGF receptor family. The human epidermal growth factor receptor (Her) family of receptor tyrosine kinases includes Her-1, Her-2, and Her-3. The overexpression of Her-1 and Her-2 have been reported previously in surface epithelial ovarian cancer. Although up to one-third of ovarian carcinomas have been found to have amplification or overexpression of Her-2, responses to trastuzumab therapy in these patients have been disappointing. In this study, we examined Her-1, Her-2, and Her- 3 protein expression as well as the frequency of Her-2 amplification in a series of 103 high-grade, advanced-stage (FIGO stage III or IV) ovarian surface epithelial carcinomas. Immunohistochemical staining using commercially available antibodies against Her-1-3 and fluorescence in situ hybridization (FISH) using probes against Her-2 and chromosome 17 centromere (CEP) were performed on a tissue microarray containing cores of tumor from 103 surface epithelial carcinomas (85 serous, 6 mixed surface epithelial, 5 clear cell, 3 endometrioid, 3 undifferentiated, 1 mucinous). Nine of 99 (9.1%) tumors were positive for Her-1 expression and 5 of 102 (4.9%) tumors were positive for Her-2 expression, with 1 showing strong immunoreactivity. None of the Her-1 positive tumors exhibited Her-2 immunoreactivity. There was no correlation between Her-1 or Her-2 expression and survival. Using Her-2:centromere fluorescence ratios of 2.0 or 1.5 as cutoffs in assessment of Her-2 amplification, 8 of 75 (10.7%) and 25 of 75 (33.3%) tumors, respectively, showed Her-2 amplification. Two of eight tumors that showed higher level (>2) Her-2 amplification by FISH also were positive for Her-2 by immunohistochemistry. Only 3 of 103 tumors expressed Her-3. Immunoreactivity for Her-1 and Her-2 was less frequently observed in this series than has been previously reported. The strong correlation between Her-2 immunostaining and amplification characteristic of breast carcinoma is not seen in ovarian carcinoma. These results indicated that few patients with ovarian carcinoma have tumors that would benefit from therapy targeted specifically against Her-1, Her-2, or Her-3. The human epidermal growth factor receptor (HER) family of receptor tyrosine kinase has been extensively studied in breast cancer; however, systematic studies of EGFR gene amplification and protein overexpression in breast carcinoma are lacking. We studied EGFR gene amplification by chromogenic in situ hybridization (CISH) and protein expression by immunohistochemistry in 175 breast carcinomas, using tissue microarrays. Tumors with >5 EGFR gene copies per nucleus were interpreted as positive for gene amplification. Protein overexpression was scored according to standardized criteria originally developed for HER-2. EGFR mRNA levels, as measured by Affymetrix U133 Gene Chip microarray hybridization, were available in 63 of these tumors. HER-2 gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemistry were also studied. EGFR gene amplification (copy number range: 7-18; median: 12) was detected in 11/175 (6%) tumors, and protein overexpression was found in 13/175 (7%) tumors. Of the 11 tumors, 10 (91%) with gene amplification also showed EGFR protein overexpression (2+ or 3+ by immunohistochemistry). The EGFR mRNA level, based on Affymetrix U133 chip hybridization data, was increased relative to other breast cancer samples in three of the five tumors showing gene amplification. Exons 19 and 21 of EGFR, the sites of hotspot mutations in lung adenocarcinomas, were screened in the 11 EGFR-amplified tumors but no mutations were found. Three of these 11 tumors also showed HER-2 overexpression and gene amplification. Approximately 6% of breast carcinomas show EGFR amplification with EGFR protein overexpression and may be candidates for trials of EGFR-targeted antibodies or small inhibitory molecules. Overexpression of the epidermal growth factor receptor family member Her-2/neu in breast cancer leads to autophosphorylation of the receptor and induction of multiple downstream signaling pathways, including the Akt kinase to nuclear factor-kappaB (NF-kappaB) cascade that is associated with poor prognosis. Previously, we showed that the green tea polyphenol epigallocatechin 3-gallate (EGCG) inhibits growth of NF639 Her-2/neu-driven breast cancer cells via reducing receptor autophosphorylation and downstream Akt and NF-kappaB activities. Interestingly, upon prolonged culture in the presence of EGCG, cells resistant to the polyphenol could be isolated. Here, we report that resistant cells have lost tyrosine phosphorylation on the Her-2/neu receptor. Surprisingly, they displayed elevated NF-kappaB activity, and inhibition of this activity sensitized cells to EGCG. Data from microarray studies of the original and resistant NF639 populations of cells were subjected to Gene Set Enrichment Analysis pathway assessment, which revealed that the mitogen activated protein kinase (MAPK) pathway was activated in the resistant cells. Treatment of the resistant cells with the MAPK inhibitor U0216 reduced growth in soft agar and invasive phenotype, whereas the combination of EGCG and U0216 resulted in cells with a cobblestone epithelial phenotype. Thus, activation of the MAPK pathway mediates resistance to EGCG. Metastatic breast cancer is an incurable disease in a very high percentage of patients. Despite new progress in endocrine and other systemic therapies, this evidence remains challenging for patients and clinicians. HER2 protein is a member of the epidermal growth factor family of transmembrane receptors. HER2 is overexpressed in approximately 20% to 30% of breast cancers. Overexpression of HER2 has been shown to be associated with increased tumor proliferation and relative resistance to some types of chemotherapy and hormonal therapies. Trastuzumab, a humanized monoclonal antibody directed against HER2 protein, has been shown to be an efficacious and well tolerated treatment for HER2-overexpressing metastatic breast cancer, both as a single agent and when it is used in combination with chemotherapy. HER-2 belongs to a family of four transmembrane receptor tyrosine kinases that mediate growth, differentiation and survival of cells. HER-2 overexpression and amplification occurs in approximately 15 to 25 % of breast cancers and is associated with aggressive tumour behaviour. Herceptin (trastuzumab), a humanized monoclonal antibody directed against the extracellular domain of the HER-2 receptor, has been shown to have clinical activity in HER-2-positive advanced breast cancer when administered alone or in combination with chemotherapy. It has been approved for HER-2-positive metastatic breast cancer by the United States Food and Drug Administration in 1998 and in the countries of the European Union in 2000. Recently, promising results of the four randomized international multicenter trials evaluating the therapeutic benefit of Herceptin in the adjuvant treatment of HER-2-positive primary breast cancer have been reported. Data of the first planned interim analysis of the studies showed significantly improved disease-free survival in patients assigned to one year of Herceptin compared to the control groups even after short term follow up. These results caused an immediate wave of demand for Herceptin in adjuvant therapy. Results of these studies are critically reviewed. Furthermore, the available preliminary results from studies using Herceptin in the primary (neoadjuvant) therapy of HER-2-positive breast cancer are addressed and possible implications for HER-2 testing are discussed. BACKGROUND: The epidermal growth factor family members: EGF, EGFR and the c-erbB-2(HER-2/neu) gene product have been found to play a role in carcinomas of the stomach, liver, breast, ovary and lungs. Recent reports have indicated that they are also involved in the growth of pancreatic ductal carcinoma, its invasiveness and metastasis. PATIENTS AND METHODS: Thirty-six patients with pancreatic ductal carcinoma were analysed with respect to sex, age, histological type, maligcy grade (G), pTN status (pTN), local lymph node involvement and distant metastasis. The tumor levels of EGF, EGFR and c-erbB-2 expression were determined immunohistochemically. RESULTS: Expression of c-erbB-2 was observed in 24/36 cases, EGF in 13/36 cases and EGFR in 18/36 cases. Overexpression of EGF and EGFR was associated with metastasis to lymph nodes and other organs. A correlation was also found between EGF expression and the presence of EGFR in the tumour. The expression of c-erbB-2 protein was not found to correlate with any parameters. CONCLUSION: EGF and EGFR play a key role in neoplastic spread through lymph node involvement and metastasis to other organs. The human epidermal growth factor (EGF) receptor (HER) family members cooperate in maligcy. Of this family, HER2 does not bind growth factors and HER3 does not encode an active tyrosine kinase. This diversity creates difficulty in creating pan-specific therapeutic HER family inhibitors. We have identified single amino acid changes in epidermal growth factor receptor (EGFR) and HER3 which create high affinity sequestration of the cognate ligands, and may be used as receptor decoys to downregulate aberrant HER family activity. In silico modeling and high throughput mutagenesis were utilized to identify receptor mutants with very high ligand binding activity. A single mutation (T15S; EGFR subdomain I) enhanced affinity for EGF (two-fold), TGF-alpha (twenty-six-fold), and heparin-binding (HB)-EGF (six-fold). This indicates that T15 is an important, previously undescribed, negative regulatory amino acid for EGFR ligand binding. Another mutation (Y246A; HER 3 subdomain II) enhanced neuregulin (NRG)1-beta binding eight-fold, probably by interfering with subdomain II-IV interactions. Further work revealed that the HER3 subunit of an EGFR:HER3 heterodimer suppresses EGFR ligand binding. Optimization required reversing this suppression by mutation of the EGFR tether domain (G564A; subdomain IV). This mutation resulted in enhanced ligand binding (EGF, ten-fold; TGF-alpha, thirty-four-fold; HB-EGF, seventeen-fold; NRG1-beta, thirty-one-fold). This increased ligand binding was reflected in improved inhibition of in vitro tumor cell proliferation and tumor suppression in a human non-small cell lung cancer xenograft model. In conclusion, amino acid substitutions were identified in the EGFR and HER3 ECDs that enhance ligand affinity, potentially enabling a pan-specific therapeutic approach for downregulating the HER family in cancer. BACKGROUND AND PURPOSE: Erb-B1 (epidermal growth factor receptor, EGFR) and Erb-B2 (HER-2) are two of the best-characterized members in the EGFR pathway. In many tumor types, overexpression of these proteins is associated with enhanced maligt potential. The aim of this study was to determine the prognostic impact of EGFR and HER-2 protein expression on colorectal cancer. METHOD: Immunohistochemistry was carried out in paraffin-embedded specimens of 115 colorectal carcinomas for the assessment of EGFR and HER-2 expression. Immunostaining for EGFR was graded negative, weak or strong according to extension and staining intensity. The results were correlated with traditional clinicopathologic parameters and patients' outcome. RESULTS: The mean survival time was 64 (range 9-78) months in the EGFR-negative group, 166 (range 2-293) months in the group with a low EGFR expression, and 51 (range 4-71) months in the group with a high EGFR expression. The median survival time was 31 (range 2-114) months in the HER-2 negative group, and 30 (range 4-293) months in the HER-2 positive group. None of the clinicopathologic parameters or patient prognoses had statistically significant association with EGFR or HER-2 expression. CONCLUSION: Conventional immunohistochemistry was unable to reveal any association between EGFR or HER-2 expression and outcome predicted by the biologic role of EGFR in tumor behavior and the established prognostic role of HER-2 in breast cancer. The human epidermal growth factor (EGF) receptor (HER) family consists of four receptors that bind to ligands sharing an EGF-like motif. The HER family of receptor tyrosine kinases and their ligands (EGF family) are known to play a significant role in gastrointestinal cancer. In particular, the EGF receptor, HER1, is one of the main candidates for the molecular-targeted therapy of colon cancer, and HER2 is a candidate for the treatment of gastric cancer which overexpresses HER2. In contrast, the role of the HER and EGF families in maligt lymphoma has not been fully elucidated. In this study, we investigated the expression and function of the HER and EGF families in lymphoma cell lines and tumor samples. Reverse transcription polymerase chain reaction revealed that the ligands for HER1 were mainly expressed in gastric cancer and colon cancer cell lines, but not in lymphoma cell lines. On the other hand, the EGF family member, neuregulin (NRG) 4, was highly expressed in lymphoma cell lines. Immunohistochemical analyses of maligt lymphoma clinical samples revealed that NRG4 and HER4 were mainly expressed in mucosa-associated lymphoid tissue (MALT) and follicular lymphoma. Immunoprecipitation of Raji and Daudi cell lines revealed that recombit NRG4 induced the tyrosine phosphorylation of HER4. Additionally, recombit NRG4 activated the proliferation of lymphoma cell lines. These findings suggest that the NRG4-HER4 axis plays a major role in the proliferation of maligt lymphoma cells in the gastrointestinal tract. Epidermal growth factor receptor (EGFR) family members (EGFR, HER2, HER3 and HER4) have been extensively investigated for its possible involvement in cancer development and progression. In colorectal cancer (CRC) EGFR family has been found frequently over-expressed, thus therapy targeting EGFR has been developed. Interestingly, it has been observed that genetic variants in these receptors may alter the therapeutic efficacy of EGFR inhibitors. Polymorphic variants in members of the EGFR family could influence different biologic activities, such as ligands affinity, dimerization efficiency, kinase activity, expression levels, with a consequent impact in signalling pathways and cell behaviour. This study aimed to verify whether single nucleotide polymorphisms (SNPs) of EGFR family members could represent susceptibility factors able to influence the risk to develop CRC. Peripheral blood of 70 Italian colon cancer patients and 72 healthy controls was used as a source of genomic DNA to investigate EGFR, HER2 and HER3 common non-synonymous SNPs. Genetic association tests were performed to verify a possible relationship with CRC. Evidence of genotype association was found for the R521K EGFR polymorphism under a domit mode of inheritance (Mid-P=0.031). Genotypes with the variant allele of EGFR R521K SNP confer a risk reduction to develop CRC. HER2, a member of the human ErbB protein family belonging to receptor tyrosine kinases, plays important roles in regulating crucial cellular processes, including cell migration, proliferation, and differentiation. A missense mutation, L755P, in the HER2 kinase domain has been involved in lung cancer in humans and exhibits reduced response to lapatinib therapy. However, the detailed mechanism of how the HER2 L755P mutation causes drug resistance to lapatinib remains elusive. Here, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations [molecular mechanics and generalized Born/surface area (MM-GBSA)] were performed to reveal the mechanism of drug resistance due to the HER2 L755P mutation. MD simulations revealed that the L755P mutation caused structural changes in the regions of helix αC, the glycine-rich loop, and the activation loop, thereby leading to the loss of interactions between the solubilizing group of lapatinib and HER2. Moreover, MM-GBSA calculations suggested that hydrophobic interactions between lapatinib and HER2 contribute most to the binding affinity, and that the L755P mutation could result in a less energetically favorable HER2/lapatinib complex. This may weaken the binding of lapatinib to the mutated HER2, thereby leading to the emergence of drug resistance. This study offers a structural explanation for the effect of the L755P mutation on the HER2/lapatinib complex. Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations. Receptor tyrosine kinases (RTKs) are a superfamily of transmembrane proteins that mediate intracellular signaling by phosphorylating substrate proteins involved in cell proliferation, survival, differentiation or migration. The Human Epidermal growth factor Receptor (HER) family belongs to the RTKs superfamily, and comprises four members: EGFR (epidermal growth factor receptor), HER2, HER3 and HER4. Physiologically, these receptors are activated by the ligands of the EGF family. In solid tumors other mechanisms of activation, such as overexpression or molecular alterations have been reported, and have been linked to tumour initiation/progression. Because of that, several strategies have been developed to target HER receptors and include i) antibody-based therapies using monoclonal antibodies against the extracellular domain of these receptors, and ii) small molecule tyrosine kinase inhibitors (TKIs) against the intracellular kinase domain. In this review we will provide basic information about biological aspects of HER receptors and their ligands as well as the therapeutic strategies to target them. We also summarize general mechanisms of resistance generated in patients to such anti-HER therapies. BACKGROUND: Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy. OBJECTIVE: In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer. CONCLUSION: Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer. HER-2 belongs to the human epidermal growth factor receptor (HER) family. Via different signal transduction pathways, HER-2 regulates normal cell proliferation, survival, and differentiation. Recently, it was reported that MCF10A, BT474, and MDA-MB-231 cells bearing the HER2 K753E mutation were resistant to lapatinib. Present study revealed that HER-2 mutant K753E showed some contrasting behaviour as compared to wild, L768S and V773L HER-2 in complex with lapatinib while similar to previously known lapatinib resistant L755S HER-2 mutant. Lapatinib showed stable but reverse orientation in binding site of K753E and the highest binding energy among studied HER2-lapatinib complexes but slightly lesser than L755S mutant. Results indicate that K753E has similar profile as L755S mutant for lapatinib. The interacting residues were also found different from other three studied forms as revealed by free energy decomposition and ligplot analysis. Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the antibiotic Tetrocarcin-A as a novel downregulator of JAM-A and human epidermal growth factor receptor-2 (HER2) protein expression in breast cancer cells. Lysosomal inhibition partially rescued the downregulation of JAM-A and HER2 caused by Tetrocarcin-A, and attenuated its cytotoxic activity. Tetrocarcin-A treatment or JAM-A silencing reduced AKT and ERK phosphorylation, inhibited c-FOS phosphorylation at Threonine-232 (its transcriptional regulation site), inhibited nuclear localization of c-FOS, and downregulated expression of the inhibitor of apoptosis proteins (IAP). This was accompanied by Tetrocarcin-A-induced caspase-dependent apoptosis. To begin evaluating the potential clinical relevance of our findings, we extended our studies to other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cells and lung cancer stem cells, and inhibited the growth of xenografts in a semi-in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A warrants future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, reduce tumorigenic signaling and induce apoptosis. HER2 (Human Epidermal Growth Factor Receptor 2), also known as ERBB2, CD340, and Neu protooncogene, is a member of the epidermal growth factor receptor (EGRF) family. Members of the ERBB family, including HER2, activate molecular cascades that stimulate proliferation and migration of cancer cells, as well as their resistance to the anticancer therapy. These proteins are often overexpressed and/or mutated in various cancer types and represent promising targets for the anti-cancer therapy. Currently, anti-HER2 drugs have been approved for the treatment of several types of solid tumors. HER2-specific therapy includes monoclonal antibodies and low-molecular weight inhibitors of tyrosine kinase receptors, such as lapatinib, neratinib, and pyrotinib. In addition to the activation of molecular pathways responsible for cell proliferation and survival under stress conditions, HER2 directly regulates programmed cell death. Here, we review the studies focused on the involvement of HER2 in various signaling pathways and its role in the regulation of apoptosis.

What is the chemical structure of Etanercept (ETN)?

Etanercept (ETN) is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites, which form a complex with the plasma membrane protein Enbrel. Etanercept has been tested for treatment of solid cancers, including glioblastoma and neuroblastoma, and liver fibrosis.

There is accumulating evidence that tumour necrosis factor (TNF) plays a major role in the pathogenesis of rheumatoid arthritis (RA). Recent biotechnological advances have allowed for the development of agents that directly target TNF, a pro-inflammatory cytokine. In the last 2 years, the US FDA and the EU's Commission of the European Communities have approved two biological agents for the treatment of refractory RA, etanercept and infliximab. Etanercept is a fusion protein, composed of the Fc portion of IgG1 and the extracellular domain of the TNF receptor (p75). Infliximab is a chimeric monoclonal antibody (mAb) composed of murine variable and human constant regions. In placebo-controlled trials, both agents have proven to be effective and well-tolerated in RA patients. This review evaluates the available TNF inhibitors, summarises pertinent clinical trials and underscores differences between the two agents in terms of molecular structure, efficacy, safety data, antigenicity and pharmacokinetics. Etanercept is a protein comprised of the extracellular domains of two TNF receptors attached to a Fc portion of an IgG. Etanercept was approved for not only reducing signs and symptoms but inhibiting of structural damage in patients with active RA who had an inadequate response to one or more DMARDs. Moreover, combination therapy with methotrexate will be attractive for severely active patients. The proportion of patients who have discontinued therapy due to adverse events is approximately 4%. Etanercept has not raised the risk for serious infections(0.04/patient-year) as well as maligcies. There are sporadic case reports of aplastic anemia, demyelination, lupus-like conditions, which are not significant so far. Etanercept may contribute rheumatologists to manage patients with RA. Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predomitly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MS(E) protocol to identify the O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics. Diabetes mellitus is known to exacerbate acute cerebral ischemic injury. Previous studies have demonstrated that infarction volumes caused by transient cerebral ischemia were greater in diabetic rats than in nondiabetic rats. Tumor necrosis factor-α (TNF-α) is a proinflammatory protein produced in the brain in response to cerebral ischemia that promotes apoptosis. Etanercept (ETN), a recombit TNF receptor (p75)-Fc fusion protein, competitively inhibits TNF-α. Therefore, we evaluated the neuroprotective effects of chronic or acute treatment with ETN on cerebral injury caused by middle cerebral artery occlusion/reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. Furthermore, we evaluated the effects of ETN against the apoptosis and myeloperoxidase activity. Single administration of ETN before MCAO significantly suppressed exacerbation of cerebral damage in nondiabetic rats, as assessed by infarct volume. In contrast, the diabetic state markedly aggravated MCAO/Re-induced cerebral damage despite ETN treatment within 24 h before MCAO. However, the damage was improved by repeated administration of ETN at 900 μg/kg/daily in rats in an induced diabetic state. These results suggested that repeated administration of ETN can prevent exacerbation of cerebral ischemic injury in the diabetic state and is mainly attributed to anti-inflammatory effects. Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety. Etanercept (ETN) (Enbrel®) is a soluble protein that binds to, and specifically inhibits, tumor necrosis factor (TNF), a proinflammatory cytokine. ETN is synthesized in Chinese hamster ovary cells by recombit DNA technology as a fusion protein, with a fully human TNFRII ectodomain linked to the Fc portion of human IgG1. Successful manufacture of biologics, such as ETN, requires sophisticated process and product understanding, as well as meticulous control of operations to maintain product consistency. The objective of this evaluation was to show that the product profile of ETN drug substance (DS) has been consistent over the course of production. Multiple orthogonal biochemical analyses, which included evaluation of attributes indicative of product purity, potency, and quality, were assessed on >2,000 batches of ETN from three sites of DS manufacture, during the period 1998-2015. Based on the key quality attributes of product purity (assessed by hydrophobic interaction chromatography HPLC), binding activity (to TNF by ELISA), potency (inhibition of TNF-induced apoptosis by cell-based bioassay) and quality (N-linked oligosaccharide map), we show that the integrity of ETN DS has remained consistent over time. This consistency was maintained through three major enhancements to the initial process of manufacturing that were supported by detailed comparability assessments, and approved by the European Medicines Agency. Examination of results for all major quality attributes for ETN DS indicates a highly consistent process for over 18 years and throughout changes to the manufacturing process, without affecting safety and efficacy, as demonstrated across a wide range of clinical trials of ETN in multiple inflammatory diseases. Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel® and its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126 kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity. Etanercept is a dimeric genetic recombit glycoprotein consisting of Fc domain of human Immunoglobulin G1 and the extracellular domain of human tumor necrosis factor (TNF) receptor type II. Etanercept exerts therapeutic effects on inflammatory diseases such as rheumatoid arthritis and juvenile idiopathic arthritis by neutralizing biological activities of TNFα/Lymphotoxin (LT) α. Mochida Pharmaceutical and LG Chem have developed syringe, pen, and vial products of Etanercept BS (biosimilar) as the first biosimilar of Enbrel in Japan. The active ingredient of those products "Etanercept biosimilar 1" has the identical primary structure to that of Enbrel. The development of the Etanercept BS, including evaluations of quality attributes, nonclinical and clinical studies was performed in accordance with "Policies on Assurance of Quality, Safety and Efficacy of Biosimilars". The quality attributes of Etanercept BS were similar to those of Enbrel, and the binding affinities to TNFα/LTα, TNFα neutralizing activity, nonclinical pharmacokinetics and toxicological profiles of Etanercept BS were comparable to Enbrel. Additionally, the pharmacokinetic profile and efficacy of Etanercept BS were equivalent to those of Enbrel and there was no clinically significant difference in safety profiles between them in Phase I and Phase III clinical studies. The marketing approval application of the Etanercept BS with the same indications as Enbrel filed by Mochida Pharmaceutical was approved in January 2018 and the products will be launched by Ayumi Pharmaceutical in the near future. The Etanercept BS, which is as highly effective as Enbrel is expected to make beneficial therapies more easily accessible to patients.

What does bDMARD stand for?

bDMARDs are biologic disease-modifying antirheumatic drugs.

Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation due to unknown causes. Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), biological DMARDs (bDMARDs), and tofacitinib, a targeted sDMARD, can be used to treat RA. In clinical trials, molecular-targeted therapies showed a significant reduction in RA symptoms and provided pain relief for patients with active RA. Even if patients did not show clinical improvement with combination therapy with a bDMARD and methotrexate (MTX), some patients showed a significant inhibition in structural damage. The clinical efficacies of tofacitinib were shown to be equivalent to adalimumab, a bDMARD, in patients with RA treated with MTX. MTX is the first-line agent for the treatment of RA. Higher doses of MTX might be needed to maintain the effects of bDMARDs. Patients receiving some bDMARDs have been shown to have a higher risk for serious infections; thus, pre-screening for infections is important before beginning treatment with bDMARDs. The rates of patients maintaining targeted levels of disease activity after stopping bDMARDs are relatively low. It is uncertain whether remission or low disease activity can be maintained after stopping molecular-targeted therapies. The development of bDMARDs and targeted-molecular sDMARDs has provided a wide range of treatment options for RA. Patients with active RA should be treated with a treat-to-target strategy after assessment of risks and benefits. Publisher: Biologika stellen eine hochwirksame Therapieoption für verschiedene nicht infektiöse Uveitisformen dar. Einziges zugelassenes Biologikum ist der TNF-α-Inhibitor Adalimumab, alle anderen Präparate müssen im Rahmen einer Off-Label-Therapie gegeben werden. Die Indikation zur Therapieinitiierung mit einem Biologikum besteht, wenn die Erkrankung nicht ausreichend anspricht auf eine Behandlung mit systemischen Steroiden und/oder csDMARDs (konventionell-synthetischen disease modifying antirheumatic drugs) oder diese aufgrund von unerwünschten Wirkungen nicht gegeben werden können. Derzeit in der klinischen Anwendung befindliche biologische DMARD-Präparate wirken über zytokinspezifische Mechanismen (TNF-α-Inhibition, Interferone, Hemmung der Signaltransduktion von Interleukin-1 [IL-1], IL-6 und IL-17) sowie Hemmung der T-Zell-Kostimulation (CTLA-4-Fusionsprotein), oder B-Zell-Depletion (Anti-CD20). Alle Präparate müssen parenteral verabreicht werden. Die Einleitung einer Biologikatherapie sollte nach interdisziplinärer Abstimmung und Ausschluss von Kontraindikationen erfolgen. Ein regelmäßiges klinisches und laborchemisches Monitoring unter der Therapie ist erforderlich. The introduction of biological disease-modifying antirheumatic drug (bDMARD) treatments for various types of autoimmune arthritis, such as rheumatoid arthritis, psoriatic arthropathy and ankylosing spondylitis, represents a new era of treatment for patients with a refractory response to conventional synthetic DMARDs (csDMARDs). Many new bDMARDs with different modalities or that target different pro-inflammatory molecules, likely cytokines, are rapidly emerging. Hence, physicians in the field may be confused about choosing appropriate bDMARDs for their patients. Considering the high cost of bDMARDs and the rapid destructive process of autoimmune arthritis in patients, the choice of optimal bDMARDs for patients who fail to respond or show an inadequate therapeutic response to csDMARDs designed to control the disease is very critical. Here, we summarize the strengths and weaknesses of bDMARDs and specifically focus on their uses in patients with comorbid conditions or with specific medical conditions, such as pregcy. This commentary provides a solid up-to-date review on commercially available bDMARDs and very useful information for physicians to facilitate the choice of more appropriate bDMARDs to treat patients with autoimmune arthritis and for basic researchers to understand the current strategies of bDMARD usage and hopefully to develop more powerful bDMARDs with fewer safety concerns.

Is there any role of genotoxic pks + E. coli in cancer?

Yes. Genotoxic pks and E. Coli are known to cause mutations in the DNA of cells, which can lead to cancer.

Should tirilazad be used for treatment of ischemic stroke?

No. Tirilazad should not be used for treatment of stroke because it does not improve disease outcomes, but may increase death and disability.

BACKGROUND: Tirilazad is a non-glucocorticoid, 21-aminosteriod that inhibits lipid peroxidation. It had neuroprotective effects in experimental ischemic stroke and reduced angiographic vasospasm after experimental subarachnoid hemorrhage (SAH). Five randomized clinical trials of tirilazad were conducted in patients with SAH. We performed a meta-analysis of these trials to assess the effect of tirilazad on unfavorable outcome, symptomatic vasospasm, and cerebral infarction after SAH. METHODS: Data from 3,797 patients were analyzed and modeled using random effect and Mantel-Haenszel meta-analyses and multivariable logistic regression to determine the effect of tirilazad on clinical outcome, symptomatic vasospasm, and cerebral infarction. Clinical outcome was assessed 3 months after SAH using the Glasgow outcome scale, and symptomatic vasospasm was defined by clinical criteria with laboratory and radiological exclusion of other causes of neurological deterioration. RESULTS: The five trials were randomized, double-blind, and placebo-controlled. Tirilazad did not significantly decrease unfavorable clinical outcome on the GOS (odds ratio [OR] 1.04, 95% confidence interval [CI] 0.89-1.20) or cerebral infarction (OR 1.04, 95% CI 0.89-1.22). There was a significant reduction in symptomatic vasospasm in patients treated with tirilazad (OR 0.80, 95% CI 0.69-0.93). There was no heterogeneity across the five trials. CONCLUSION: Tirilazad had no effect on clinical outcome but did decrease symptomatic vasospasm in five trials of aneurysmal SAH. The dissociation between clinical outcome and symptomatic vasospasm deserves further investigation.

List the major families of Histones.

Five histone families (H1, H2A, H2B, H3, and H4).

The rich diversity within each of the five histone families (H1, H2A, H2B, H3, and H4) can hardly be reconciled with the notion of homogenizing evolution. The prevalence of birth-and-death long-term evolution over concerted evolution has already been demonstrated in the linker histone H1 family as well as for the H2A, H3, and H4 core histone families. However, information about histone H2B is lacking. In the present work, we have analyzed the diversity of the members of this histone family across different eukaryotic genomes and have characterized the mechanisms involved in their long-term evolution. Our results reveal that, quite in contrast with other histones, H2B variants are subject to a very rapid process of diversification that primarily affects the male germinal cell lineage and involves their functional specialization probably as a consequence of neofunctionalization and subfunctionalization events after gene duplication. The overall parallelism observed between the molecular phylogenies and the relationships among the electrostatic potentials of the different variants suggests that the latter may have played a major structural selective constraint during H2B evolution. It thus seems that the reorganization of chromatin structure during spermiogenesis might have affected the evolutionary constraints driving histone H2B evolution, leading to an increase in diversity. N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs), which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/), is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm.

Is the Paramyxovirus geneome segmented, negative-sense RNA?

The paramyxovirus family has a genome consisting of a SINGLE STRAND of negative sense RNA

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of approximately 15 kb, contains six transcription units flanked at the 3' and 5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3' end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses. Simian parainfluenza virus 5 (SV5) is a prototype of the Paramyxoviridae family of nonsegmented negative-sense RNA viruses. The single-stranded RNA genomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences flanked by gene-end (GE) and gene-start (GS) sequences. The viral RNA polymerase (vRNAP) complex is thought to enter the genome at its 3' end, and synthesis of mRNAs is thought to occur by a stop-start mechanism in a sequential and polar manner, with transcriptional attenuation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNAP. To investigate the functions of GE, IG, and GS sequences in transcription, we constructed plasmids containing cDNAs of the full-length SV5 genome in which the gene junction sequences (GE, IG, and GS sequences) located between the hemagglutinin-neuraminidase (HN) and the polymerase (L) genes were replaced with the counterpart sequences from other gene junctions. By using reverse genetics, we recovered viable viruses from each cDNA construct, although their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of HN to L mRNAs varied over a fourfold range. The alteration of the gene junction sequences also permitted examination of the hypothesized requirement for hexamer nucleotide position of the GS sites. The recovery of infectious viruses with transcription initiation sites that occurred at nucleotide positions 1, 2, 3, 5, and 6 of the hexamer suggest that the requirement is nonstringent. During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3' half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV. The avian paramyxovirus type 1 (APMV-1), or Newcastle disease virus (NDV), comprise a diverse group of viruses with a single-stranded, negative-sense RNA genome. Historically, two systems have been simultaneously used to classify NDV isolates into lineages or genotypes, generating confusion in the nomenclature and discrepancies in the assignment of genetic groups. In the present study we assessed the genetic diversity of the avian paramyxovirus type-1 (APMV-1) and propose a unified nomenclature and a classification system based on objective criteria to separate NDV into genotypes. Complete F gene sequences of class I (n = 110) and class II (n = 602) viruses were used for the phylogenetic reconstruction and to identify distinct taxonomic groups. The mean interpopulational evolutionary distance was estimated (10%) and set as the cutoff value to assign new genotypes. Results of our study revealed that class I viruses comprise a single genotype, while class II contains 15 genetic groups including 10 previously established (I-IX, and XI) and five new genotypes (X, XII, XIII, XIV and XV). Sub-genotypes were identified among class I and class II genotypes. Adoption of a unified nomenclature and of objective criteria to classify NDV isolates will facilitate studies on NDV epidemiology, evolution, disease control and diagnostics. Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the "rule of six" that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure. Human metapneumovirus (HMPV), a single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae, is associated with respiratory tract illness, primarily in young children and persons with underlying disease. Based on genetic and antigenic variation, HMPV strains are classified into two serotypes, with isolates NL/1/00 and NL/1/99 as prototypes for serotypes A and B, respectively. The development of plasmid-based reverse genetics systems for both serotypes has resulted in developments of a wide range of vaccine candidates against HMPV infection. The approach to virus rescue of HMPV is similar to that used for other paramyxoviruses, starting with mini-replicon assays for optimizations of the rescue protocols and subsequent replacement of the mini genome with a plasmid expressing the cDNA of the full-length viral RNA genome. Here, we provide detailed information on the reverse genetics systems for HMPV. The paramyxovirus family comprises major human and animal pathogens such as measles virus (MeV), mumps virus (MuV), the parainfluenzaviruses, Newcastle disease virus (NDV), and the highly pathogenic zoonotic hendra (HeV) and nipah (NiV) viruses. Paramyxovirus particles are pleomorphic, with a lipid envelope, nonsegmented RNA genomes of negative polarity, and densely packed glycoproteins on the virion surface. A number of crystal structures of different paramyxovirus proteins and protein fragments were solved, but the available information concerning overall virion organization remains limited. However, recent studies have reported cryo-electron tomography-based reconstructions of Sendai virus (SeV), MeV, NDV, and human parainfluenza virus type 3 (HPIV3) particles and a surface assessment of NiV-derived virus-like particles (VLPs), which have yielded innovative hypotheses concerning paramyxovirus particle assembly, budding, and organization. Following a summary of the current insight into paramyxovirus virion morphology, this review will focus on discussing the implications of these particle reconstructions on the present models of paramyxovirus assembly and infection.

Approximately how many genes are contained in the X chromosome's non-pseudoautosomal region (non-PAR)?

The total number of genes contained in the X chromosome's non- pseudoautosomal region (PAR) is 783.

There is a striking and unexplained male predomice across many cancer types. A subset of X-chromosome genes can escape X-inactivation, which would protect females from complete functional loss by a single mutation. To identify putative 'escape from X-inactivation tumor-suppressor' (EXITS) genes, we examined somatic alterations from >4,100 cancers across 21 tumor types for sex bias. Six of 783 non-pseudoautosomal region (PAR) X-chromosome genes (ATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3) harbored loss-of-function mutations more frequently in males (based on a false discovery rate < 0.1), in comparison to zero of 18,055 autosomal and PAR genes (Fisher's exact P < 0.0001). Male-biased mutations in genes that escape X-inactivation were observed in combined analysis across many cancers and in several individual tumor types, suggesting a generalized phenomenon. We conclude that biallelic expression of EXITS genes in females explains a portion of the reduced cancer incidence in females as compared to males across a variety of tumor types.

Does the use of bDMARDs during pregnancy impact neonatal development?

Exposure to bDMARDs during pregnancy does not seem to interfere with post-natal development up to infancy.

Do exon 38 or 39 KMT2D missense variants cause Kabuki syndrome type 1 (KS1)?

No. The KMT2D missense variants do not cause KS1, they cause a different type of malformations disorder distinct from Kabuki syndrome.

The NoSAS Score can be used for screening of which disorders?

The NoSAS score can be used for screening of obstructive sleep apnea syndrome, Sleep-Disordered Breathing and obstructive sleep apnea-hypopnea syndrome.

BACKGROUND: Diagnosis of sleep-disordered breathing requires overnight recordings, such as polygraphy or polysomnography. Considering the cost and low availability of these procedures, preselection of patients at high risk is recommended. We aimed to develop a screening tool allowing identification of individuals at risk of sleep-disordered breathing. METHODS: We used the participants from the population-based HypnoLaus cohort in Lausanne, Switzerland, who had a clinical assessment and polysomnography at home, to build a clinical score (the NoSAS score) using multiple factor analysis and logistic regression to identify people likely to have clinically significant sleep-disordered breathing. The NoSAS score was externally validated in an independent sleep cohort (EPISONO). We compared its performance to existing screening scores (STOP-Bang and Berlin scores). FINDINGS: We used the 2121 participants from the HypnoLaus cohort who were assessed between Sept 1, 2009, and June 30, 2013. The NoSAS score, which ranges from 0 to 17, allocates 4 points for having a neck circumference of more than 40 cm, 3 points for having a body-mass index of 25 kg/m(2) to less than 30 kg/m(2) or 5 points for having a body-mass index of 30 kg/m(2) or more, 2 points for snoring, 4 points for being older than 55 years of age, and 2 points for being male. Using a threshold of 8 points or more, the NoSAS score identified individuals at risk of clinically significant sleep-disordered breathing, with an area under the curve (AUC) of 0·74 (95% CI 0·72-0·76). It showed an even higher performance in the EPISONO cohort, with an AUC of 0·81 (0·77-0·85). The NoSAS score performed significantly better than did the STOP-Bang (AUC 0·67 [95% CI 0·65-0·69]; p<0·0001) and Berlin (0·63 [0·61-0·66]; p<0·0001) scores. INTERPRETATION: The NoSAS score is a simple, efficient, and easy to implement score enabling identification of individuals at risk of sleep-disordered breathing. Because of its high discrimination power, the NoSAS score can help clinicians to decide which patients to further investigate with a nocturnal recording. FUNDING: Faculty of Biology and Medicine of the University of Lausanne, Lausanne University Hospital, Swiss National Science Foundation, Leenaards Foundation, GlaxoSmithKline, and Vaud Pulmonary League. PURPOSE: The NoSAS score was developed to identify subjects at high risk of sleep-disordered breathing (SDB). We aimed to validate the NoSAS score in a multiethnic Asian cohort and compare its performance to the STOP-Bang and Berlin questionnaires. METHODS: A sample of 242 subjects selected from a population-based cohort in Singapore completed home-based sleep testing with an Embletta device (type 3 monitor). All subjects were given the STOP-Bang and Berlin questionnaires for self-administration prior to the sleep study. The NoSAS score was subsequently calculated based on available demographic data and Berlin questionnaire responses. RESULTS: The prevalence of severe SDB, defined as an apnea-hypopnea index cutoff of ≥30 events/h, was 10.7%. The number of subjects who were classified as high risk by the NoSAS score and STOP-Bang and Berlin questionnaires were 76 (31.4%), 89 (36.8%), and 79 (32.6%), respectively. The sensitivity, specificity, and negative and positive predictive values of the NoSAS score to predict severe SDB were 69.2, 73.1, 95.2, and 23.7%, respectively. The STOP-Bang and Berlin questionnaires performed similarly to the NoSAS score, with area under the curve (AUC) values of all three questionnaires clustered around 0.682-0.748. Compared to the STOP-Bang (94.8%) and Berlin questionnaires (96.3%), the NoSAS score (95.2%) had equally high negative predictive value in ruling out severe SDB. CONCLUSIONS: The NoSAS score performed similarly to the STOP-Bang and Berlin questionnaires in a multiethnic Asian cohort. All three questionnaires had high negative predictive values in ruling out severe SDB and may have utility as screening tools. BACKGROUND: Since the clinical presentation of obstructive sleep apnea syndrome (OSAS) shares common features with major depressive (MDE), the screening of OSAS is challenging in this population. The aim of this study was to assess the effectiveness of the NoSAS score in predicting the presence of OSAS among participants with current MDE and to compare it with the performance of existing screening tools. METHODS: A random sample of the population-based cohort CoLaus (Lausanne, Switzerland) underwent a psychiatric evaluation (PsyCoLaus) and a complete polysomnography at home (HypnoLaus). The effectiveness of the NoSAS score in detecting the risk of significant OSAS among current MDE participants was assessed and compared with STOP-BANG and Berlin scores. RESULTS: Among the 1761 subjects (58,75 ± 11y.o.; 47,8%men) who underwent polysomnography, significant OSAS was present in 24.0% with and 26.1% without current MDE. Using a threshold of ≥ 8 points, the NoSAS score identified OSAS in MDE participants with a sensitivity of 0.79, a specificity of 0.66, a negative predictive value of 0.91, and a positive predictive value of 0.41. The area under the ROC curve was 0.72 for NoSAS, 0.66 for STOP-BANG and 0.69 for the Berlin score (NS). LIMITATIONS: Only 44% of the PsyCoLaus participants had a polysomnography. The studied population was mainly of Caucasian ancestry and above 40 years of age. CONCLUSIONS: This is the first study assessing the performance of screening tools for OSAS in MDE. The NoSAS score is a simple and efficient screening tool for OSAS in this population, and may be a helpful instrument for clinicians. STUDY OBJECTIVES: This study was conducted to validate the NoSAS score in clinical populations and to compare it with the Berlin, STOP, and STOP-Bang questionnaires, as well as the Epworth Sleepiness Scale (ESS), in screening for sleep-disordered breathing (SDB). METHODS: A retrospective analysis was conducted of all patients aged 18 to 80 years who had completed a full-night polysomnography (PSG) at the sleep center of the First Affiliated Hospital of Guangzhou Medical University from October 2012 to November 2016. Patients who had incomplete or uswered questionnaires were excluded. The data for the NoSAS score, ESS, STOP, STOP-Bang, and Berlin questionnaires were collected, after which the NoSAS score was compared against the other questionnaires for SDB screening. RESULTS: A total of 2,208 participants were enrolled in this study. The NoSAS scores, which ranged from 0 to 17 and allocated a threshold of 8 points, identified individuals at risk of clinically significant SDB (defined as an apnea-hypopnea index [AHI] cutoff of ≥ 20 events/h), with an area under the curve (AUC) of 0.707. The NoSAS score performed significantly better than the STOP (AUC 0.655) and STOP-Bang (AUC 0.704) questionnaires and the ESS (AUC 0.642), and it was at par with the Berlin (AUC 0.697) scores for SDB screening. A significant correlation was found between the AHI and NoSAS score (r = .386, P < .001). CONCLUSIONS: The NoSAS score is a simple, efficient, and easy method for screening SDB in the clinical setting, especially in moderate to severe SDB. It demonstrates a moderately high level of sensitivity for SDB. Objective: To evaluate the clinical utility of the NoSAS score in the screening of patients with obstructive sleep apnea-hypopnea syndrome(OSAHS), and to compare the performance of the NoSAS score with other tools including Epworth Sleepiness Scale(ESS), STOP, STOP-Bang(SBQ) and Berlin questionnaires. Methods: A total of 444 consecutive patients(328 males and 116 females) with suspected OSAHS who underwent an overnight polysomnography(PSG) were recruited into this study. Five questionnaires including the NoSAS score, ESS, STOP, SBQ and Berlin were completed. Based on the severity of OSAHS which was determined by apnea-hypopnea index(AHI), the patients were classified into 4 groups: normal(<5 events/h), mild(5-15 events/h), moderate(15-30 events/h) and, severe (≥30 events/h) OSA.Sensitivity, specificity, positive predictive values, negative predictive values and the area under the receiver operating characteristics curve of 5 questionnaires were calculated. Results: With AHI≥5 events/h as the standard diagnosis of OSAHS, the NoSAS score and SBQ questionnaire showed a moderate performance, with the NoSAS score having the largest area under the ROC curve(0.753, P<0.001), followed by the SBQ questionnaire (0.727, P<0.001). The performance of the ESS, Berlin, and the STOP questionnaire was not high. Using mild moderate-severe(≥5 events/h), moderate-severe(≥15 events/h), and severe(≥30 events/h)OSAHS as cutoffs, NoSAS had the highest specificity and positive predictive values(80.2% and 88%, 72% and 69.8%, 66.3% and 50.5%), and the sensitivity and negative predictive values were (51.5% and 36.9%, 56.5% and 59.1%, 66.3% and 74.2%) .SBQ had the highest sensitivity and the negative predictive values(80.2% and 88%, 72% and 69.8%, 66.3% and 50.5%), and the specific and positive predictive values were (45.7% and 81.0%, 39.1% and 61.9%, 34.8% and 44.4%). The NoSAS score ≥ 7 had higher sensitivity and negative predictive value(75.0% and 47.1%, 78.1% and 66.5%, 82.7% and 81.9%)than the NoSAS socre ≥ 8. With AHI≥5 events/h as the standard diagnosis of OSAHS, the NoSAS score and the SBQ questionnaire had a higher accuracy than the other 3 questionnaires as screening questionnaires for diagnosing OSAHS, and the value of DOR were 4.298 and 3.758 respectively. Conclusions: The NoSAS score and the SBQ questionnaire have a moderate performance in diagnosing OSAHS. The NoSAS score is a new screening tool, and it is similar to the SBQ questionnaire, being also simple and effective. While the SBQ questionnaire is more widely used, it is necessary to further evaluate the diagnostic value of NoSAS score. Obstructive sleep apnoea (OSA) is the main secondary form associated with resistant hypertension (RH), but it is largely underdiagnosed and consequently undertreated in clinical practice. The Berlin questionnaire (BQ) is a useful tool among general population, but seems to not perform well among patients with RH. Recently, NoSAS score was validated in a large population, however, has not been tested in the cardiovascular scenario. Thus, we aimed to compare BQ versus the NoSAS score as screening tools for OSA in RH. In the present study, patients with confirmed diagnosis of RH were invited to perform polysomnography. OSA was diagnosed by an apnoea-hypopnoea index (AHI) ≥15 events/h. BQ and NoSAS were applied in a blinded way. We calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the curve (AUC) of the two sleep questionnaires to detect OSA in RH. The frequency of OSA was 64%. The BQ presented a better sensitivity (91 vs. 72%) and higher values of NPV (67 vs. 54%) than NoSAS score. In contrast, the NoSAS score had higher specificity for excluding OSA (58 vs. 33%) and higher PPV (75 vs. 70%). Compared to the BQ, NoSAS score had a better AUC (0.55 vs. 0.64) but these values are in the fail to poor accuracy range. In conclusion, both BQ and NoSAS score had low accuracy for detecting OSA in RH. Considering the high frequency of OSA, objective sleep study may be considered in these patients. The use of metabolomic and lipidomic strategies for selecting potential biomarkers for obstructive sleep apnoea (OSA) has been little explored. We examined adult male patients with OSA (defined by an apnoea-hypopnoea index ≥15 events/hour), as well as age-, gender-, and fat-composition-matched volunteers without OSA. All subjects were subjected to clinical evaluation, sleep questionnaires for detecting the risk of OSA (Berlin and NoSAS score), metabolomic analysis by gas chromatography coupled to mass spectrometry and lipidomic analysis with liquid chromatography followed by detection by MALDI-MS. This study included 37 patients with OSA and 16 controls. From the 6 metabolites and 22 lipids initially selected, those with the best association with OSA were glutamic acid, deoxy sugar and arachidonic acid (metabolites), and glycerophosphoethanolamines, sphingomyelin and lyso-phosphocholines (lipids). For the questionnaires, the NoSAS score performed best with screening for OSA (area under the curve [AUC] = 0.724, p = 0.003). The combination of the NoSAS score with metabolites or lipids resulted in an AUC for detecting OSA of 0.911 and 0.951, respectively. In conclusion, metabolomic and lipidomic strategies suggested potential early biomarkers in OSA that could also be helpful in screening for this sleep disorder beyond traditional questionnaires. Objective: To evaluate the effect of arterial blood HCO3- level on the accuracy of NoSAS questionnaire screening for obstructive sleep apnea hypopnea syndrome (OSAHS). Methods: The hospitalized patients with suspected OSAHS were recruited from March 2016 to December 2017 in the First Affiliated Hospital of Guangzhou Medical University. NoSAS scores, blood gas analysis and polysomnography (PSG) were performed in these patients. Patients were divided into non OSAHS group and mild, moderate and severe OSAHS group according to the PSG results. According to the NoSAS questionnaire score, the patients were divided into OSAHS high-risk group and low risk group. The correlation between arterial blood HCO3- level and apnea hypopnea index (AHI) was analyzed. The receiver operating characteristic (ROC) curve was plotted to analyze the accuracy of HCO3- prediction OSAHS. Predictive parameters(sensitivity, specificity, positive and negative predictive values)for NoSAS scores and HCO3- level were calculated. Results: A total of 243 patients with suspected OSAHS were included, including 186 males (76.5%), 57 females (23.5%), age (49±13) years, body mass index (BMI) (26.9±4.4) kg/m2, and neck circumference (38.6±4.5) cm. The HCO3- level was positively correlated with AHI (r=0.206, P=0.001). The proportion of patients with HCO3- level ≥26 mmol/L in non-OSAHS group was lower than that in OSAHS group (13.0% vs 34.5%, P=0.004); the proportion of patients with HCO3- level ≥26 mmol/L in severe OSAHS group was higher than that in mild OSAHS group (37.7% vs 15.0%, P=0.008), and there was no difference in the ratio of patients with severe OSAHS and moderate OSAHS (37.7% vs 35.3%, P=0.767). The specificity of OSAHS predicted by HCO3- level 25 and 26 mmol/L was 69.6% and 87.0%, respectively. With the NoSAS score of 8 or 7 as cutoffs for analysis, the sensitivity for OSAHS was 61.9% and 79.2%, the specificity for OSAHS was 57.4% and 40.4%, respectively. With the addition of HCO3- level ≥ 26 mmol/L to the NoSAS score ≥ 7, the specificity for OSAHS improved to 93.6%, while the sensitivity decreased to 27.4%. Conclusion: Combined with the arterial blood HCO3- level, the specificity of the NoSAS questionnaire increases and the sensitivity decreases. BACKGROUND: Diagnosis of sleep-disordered breathing (SDB) requires overnight polysomnography (PSG). Because of the cost and low availability of these procedures, the NoSAS score was developed to identify subjects at high risk of SDB. To evaluate the clinical utility of the NoSAS score for screening patients with SDB in China and to compare the predictive value of the NoSAS score with the Epworth Sleepiness Scale (ESS), we used the STOP-Bang questionnaire and the Berlin questionnaire. METHODS: In our study, we retrospectively reviewed the existing clinical data of patients who underwent an overnight PSG for suspected SDB from June 2014 to September 2017 at the sleep medical center of Guangdong Medical University Affiliated Second Hospital. The information we collected included all parts of the NoSAS score, the ESS, the STOP-Bang questionnaire and the Berlin questionnaire. Based on the severity of SDB determined by the apnea-hypopnea index (AHI), the patients were classified into four groups of primary snoring (<5 events/h), mild SBD (AHI ≥5 and <15 events/h), moderate SBD (AHI ≥15 and ≤30 events/h) and severe SBD (>30 events/h). We calculated the sensitivity, specificity, positive predictive value, negative predictive value and area under the receiver operating characteristic curve of the five questionnaires to compare their relative efficacy for screening SDB. RESULTS: A total of 479 consecutive patients (374 males and 105 females) ranging in age from 18 to 80 years old (mean ± SD, 48.9±14.4 years old) were recruited into this study. When using the standard of AHI ≥5 for diagnosing SDB, the NoSAS score had the largest area under the curve (AUC) (AUC =0.734), and the Berlin questionnaire (AUC =0.732) came second. Both exhibited a better predictive value than the ESS score and the STOP-Bang questionnaire. Using NoSAS ≥8 to predict AHI ≥5 events/h, AHI ≥15 events/h and AHI >30 events/h, the sensitivity and specificity were 0.590 and 0.707, 0.649 and 0.626, and 0.644 and 0.562, respectively; for the STOP-Bang questionnaire, the values were 0.721 and 0.512, 0.752 and 0.440, and 0.763 and 0.399, respectively; and for the Berlin questionnaire, the values were 0.721 and 0.512, 0.752 and 0.440, and 0.763 and 0.399, respectively. CONCLUSIONS: The NoSAS score and the Berlin questionnaire both exhibited good predictive value for SDB patients. NoSAS is a more suitable questionnaire to use in clinic for the conveniences but the similar performance with another questionnaire. INTRODUCTION: Screening methods have become increasingly important due to the growing number of patients suspected of having obstructive sleep apnea (OSA) being referred to sleep clinics. The Lausanne NoSAS (Neck circumference, Obesity, Snoring, Age, Sex) score test is a simple, efficient, and easily employed tool enabling identification of individuals at risk for the disease. The score ranges from 0 to 17 and the patient has a high probability of OSA if they have a NoSAS score of 8 or higher. OBJECTIVES: To evaluate the performance of the NoSAS score as a screening tool for the diagnosis of OSA in a sleep clinic. METHODS: Prospectively, for 12 months, we included all the patients referred by primary care physicians to our sleep unit for clinical evaluation who had undergone in-lab polysomnography (PSG) and completed the NoSAS score. This test assigns 4 points for a neck circumference of more than 40cm, 3 points for a body-mass index of 25kg/m2 to less than 30kg/m2 or 5 points for having a body-mass index of 30kg/m2 or more, 2 points for snoring, 4 points for being older than 55 years of age and 2 points for being male. RESULTS: Of the 294 patients, 70.7% were male, aged 53.5±12.1 years, with a neck circumference of 41.0±3.6cm and a BMI of 30.8±5.1kg/m2. OSA was present in 84.0% of the patients, 34.8% with moderate OSA and 36.4% severe OSA. Using the NoSAS model for the prediction of all OSA, moderate/severe OSA and severe OSA, the area under the ROC (Receiver Operating Characteristic) was 0.770 (IC95%: (0.703; 0.837), p<0.001), 0.746 (IC95%: (0.691; 0.802), p<0.001) and 0.686 (IC95%: (0.622; 0.749), p<0.001), respectively, thus confirming the diagnostic ability of the NoSAS model. With a NoSAS score ≥7, the sensitivity and positive predictive value (PPV) were 94.3% and 87.6% for all OSA, 94.9% and 62.8% for moderate/severe OSA and 100% and 33.8% for severe OSA, respectively. With the same cut-off, the negative predictive value (NPV) for moderate/severe and severe OSA were 67.9% and 100%, respectively. Each increase in the NoSAS score was associated with an increase in the probability of OSA, reaching a 97% OSA probability for a score of 17. CONCLUSIONS: The NoSAS score showed high sensitivity and PPV for OSA with specificity and diagnostic accuracy steadily increasing with higher scores. Furthermore, a low score showed high predictive value for the exclusion of moderate/severe OSA. Overall, our results suggest that, in primary care, this score can be a powerful tool for stratifying and prioritizing patients in the diagnosis of OSA. Nevertheless, more studies are needed to evaluate the efficacy of this score in hospital health care, in younger populations, with a predomice of female and non-obese individuals or in cardiovascular disease. BACKGROUND: There is a growing number of patients with sleep-disordered breathing (SDB) referred to sleep clinics. Therefore, a simple but useful screening tool is urgent. The NoSAS score, containing only five items, has been developed and validated in population-based studies. AIM: To evaluate the performance of the NoSAS score for the screening of SDB patients from a sleep clinic in China, and to compare the predictive value of the NoSAS score with the STOP-Bang questionnaire. METHODS: We enrolled consecutive patients from a sleep clinic who had undergone apnea-hypopnea index (AHI) testing by type III portable monitor device at the hospital and completed the STOP-Bang questionnaire. The NoSAS score was assessed by reviewing medical records. Sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve (AUC) of both screening tools were calculated at different AHI cutoffs to compare the performance of SDB screening. RESULTS: Of the 596 eligible patients (397 males and 199 female), 514 were diagnosed with SDB. When predicting overall (AHI ≥ 5), moderate-to-severe (AHI ≥ 15), and severe (AHI ≥ 30) SDB, the sensitivity and specificity of the NoSAS score were 71.2, 80.4, and 83.1% and 62.4, 49.3, and 40.7%, respectively. At all AHI cutoffs, the AUC ranged from 0.688 to 0.715 for the NoSAS score and from 0.663 to 0.693 for the STOP-Bang questionnaire. The NoSAS score had the largest AUC (0.715, 95% CI: 0.655-0.775) of diagnosing SDB at AHI cutoff of ≥5 events/h. NoSAS performed better in discriminating moderate-to-severe SDB than STOP-Bang with a marginally significantly higher AUC (0.697 vs. 0.663, P=0.046). CONCLUSION: The NoSAS score had good performance on the discrimination of SDB patients in sleep clinic and can be utilized as an effective screening tool in clinical practice. Objective:To evaluate the value of improved Mallampati grading combined with NoSAS questionnaire in screening for obstructive sleep apnea （OSA）. Method:A total of 344 patients admitted to our hospital for sleep disorders were studied. All patients were measured for their height, weight, neck circumference and other parameters. NoSAS scores, improved Mallampati grading and polysomnography （PSG） were performed in these patients. According to AHI in PSG monitoring results, patients were divided into non-osa group （AHI<5） 93 cases and OSA group 251 cases. The OSA group were divided into mild （AHI 5-15）, moderate（AHI 16-30） and severe OSA group（AHI>30） according to the PSG result. The ROC curve was plotted to evaluate the screening value of NoSAS and improved Mallampati grading combined with NoSAS for OSA. Result:With the NoSAS score of 8 or 9 as cutoffs for analysis, the sensitivity for OSA was 0.733 and 0.701; the specificity for OSA was 0.538 and 0.624, respectively. The sensitivity and specificity of NoSAS combined with improved Mallampati grading for screening OSA were 0.813 and 0.710, respectively. Conclusion:As a new screening tool, NoSAS questionnaire is simple and convenient, and has certain screening value to OSA. The improved Mallampati grading combined with NoSAS questionnaire can obviously improve the screening sensitivity and specificity of Osa, and has higher application value. BACKGROUND/AIM: The NoSAS score is a new tool for the identification of high-risk patients for sleep-disordered breathing (SDB). The aim of this study was to validate the NoSAS score in a sleep clinical population in Turkey and compare its performance with the Epworth Sleepiness Scale (ESS), STOP-Bang, and Berlin questionnaires for high-risk SDB. MATERIALS AND METHODS: This was a retrospective study. Patients who had a full-night PSG examination between 01.03.2017 and 01.01.2018 at the sleep center of our hospital were included in the study. Demographic characteristics, anthropometrics measurements, ESS, STOP-Bang, and Berlin scores were collected from the existing data of the patients. The NoSAS score was subsequently calculated based on available data. Predictive parameters for each screening questionnaires were calculated to compare the discriminative power of those for high-risk SDB. RESULTS: A total of 450 patients were included in the study. The sensitivity, specificity, PPV, and NPV of the NoSAS score were 81%, 51.2%, 88.2%, and 37.5% for an AHI (apnea–hypopnea index) ≥ 5 event/h and 84.5%, 38.2%, 66%, and 63.4% for an AHI ≥ 15 event/h, respectively. AUC percentages for the NoSAS score, STOP-Bang questionnaire, Berlin questionnaire, and ESS were 0.740, 0.737, 0.626, and 0.571 for an AHI ≥ 5 events/h and 0.715, 0.704, 0.574, and 0.621 for an AHI ≥ 30 events/h. The NoSAS score had a false negative rate of 2.9% for severe SDB. CONCLUSION: The NoSAS score had a good degree of differentiation for SDB and can be used as an easily applicable, subjective, and effective screening tool in a sleep clinical population in Turkey. Not only in moderate to severe SDB but also in mild SDB, the NoSAS score performed better than the other 3 screening tools. OBJECTIVES: The NoSAS score has been shown to be a reliable screening tool for obstructive sleep apnea (OSA) in overall populations. This study aimed to explore the effects of age and sex on the predicting performance of this score. METHODS: A retrospective analysis was conducted on 1119 subjects aged ≥ 18 years and with a total sleep time of ≥ 4 h during overnight polysomnography. Discrimination was assessed by using areas under receiver operating characteristic curve (AUCs), while predictive parameters were calculated by using contingency tables. RESULTS: Overall, a NoSAS score of 8 points or higher resulted in sensitivity, specificity, and AUC for predicting an apnea-hypopnea index (AHI) of ≥ 20 events/h of 74%, 36%, and 0.63 (in non-elderly 73%, 46%, and 0.65; in elderly 91%, 17%, and 0.59; in men 85%, 18%, and 0.56; in women 52%, 76%, and 0.71, respectively). The AUCs at all AHI cutoffs were significantly lower in men than in women (all with p < 0.01), while the AUCs at AHI cutoff of 5, 15, and 30 events/h were significantly lower in elderly than in non-elderly (p < 0.01, 0.05, and 0.05, respectively). In non-elderly, a conventional NoSAS with cutoff of 7 or a modified NoSAS with age cutoff of 50 years provided sensitivity and specificity for predicting an AHI of ≥ 20 events/h of 87%, 37% and 80%, 36%, respectively, with comparable AUCs. In women, a conventional NoSAS with cutoff of 6 or a modified NoSAS with neck circumference cutoff of 35 cm provided sensitivity and specificity for predicting an AHI of ≥ 20 events/h of 85%, 39% and 79%, 52%, respectively, with comparable AUCs. CONCLUSIONS: NoSAS score has better discrimination but lower sensitivity for predicting OSA in non-elderly and women than in their counterparts. Age- and sex-specific cutoff values reverse this imbalance. Our results underline the preference of age- and sex-specific cutoff values and the need for better age- and sex-specific screening algorithms.

What is the chromosomal abnormality associated with Klinefelter Syndrome

About 1 in 650 boys are born with an extra X chromosome (47,XXY or Klinefelter syndrome). 47,XXY

A case of Klinefelter's syndrome with the development of a mediastinal teratocarcinoma is reported suggesting that the association of a gonadotropin-secreting tumor with the XXY chromosomal abnormality may be more than coincidental. Whereas this child appeared to survive the effects of the teratocarcinoma, he succumbed to acute leukemia two years later. This prompted a review of secondary leukemias in children following chemotherapy/radiotherapy for another primary maligcy. These patients responded poorly to treatment of the secondary leukemia with a median survival of about four months. The incidence of secondary leukemias might be expected to be on the rise as increasing numbers of pediatric cancer patients are surviving longer after treatment with agents that are potentially leukemogenic or carcinogenic themselves. Children who have survived cancer and its therapy present special problems and it will be necessary for the pediatrician and practitioner to monitor these children. In this communication, a case of Klinefelter syndrome associated with a 13/14 translocation is described. Such a rare occurrence is most probably due to the de novo arrangement of chromosomes related to the advanced ages of both parents at conception. Klinefelter syndrome is the most commonly diagnosed sex chromosome disorder among males. It is usually associated with 47 chromosomes, including two Xs and one Y. The formal cytogenetic designation for Klinefelter syndrome is 47, XXY; the extra sex chromosome is due to meiotic chromosomal nondisjunction. Increased risk of various maligt diseases has been recognized among patients with different congenital chromosomal abnormalities. Since the early 1960s, numerous reports have appeared of an increased risk of maligt neoplasms among patients with Klinefelter syndrome. Evidence suggests a correlation with increased incidences of germ cell tumors and breast cancers. Whether these patients are at an increased risk of hematologic maligt disease, especially acute leukemia, is still uncertain. This report describes a patient with agnogenic myeloid metaplasia and Klinefelter syndrome, an association not previously reported. Chromosomal abnormality and Y chromosome microdeletion are regarded as two frequent genetic causes associated with spermatogenic failure in Caucasian population. To investigate the distribution of the two genetic defects in Chinese patients with azoospermia or severe oligozoospermia, karyotype analysis by G-banding was carried out in 358 idiopathic infertile men, including 256 patients with azoospermia and 102 patients with severe oligozoospermia, and screening of AZF region microdeletion of Y chromosome by multiplex PCR was performed in those patients without detectable chromosomal abnormality and 100 fertile controls. Of 358 patients, 39(10.9%) were found to have chromosomal abnormalities in which Klinefelter's syndrome (47, XXY) was the most common chromosomal aberration. The incidence of sex chromosomal abnormality in patients with azoospermia was significantly higher than that in patients with severe oligozoospermia (12.1% vs 1%). Among the rest of the 319 patients with normal karyotype, 46 (14.4%) were found to have microdeletions in AZF region. The prevalence rates of AZF microdeletion was 15% and 13.1% in patients with azoospermia and severe oligozoospermia respectively. The microdeletion in AZFc was the most frequent deletion and all the microdeletions in AZFa were found in azoospermic patients. No microdeletion in AZF region was detected in fertile controls. In conclusion, chromosomal abnormality and AZF region microdeletion of Y chromosome might account for about 25% of Chinese infertile patients with azoospermia or severe oligozoospermia, suggesting the two abnormalities are important genetic etiology of spermatogenic failure in Chinese population and it is essential to screen them during diagnosis of male infertility before in vitro assisted fertilization by introcytoplasmic sperm injection. Klinefelter's syndrome, characterised by a 47,XXY chromosomal pattern, has largely been associated with physical abnormalities. Here, we report high levels of schizophrenia-spectrum pathology in 32 men with this syndrome in comparison with 26 healthy controls. This may have implications for treatment of have implications for treatment of Klinefelter's syndrome and suggests that the X chromosome may be involved in the aetiology of schizophrenia. Klinefelter syndrome is caused by the presence of one or more additional X chromosomes in an affected male. Patients often exhibit gynecomastia, small testes, and infertility. Though the characteristics of Klinefelter have been well-documented, associated ocular abnormalities have been only occasionally reported. Here we present a 2-month-old infant with Klinefelter syndrome and a unique combination of ocular abnormalities including microphthalmia, cataracts, and malformed pupils. Klinefelter syndrome (KS), a 47,XXY chromosomal abnormality, has been shown to be associated with a number of maligcies, but has not been linked to acute leukemias to date. We present a case of a 54-year-old male diagnosed with acute myeloid leukemia (AML) with monocytic differentiation, whose cytogenetic and subsequent FISH analyses revealed a constitutional 47,XXY karyotype. We also review and discuss relevant prior literature. Klinefelter syndrome (KS) is a chromosome abnormality characterized by a 47, XXY karyotype associated with hypogonadism and infertility. We present two cases of leukemia in patients with KS. The first patient presented with acute promyelocytic leukemia. He relapsed after the end of treatment. The second patient was diagnosed with chronic myeloid leukemia. Treatment with imatinib failed and the patient presented with myeloid blast crisis. BACKGROUND: Klinefelter's syndrome is a sex chromosome abnormality affecting approximately 1 in 1000 men. There have been suggestions that it is associated with a higher than average prevalence of sexual offending but to what extent does research evidence support this assertion? AIMS: This study aimed to conduct a systematic review of published research to establish the prevalence of sexual offending in men with Klinefelter's syndrome. METHOD: The databases MEDLINE, PsycINFO and EMBASE were searched from inception until 31 December 2016 by using a range of terms for Klinefelter's syndrome and for sexual offending. All selected papers were examined for quality by using the Strengthening the Reporting of Observational Studies in Epidemiology checklist. RESULTS: We identified 53 relevant papers of which 10 met our inclusion criteria. All but one were prevalence studies conducted in a prison or hospital setting. The one, Danish, register-based cohort study did suggest an increased risk of sex offending among Klinefelter men, probably established before the diagnosis was made and, therefore, any hormone replacement instituted. CONCLUSION: There is insufficient evidence to date to support concerns about exceptional risk of sex offending among men with Klinefelter's syndrome. Rather, it is arguable that there is a research gap in understanding how the experience of and treatment for their condition may affect them. Copyright © 2017 John Wiley & Sons, Ltd. About 1 in 650 boys are born with an extra X chromosome (47,XXY or Klinefelter syndrome). 47,XXY is associated with vulnerabilities in socio-emotional development. This study was designed to assess types of cognitive deficits in individuals with 47,XXY that may contribute to social-emotional dysfunction, and to evaluate the nature of such deficits at various levels: ranging from basic visuospatial processing deficits, impairments in face recognition (FR), to emotion expression impairments. A total of 70 boys and men with 47,XXY, aged 8 to 60 years old, participated in the study. The subtests feature identification, FR and identification of facial emotions of the Amsterdam Neuropsychological Tasks were used. Level of intellectual functioning was assessed with the child and adult versions of the Wechsler Intelligence Scales. Reaction time data showed that in the 47,XXY group, 17% had difficulties in visuospatial processing (no social load), 26% had difficulties with FR (medium social load) and an even higher number of 33% had difficulties with facial expressions of emotions (high-social load). Information processing impairments increased as a function of "social load" of the stimuli, independent of intellectual functioning. Taken together, our data suggest that on average individuals with XXY may have more difficulties in information processing when "social load" increases, suggesting a specific difficulty in the higher-order labeling and interpretation of social cues, which cannot be explained by more basic visuospatial perceptual skills. Considering the increased risk for social cognitive impairments, routine assessment of social cognitive functioning as part of neuropsychological screening is warranted. 47,XXY (KS) occurs in 1:650 male births, though less than 25% are ever identified. We assessed stability of neurocognitive features across diverse populations and quantified factors mediating outcome. Forty-four boys from the Netherlands (NL) and 54 boys from the United States (US) participated. The Wechsler Intelligence Scales assessed intellectual functioning; the ANT program evaluated cognitive function; and the CBCL assessed behavioral functioning. ANOVA was used for group comparisons. Hierarchical regressions assessed variance explained by each independent variable: parental education, timing of diagnosis, testosterone, age, and nationality. Parental education, timing of diagnosis, and hormonal treatment all played an important role in neurocognitive performance. The observed higher IQ and better attention regulation in the US group as compared to the NL group was observed with decreased levels of behavioral problems in the US group. Cognitive measures that were different between the NL and US groups, i.e., attention regulation and IQ scores, were also significantly influenced by external factors including timing of diagnosis, testosterone treatment, and parental education. On the ANT, a cognitive phenotype of 47,XXY was observed, with similar scores on 9 out of the 10 ANT subtests for the NL and US groups. This study lays additional features to the foundation for an algorithm linking external variables to outcome on various neurodevelopmental measures. Klinefelter syndrome is a condition in which a male patient has one Y chromosome and one or more extra X chromosomes. It is the most common sex chromosome disorder. Klinefelter syndrome is distinguished by many clinical features, such as infertility, high gonadotropin and low testosterone levels, increased height, and sparse body and facial hair. We report the case of a 32-year-old man who visited our hospital complaining of male infertility. Semen analysis showed azoospermia, and chromosomal analysis revealed a 47,XY,i(X)(q10) karyotype, which is a rare variant of Klinefelter syndrome. No spermatozoon was found on microdissection testicular sperm extraction, and the testis biopsy histology showed only Sertoli cells and hyalinised seminiferous tubules. 47,XY, i(X)(q10) has an additional isochromosome made of the long arm of the X chromosome, which shares some features of classical Klinefelter syndrome in many aspects, but patients are usually shorter than average height and have normal intelligence. In addition, to the best of our knowledge, no successful sperm extractions from 47,XY, i(X)(q10) patients were reported in the literature. The reports of patients who have undergone microdissection testicular sperm extraction are very rare. Further reports and studies of this chromosomal abnormality are needed. Özyörük D, Kocayozgat A, Yaman-Bajin İ, Çetindağ F, Oğuz- Erdoğan AS, Güneş A. A synchronous occurrence of bifocal intracranial germinoma and bilateral testicular epidermoid cyst in an adolescent patient with Klinefelter`s syndrome. Turk J Pediatr 2019; 61: 456-459. Klinefelter syndrome (KS) is characterized by an additional X chromosome in males leading to a karyotype of 47,XXY. It is associated with an increased risk of certain maligcies, including leukemia, breast cancer and extragonadal germ cell tumor such as mediastinal germ cell tumors and rarely intracranial germ cell tumors. It is possible that the increased risk of developing certain cancers can be attributed to a direct effect of the chromosomal abnormality or the combined action of the abnormal chromosomes and hormonal imbalances. Here we describe a synchronous occurrence of bifocal intracranial germinoma and bilateral testicular epidermoid cyst in an adolescent patient with Klinefelter`s syndrome. The synchronous occurrence of the dual tumors in this patient with Klinefelter`s syndrome might be resulted from the migration defect during embriyogenesis due to underlying genetic disease or it is a coincidental condition, yet there has been no case reported in the literature, so far.

What distinguishes RIDLs from other transpozable elements?

Here, we link these two concepts by proposing that exonic TEs act as RNA domains that are essential for lncRNA function. We term such elements Repeat Insertion Domains of LncRNAs (RIDLs).

Our genome contains tens of thousands of long noncoding RNAs (lncRNAs), many of which are likely to have genetic regulatory functions. It has been proposed that lncRNA are organized into combinations of discrete functional domains, but the nature of these and their identification remain elusive. One class of sequence elements that is enriched in lncRNA is represented by transposable elements (TEs), repetitive mobile genetic sequences that have contributed widely to genome evolution through a process termed exaptation. Here, we link these two concepts by proposing that exonic TEs act as RNA domains that are essential for lncRNA function. We term such elements Repeat Insertion Domains of LncRNAs (RIDLs). A growing number of RIDLs have been experimentally defined, where TE-derived fragments of lncRNA act as RNA-, DNA-, and protein-binding domains. We propose that these reflect a more general phenomenon of exaptation during lncRNA evolution, where inserted TE sequences are repurposed as recognition sites for both protein and nucleic acids. We discuss a series of genomic screens that may be used in the future to systematically discover RIDLs. The RIDL hypothesis has the potential to explain how functional evolution can keep pace with the rapid gene evolution observed in lncRNA. More practically, TE maps may in the future be used to predict lncRNA function.

What indication has FTY720 been approved for by the FDA?

FTY720 has been pproved (September 2010) by the U.S. FDA as a new treatment for multiple sclerosis (MS).

Is fingolimod a drug or a pro-drug?

FTY720 is a prodrug.

FTY720 is a prodrug for FTY-phosphate, an agonist at four of the five known receptors for sphingosine-1-phosphate (S1P). We show that administration of either FTY720 or FTY-P to SJL mice with established relapsing-remitting experimental autoimmune encephalitis (EAE) results in a rapid and sustained improvement in their clinical status, and a reversal of changes in expression of mRNAs encoding some myelin proteins and inflammatory mediators. EAE produced by adoptively transferring lymph node cells from immunized mice to naïve hosts is similarly ameliorated by FTY-P. Treatment with FTY-P is accompanied by a dose-responsive peripheral lymphopoenia.

Which conditions is caused by mutations in HFE?

Mutations in the HFE gene, encoding the syntaxin binding protein HFE1, are the cause of hereditary hemochromatosis.

Recent studies have shown that hereditary hemochromatosis (HH) is likely to be caused by homozygosity for a Cys282Tyr mutation in the HFE gene located 4.5 Mb telomeric to HLA-A. Population studies of this polymorphism are facilitated by the fact that the Cys282Tyr mutation creates a Rsal restriction site. We have studied the codon 282 (Cys/Tyr) polymorphism in different ethnic groups. In agreement with previous observations the Tyr allele appeared to be rare or absent in Asiatic (Indian, Chinese) populations. The highest allele frequency (7.5%) was found in Swedes. Saamis (2%) and Mordvinians (1.8%) had significantly lower frequencies of the Tyr allele. Comparisons with allele frequencies based on prevalence estimates of HH showed some disagreements with the RFLP data, particularly in Finns. The newly described HFE marker provides a new approach to the screening of HH as well as studies of the relationship between the HFE Tyr allele and different disorders including cancer. OBJECTIVE: The aim of the study was to investigate the molecular basis of hereditary haemochromatosis (HH) in South Africa in order to establish a reliable, cost-effective molecular diagnostic service for this potentially lethal disorder. DESIGN: DNA samples of patient and control groups were screened for two common haemochromatosis (HFE) gene mutations. The local frequencies of mutations C282Y and H63D were determined and the DNA results correlated with biochemical parameters. SETTING: Patients were referred from private practitioners, health workers and pathologists for a molecular diagnosis of HH at the University of Stellenbosch Medical School. Twenty-two of the 244 referrals were clinically diagnosed with HH, while the remaining patients were family members of the probands or unrelated subjects referred solely on the basis of an abnormal iron profile. RESULTS: Seventeen of the 22 patient referrals (77%) diagnosed with HH were homozygous for the C282Y mutation, 3 (14%) were compound heterozygotes for mutations C282Y and H63D, and 2 patients (9%) did not exhibit either mutation. Screening of 458 control individuals from the general South African population demonstrated a carrier frequency of approximately 17% for the C282Y mutation among whites, implying that up to 1 out of every 115 South Africans of European descent may be homozygous for this founder-type mutation. Among 64 healthy blood donors of mixed ancestry, we detected 2 individuals heterozygous and 1 homozygous for the C282Y mutation. CONCLUSIONS: The detection of mutations C282Y and H63D at a high frequency in the majority of affected South African patients facilitates accurate pre-clinical and confirmatory diagnosis of HH in South Africa. Early detection by DNA screening and subsequent treatment by repeated phlebotomy can prevent disease onset in affected individuals. DNA diagnosis is particularly applicable to a common genetic disease such as HH, which is underdiagnosed and potentially lethal, but treatable. BACKGROUND: Hitherto studies on the ethiopathogenesis of porphyria cutanea tarda (PCT) show that the major pathogenic factor is iron ion, which acts via inhibition of the uroporphyrinogen decarboxylase. New speculations have appeared on the possible relation of this role of iron and the occurrence of mutation of the recently discovered gene of the hereditary hemochromatosis HFE, which may cause the iron overloading of the organism. Our paper describes prevalence of the C282Y gene mutation (HFE) together with the clinical and laboratory record in PCT patients. METHODS AND RESULTS: PCT was diagnosed mostly on the basis of clinical finding of actinic-traumatic vesicular dermatitis and the typical laboratory record of elevated higher-carboxylic porphyrines in urine and stool. Other laboratory methods tested the liver functions, plasma iron level and its binding capacity, ferritine level. All patient underwent routine haematological testing. Presence of antibodies against hepatitis C was also assayed (Elisa test 2nd generation, Sanofi Pasteur). In patients with prominent laboratory alterations showing possibility of the hepatic structural lesion, histology from the liver punctate was done. Frequency data of the C282Y gene mutation (HFE) in PCT patients was estimated on the basis of the genetic testing using PCR reaction of our own system. Group of PCT patients had 69 persons (63 patients with the sporadic form and 6 patients with familiar form of the disease). Hereditary haemochromatosis C282Y gene mutation (HH) was found in 15 patients, three of them were homozygotes and twelve heterozygotes (three heterozygotes had the familiar form of the disease). Nobody in this group was positive in the HIV antibody testing. In all porphyria patients with the presence of mutated gene who underwent liver biopsy, siderosis of different degrees was identified. In three patients neither the phenotypic observation nor the laboratory testing have shown haemochromatosis. Prevalence of C282Y gene mutation HFE in patients with porphyria cutanea tarda was studied. Such mutation was found in 15 persons (12 heterozygotes and 3 homozygotes) from the group of 69 tested patients (21.7%). Such frequency is significantly higher than in the control--nonporphyric--persons (10%). Patients were without clinical symptoms. Laboratory haematological changes, typical for HH, manifested in some of them only (elevated level of ferritine was found in 10 from 15 porphyria patients, elevated sideremia in one of them). Red blood cell counts were in both homo- and heterozygotes normal. Concurrence of the two porphyrinogenic factors--presence of gene mutation HFE and hepatitis C infection--was not proved. Antibodies against hepatitis C virus were not identified in any of the patients. Siderosis was found to be only a symptomatic sign, which was pronounced in different degree in all 9 porphyria patients with C282Y gene mutation who underwent liver biopsy. CONCLUSIONS: Frequency of C282Y gene mutation in our patients with porphyria cutanea tarda appears similar to that in other Middle European countries. It differs significantly from the frequency found in South European and North European countries (British). Comparative analysis of the hemochromatosis-associated mutations C282Y, H63D and S65C in the HFE gene in 51 patients using three different methods is reported. One PCR-RFLP method was based on general primers, whereas another employed mutation-specific mismatched primers. The third method was a new PCR-based reverse hybridisation line probe assay (LiPA), comprising DNA amplification by general primers followed by a single step reverse hybridization to specific probes, immobilized on a nitrocellulose strip. Forty-eight (94%) of the 51 samples yielded identical results by all three methods. Three discrepant results were obtained, caused by polymorphisms in the primer binding region, resulting in no amplification at all or selective amplification, leading to misinterpretation of the HFE genotype by PCR-RFLP. The design of the assay and the stringency of the reaction conditions used are crucial to obtain a correct HFE genotype. PCR-LiPA offers an easy and reliable alternative to currently used conventional methods. Hemochromatosis is a common disorder characterized by excess iron absorption and accumulation of iron in tissues. Usually hemochromatosis is inherited in an autosomal recessive pattern and is caused by mutations in the HFE gene. Less common non-HFE-related forms of hemochromatosis have been reported and are caused by mutations in the transferrin receptor 2 gene and in a gene localized to chromosome 1q. Autosomal domit forms of hemochromatosis have also been described. Recently, 2 mutations in the ferroportin1 gene, which encodes the iron transport protein ferroportin1, have been implicated in families with autosomal domit hemochromatosis from the Netherlands and Italy. We report the finding of a novel mutation (V162del) in ferroportin1 in an Australian family with autosomal domit hemochromatosis. We propose that this mutation disrupts the function of the ferroportin1 protein, leading to impaired iron homeostasis and iron overload. The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies using solubilized cell extracts demonstrated that transferrin (Tf) competed with HFE for binding to the transferrin receptor (TfR) similar to previous in vitro studies using soluble truncated forms of HFE and the TfR. At concentrations of Tf approaching those found in the blood, no differences in Tf binding to cells were detected, which is consistent with the lower binding constant of HFE for TfR versus Tf. However, cells expressing HFE still showed a decrease in Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE from the TfR. These results indicate that the association of HFE with TfR is not essential for its ability to lower intracellular iron stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa cells expressing fW81AHFE behaved in a similar manner to cells expressing wild-type HFE with respect to decreased intracellular iron levels measured by iron regulatory protein gel-shift assays and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its interaction with the TfR. The classification of hereditary abnormalities of iron metabolism was recently expanded and diversified. Genetic hemochromatosis now corresponds to six diseases, namely classical hemochromatosis HFE 1; juvenile hemochromatosis HFE 2 due to mutations in an unidentified gene on chromosome 1; hemochromatosis HFE 3 due to mutations in the transferrin receptor 2 (TfR2); hemochromatosis HFE 4 caused by a mutation in the H subunit of ferritin; and hemochromatosis HFE 6 whose gene is hepcidine (HAMP). Systemic iron overload is also associated with aceruloplasminemia, atransferrinemia and the "Gracile" syndrome caused by mutations in BCS1L. The genes responsible for neonatal and African forms of iron overload are unknown. Other genetic diseases are due to localized iron overload: Friedreich's ataxia results from the expansion of triple nucleotide repeats within the frataxin (FRDA) gene; two forms of X-linked sideroblastic anemia are due to mutations within the delta aminolevulinate synthetase (ALAS 2) or ABC-7 genes; Hallervorden-Spatz syndrome is caused by a pantothenate kinase 2 gene (PANK-2) defect; neuroferritinopathies; and hyperferritinemia--cataract syndrome due to a mutation within the L-ferritin gene. In addition to this wide range of genetic abnormalities, two other features characterize these iron disorders: 1) most are transmitted by an autosomal recessive mechanism, but some, including hemochromatosis type 4, have domit transmission; and 2) most correspond to cytosolic iron accumulation while some, like Friedreich's ataxia, are disorders of mitochondrial metabolism. The etiology of amyotrophic lateral sclerosis (ALS) is unknown. The presence of mutations in the superoxide dismutase gene (SOD1) has led to theories regarding a role for oxidative stress in the pathogenesis of this disease. A primary cause of oxidative stress is perturbations in cellular iron homeostasis. Cellular iron mismanagement and oxidative stress are associated with a number of neurodegenerative diseases. One mechanism by which cells fail to properly regulate their iron status is through a mutation in the Hfe gene. Mutations in the Hfe gene are associated with the iron overload disease, hemochromatosis. In the current study, 31% of patients with sporadic ALS carried a mutation in the Hfe gene, compared to only 14% of patients without identifiable neuromuscular disease, or with neuromuscular diseases other than ALS (p<0.005). To determine the cellular consequences of carrying an Hfe mutation, a human neuronal cell line was transfected with genes carrying the Hfe mutation. The presence of the Hfe mutation disrupted expression of tubulin and actin at the protein levels potentially consistent with the disruption of axonal transport seen in ALS and was also associated with a decrease in CuZnSOD1 expression. These data provide compelling evidence for a role for the Hfe mutation in etiopathogenesis of ALS and warrant further investigation. The mechanism of excessive iron storage in patients with hereditary hemochromatosis caused by mutations of the HFE gene seems to be a failure to up-regulate hepcidin in the face of increased body iron. Since the cytokines IL-1 and IL-6 stimulate hepcidin transcription in the absence of HFE, chronic inflammatory states might counteract the effect of HFE mutations. We measured the pre-phlebotomy plasma levels of C reactive protein (CRP) and of interleukin 6 (IL-6) in homozygotes for the C282Y mutation of HFE. There was no difference in these levels in subjects with high iron stores than in those with low iron stores, suggesting that the phenotypic differences between such homozygotes is not appreciably affected by ongoing chronic inflammation. OBJECTIVE: To assess geographic differences in the frequencies of HFE C282Y and H63D genotypes in six racial/ethnic groups recruited in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. DESIGN: HFE C282Y and H63D genotypes of 97,551 participants, ages > or = 25 years, who reported that they belonged to one of six racial/ethnic groups, were analyzed. HFE genotype frequencies were compared among the racial/ethnic groups and among the HEIRS Study field centers within each racial/ethnic group. RESULTS: The distribution of HFE C282Y and H63D genotypes differed among racial/ethnic groups (P<.0001) and among field centers in Hispanics, Asians, Whites, and Blacks (each P<.05). Genotype frequencies were similar among field centers in Native Americans and Pacific Islanders. Frequencies of C282Y and H63D genotypes were greatest in Whites. The lowest frequencies of C282Y genotypes were observed in Asians; Blacks had the lowest H63D genotype frequencies and the highest frequency of the wild-type genotype. Among racial/ethnic groups, Hispanics had the greatest variation in HFE genotypes across geographic regions. CONCLUSION: HFE C282Y and H63D genotype frequencies vary significantly between racial/ethnic groups and within some racial/ethnic groups across geographic regions. Iron is a potent generator of oxidative damage whose levels increase with age, potentially exacerbating age-related diseases. Several lines of evidence suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). AMD retinas have more iron within the photoreceptors, RPE, and drusen than do age-matched control retinas. Accelerated AMD-like maculopathy develops in patients with retinal iron overload from the hereditary disease aceruloplasminemia. Mice with retinal iron overload resulting from knockout of ceruloplasmin and its homologue hephaestin exhibit retinal degeneration with some features of AMD, including subretinal neovascularization, accumulation of RPE lipofuscin and sub-RPE deposits, and RPE/photoreceptor death. Increased understanding of the mechanisms of retinal iron homeostasis may help in the development of therapies to prevent iron overload. For example, herein it is shown that one regulator of systemic iron homeostasis, HFE, is expressed in the RPE. Thus, patients with the common disease hereditary hemochromatosis, which is often caused by an HFE mutation, may have retinal iron overload predisposing to AMD. Preliminary data suggest that iron chelation can reduce RPE iron overload in mice and protect them from degeneration, suggesting that iron-binding drugs may one day prove useful in reducing RPE oxidative stress and decreasing the risk of AMD progression. Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-alpha, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-alpha. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease. Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by systemic iron overload with consequent tissue damage. The vast majority of HH patients are homozygous for the C282Y mutation in HFE, a non-classical MHC class-I gene located in chromosome 6, whose role in the regulation of systemic iron metabolism is still not completely understood. Iron enhances the formation of reactive oxygen species, with increasing risk of DNA damage induced by oxidative stress, and consequently an increased susceptibility to chromosome instability. In the present work we examined spontaneous and diepoxybutane (DEB)-induced chromosome instability in PHA-stimulated lymphocyte cultures from 23 HH patients, all homozygous for the C282Y HFE mutation, in comparison to 29 normal controls. In addition, three patients with secondary forms of iron overload, not related to HFE, were studied as controls to test the role of iron overload on DEB-induced chromosome instability. Our results show a significantly higher frequency of spontaneous chromosome breaks in lymphocytes from the HH patients, when compared with lymphocytes from normal controls (p<0.0001). In addition, there is a significant correlation between the percentage of cells with spontaneous chromosomal breaks and the transferrin saturation (r=0.53, p=0.0041) or serum-ferritin levels (r=0.66, p=0.0001) in patients. Surprisingly, the frequency of DEB-induced chromosome breaks was significantly lower in lymphocytes from HH patients than in lymphocytes from both controls and patients with secondary forms of hemochromatosis (p=0.0029). No correlation was observed between the percentage of cells with DEB-induced chromosome breaks and the transferrin saturation or serum-ferritin values. These results suggest that lymphocytes from HH patients may have an increased capacity to respond to DEB-induced chromosome breakage, and that this capacity is somehow related to the presence of the C282Y HFE mutation. On admission to hospital Caucasian 61 year old male with jaundice was found to have unexplained increased serum iron indices. He had bilateral peripheral arthritis. On further investigation he had grade II hepatocellular iron staining and a hepatic index of 5.4 leading to a diagnosis of hereditary hemochromatosis. He lacked the common C282Y HFE mutation. We sequenced the complete HFE gene and found that he was heterozygous for a novel single nucleotide deletion (c.del478) in exon 3 of HFE. He lacks any other mutation in HFE or HJV, TFR2, HAMP and SLC40A1. The HFE mutation causes a frameshift (p.P160fs) that introduces a premature termination codon leading to mRNA degradation by nonsense-mediated decay. Haploinsufficiency of HFE may be one possible explanation for hemochromatosis in this patient. CONTEXT: Most cases of primary hypertriglyceridemia (HTG) are caused by the interaction of unknown polygenes and environmental factors. Elevated iron storage is associated with metabolic syndrome, diabetes, and obesity, and all of them are associated with HTG. OBJECTIVE: The aim of the study was to analyze whether HFE mutations causing hereditary hemochromatosis (HH) are associated with primary HTG. DESIGN: Genetic predisposition to HH was analyzed in a case-control study. SETTING: The study was conducted at University Hospital Lipid Clinic. PARTICIPANTS: We studied two groups: 1) the HTG group, composed of 208 patients; and 2) the control group, composed of 215 normolipemic subjects and 161 familial hypercholesterolemia patients. INTERVENTION: Two HFE mutations (C282Y and H63D) were analyzed. MAIN OUTCOME MEASURE: We measured HH genetic predisposition difference between groups. RESULTS: HH genetic predisposition was 5.9 and 4.4 times higher in the HTG group than in the normolipemic (P = 0.02) and FH (P = 0.05) subjects, respectively. There were 35 cases (16.8%) of iron overload in the primary HTG group, 14 (6.5%) and nine (5.6%) in the normolipidemic and FH groups, respectively. A higher HH genetic predisposition and a different prevalence of iron overload in subjects with HH genetic predisposition among groups contributed to this higher prevalence. None of the four cases with the HFE genotype associated with high risk of HH in the control groups presented iron overload; however, in eight of 11 subjects (72.7%) with primary HTG and HH genetic predisposition, the iron overload was present. CONCLUSION: Mutations in HFE gene, favoring iron overload and causing HH, could play an important role in the development of several phenotypes of primary HTG. The most common form of hemochromatosis is caused by mutations in the HFE gene. Rare forms of the disease are caused by mutations in other genes. We present a patient with hyperferritinemia and iron overload, and facial flushing. Magnetic resoce imaging was performed to measure hepatic iron overload, and a molecular study of the genes involved in iron metabolism was undertaken. The iron overload was similar to that observed in HFE hemochromatosis, and the patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G (p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively. Hyperferritinemia and facial flushing improved after phlebotomy. Two of the patient's children were also studied, and the daughter was heterozygous for the mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The patient presented a mild iron overload phenotype probably because of the two novel mutations in the HFE and SLC40A1 genes. Elevated body iron stores are associated with morbidity and mortality due to oxidative stress. Hereditary hemochromatosis, a common condition caused by HFE gene mutations, can lead to excess iron storage and disease but clinical penetrance of HFE gene mutations is low and many people with elevated iron stores lack HFE mutations. We analyzed data from the Hemochromatosis and Iron Overload Screening Study to assess the relationship among HFE genotype (individuals with either homozygous or compound heterozygous status for C282Y and/or H63D HFE mutations were defined as genotype positive, or G+), elevated iron phenotype (individuals exceeding gender-specific transferrin saturation and serum ferritin threshold levels were considered phenotype positive, or P+), and leukocyte telomere length, a marker of biological aging and cumulative oxidative stress. In unadjusted analyses in comparison to individuals who were G-P-, G+P- were not significantly different (OR 0.74; 95% CI 0.26-2.04), while the G+P+ (OR 2.03; 95% CI 1.15-3.56), and G-P+ (OR 2.24; 95% CI 1.5-3.29) had increased risk of short telomeres (<=25th percentile) rather than long telomeres (>=75th percentile). In analyses adjusting for age, gender, and race/ethnicity, the effect of individuals with elevated iron phenotypes having short telomeres persisted with G+P+ individuals (OR 1.94; 95% CI 1.02-3.72), and G-P+ individuals (OR 2.17; 95% CI 1.39-3.39) being significantly different from the G-P- group. In conclusion, elevated iron phenotype, but not HFE genotype, was associated with shortened telomeres. Further studies will be needed to determine whether telomere length provides a marker for morbidities specifically associated with iron overload. Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface. Iron overload is associated with acquired and genetic conditions, the most common being hereditary hemochromatosis (HH) type-I, caused by HFE mutations. Here, we conducted a hospital-based case-control study of 41 patients from the São Miguel Island (Azores, Portugal), six belonging to a family with HH type-I pseudodomit inheritance, and 35 unrelated individuals fulfilling the biochemical criteria of iron overload compatible with HH type-I. For this purpose, we analyzed the most common HFE mutations- c.845G>A [p.Cys282Tyr], c.187C>G [p.His63Asp], and c.193A>T [p.Ser65Cys]. Results revealed that the family's HH pseudodomit pattern is due to consanguineous marriage of HFE-c.845G>A carriers, and to marriage with a genetically unrelated spouse that is a -c.187G carrier. Regarding unrelated patients, six were homozygous for c.845A, and three were c.845A/c.187G compound heterozygous. We then performed sequencing of HFE exons 2, 4, 5 and their intron-flanking regions. No other mutations were observed, but we identified the -c.340+4C [IVS2+4C] splice variant in 26 (74.3%) patients. Functionally, the c.340+4C may generate alternative splicing by HFE exon 2 skipping and consequently, a protein missing the α1-domain essential for HFE/ transferrin receptor-1 interactions. Finally, we investigated HFE mutations configuration with iron overload by determining haplotypes and genotypic profiles. Results evidenced that carriers of HFE-c.187G allele also carry -c.340+4C, suggesting in-cis configuration. This data is corroborated by the association analysis where carriers of the complex allele HFE-c.[187C>G;340+4T>C] have an increased iron overload risk (RR = 2.08, 95% CI = 1.40-2.94, p<0.001). Therefore, homozygous for this complex allele are at risk of having iron overload because they will produce two altered proteins--the p.63Asp [c.187G], and the protein lacking 88 amino acids encoded by exon 2. In summary, we provide evidence that the complex allele HFE-c.[187C>G;340+4T>C] has a role, as genetic predisposition factor, on iron overload in the São Miguel population. Independent replication studies in other populations are needed to confirm this association. We report a family case of type II early-onset Alzheimer's disease (AD) inherited over three generations. None of the patients in the family had mutations in the genes believed to be the major risk factors for AD, such as APP, presenilin 1 or 2. Targeted exome sequencing of 249 genes that were previously reported to be associated with AD revealed a rare mutation in hemochromatosis (HFE) gene known to be associated with hemochromotosis. Compared to previous studies, we show that HFE mutation can possess the risk of AD in transferrin-, APOE- and APP-normal patients. Hereditary Hemochromatosis (HH) is a genetically heterogeneous disorder caused by mutations in at least five different genes (HFE, HJV, TFR2, SLC40A1, HAMP) involved in the production or activity of the liver hormone hepcidin, a key regulator of systemic iron homeostasis. Nevertheless, patients with an HH-like phenotype that remains completely/partially unexplained despite extensive sequencing of known genes are not infrequently seen at referral centers, suggesting a role of still unknown genetic factors. A compelling candidate is Bone Morphogenetic Protein 6 (BMP6), which acts as a major activator of the BMP-SMAD signaling pathway, ultimately leading to the upregulation of hepcidin gene transcription. A recent seminal study by French authors has described three heterozygous missense mutations in BMP6 associated with mild to moderate late-onset iron overload (IO). Using an updated next-generation sequencing (NGS)-based genetic test in IO patients negative for the classical HFE p.Cys282Tyr mutation, we found three BMP6 heterozygous missense mutations in four patients from three different families. One mutation (p.Leu96Pro) has already been described and proven to be functional. The other two (p.Glu112Gln, p.Arg257His) were novel, and both were located in the pro-peptide domain known to be crucial for appropriate BMP6 processing and secretion. In silico modeling also showed results consistent with their pathogenetic role. The patients' clinical phenotypes were similar to that of other patients with BMP6-related IO recently described. Our results independently add further evidence to the role of BMP6 mutations as likely contributing factors to late-onset moderate IO unrelated to mutations in the established five HH genes. The three main mutations in gene HFE (C282Y, H63D, S65C) are the cause of development of 97% of cases of inherent hemochromatosis. It is known that about 85% of patients with inherent hemochromatosis are either homo-zygotic agents of mutation C282Y or carry compound-heterozygote C282Y/H63D. Therefore, the molecular genetic study intended for detection of these three mutations in gene HFE takes important place in diagnostic of inherent hemochromatosis. The study was organized to develop methods for detection of mutations C282Y, H63D, S65C on the basis of two molecular genetic methods - polymerase chain reaction in real-time and pyrosequenation. As reference method was used published method by Moyses C.B. et al. (2008). These methods were applied to analyzing 129 DNA samples. There were no discordant results. Among analyzed clinical DNA samples, mutant alleles of gene HFE were detected in 42 samples (32.5%)ю The mutation C282Y is detected in heterozygotic condition in 4 samples (3.1%); mutation H63D was detected in heterozygotic condition in 31 samples (24%) and in homo-zygotic condition in 4 samples (4%). The mutation S65C encountered in heterozygotic condition in one sample (0.8%) and in one sample compound-heterozygote H63D/S65C was detected (0.8%). The comparative characteristic of these three methods was made according the following parameters: time, number of analysis stages and convenience of interpretation of results. The main merit of method based on polymerase chain reaction in real-time is time of analysis implementation. The main merit of method based on pyrosequenation is automatic identification of genotype.

What is the reason for the abundance of operons in the genome of C. elegans?

Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Recent hypothesis proposes that operons provide an evolutionary advantage via the conservation of machinery during recovery from growth arrested states.

A new measure for assessing codon bias of one group of genes with respect to a second group of genes is introduced. In this formulation, codon bias correlations for Escherichia coli genes are evaluated for level of expression, for contrasts along genes, for genes in different 200 kb (or longer) contigs around the genome, for effects of gene size, for variation over different function classes, for codon bias in relation to possible lateral transfer and for dicodon bias for some gene classes. Among the function classes, codon biases of ribosomal proteins are the most deviant from the codon frequencies of the average E. coli gene. Other classes of 'highly expressed genes' (e.g. amino acyl tRNA synthetases, chaperonins, modification genes essential to translation activities) show less extreme codon biases. Consistently for genes with experimentally determined expression rates in the exponential growth phase, those of highest molar abundances are more deviant from the average gene codon frequencies and are more similar in codon frequencies to the average ribosomal protein gene. Independent of gene size, the codon biases in the 5' third of genes deviate by more than a factor of two from those in the middle and 3' thirds. In this context, there appear to be conflicting selection pressures imposed by the constraints of ribosomal binding, or more generally the early phase of protein synthesis (about the first 50 codons) may be more biased than the complete nascent polypeptide. In partitioning the E. coli genome into 10 equal lengths, pronounced differences in codon site 3 G+C frequencies accumulate. Genes near to oriC have 5% greater codon site 3 G+C frequencies than do genes from the ter region. This difference also is observed between small (100-300 codons) and large (>800 codons) genes. This result contrasts with that for eukaryotic genomes (including human, Caenorhabditis elegans and yeast) where long genes tend to have site 3 more AT rich than short genes. Many of the above results are special for E. coli genes and do not apply to genes of most bacterial genomes. A gene is defined as alien (possibly horizontally transferred) if its codon bias relative to the average gene exceeds a high threshold and the codon bias relative to ribosomal proteins is also appropriately high. These are identified, including four clusters (operons). The bulk of these genes have no known function. The current Caenorhabditis elegans genomic annotation has many genes organized in operons. Using directionally stitched promoterGFP methodology, we have conducted the largest survey to date on the regulatory regions of annotated C. elegans operons and identified 65, over 25% of those studied, with internal promoters. We have termed these operons "hybrid operons." GFP expression patterns driven from internal promoters differ in tissue specificity from expression of operon promoters, and serial analysis of gene expression data reveals that there is a lack of expression correlation between genes in many hybrid operons. The average length of intergenic regions with putative promoter activity in hybrid operons is larger than previous estimates for operons as a whole. Genes with internal promoters are more commonly involved in gene duplications and have a significantly lower incidence of alternative splicing than genes without internal promoters, although we have observed almost all trans-splicing patterns in these two distinct groups. Finally, internal promoter constructs are able to rescue lethal knockout phenotypes, demonstrating their necessity in gene regulation and survival. Our work suggests that hybrid operons are common in the C. elegans genome and that internal promoters influence not only gene organization and expression but also operon evolution. The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and "spliced leader" (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

Which chromosome contains the TLR7 locus in the human genome?

The TLR7 locus acts in vivo as a tumor suppressor gene and is located on chromosome X (X chromosome).

Antibodies against nuclear self-antigens are characteristic of systemic autoimmunity, although mechanisms promoting their generation and selection are unclear. Here, we report that B cells containing the Y-linked autoimmune accelerator (Yaa) locus are intrinsically biased toward nucleolar antigens because of increased expression of TLR7, a single-stranded RNA-binding innate immune receptor. The TLR7 gene is duplicated in Yaa mice because of a 4-Megabase expansion of the pseudoautosomal region. These results reveal high divergence in mouse Y chromosomes and represent a good example of gene copy number qualitatively altering a polygenic disease manifestation. The y-linked autoimmune accelerating (yaa) locus is a potent autoimmune disease allele. Transcription profiling of yaa-bearing B cells revealed the overexpression of a cluster of X-linked genes that included Tlr7. FISH analysis demonstrated the translocation of this segment onto the yaa chromosome. The resulting overexpression of Tlr7 increased in vitro responses to Toll-like receptor (TLR) 7 signaling in all yaa-bearing males. B6.yaa mice are not overtly autoimmune, but the addition of Sle1, which contains the autoimmune-predisposing Slam/Cd2 haplotype, causes the development of fatal lupus with numerous immunological aberrations. B6.Sle1yaa CD4 T cells develop the molecular signature for T(FH) cells and also show expression changes in numerous cytokines and chemokines. Disease development and all component autoimmune phenotypes were inhibited by Sles1, a potent suppressor locus. Sles1 had no effect on yaa-enhanced TLR7 signaling in vitro, and these data place Sles1 downstream from the lesion in innate immune responses mediated by TLR7, suggesting that Sles1 modulates the activation of adaptive immunity in response to innate immune signaling. The Y-linked autoimmune accelerating (Yaa) locus drives the transition to fatal lupus nephritis when combined with B6.Sle1 in our C57BL/6J (B6)-congenic model of systemic autoimmunity. We and others recently demonstrated that the translocation of a cluster of X-linked genes onto the Y chromosome is the genetic lesion underlying Yaa (Subramanian, S. et al., Proc. Natl. Acad. Sci. USA 2006. 103: 9970-9975; Pisitkun, P. et al., Science 2006. 312: 1669-1672). In male mice carrying Yaa, the transcription of several genes within the translocated segment is increased roughly twofold. Although the translocated X chromosome segment in Yaa may contain as many as 16 genes, the major candidate gene for causation of the Yaa-associated autoimmune phenotypes has been TLR7. To confirm the role of TLR7 in Yaa-mediated autoimmune phenotypes, we introgressed a targeted disruption of TLR7 (TLR7(-)) onto B6.Sle1Yaa to produce B6.Sle1YaaTLR7(-) and examined evidence of disease at 6 and 9 months of age. Our results demonstrate that the up-regulation of TLR7 in the B6.Sle1Yaa strain is responsible for splenomegaly, glomerular nephritis and the majority of the cellular abnormalities of B, T and myeloid cells. The up-regulation of TLR7 was also responsible for driving the infiltration and activation of leukocytes in the kidney, in which activated T cells were a primary component. However, the resolution of TLR7 up-regulation did not eliminate the enhanced humoral autoimmunity observed in B6.SleYaa, suggesting that additional elements in the translocation may contribute to the disease phenotype. BACKGROUND: Toll-like receptors (TLRs) are structurally and functionally related and play important roles in the innate and adaptive immune system. By genome scanning, evidence of linkage between chromosome Xp22 and asthma and related atopic disorders has previously been obtained. Xp22 harbours the TLR7 and TLR8 genes. METHODS: The involvement of TLR7 and TLR8 in the aetiology of asthma and related disorders was investigated by a family based association analysis of two independently ascertained family samples comprising 540 and 424 individuals from 135 and 100 families, respectively. Ten affected individuals from families showing evidence of linkage to Xp22 were screened for sequence variations in TLR7 and 8, and nine single nucleotide polymorphisms (SNPs) identified were tested for association. RESULTS: In both samples, significant associations were observed for single SNPs and haplotypes of both TLR7 and 8 in all four phenotypes investigated: asthma, rhinitis, atopic dermatitis and increased specific IgE. The most significant association was seen for rs2407992 (TLR8) in asthma (p = 0.00023, sample A and B combined, recessive model). In TLR7, rs179008 showed the strongest association. Both rs179008 and rs2407992 are of putative functional significance, potentially affecting TLR7 processing and TLR8 splicing, respectively. Haplotypes comprising the major alleles of these two SNPs were overtransmitted to the affected offspring (eg, p = 0.00012 in asthma, combined sample, additive model). CONCLUSION: The results provide strong evidence that TLR7 and 8 may confer susceptibility to asthma and related atopic disorders and highlight these receptors as interesting targets for individualised, causally directed treatment. INTRODUCTION: The Toll-like receptor 7 (TLR7) gene, encoded on human chromosome Xp22.3, is crucial for type I interferon production. A recent multicenter study in East Asian populations, comprising Chinese, Korean and Japanese participants, identified an association of a TLR7 single-nucleotide polymorphism (SNP) located in the 3' untranslated region (3' UTR), rs3853839, with systemic lupus erythematosus (SLE), especially in males, although some difference was observed among the tested populations. To test whether additional polymorphisms contribute to SLE in Japanese, we systematically analyzed the association of TLR7 with SLE in a Japanese female population. METHODS: A case-control association study was conducted on eight tag SNPs in the TLR7 region, including rs3853839, in 344 Japanese females with SLE and 274 healthy female controls. RESULTS: In addition to rs3853839, two SNPs in intron 2, rs179019 and rs179010, which were in moderate linkage disequilibrium with each other (r2 = 0.53), showed an association with SLE (rs179019: P = 0.016, odds ratio (OR) 2.02, 95% confidence interval (95% CI) 1.15 to 3.54; rs179010: P = 0.018, OR 1.75, 95% CI 1.10 to 2.80 (both under the recessive model)). Conditional logistic regression analysis revealed that the association of the intronic SNPs and the 3' UTR SNP remained significant after we adjusted them for each other. When only the patients and controls carrying the risk genotypes at the 3' UTR SNP position were analyzed, the risk of SLE was significantly increased when the individuals also carried the risk genotypes at both of the intronic SNPs (P = 0.0043, OR 2.45, 95% CI 1.31 to 4.60). Furthermore, the haplotype containing the intronic risk alleles in addition to the 3' UTR risk allele was associated with SLE under the recessive model (P = 0.016, OR 2.37, 95% CI 1.17 to 4.80), but other haplotypes were not associated with SLE. CONCLUSIONS: The TLR7 intronic SNPs rs179019 and rs179010 are associated with SLE independently of the 3' UTR SNP rs3853839 in Japanese women. Our findings support a role of TLR7 in predisposition for SLE in Asian populations. Human plasmacytoid dendritic cells (pDCs) play a major role in innate immunity through the production of type I IFNs after TLR engagement by pathogens. Sex-based differences in the innate function of human pDCs have been established, with pDCs from women exhibiting enhanced TLR7-mediated IFN-α production as compared with pDCs from males. In mice, we recently provided evidence for a role of estrogens as a positive regulator of pDC innate functions through cell-intrinsic estrogen receptor α signaling, but did not exclude a role for other X-linked factors, particularly in human pDCs. In this study, we investigated the respective contribution of X chromosome dosage and sex hormones using a humanized mouse model in which male or female NOD-SCID-β2m(-/-) were transplanted with human progenitor cells purified from either male or female cord blood cells. We showed that, in response to TLR7 ligands, the frequency of IFN-α- and TNF-α-producing pDCs from either sex was greater in female than in male host mice, suggesting a positive role for estrogens. Indeed, blockade of estrogen receptor signaling during pDC development in vitro inhibited TLR7-mediated IFN-α production by human pDCs, which expressed both ESR1 and ESR2 genes. Interestingly, we also found that X chromosome dosage contributed to this sex bias as female pDCs have an enhanced TLR7-mediated IFN-α response as compared with male ones, irrespective of the sex of the recipient mice. Together, these results indicate that female sex hormones, estrogens, and X chromosome complement independently contribute to the enhanced TLR7-mediated IFN-α response of pDCs in women. PURPOSE: The purpose of this study was to test whether gene copy number variations (CNVs) of Toll-like receptors (TLRs) are associated with uveitis. METHODS: Copy number variations of TLRs were detected by real-time PCR. The first stage of the study consisted of enrolling 400 Behçet's disease (BD) patients, 400 Vogt-Koyanagi-Harada syndrome patients, 400 patients with acute anterior uveitis associated with or without ankylosing spondylitis, and 600 healthy subjects. The second stage included another set of 578 BD patients and 1000 healthy controls. The frequencies of TLR gene copy number types (TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR9, TLR10) were compared among patients and controls by using the χ(2) test. Real-time PCR was used to detect mRNA expression from peripheral blood mononuclear cells (PBMCs) obtained from healthy controls following stimulation with the TLR7 agonist R848. Levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured by ELISA. RESULTS: All TLRs tested, except for TLR7, had a gene copy number of two in more than 98% of individuals tested. In the first stage, we found a significantly increased frequency of more than one copy of TLR7 (located on the X chromosome) in male BD patients and more than two copies in female patients (correction of P value [PC] = 0.021; PC = 0.048, respectively). A second stage and combined study confirmed the association (PC = 1.14 × 10(-6); PC = 9.12 × 10(-5), respectively). TLR7 mRNA expression in PBMCs was increased in healthy male carriers having more than one copy of TLR7 or females having more than two copies following stimulation with R848 (P = 0.021, P = 0.006, respectively). No effect of the various TLR7 copies on the release of TNF-α, IL-6, IL-1β, and IFN-β could be detected. CONCLUSIONS: This study provides evidence that a high copy number of TLR7 confers risk for BD in a Chinese Han population. (http://www.chictr.org number, ChiCTR-CCC-12002184.). Cutaneous melanoma is a life-threatening skin cancer. Its incidence is rapidly increasing, and early diagnosis is the main factor able to improve its poor prognosis. Toll-like receptors (TLRs) are transmembrane glycoproteins that recognize pathogen- and damage-associated molecular patterns, against which TLRs activate the innate immune response and initiate the adaptive immune response. Genetic variations of these receptors may alter the immune system, and are involved in evolution and susceptibility to various diseases, including cancer. The aim of the present study was to evaluate whether the presence of TLR7 glutamine (Gln) 11 leucine (Leu) polymorphism confers an increased susceptibility to cutaneous melanoma. For that purpose, a case-control study was performed with 182 melanoma cases and 89 controls. To highlight the possible association between the aforementioned polymorphism and the susceptibility to melanoma, 93 cases of single melanoma and 89 cases of multiple primary melanoma (MPM) were compared in the present study. Since the TLR7 gene is localized on the chromosome X, the allelic frequency of the Gln11Leu polymorphism was analyzed separately in males and females. The distribution of allele frequencies between melanoma cases and controls (P=0.245) and between single melanoma and MPM cases (P=0.482) was not significant. Therefore, the present results do not suggest an association between TLR7 Gln11Leu polymorphism and susceptibility to cutaneous melanoma. Further studies are required to analyze the influence of other TLR polymorphisms on the susceptibility to maligt melanoma and the involvement of innate immunity in this maligcy. Toll-like receptor 7 (TLR7) is critical to the induction of antiviral immunity, but TLR7 dosage is also a key pathogenic factor in systemic lupus erythematosus (SLE), an autoimmune disease with strong female bias. SLE prevalence is also elevated in individuals with Klinefelter syndrome, who carry one or more supernumerary X chromosomes, suggesting that the X chromosome complement contributes to SLE susceptibility. TLR7 is encoded by an X chromosome locus, and we examined here whether the TLR7 gene evades silencing by X chromosome inactivation in immune cells from women and Klinefelter syndrome males. Single-cell analyses of TLR7 allelic expression demonstrated that substantial fractions of primary B lymphocytes, monocytes, and plasmacytoid dendritic cells not only in women but also in Klinefelter syndrome males express TLR7 on both X chromosomes. Biallelic B lymphocytes from women displayed greater TLR7 transcriptional expression than the monoallelic cells, correlated with higher TLR7 protein expression in female than in male leukocyte populations. Biallelic B cells were preferentially enriched during the TLR7-driven proliferation of CD27+ plasma cells. In addition, biallelic cells showed a greater than twofold increase over monoallelic cells in the propensity to immunoglobulin G class switch during the TLR7-driven, T cell-dependent differentiation of naive B lymphocytes into immunoglobulin-secreting cells. TLR7 escape from X inactivation endows the B cell compartment with added responsiveness to TLR7 ligands. This finding supports the hypothesis that enhanced TLR7 expression owing to biallelism contributes to the higher risk of developing SLE and other autoimmune disorders in women and in men with Klinefelter syndrome.

What is marked by DNaseI hypersensitive sites?

Hypersensitive sites are chromosomal regions up to 2kb distant to known genomic regulatory regions and 5 kb from known regulatory regions.

We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function. Mammalian beta-globin loci are composed of multiple orthologous genes whose expression is erythroid specific and developmentally regulated. The expression of these genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR). The LCR encompasses 5 major HSs, each of which is highly homologous among humans, mice, and other mammals. To analyze the function of individual HSs in the endogenous murine beta-globin LCR, we have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In this report, we demonstrate that deletion of the conserved region of 5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion site but has no influence on the formation of the remaining HSs in the LCR. Therefore, in the endogenous murine locus, there is no domit or initiating site whose formation must precede the formation of the other HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of beta-globin regulation. Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue-specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue-specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta-specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis-regulatory elements, an E-box in HS1 and a trophoblast-specific element-like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta-derived nuclear factors. In conclusion, observed tissue-specific differences of the chromatin structure which correlate with tissue-specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle. A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (-5262 to -899) that is required for high level expression of human apoB transgenes in the livers of mice. These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei. Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators. The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database. Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints. The mammalian beta-globin locus is a multigenic, developmentally regulated, tissue-specific locus from which gene expression is regulated by a distal regulatory region, the locus control region (LCR). The functional mechanism by which the beta-globin LCR stimulates transcription of the linked beta-like globin genes remains unknown. The LCR is composed of a series of 5 DNaseI hypersensitive sites (5'HSs) that form in the nucleus of erythroid precursors. These HSs are conserved among mammals, bind transcription factors that also bind to other parts of the locus, and compose the functional components of the LCR. To test the hypothesis that individual HSs have unique properties, homologous recombination was used to construct 5 lines of mice with individual deletions of each of the 5'HSs of the endogenous murine beta-globin LCR. Here it is reported that deletion of 5'HS1 reduces expression of the linked genes by up to 24%, while deletion of 5'HS4 leads to reductions of up to 27%. These deletions do not perturb the normal stage-specific expression of genes from this multigenic locus. In conjunction with previous studies of deletions of the other HSs and studies of deletion of the entire LCR, it is concluded that (1) none of the 5'HSs is essential for nearly normal expression; (2) none of the HSs is required for proper developmental expression; and (3) the HSs do not appear to synergize either structurally or functionally, but rather form independently and appear to contribute additively to the overall expression from the locus. BACKGROUND: Transposable elements (TEs) are abundant genomic sequences that have been found to contribute to genome evolution in unexpected ways. Here, we characterize the evolutionary and functional characteristics of TE-derived human genome regulatory sequences uncovered by the high throughput mapping of DNaseI-hypersensitive (HS) sites. RESULTS: Human genome TEs were found to contribute substantially to HS regulatory sequences characterized in CD4+ T cells: 23% of HS sites contain TE-derived sequences. While HS sites are far more evolutionarily conserved than non HS sites in the human genome, consistent with their functional importance, TE-derived HS sites are highly divergent. Nevertheless, TE-derived HS sites were shown to be functionally relevant in terms of driving gene expression in CD4+ T cells. Genes involved in immune response are statistically over-represented among genes with TE-derived HS sites. A number of genes with both TE-derived HS sites and immune tissue related expression patterns were found to encode proteins involved in immune response such as T cell specific receptor antigens and secreted cytokines as well as proteins with clinical relevance to HIV and cancer. Genes with TE-derived HS sites have higher average levels of sequence and expression divergence between human and mouse orthologs compared to genes with non TE-derived HS sites. CONCLUSION: The results reported here support the notion that TEs provide a specific genome-wide mechanism for generating functionally relevant gene regulatory divergence between evolutionary lineages. REVIEWERS: This article was reviewed by Wolfgang J. Miller (nominated by Jerzy Jurka), Itai Yanai and Mikhail S.Gelfand. The identification of cis-regulatory elements is central to understanding gene transcription. Hypersensitivity of cis-regulatory elements to digestion with DNaseI remains the gold-standard approach to locating such elements. Traditional methods used to identify DNaseI hypersensitive sites are cumbersome and can only be applied to short stretches of DNA at defined locations. Here we report the development of a novel genomic array-based approach to DNaseI hypersensitive site mapping (ADHM) that permits precise, large-scale identification of such sites from as few as 5 million cells. Using ADHM we identified all previously recognized hematopoietic regulatory elements across 200 kb of the mouse T-cell acute lymphocytic leukemia-1 (Tal1) locus, and, in addition, identified two novel elements within the locus, which show transcriptional regulatory activity. We further validated the ADHM protocol by mapping the DNaseI hypersensitive sites across 250 kb of the human TAL1 locus in CD34+ primary stem/progenitor cells and K562 cells and by mapping the previously known DNaseI hypersensitive sites across 240 kb of the human alpha-globin locus in K562 cells. ADHM provides a powerful approach to identifying DNaseI hypersensitive sites across large genomic regions. Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high-throughput sequencing and genomic array tiling paths. In this article, we describe a novel protocol to use real-time PCR to map DHS. Advantages of the technique reported here include the small cell numbers required for each analysis, rapid, relatively low-cost experiments with minimal need for specialist equipment. Presented examples include comparative DHS mapping of known TAL1/SCL regulatory elements between human embryonic stem cells and K562 cells. Embryonic stem (ES) cells offer insight into early developmental fate decisions, and their controlled differentiation may yield vast regenerative potential. The molecular determits supporting ES cell self-renewal are incompletely understood. The homeodomain proteins Nanog and Oct4 are essential for mouse ES cell self-renewal. Using a high-throughput approach, we discovered DNaseI hypersensitive sites and potential regulatory elements along a 160-kb region of the genome that includes GDF3, Dppa3, and Nanog. We analyzed gene expression, chromatin occupancy, and higher-order chromatin structure throughout this gene locus and found that expression of the reprogramming factor Oct4 is required to maintain its integrity. Historically, the simplest method to robustly identify active gene regulatory elements has been enzymatic digestion of nuclear DNA by nucleases such as DNaseI. Regions of extreme chromatin accessibility to DNaseI, commonly known as DNaseI hypersensitive sites, have been repeatedly shown to be markers for all types of active cis-acting regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. However, the original classical method, which for over 25 years relied on Southern blot, was limited to studying only small regions of the genome. Here we describe the detailed protocol for DNase-chip, a high-throughput method that allows for a targeted or genome-wide identification of cis-acting gene regulatory elements. BACKGROUND: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems. RESULTS: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method. CONCLUSION: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions. To understand the molecular mechanisms that underlie global transcriptional regulation, it is essential to first identify all the transcriptional regulatory elements in the human genome. The advent of next-generation sequencing has provided a powerful platform for genome-wide analysis of different species and specific cell types; when combined with traditional techniques to identify regions of open chromatin [DNaseI hypersensitivity (DHS)] or specific binding locations of transcription factors [chromatin immunoprecipitation (ChIP)], and expression data from microarrays, we become uniquely poised to uncover the mysteries of the genome and its regulation. To this end, we have performed global meta-analysis of the relationship among data from DNaseI-seq, ChIP-seq and expression arrays, and found that specific correlations exist among regulatory elements and gene expression across different cell types. These correlations revealed four distinct modes of chromatin domain structure reflecting different functions: repressive, active, primed and bivalent. Furthermore, CCCTC-binding factor (CTCF) binding sites were identified based on these integrative data. Our findings uncovered a complex regulatory process involving by DNaseI HS sites and histone modifications, and suggest that these dynamic elements may be responsible for maintaining chromatin structure and integrity of the human genome. Our integrative approach provides an example by which data from diverse technology platforms may be integrated to provide more meaningful insights into global transcriptional regulation. Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.

Do polycomb group proteins (PcG) mediate the formation of chromatin loops?

Yes. The polycomb group proteins (PcG) mediate the formation of chromatin loops by facilitating co-localization of heterochromatin loops.

CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes. Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream ( approximately 30 kb) of and extending approximately 60 kb around one such gene, GATA-4, is organized-in Tera-2 undifferentiated embryonic carcinoma (EC) cells-in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)-mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a approximately 60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells. BACKGROUND: The INK4b-ARF-INK4a tumour suppressor locus controls the balance between progenitor cell renewal and cancer. In this study, we investigated how higher-order chromatin structure modulates differential expression of the human INK4b-ARF-INK4a locus during progenitor cell differentiation, cellular ageing and senescence of cancer cells. RESULTS: We found that INK4b and INK4a, but not ARF, are upregulated following the differentiation of haematopoietic progenitor cells, in ageing fibroblasts and in senescing maligt rhabdoid tumour cells. To investigate the underlying molecular mechanism we analysed binding of polycomb group (PcG) repressive complexes (PRCs) and the spatial organization of the INK4b-ARF-INK4a locus. In agreement with differential derepression, PcG protein binding across the locus is discontinuous. As we described earlier, PcG repressors bind the INK4a promoter, but not ARF. Here, we identified a second peak of PcG binding that is located approximately 3 kb upstream of the INK4b promoter. During progenitor cell differentiation and ageing, PcG silencer EZH2 attenuates, causing loss of PRC binding and transcriptional activation of INK4b and INK4a. The expression pattern of the locus is reflected by its organization in space. In the repressed state, the PRC-binding regions are in close proximity, while the intervening chromatin harbouring ARF loops out. Down regulation of EZH2 causes release of the approximately 35 kb repressive chromatin loop and induction of both INK4a and INK4b, whereas ARF expression remains unaltered. CONCLUSION: PcG silencers bind and coordinately regulate INK4b and INK4a, but not ARF, during a variety of physiological processes. Developmentally regulated EZH2 levels are one of the factors that can determine the higher order chromatin structure and expression pattern of the INK4b-ARF-INK4a locus, coupling human progenitor cell differentiation to proliferation control. Our results revealed a chromatin looping mechanism of long-range control and argue against models involving homogeneous spreading of PcG silencers across the INK4b-ARF-INK4a locus. Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology. The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or "anchors" are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of polycomb repressive complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, our results indicate that loops are also associated with gene repression.

Which type of analysis does DeSeq2 perform?

DeSeq2 is a software for differential gene expression analysis of RNA sequencing data.

In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html webcite. Background. A common research goal in transcriptome projects is to find genes that are differentially expressed in different phenotype classes. Biologists might wish to validate such gene candidates experimentally, or use them for downstream systems biology analysis. Producing a coherent differential gene expression analysis from RNA-seq count data requires an understanding of how numerous sources of variation such as the replicate size, the hypothesized biological effect size, and the specific method for making differential expression calls interact. We believe an explicit demonstration of such interactions in real RNA-seq data sets is of practical interest to biologists. Results. Using two large public RNA-seq data sets-one representing strong, and another mild, biological effect size-we simulated different replicate size scenarios, and tested the performance of several commonly-used methods for calling differentially expressed genes in each of them. We found that, when biological effect size was mild, RNA-seq experiments should focus on experimental validation of differentially expressed gene candidates. Importantly, at least triplicates must be used, and the differentially expressed genes should be called using methods with high positive predictive value (PPV), such as NOISeq or GFOLD. In contrast, when biological effect size was strong, differentially expressed genes mined from unreplicated experiments using NOISeq, ASC and GFOLD had between 30 to 50% mean PPV, an increase of more than 30-fold compared to the cases of mild biological effect size. Among methods with good PPV performance, having triplicates or more substantially improved mean PPV to over 90% for GFOLD, 60% for DESeq2, 50% for NOISeq, and 30% for edgeR. At a replicate size of six, we found DESeq2 and edgeR to be reasonable methods for calling differentially expressed genes at systems level analysis, as their PPV and sensitivity trade-off were superior to the other methods'. Conclusion. When biological effect size is weak, systems level investigation is not possible using RNAseq data, and no meaningful result can be obtained in unreplicated experiments. Nonetheless, NOISeq or GFOLD may yield limited numbers of gene candidates with good validation potential, when triplicates or more are available. When biological effect size is strong, NOISeq and GFOLD are effective tools for detecting differentially expressed genes in unreplicated RNA-seq experiments for qPCR validation. When triplicates or more are available, GFOLD is a sharp tool for identifying high confidence differentially expressed genes for targeted qPCR validation; for downstream systems level analysis, combined results from DESeq2 and edgeR are useful. BACKGROUND: Differential expression (DE) analysis of RNA-seq data still poses inferential challenges, such as handling of transcripts characterized by low expression levels. In this study, we use a plasmode-based approach to assess the relative performance of alternative inferential strategies on RNA-seq transcripts, with special emphasis on transcripts characterized by a small number of read counts, so-called low-count transcripts, as motivated by an ecological application in prairie grasses. Big bluestem (Andropogon gerardii) is a wide-ranging domit prairie grass of ecological and agricultural importance to the US Midwest while edaphic subspecies sand bluestem (A. gerardii ssp. Hallii) grows exclusively on sand dunes. Relative to big bluestem, sand bluestem exhibits qualitative phenotypic divergence consistent with enhanced drought tolerance, plausibly associated with transcripts of low expression levels. Our dataset consists of RNA-seq read counts for 25,582 transcripts (60% of which are classified as low-count) collected from leaf tissue of individual plants of big bluestem (n = 4) and sand bluestem (n = 4). Focused on low-count transcripts, we compare alternative ad-hoc data filtering techniques commonly used in RNA-seq pipelines and assess the inferential performance of recently developed statistical methods for DE analysis, namely DESeq2 and edgeR robust. These methods attempt to overcome the inherently noisy behavior of low-count transcripts by either shrinkage or differential weighting of observations, respectively. RESULTS: Both DE methods seemed to properly control family-wise type 1 error on low-count transcripts, whereas edgeR robust showed greater power and DESeq2 showed greater precision and accuracy. However, specification of the degree of freedom parameter under edgeR robust had a non-trivial impact on inference and should be handled carefully. When properly specified, both DE methods showed overall promising inferential performance on low-count transcripts, suggesting that ad-hoc data filtering steps at arbitrary expression thresholds may be unnecessary. A note of caution is in order regarding the approximate nature of DE tests under both methods. CONCLUSIONS: Practical recommendations for DE inference are provided when low-count RNA-seq transcripts are of interest, as is the case in the comparison of subspecies of bluestem grasses. Insights from this study may also be relevant to other applications focused on transcripts of low expression levels. myPhyloDB v.1.1.2 is a user-friendly personal database with a browser-interface designed to facilitate the storage, processing, analysis, and distribution of microbial community populations (e.g. 16S metagenomics data). MyPhyloDB archives raw sequencing files, and allows for easy selection of project(s)/sample(s) of any combination from all available data in the database. The data processing capabilities of myPhyloDB are also flexible enough to allow the upload and storage of pre-processed data, or use the built-in Mothur pipeline to automate the processing of raw sequencing data. myPhyloDB provides several analytical (e.g. analysis of covariance,t-tests, linear regression, differential abundance (DESeq2), and principal coordinates analysis (PCoA)) and normalization (rarefaction, DESeq2, and proportion) tools for the comparative analysis of taxonomic abundance, species richness and species diversity for projects of various types (e.g. human-associated, human gut microbiome, air, soil, and water) for any taxonomic level(s) desired. Finally, since myPhyloDB is a local web-server, users can quickly distribute data between colleagues and end-users by simply granting others access to their personal myPhyloDB database. myPhyloDB is available athttp://www.ars.usda.gov/services/software/download.htm?softwareid=472 and more information along with tutorials can be found on our websitehttp://www.myphylodb.org. Database URL:http://www.myphylodb.org. BACKGROUND: Several R packages exist for the detection of differentially expressed genes from RNA-Seq data. The analysis process includes three main steps, namely normalization, dispersion estimation and test for differential expression. Quality control steps along this process are recommended but not mandatory, and failing to check the characteristics of the dataset may lead to spurious results. In addition, normalization methods and statistical models are not exchangeable across the packages without adequate transformations the users are often not aware of. Thus, dedicated analysis pipelines are needed to include systematic quality control steps and prevent errors from misusing the proposed methods. RESULTS: SARTools is an R pipeline for differential analysis of RNA-Seq count data. It can handle designs involving two or more conditions of a single biological factor with or without a blocking factor (such as a batch effect or a sample pairing). It is based on DESeq2 and edgeR and is composed of an R package and two R script templates (for DESeq2 and edgeR respectively). Tuning a small number of parameters and executing one of the R scripts, users have access to the full results of the analysis, including lists of differentially expressed genes and a HTML report that (i) displays diagnostic plots for quality control and model hypotheses checking and (ii) keeps track of the whole analysis process, parameter values and versions of the R packages used. CONCLUSIONS: SARTools provides systematic quality controls of the dataset as well as diagnostic plots that help to tune the model parameters. It gives access to the main parameters of DESeq2 and edgeR and prevents untrained users from misusing some functionalities of both packages. By keeping track of all the parameters of the analysis process it fits the requirements of reproducible research. Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted P<0.1). In depression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted P<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted P<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders. In the past 5 years, RNA-Seq has become a powerful tool in transcriptome analysis even though computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. It is, however, now commonly accepted that the choice of a normalization procedure is an important step in such a process, for example in differential gene expression analysis. The present article highlights the similarities between three normalization methods: TMM from edgeR R package, RLE from DESeq2 R package, and MRN. Both TMM and DESeq2 are widely used for differential gene expression analysis. This paper introduces properties that show when these three methods will give exactly the same results. These properties are proven mathematically and illustrated by performing in silico calculations on a given RNA-Seq data set. The current DNA sequencing technologies and their high-throughput yield, allowed the thrive of genomic and transcriptomic experiments but it also have generated big data problem. Due to this exponential growth of sequencing data, also the complexity of managing, processing and interpreting it in order to generate results, has raised. Therefore, the demand of easy-to-use friendly software and websites to run bioinformatic tools is imminent. In particular, RNA-Seq and differential expression analysis have become a popular and useful method to evaluate the genetic expression change in any organism. However, many scientists struggle with the data analysis since most of the available tools are implemented in a UNIX-based environment. Therefore, we have developed the web server IDEAMEX (Integrative Differential Expression Analysis for Multiple EXperiments). The IDEAMEX pipeline needs a raw count table for as many desired replicates and conditions, allowing the user to select which conditions will be compared, instead of doing all-vs.-all comparisons. The whole process consists of three main steps (1) Data Analysis: that allows a preliminary analysis for quality control based on the data distribution per sample, using different types of graphs; (2) Differential expression: performs the differential expression analysis with or without batch effect error awareness, using the bioconductor packages, NOISeq, limma-Voom, DESeq2 and edgeR, and generate reports for each method; (3) Result integration: the obtained results the integrated results are reported using different graphical outputs such as correlograms, heatmaps, Venn diagrams and text lists. Our server allows an easy and friendly visualization for results, providing an easy interaction during the analysis process, as well as error tracking and debugging by providing output log files. The server is currently available and can be accessed at http://www.uusmb.unam.mx/ideamex/ where the documentation and example input files are provided. We consider that this web server can help other researchers with no previous bioinformatic knowledge, to perform their analyses in a simple manner. The rapid expansion of transcriptomics and affordability of next-generation sequencing (NGS) technologies generate rocketing amounts of gene expression data across biology and medicine, including cancer research. Concomitantly, many bioinformatics tools were developed to streamline gene expression and quantification. We tested the concordance of NGS RNA sequencing (RNA-seq) analysis outcomes between two predomit programs for read alignment, HISAT2, and STAR, and two most popular programs for quantifying gene expression in NGS experiments, edgeR and DESeq2, using RNA-seq data from breast cancer progression series, which include histologically confirmed normal, early neoplasia, ductal carcinoma in situ and infiltrating ductal carcinoma samples microdissected from formalin fixed, paraffin embedded (FFPE) breast tissue blocks. We identified significant differences in aligners' performance: HISAT2 was prone to misalign reads to retrogene genomic loci, STAR generated more precise alignments, especially for early neoplasia samples. edgeR and DESeq2 produced similar lists of differentially expressed genes, with edgeR producing more conservative, though shorter, lists of genes. Gene Ontology (GO) enrichment analysis revealed no skewness in significant GO terms identified among differentially expressed genes by edgeR versus DESeq2. As transcriptomics of FFPE samples becomes a vanguard of precision medicine, choice of bioinformatics tools becomes critical for clinical research. Our results indicate that STAR and edgeR are well-suited tools for differential gene expression analysis from FFPE samples. RNA-Seq examines global gene expression to provide insights into cellular processes, and it can be particularly informative when comparing contrasting physiological states or strains. Although relatively routine in many laboratories, there are many steps involved in performing a transcriptomics experiment to ensure representative and high-quality results are generated for analysis. In this chapter, we present the application of widely used bioinformatic methodologies to assess, trim, and filter RNA-seq reads for quality using FastQC and Trim Galore, respectively. High-quality reads are mapped using Bowtie2 and differentially expressed genes across different groups were estimated using the DEseq2 R-Bioconductor package. In addition, we describe the various steps to perform the sample-wise data quality assessment by generating exploratory plots through the DESeq2 package. Simple steps to calculate the significant differentially expressed genes, up- and down-regulated genes, and exporting the data and images are also included. A Venn diagram is a useful method to compare the differentially expressed genes across various comparisons and steps to generate the Venn diagram from DESeq2 results are provided. Finally, the output from DESeq2 is compared to published results from EdgeR. The Clostridium autoethanogenum data are published and publicly available. Background: Melanoma is highly immunogenic and therefore suitable for immunotherapy, but the efficacy is limited by response rate. In several types of tumor, tumor mutation burden (TMB) and immune infiltration have been reported to predict the response to immunotherapy, although each has its limitations. In the current study, we aimed to explore the association of TMB with immune infiltration and prognosis in cutaneous melanoma. Methods: The data of cutaneous melanoma used for analyses was downloaded from The Cancer Genome Atlas (TCGA) database. The mutation data was sorted using "maftools" R package. TMB was estimated and then patients were divided into two groups based on TMB. The association of TMB with prognosis and clinical characteristics was explored. Differential analysis between two TMB groups was performed using "DESeq2" R package to identify differentially expressed genes (DEGs). The function enrichment analyses of DEGs were conducted to screen critical pathways. Besides, DEGs were further filtered to identify two hub genes, based on which a risk score model and nomogram for predicting prognosis were conducted, and the validation was performed using three datasets from Gene Expression Omnibus (GEO) database. Finally, CIBERSORT algorithm and TIMER database were used to assess the effect of TMB and hub genes on immune infiltration. Results: The most common mutation was C > T, and the top three frequently mutated genes were TTN, MUC16, and BRAF. Higher TMB indicated better survival outcomes and lower pathological stages. 735 DEGs were identified and mainly involved in immune-related and adhesion-related pathways. The risk score model and nomogram were validated using receiver operating characteristic (ROC) curves and calibration curves, and exhibited relatively high predictive capability. Decision curve analysis (DCA) was used to assess clinical benefit. As for immune infiltration, the proportion was higher for macrophages M1 and M2 in the high-TMB group, while lower for memory B cells and regulatory T cells. Conclusions: In cutaneous melanoma, TMB was positively correlated with prognosis. The risk score model and nomogram can be conveniently used to predict prognosis. The association of TMB with immune infiltration can help improve the predicting methods for the response to immunotherapy. Cardiovascular disease accounts for millions of deaths each year and is currently the leading cause of mortality worldwide. The aging process is clearly linked to cardiovascular disease, however, the exact relationship between aging and heart function is not fully understood. Furthermore, a holistic view of cardiac aging, linking features of early life development to changes observed in old age, has not been synthesized. Here, we re-purpose RNA-sequencing data previously-collected by our group, investigating gene expression differences between wild-type mice of different age groups that represent key developmental milestones in the murine lifespan. DESeq2's generalized linear model was applied with two hypothesis testing approaches to identify differentially-expressed (DE) genes, both between pairs of age groups and across mice of all ages. Pairwise comparisons identified genes associated with specific age transitions, while comparisons across all age groups identified a large set of genes associated with the aging process more broadly. An unsupervised machine learning approach was then applied to extract common expression patterns from this set of age-associated genes. Sets of genes with both linear and non-linear expression trajectories were identified, suggesting that aging not only involves the activation of gene expression programs unique to different age groups, but also the re-activation of gene expression programs from earlier ages. Overall, we present a comprehensive transcriptomic analysis of cardiac gene expression patterns across the entirety of the murine lifespan.

What is the function of a protein degron?

Protein degrons are part of the DNA damage response triggered by dysfunctional transcription factors. Proteins that are destined for proteasome-mediated degradation are usually tagged with a chain of ubiquitin linked via lysine residues that targets them to the proteolytic machinery. Disruption of one degron by a RNA-binding protein causes it to become ubiquitinated, dimerizes and translocates to the nucleus.

Protein degradation is employed in both regulation and quality control. Regulated degradation of specific proteins is often mediated by discrete regions of primary sequence known as degrons, whereas protein quality control involves recognition of structural features common to damaged or misfolded proteins, rather than specific features of an individual protein. The yeast HMG-CoA reductase isozyme Hmg2p undergoes stringently regulated degradation by machinery that is also required for ER quality control. The 523 residue N-terminal transmembrane domain of Hmg2p is necessary and sufficient for regulated degradation. To understand how Hmg2p undergoes regulated degradation by the ER quality control pathway, we analyzed over 300 mutants of Hmg2p. Regulated degradation of Hmg2p requires information distributed over the entire transmembrane domain. Accordingly, we refer to this determit as a 'distributed' degron, which has functional aspects consistent with both regulation and quality control. The Hmg2p degron functions in the specific, regulated degradation of Hmg2p and can impart regulated degradation to fusion proteins. However, its recognition is based on dispersed structural features rather than primary sequence motifs. This mode of targeting has important consequences both for the prediction of degradation substrates and as a potential therapeutic strategy for targeted protein degradation using endogenous degradation pathways. The N-end rule pathway is a ubiquitin-dependent proteolytic system, in which destabilizing N-terminal residues of short-lived proteins function as an essential determit of an N-terminal degradation signal (N-degron). An N-degron can be created from a pre-N-degron through specific N-terminal modifications, providing a means conditionally to destabilize otherwise stable polypeptides. The pathway has been found in all organisms examined, from prokaryotes to eukaryotes. Recent biochemical and proteomic studies identified many components of the mammalian N-end rule pathway, including a family of substrate recognition ubiquitin ligases and their substrates. The genetic dissection in animals and humans revealed its essential role in various vital physiological processes, ranging from cardiovascular development and meiosis to the pathogenesis of human genetic diseases. These discoveries have provided new insights into the components, functions and mechanics of this unique proteolytic system. Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology. Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring. Author information: (1)Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. (2)Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. (3)Renal Division, Brigham and Women's Hospital, Boston, Massachusetts, USA. (4)Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. (5)McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. (6)Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. (7)Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Methods to induce proteasomal degradation of unwanted proteins are valuable in biomedical studies and thus receive increasing attention. For efficient degradation, the proteasome requires both a ubiquitin tag, which delivers substrates to the proteasome, and an unstructured region, where the proteasome engages the substrate for unfolding and degradation. We fused two degron components into a single molecule to create a fusion protein comprising ubiquitin and Rpn4-derived unstructured region. We demonstrated that the fusion protein retained its function to polyubiquitinate target proteins, thereby inducing more efficient proteasomal target degradation than wild-type ubiquitin in vitro and in cells. These results provide novel strategies for robust degradation enhancement of polyubiquitinated proteins.

Which computational methods are used for the definition of synteny?

The computational methods which are used for the definition of synteny are:1) multisyn, 2) poff, 3) orthocluster, 4) phyldiag, 5) synblast, 6) cinteny, 7) domain team, 8)mcscanx and 9) run Orthoclusterdb and 10) view synteny.

BACKGROUND: Identifying syntenic regions, i.e., blocks of genes or other markers with evolutionary conserved order, and quantifying evolutionary relatedness between genomes in terms of chromosomal rearrangements is one of the central goals in comparative genomics. However, the analysis of synteny and the resulting assessment of genome rearrangements are sensitive to the choice of a number of arbitrary parameters that affect the detection of synteny blocks. In particular, the choice of a set of markers and the effect of different aggregation strategies, which enable coarse graining of synteny blocks and exclusion of micro-rearrangements, need to be assessed. Therefore, existing tools and resources that facilitate identification, visualization and analysis of synteny need to be further improved to provide a flexible platform for such analysis, especially in the context of multiple genomes. RESULTS: We present a new tool, Cinteny, for fast identification and analysis of synteny with different sets of markers and various levels of coarse graining of syntenic blocks. Using Hannenhalli-Pevzner approach and its extensions, Cinteny also enables interactive determination of evolutionary relationships between genomes in terms of the number of rearrangements (the reversal distance). In particular, Cinteny provides: i) integration of synteny browsing with assessment of evolutionary distances for multiple genomes; ii) flexibility to adjust the parameters and re-compute the results on-the-fly; iii) ability to work with user provided data, such as orthologous genes, sequence tags or other conserved markers. In addition, Cinteny provides many annotated mammalian, invertebrate and fungal genomes that are pre-loaded and available for analysis at http://cinteny.cchmc.org. CONCLUSION: Cinteny allows one to automatically compare multiple genomes and perform sensitivity analysis for synteny block detection and for the subsequent computation of reversal distances. Cinteny can also be used to interactively browse syntenic blocks conserved in multiple genomes, to facilitate genome annotation and validation of assemblies for newly sequenced genomes, and to construct and assess phylogenomic trees. We present here a method to identify microsyntenies across several genomes. This method adopts the innovative approach of deconstructing proteins into their domains. This allows the detection of strings of domains that are conserved in their content, but not necessarily in their order, that we refer to as domain teams or syntenies of domains. The prominent feature of the method is that it relaxes the rigidity of the orthology criterion and avoids many of the pitfalls of gene families identification methods, often hampered by multidomain proteins or low levels of sequence similarity. This approach, that allows both inter- and intrachromosomal comparisons, proves to be more sensitive than the classical methods based on pairwise sequence comparisons, particularly in the simultaneous treatment of many species. The automated and fast detection of domain teams is implemented in the DomainTeam software. In this chapter, we describe the procedure to run DomainTeam. After formatting the input and setting up the parameters, running the algorithm produces an output file comprising all the syntenies of domains shared by two or more (sometimes all) of the compared genomes. BACKGROUND: The recent availability of an expanding collection of genome sequences driven by technological advances has facilitated comparative genomics and in particular the identification of synteny among multiple genomes. However, the development of effective and easy-to-use methods for identifying such conserved gene clusters among multiple genomes-synteny blocks-as well as databases, which host synteny blocks from various groups of species (especially eukaryotes) and also allow users to run synteny-identification programs, lags behind. DESCRIPTIONS: OrthoClusterDB is a new online platform for the identification and visualization of synteny blocks. OrthoClusterDB consists of two key web pages: Run OrthoCluster and View Synteny. The Run OrthoCluster page serves as web front-end to OrthoCluster, a recently developed program for synteny block detection. Run OrthoCluster offers full control over the functionalities of OrthoCluster, such as specifying synteny block size, considering order and strandedness of genes within synteny blocks, including or excluding nested synteny blocks, handling one-to-many orthologous relationships, and comparing multiple genomes. In contrast, the View Synteny page gives access to perfect and imperfect synteny blocks precomputed for a large number of genomes, without the need for users to retrieve and format input data. Additionally, genes are cross-linked with public databases for effective browsing. For both Run OrthoCluster and View Synteny, identified synteny blocks can be browsed at the whole genome, chromosome, and individual gene level. OrthoClusterDB is freely accessible. CONCLUSION: We have developed an online system for the identification and visualization of synteny blocks among multiple genomes. The system is freely available at (http://genome.sfu.ca/orthoclusterdb/). Synteny blocks are composed of two or more orthologous genes conserved among species, resulting from speciation from their last common ancestor. OrthoCluster (Zeng et al., 2008) is a fast and easy-to-use program for the identification of synteny blocks among multiple genomes. It allows users to identify synteny blocks that contain different types of mismatches, and to decide whether they require conservation of gene orientation and conservation of gene order within the blocks. OrthoCluster can also be used to find duplicated blocks within genomes. Although genes and their correspondence are usually used as input for OrthoCluster, in fact, OrthoCluster can be applied using any type of markers as input as long as their relationships can be established. OrthoClusterDB provides a Web interface for running OrthoCluster with user-defined datasets and parameters, as well as for browsing and downloading precomputed synteny blocks for different groups of genomes. MCScan is an algorithm able to scan multiple genomes or subgenomes in order to identify putative homologous chromosomal regions, and align these regions using genes as anchors. The MCScanX toolkit implements an adjusted MCScan algorithm for detection of synteny and collinearity that extends the original software by incorporating 14 utility programs for visualization of results and additional downstream analyses. Applications of MCScanX to several sequenced plant genomes and gene families are shown as examples. MCScanX can be used to effectively analyze chromosome structural changes, and reveal the history of gene family expansions that might contribute to the adaptation of lineages and taxa. An integrated view of various modes of gene duplication can supplement the traditional gene tree analysis in specific families. The source code and documentation of MCScanX are freely available at http://chibba.pgml.uga.edu/mcscan2/. Information on multiple synteny between plants and/or within a plant is key information to understand genome evolution. In addition, visualization of multiple synteny is helpful in interpreting evolution. So far, some web applications have been developed to determine and visualize multiple homology regions at once. However, the applications are not fully convenient for biologists because some of them do not include the function of synteny determination but visualize the multiple synteny plots by allowing users to upload their synteny data by determining the synteny based only on BLAST similarity information, with some algorithms not designed for synteny determination. Here, we introduce a web application that determines and visualizes multiple synteny from two types of files, simplified browser extensible data and protein sequence file by MCScanX algorithm, which have been used in many synteny studies.

Can ATAC-Seq be employed in single-cell mode?

Single-cell ATAC-seq detects open chromatin in individual cells.

Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'. Author information: (1)Department of Human Genetics, The University of Chicago, 920 E. 58th Street, Chicago, IL, 60637, USA. [email protected]. (2)Department of Human Genetics, The University of Chicago, 920 E. 58th Street, Chicago, IL, 60637, USA. [email protected]. Most genomes to date have been sequenced without taking into account the diploid nature of the genome. However, the distribution of variants on each individual chromosome can (1) significantly impact gene regulation and protein function, (2) have important implications for analyses of population history and medical genetics, and (3) be of great value for accurate interpretation of medically relevant genetic variation. Here, we describe a comprehensive and detailed protocol for an ultra fast (<3 h library preparation), cost-effective, and scalable haplotyping method, named Contiguity Preserving Transposition sequencing or CPT-seq (Amini et al., Nat Genet 46(12):1343-1349, 2014). CPT-seq accurately phases >95 % of the whole human genome in Mb-scale phasing blocks. Additionally, the same workflow can be used to aid de novo assembly (Adey et al., Genome Res 24(12):2041-2049, 2014), detect structural variants, and perform single cell ATAC-seq analysis (Cusanovich et al., Science 348(6237):910-914, 2015). Author information: (1)Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA. (2)Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. (3)Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. (4)Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA. (5)Harvard Society of Fellows, Harvard University, Cambridge, MA, USA. (6)Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA. (7)Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA. (8)Biophysics Program, Stanford University School of Medicine, Stanford, CA, USA. (9)Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. (10)Department of Applied Physics, Stanford University, Stanford, CA, USA. (11)Chan Zuckerberg Biohub, San Francisco, CA, USA. (12)Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. [email protected]. (13)Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. [email protected]. (14)Institute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA, USA. [email protected]. (15)Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA. [email protected]. (16)Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA. [email protected]. (17)Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA. [email protected]. (#)Contributed equally ATAC-seq is a recently developed method to identify the areas of open chromatin in a cell. These regions usually correspond to active regulatory elements and their location profile is unique to a given cell type. When done at single-cell resolution, ATAC-seq provides an insight into the cell-to-cell variability that emerges from otherwise identical DNA sequences by identifying the variability in the genomic location of open chromatin sites in each of the cells. This paper presents Scasat (single-cell ATAC-seq analysis tool), a complete pipeline to process scATAC-seq data with simple steps. Scasat treats the data as binary and applies statistical methods that are especially suitable for binary data. The pipeline is developed in a Jupyter notebook environment that holds the executable code along with the necessary description and results. It is robust, flexible, interactive and easy to extend. Within Scasat we developed a novel differential accessibility analysis method based on information gain to identify the peaks that are unique to a cell. The results from Scasat showed that open chromatin locations corresponding to potential regulatory elements can account for cellular heterogeneity and can identify regulatory regions that separates cells from a complex population. Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1+ CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) is widely used to identify regulatory regions throughout the genome. However, very few studies have been performed at the single cell level (scATAC-seq) due to technical challenges. Here we developed a simple and robust plate-based scATAC-seq method, combining upfront bulk Tn5 tagging with single-nuclei sorting. We demonstrate that our method works robustly across various systems, including fresh and cryopreserved cells from primary tissues. By profiling over 3000 splenocytes, we identify distinct immune cell types and reveal cell type-specific regulatory regions and related transcription factors. Author information: (1)Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. (2)Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA 94305, USA. (3)Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. (4)Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA. (5)Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Applied Physics, Stanford University, Stanford, CA 94305, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. (6)Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: [email protected]. (7)Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304, USA. Electronic address: [email protected]. Here we present a comprehensive map of the accessible chromatin landscape of the mouse hippocampus at single-cell resolution. Substantial advances of this work include the optimization of a single-cell combinatorial indexing assay for transposase accessible chromatin (sci-ATAC-seq); a software suite, scitools, for the rapid processing and visualization of single-cell combinatorial indexing data sets; and a valuable resource of hippocampal regulatory networks at single-cell resolution. We used sci-ATAC-seq to produce 2346 high-quality single-cell chromatin accessibility maps with a mean unique read count per cell of 29,201 from both fresh and frozen hippocampi, observing little difference in accessibility patterns between the preparations. By using this data set, we identified eight distinct major clusters of cells representing both neuronal and nonneuronal cell types and characterized the driving regulatory factors and differentially accessible loci that define each cluster. Within pyramidal neurons, we identified four major clusters, including CA1 and CA3 neurons, and three additional subclusters. We then applied a recently described coaccessibility framework, Cicero, which identified 146,818 links between promoters and putative distal regulatory DNA. Identified coaccessibility networks showed cell-type specificity, shedding light on key dynamic loci that reconfigure to specify hippocampal cell lineages. Lastly, we performed an additional sci-ATAC-seq preparation from cultured hippocampal neurons (899 high-quality cells, 43,532 mean unique reads) that revealed substantial alterations in their epigenetic landscape compared with nuclei from hippocampal tissue. This data set and accompanying analysis tools provide a new resource that can guide subsequent studies of the hippocampus. Author information: (1)MOE Key Laboratory of Bioinformatics, Beijing Advanced Innovation Center for Structural Biology, Center for Synthetic and Systems Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing, China. (2)Beijing Advanced Innovation Center for Genomics (ICG), Biomedical Pioneering Innovation Center (BIOPIC), Peking University, 100871, Beijing, China. (3)State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Center for Bioinformatics, Peking University, 100871, Beijing, China. (4)Bioinformatics Division, BNRist, Department of Automation, Tsinghua University, 100084, Beijing, China. (5)Department of Biological Sciences, Center for Systems Biology, The University of Texas, Dallas 800 West Campbell Road, RL11, Richardson, TX, 75080-3021, USA. (6)MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Medicine, Tsinghua University, 100084, Beijing, China. (7)Department of Computer Science and Engineering, University of California, Riverside, CA, 92521, USA. (8)Bioinformatics Division, BNRIST; Department of Computer Science and Technology, Tsinghua University, 100084, Beijing, China. (9)MOE Key Laboratory of Bioinformatics, Beijing Advanced Innovation Center for Structural Biology, Center for Synthetic and Systems Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing, China. [email protected]. OBJECTIVE: Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome-wide association studies (GWAS) have identified > 400 independent signals that encode genetic predisposition. More than 90% of associated single-nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity. METHODS: We present genome-wide single-cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on U-Net architecture to accurately predict open chromatin peak calls in rare cell populations. RESULTS: We show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, T2D GWAS SNPs are significantly enriched in beta cell-specific and across cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals. CONCLUSIONS: Collectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways. ATAC-seq has become a leading technology for probing the chromatin landscape of single and aggregated cells. Distilling functional regions from ATAC-seq presents diverse analysis challenges. Methods commonly used to analyze chromatin accessibility datasets are adapted from algorithms designed to process different experimental technologies, disregarding the statistical and biological differences intrinsic to the ATAC-seq technology. Here, we present a Bayesian statistical approach that uses latent space models to better model accessible regions, termed ChromA. ChromA annotates chromatin landscape by integrating information from replicates, producing a consensus de-noised annotation of chromatin accessibility. ChromA can analyze single cell ATAC-seq data, correcting many biases generated by the sparse sampling inherent in single cell technologies. We validate ChromA on multiple technologies and biological systems, including mouse and human immune cells, establishing ChromA as a top performing general platform for mapping the chromatin landscape in different cellular populations from diverse experimental designs. Most genetic variations associated with human complex traits are located in non-coding genomic regions. Therefore, understanding the genotype-to-phenotype axis requires a comprehensive catalog of functional non-coding genomic elements, most of which are involved in epigenetic regulation of gene expression. Genome-wide maps of open chromatin regions can facilitate functional analysis of cis- and trans-regulatory elements via their connections with trait-associated sequence variants. Currently, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) is considered the most accessible and cost-effective strategy for genome-wide profiling of chromatin accessibility. Single-cell ATAC-seq (scATAC-seq) technology has also been developed to study cell type-specific chromatin accessibility in tissue samples containing a heterogeneous cellular population. However, due to the intrinsic nature of scATAC-seq data, which are highly noisy and sparse, accurate extraction of biological signals and devising effective biological hypothesis are difficult. To overcome such limitations in scATAC-seq data analysis, new methods and software tools have been developed over the past few years. Nevertheless, there is no consensus for the best practice of scATAC-seq data analysis yet. In this review, we discuss scATAC-seq technology and data analysis methods, ranging from preprocessing to downstream analysis, along with an up-to-date list of published studies that involved the application of this method. We expect this review will provide a guideline for successful data generation and analysis methods using appropriate software tools and databases for the study of chromatin accessibility at single-cell resolution.

How can B-cells transdifferentiate into macrophages?

Inflammatory macrophages can transdifferentiate into myofibroblasts during renal fibrosis . Vascular endothelial growth factor modified macrophage transdifferentiates into endothelial-like cells and decrease foam cell formation . Human cancer cells can be induced by C/EBPα to transdifferentiated into seemingly normal cells at high frequencies .

Starting with multipotent progenitors, hematopoietic lineages are specified by lineage-restricted transcription factors. The transcription factors that determine the decision between lymphoid and myeloid cell fates, and the underlying mechanisms, remain largely unknown. Here, we report that enforced expression of C/EBPalpha and C/EBPbeta in differentiated B cells leads to their rapid and efficient reprogramming into macrophages. C/EBPs induce these changes by inhibiting the B cell commitment transcription factor Pax5, leading to the downregulation of its target CD19, and synergizing with endogenous PU.1, an ETS family factor, leading to the upregulation of its target Mac-1 and other myeloid markers. The two processes can be uncoupled, since, in PU.1-deficient pre-B cells, C/EBPs induce CD19 downregulation but not Mac-1 activation. Our observations indicate that C/EBPalpha and beta remodel the transcription network of B cells into that of macrophages through a series of parallel and sequential changes that require endogenous PU.1. Earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor CCAAT/enhancer binding protein α at very high frequencies. Using this system, we performed a systematic analysis of whether during transdifferentiation the cells transiently reactivate progenitor-restricted genes or even retrodifferentiate. A transcriptome analysis of transdifferentiating cells showed that most genes are up- or down-regulated continuously, acquiring a macrophage phenotype within 5 d. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and FMS-like tyrosine kinase 3 and a few lineage-inappropriate genes. Importantly, however, we were unable to observe the reexpression of cell-surface marker combinations that characterize hematopoietic stem and progenitor cells, including c-Kit and FMS-like tyrosine kinase 3, even when CAAT/enhancer binding protein α was activated in pre-B cells under culture conditions that favor growth of hematopoietic stem and progenitor cells or when the transcription factor was activated in a time-limited fashion. Together, our findings are consistent with the notion that the conversion from pre-B cells to macrophages is mostly direct and does not involve overt retrodifferentiation. Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role. The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid maligcies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages. Earlier work demonstrated that the transcription factor C/EBPα can convert immature and mature murine B lineage cells into functional macrophages. Testing >20 human lymphoma and leukemia B cell lines, we found that most can be transdifferentiated at least partially into macrophage-like cells, provided that C/EBPα is expressed at sufficiently high levels. A tamoxifen-inducible subclone of the Seraphina Burkitt lymphoma line, expressing C/EBPαER, could be efficiently converted into phagocytic and quiescent cells with a transcriptome resembling normal macrophages. The converted cells retained their phenotype even when C/EBPα was inactivated, a hallmark of cell reprogramming. Interestingly, C/EBPα induction also impaired the cells' tumorigenicity. Likewise, C/EBPα efficiently converted a lymphoblastic leukemia B cell line into macrophage-like cells, again dramatically impairing their tumorigenicity. Our experiments show that human cancer cells can be induced by C/EBPα to transdifferentiate into seemingly normal cells at high frequencies and provide a proof of principle for a potential new therapeutic strategy for treating B cell maligcies. CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM). However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial-mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a 'path-breaker' as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications. MicroRNAs (miRNAs) exert negative effects on gene expression and influence cell lineage choice during hematopoiesis. C/EBPa-induced pre-B cell-to-macrophage transdifferentiation provides an excellent model to investigate the contribution of miRNAs to hematopoietic cell identity, especially because the two cell types involved fall into separate lymphoid and myeloid branches. In this process, efficient repression of the B cell-specific program is essential to ensure transdifferentation and macrophage function. miRNA profiling revealed that upregulation of miRNAs is highly predomit compared with downregulation and that C/EBPa directly regulates several upregulated miRNAs. We also determined that miRNA 34a (miR-34a) and miR-223 sharply accelerate C/EBPa-mediated transdifferentiation, whereas their depletion delays this process. These two miRNAs affect the transdifferentiation efficiency and activity of macrophages, including their lipopolysaccharide (LPS)-dependent inflammatory response. miR-34a and miR-223 directly target and downregulate the lymphoid transcription factor Lef1, whose ectopic expression delays transdifferentiation to an extent similar to that seen with miR-34a and miR-223 depletion. In addition, ectopic introduction of Lef1 in macrophages causes upregulation of B cell markers, including CD19, Pax5, and Ikzf3. Our report demonstrates the importance of these miRNAs in ensuring the erasure of key B cell transcription factors, such as Lef1, and reinforces the notion of their essential role in fine-tuning the control required for establishing cell identity. Accumulating evidence suggests that Pax5 plays essential roles in B cell lineage commitment. However, molecular mechanisms of B cell-specific expression of Pax5 are not fully understood. Here, we applied insertional chromatin immunoprecipitation (iChIP) combined with stable isotope labeling using amino acids in cell culture (SILAC) (iChIP-SILAC) to direct identification of proteins interacting with the promoter region of the endogenous single-copy chicken Pax5 gene. By comparing B cells with macrophage-like cells trans-differentiated by ectopic expression of C/EBPβ, iChIP-SILAC detected B cell-specific interaction of a nuclear protein, Thy28/Thyn1, with the Pax5 1A promoter. Trans-differentiation of B cells into macrophage-like cells caused down-regulation of Thy28 expression. Loss-of-function of Thy28 induced decrease in Pax5 expression and recruitment of myosin-9 (MYH9), one of Thy28-interacting proteins, to the Pax5 1A promoter. Loss-of-function of MYH9 also induced decrease in Pax5 expression. Thus, our analysis revealed that Thy28 is functionally required for B cell-specific expression of Pax5 via recruitment of MYH9 to the Pax5 locus in chicken B cells. The concept of unidirectional differentiation of the haematopoietic stem cell has been challenged after recent findings that human B cell progenitors and even mature B cells can be reprogrammed into histiocytic/dendritic cells by altering expression of lineage-associated transcription factors. The conversion of mature B cell lymphomas to Langerhans cell neoplasms is not well documented. Three previous reports have described clonally related follicular lymphoma and Langerhans cell tumours, whereas no case has been published of clonally related marginal zone lymphoma and Langerhans cell sarcoma. We describe the case of a 77-year-old patient who developed a Langerhans cell sarcoma and 6 years later a nodal marginal zone lymphoma. Mutation status examination showed 100 % gene identity to the germline sequence, suggesting direct trans-differentiation or dedifferentiation of the nodal marginal zone lymphoma to the Langerhans cell sarcoma rather than a common progenitor. We found inactivation of paired box 5 (PAX-5) in the lymphoma cells by methylation, along with duplication of part of the long arm of chromosomes 16 and 17 in the sarcoma cells. The absence of PAX-5 could have triggered B cells to differentiate into macrophages and dendritic cells. On the other hand, chromosomal imbalances might have activated genes involved in myeloid lineage maturation, transcription activation and oncogenesis. We hypothesize that this occurred because of previous therapies for nodal marginal zone lymphoma. Better understanding of this phenomenon may help in unravelling the molecular interplay between transcription factors during haematopoietic lineage commitment and may expand the spectrum of clonally related mature B cell neoplasms and Langerhans cell tumours. The lymphoid-myeloid transdifferentiation potentials of members of the C/EBP family (C/EBPα, β, δ, and ε) were compared in v-Abl-immortalized primary B cells. Conversion of B cells to macrophages was readily induced by the ectopic expression of any C/EBP, and enhanced by endogenous C/EBPα and β activation. High transgene expression of C/EBPβ or C/EBPε, but not of C/EBPα or C/EBPδ, also induced the formation of granulocytes. Granulocytes and macrophages emerged in a mutually exclusive manner. C/EBPβ-expressing B cells produced granulocyte-macrophage progenitor (GMP)-like progenitors when subjected to selective pressure to eliminate lymphoid cells. The GMP-like progenitors remained self-renewing and cytokine-independent, and continuously produced macrophages and granulocytes. In addition to their suitability to study myelomonocytic lineage bifurcation, lineage-switched GMP-like progenitors could reflect the features of the lympho-myeloid lineage switch observed in leukemic progression. The generation of induced pluripotent stem cells (iPSCs) achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc, transformed our classical views of the cellular epigenetic landscape and delivered a new concept for cell and tissue engineering. In addition to iPSCs, several other cell types have also been generated by master transcription factor (TF)-mediated transdifferentiation. However, the critical molecular mechanisms amongst diverse cellular identity changes are not well understood. Through the investigation of reprogramming mechanisms, we recently revealed that over-expression of constitutive active Smad3 boosted not only iPSC generation, but also 3 other master TF-mediated conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. This demonstrated that there were common mechanisms underlying different master TF-mediated cell conversions. To illuminate such mechanisms further, we have recently performed CRISPR/Cas9-mediated genome-wide knockout screening during reprogramming with a lentiviral gRNA library containing 90,000 gRNAs. This screening provided us with ~15 novel reprogramming roadblock genes as well as ~20 candidate genes essential for the reprogramming process but not for ES cell self-renewal. This data set will be a valuable resource to further understand how overexpression of master TFs alters cellular identity, and to achieve more faithful, efficient cell conversions for regenerative medicine.(Presented at the 1934th Meeting, March 17, 2017). Blood cells are derived from a common set of hematopoietic stem cells, which differentiate into more specific progenitors of the myeloid and lymphoid lineages, ultimately leading to differentiated cells. This developmental process is controlled by a complex regulatory network involving cytokines and their receptors, transcription factors, and chromatin remodelers. Using public data and data from our own molecular genetic experiments (quantitative PCR, Western blot, EMSA) or genome-wide assays (RNA-sequencing, ChIP-sequencing), we have assembled a comprehensive regulatory network encompassing the main transcription factors and signaling components involved in myeloid and lymphoid development. Focusing on B-cell and macrophage development, we defined a qualitative dynamical model recapitulating cytokine-induced differentiation of common progenitors, the effect of various reported gene knockdowns, and the reprogramming of pre-B cells into macrophages induced by the ectopic expression of specific transcription factors. The resulting network model can be used as a template for the integration of new hematopoietic differentiation and transdifferentiation data to foster our understanding of lymphoid/myeloid cell-fate decisions.

What is the function of the chromHMM software?

ChromHMM learns chromatin-state signatures using a multivariate hidden Markov model (HMM) that explicitly models the combinatorial presence or absence of each mark . It uses these signatures to generate a genome-wide annotation for each cell type by calculating the most probable state for each genomic segment . Chromatin states are learned, annotations are produced, and enrichments are computed within 1 d .

BACKGROUND: Epigenetic modifications are essential for controlling gene expression. Recent studies have shown that not only single epigenetic modifications but also combinations of multiple epigenetic modifications play vital roles in gene regulation. A striking example is the long hypomethylated regions enriched with modified H3K27me3 (called, "K27HMD" regions), which are exposed to suppress the expression of key developmental genes relevant to cellular development and differentiation during embryonic stages in vertebrates. It is thus a biologically important issue to develop an effective optimization algorithm for detecting long DNA regions (e.g., >4 kbp in size) that harbor a specific combination of epigenetic modifications (e.g., K27HMD regions). However, to date, optimization algorithms for these purposes have received little attention, and available methods are still heuristic and ad hoc. RESULTS: In this paper, we propose a linear time algorithm for calculating a set of non-overlapping regions that maximizes the sum of similarities between the vector of focal epigenetic states and the vectors of raw epigenetic states at DNA positions in the set of regions. The average elapsed time to process the epigenetic data of any of human chromosomes was less than 2 seconds on an Intel Xeon CPU. To demonstrate the effectiveness of the algorithm, we estimated large K27HMD regions in the medaka and human genomes using our method, ChromHMM, and a heuristic method. CONCLUSIONS: We confirmed that the advantages of our method over those of the two other methods. Our method is flexible enough to handle other types of epigenetic combinations. The program that implements the method is called "CSMinfinder" and is made available at: http://mlab.cb.k.u-tokyo.ac.jp/~ichikawa/Segmentation/ We analyzed the publicly available ChromHMM BED files of the ENCODE project and tested the Markov properties of the different chromatin states in the human genome. Nucleotide frequency profiles of regional chromatin segmentations were analyzed, and Markov chains were built to detect Markov properties in the chromatin states of different ChromHMM regions. By estimating the transition probabilities of 200-base pair nucleotide sequences of the human genome, we constructed a nucleotide-sequence-based Markovian chromatin map called SeqChromMM. Noncoding DNA regions have central roles in human biology, evolution, and disease. ChromHMM helps to annotate the noncoding genome using epigenomic information across one or multiple cell types. It combines multiple genome-wide epigenomic maps, and uses combinatorial and spatial mark patterns to infer a complete annotation for each cell type. ChromHMM learns chromatin-state signatures using a multivariate hidden Markov model (HMM) that explicitly models the combinatorial presence or absence of each mark. ChromHMM uses these signatures to generate a genome-wide annotation for each cell type by calculating the most probable state for each genomic segment. ChromHMM provides an automated enrichment analysis of the resulting annotations to facilitate the functional interpretations of each chromatin state. ChromHMM is distinguished by its modeling emphasis on combinations of marks, its tight integration with downstream functional enrichment analyses, its speed, and its ease of use. Chromatin states are learned, annotations are produced, and enrichments are computed within 1 d. The non-coding DNA in eukaryotic genomes encodes a language which programs chromatin accessibility, transcription factor binding, and various other activities. The objective of this short report was to determine the impact of primary DNA sequence on the epigenomic landscape across 200-base pair genomic units by integrating nine publicly available ChromHMM Browser Extensible Data files of the Encyclopedia of DNA Elements (ENCODE) project. The nucleotide frequency profiles of nine chromatin annotations with the units of 200 bp were analyzed and integrative Markov chains were built to detect the Markov properties of the DNA sequences in some of the active chromatin states of different ChromHMM regions. Our aim was to identify the possible relationship between DNA sequences and the newly built chromatin states based on the integrated ChromHMM datasets of different cells and tissue types.

What is FeatureCounts used for?

featureCounts is a general purpose program for assigning sequence reads to genomic features. It is a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments.

RNA-Seq technology has been gradually becoming a routine approach for characterizing the properties of transcriptome in terms of organisms, cell types and conditions and consequently a big burden has been put on the facet of data analysis, which calls for an easy-to-learn workflow to cope with the increased demands from a large number of laboratories across the world. We report a one-in-all solution called hppRNA, composed of four scenarios such as pre-mapping, core-workflow, post-mapping and sequence variation detection, written by a series of individual Perl and R scripts, counting on well-established and preinstalled software, irrespective of single-end or paired-end, unstranded or stranded sequencing method. It features six independent core-workflows comprising the state-of-the-art technology with dozens of popular cutting-edge tools such as Tophat-Cufflink-Cuffdiff, Subread-featureCounts-DESeq2, STAR-RSEM-EBSeq, Bowtie-eXpress-edgeR, kallisto-sleuth, HISAT-StringTie-Ballgown, and embeds itself in Snakemake, which is a modern pipeline management system. The core function of this pipeline is turning the raw fastq files into gene/isoform expression matrix and differentially expressed genes or isoforms as well as the identification of fusion genes, single nucleotide polymorphisms, long noncoding RNAs and circular RNAs. Last but not least, this pipeline is specifically designed for performing the systematic analysis on a huge set of samples in one go, ideally for the researchers who intend to deploy the pipeline on their local servers. The scripts as well as the user manual are freely available at https://sourceforge.net/projects/hpprna/. BACKGROUND: RNA-Seq is currently used routinely, and it provides accurate information on gene transcription. However, the method cannot accurately estimate duplicated genes expression. Several strategies have been previously used (drop duplicated genes, distribute uniformly the reads, or estimate expression), but all of them provide biased results. RESULTS: We provide here a tool, called mmquant, for computing gene expression, included duplicated genes. If a read maps at different positions, the tool detects that the corresponding genes are duplicated; it merges the genes and creates a merged gene. The counts of ambiguous reads is then based on the input genes and the merged genes. CONCLUSION: mmquant is a drop-in replacement of the widely used tools htseq-count and featureCounts that handles multi-mapping reads in an unabiased way.

Which is the main difference in the roles of Otx2 and Nanog during development?

Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition . The transcription factor Otx2 acts as a negative switch in the regulation of transition from naive to primed pluripotency in mouse pluripotent stem cells . OTX2-mediated Nanog regulation contributes to the integrity of the ESC state and cell lineage specification in preimplantation development .

Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development. The transcription factor Otx2 acts as a negative switch in the regulation of transition from naive to primed pluripotency in mouse pluripotent stem cells. However, the molecular features and function of porcine OTX2 have not been well elucidated in porcine-induced pluripotent stem cells (piPSCs). By studying high-throughput transcriptome sequencing and interfering endogenous OTX2 expression, we demonstrate that OTX2 is able to downgrade the self-renewal of piPSCs. OTX2 is highly expressed in porcine brain, reproductive tissues, and preimplantation embryos, but is undetectable in fibroblasts and most somatic tissues. However, the known piPSC lines reported previously produced different levels of OTX2 depending on the induction procedures and culture conditions. Overexpression of porcine OTX2 can reduce the percentage of alkaline phosphatase-positive colonies and downregulate NANOG and OCT4 expression. In contrast, knockdown of OTX2 can significantly increase endogenous expressions of NANOG, OCT4, and ESRRB, and stabilize the pluripotent state of piPSCs. On the other hand, NANOG can directly bind to the OTX2 promoter as shown in ChIP-seq data and repress OTX2 promoter activity in a dose-dependent manner. These observations indicate that OTX2 and NANOG can form a negative feedback circuitry to regulate the pluripotency of porcine iPS cells. Embryonic stem cells (ESCs) cultured in leukemia inhibitory factor (LIF) plus fetal bovine serum (FBS) exhibit heterogeneity in the expression of naive and primed transcription factors. This heterogeneity reflects the dynamic condition of ESCs and their versatility to promptly respond to signaling effectors promoting naive or primed pluripotency. Here, we report that ESCs lacking Nanog or overexpressing Otx2 exhibit an early primed identity in LIF + FBS and fail to convert into 2i-induced naive state. Conversely, Otx2-null ESCs possess naive identity features in LIF + FBS similar to Nanog-overexpressing ESCs and convert poorly into FGF-induced early primed state. When both Nanog and Otx2 are inactivated, ESCs cultured in LIF + FBS exhibit primed identity and weakened ability to convert into naive state. These data suggest that, through mutual antagonism, NANOG and OTX2 specify the heterogeneous identity of ESCs cultured in LIF + FBS and individually predispose them for optimal response to naive or primed inducing factors. During neurogenesis, neural patterning is a critical step during which neural progenitor cells differentiate into neurons with distinct functions. However, the molecular determits that regulate neural patterning remain poorly understood. Here we optimized the "dual SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain neural progenitor cells along the rostral-caudal axis. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that domitly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3β (GSK3β) through CHIR99021 application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans.

Are super enhancers structurally insulated in chromatin loops?

Although there is evidence that chromatin neighbourhoods, formed by the zinc-finger protein CTCF, can sequester enhancers and their target genes, there is limited in vivo evidence for CTCF demarcating super-enhancers and preventing cross talk between distinct regulatory elements. CTCF sites are porous borders, allowing a super-enhancer to activate a secondary target.

Precise spatiotemporal gene regulation is paramount for the establishment and maintece of cell-specific programmes. Although there is evidence that chromatin neighbourhoods, formed by the zinc-finger protein CTCF, can sequester enhancers and their target genes, there is limited in vivo evidence for CTCF demarcating super-enhancers and preventing cross talk between distinct regulatory elements. Here, we address these questions in the Wap locus with its mammary-specific super-enhancer separated by CTCF sites from widely expressed genes. Mutational analysis demonstrates that the Wap super-enhancer controls Ramp3, despite three separating CTCF sites. Their deletion in mice results in elevated expression of Ramp3 in mammary tissue through augmented promoter-enhancer interactions. Deletion of the distal CTCF-binding site results in loss of Ramp3 expression in non-mammary tissues. This suggests that CTCF sites are porous borders, allowing a super-enhancer to activate a secondary target. Likewise, CTCF sites shield a widely expressed gene from suppressive influences of a silent locus. Author information: (1)Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, MA, 02215, USA. (2)Division of Hematology/Oncology, Boston Childrens Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02215, USA. (3)Department of Pediatrics, Childrens Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. (4)Howard Hughes Medical Institute, Boston, MA, 02215, USA. (5)Department of Pediatrics, Childrens Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. [email protected]. (6)Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, MA, 02215, USA. [email protected]. The mammary luminal lineage relies on the common cytokine-sensing transcription factor STAT5 to establish super-enhancers during pregcy and initiate a genetic program that activates milk production. As pups grow, the greatly increasing demand for milk requires progressive differentiation of mammary cells with advancing lactation. Here we investigate how persistent hormonal exposure during lactation shapes an evolving enhancer landscape and impacts the biology of mammary cells. Employing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggesting that their activities evolve with advancing differentiation. Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses. Most profoundly, a seed enhancer, which is mandatory for the activation of the Wap super-enhancer during pregcy, is not required during lactation, suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent enhancers demonstrated differentiation-specific compensatory activities during lactation. We also demonstrate that the Wap super-enhancer, which is built on STAT5 and other common transcription factors, retains its exquisite mammary specificity when placed into globally permissive chromatin, suggesting a limited role of chromatin in controlling cell specificity. Our studies unveil a previously unrecognized progressive enhancer landscape where structurally equivalent components serve unique and differentiation-specific functions.

Does IL18 signaling have a role in thymus?

Yes. IL18 signaling promotes homing of mature Tregs into the thymus.

Foxp3+ regulatory T cells (Tregs) are potent suppressor cells, essential for the maintece of immune homeostasis. Most Tregs develop in the thymus and are then released into the immune periphery. However, some Tregs populate the thymus and constitute a major subset of yet poorly understood cells. Here we describe a subset of thymus recirculating IL18R+ Tregs with molecular characteristics highly reminiscent of tissue-resident effector Tregs. Moreover, we show that IL18R+ Tregs are endowed with higher capacity to populate the thymus than their IL18R- or IL18R-/- counterparts, highlighting the key role of IL18R in this process. Finally, we demonstrate that IL18 signaling is critical for the induction of the key thymus-homing chemokine receptor - CCR6 on Tregs. Collectively, this study provides a detailed characterization of the mature Treg subsets in the mouse thymus and identifies a key role of IL18 signaling in controlling the CCR6-CCL20-dependent migration of Tregs into the thymus.

Does an antiphlogistic promotes inflammation?

Antiinflammatory agents: new series of N-substituted amino acids with complex pyrimidine structures endowed with antiphlogistic activity.

The binding of sodium salicylate, sodium mefenamate, ortophen and piroxicam to membranes of erythrocyte ghosts and the effects of the drugs on the membrane microviscosity in carragee-induced inflammation were studied by the fluorescent probe method. It was shown that under inflammation the membrane microviscosity decreases and concurrently the affinity of the studied compounds to them increases. Antiphlogistics were found to enhance the membrane viscosity both in control and under inflammation. The possible mechanisms of the described effects of non-steroidal anti-inflammatory agents on biomembranes are discussed. It has been shown in experiments on mice that sodium diclofenac and indomethacin inhibited antibody titers and the number of rosette-forming cells (RFC) in mice immunized with sheep red blood cells. Sodium salicylate in all test doses did not produce any shifts in rosette-formation but lowered the number of RFC. As regards the immunodepressant activity, sodium diclofenac compares very favourably with indomethacin and sodium salicylate. Enhancement of the immunoreactivity inhibition caused by the drugs was not proportional to the increase in their antiphlogistic effects determined by the Selye model of inflammation. The antiphlogistic Ibuprofen incorporated in liposomes caused a decrease of the inflammatory edema induced by Carragee in the distal part of the rat's hind leg after both the intramuscular and percutaneous administration. The antiphlogistic effect of free Ibuprofen in the cream was weaker. Intramuscular administration of empty liposomes slowed down in the initial stages the development of inflammation and slightly diminished the size of edema. Yarrow (Achillea millefolium L. s.l.) is traditionally used in the treatment of inflammatory and spasmodic gastro-intestinal disorders, hepato-biliary complaints and inflammation. Now we could show that the flavonoids mediated the antispasmodic properties of yarrow, whereas the dicaffeoylquinic acids caused the choleretic effects. Moreover, we observed an in vitro-inhibition of human neutrophil elastase, a protease involved in the inflammatory process, by extracts and fractions from yarrow, which suggests additional mechanisms of antiphlogistic action. The presented results confirm the traditional use of yarrow. During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation. BACKGROUND: Today, the sun protection factor (SPF) value of sunscreen products is determined in vivo with a standardized protocol (EN ISO 24444:2010), and the measured SPF biological end point is the visible skin erythema. However, many of the sunscreen products currently available on the market have antiphlogistic ingredients, which may potentially result in an overestimated SPF of the sunscreen. AIMS: To investigate the potential influence of the antiphlogistic ingredients panthenol and bisabolol in sunscreens on the determined SPF value in vivo. METHODS: Formulations with different concentrations of the antiphlogistic ingredients bisabolol or panthenol were tested. As a reference, a base formulation (vehicle) without antiphlogistic ingredients was used. First, the SPF of the sunscreen formulas with and without antiphlogistic ingredients was analyzed in vitro. To investigate whether the antiphlogistic ingredient may suppress the inflammatory response to ultraviolet (UV) irradiation, the SPF was determined in vivo. Finally, selected formulations were also analyzed in an erythema model for testing formulations on UV-induced inflammation. RESULTS: It could be confirmed that no differences between the formula with and that without the active antiphlogistic ingredients bisabolol or panthenol exist when measured in vitro. However, there was also no statistically significant difference in the erythemal response between the vehicle (without an antiphlogistic active ingredient) and the test formulations with different concentrations of the antiphlogistic active ingredients in the in vivo determination of the SPF. Evidence of anti-inflammatory activity of the sunscreen antiphlogistics bisabolol and panthenol was also not apparent in the UV model over a time course of 48 h. Conlusion: The antiphlogistic ingredients panthenol and bisabolol incorporated in the tested sunscreen formula do not interfere with erythema reddening and thus do not affect the SPF value in vivo.

Which interleukin receptors are targeted with rilonacept?

Rilonacept inhibits interleukin-1α and interleukin-1β. It has a role for treatment of pericarditis.

The intracellular sensing protein termed NLRP3 (for NACHT, LRR, and PYD domains-containing protein 3) forms a macromolecular structure called the NLRP3 inflammasome. The NLRP3 inflammasome plays a major role in inflammation, particularly in the production of IL (interleukin)-1β. IL-1β is the most studied of the IL-1 family of cytokines, including 11 members, among which are IL-1α and IL-18. Here, we summarize preclinical and clinical findings supporting the key pathogenetic role of the NLRP3 inflammasome and IL-1 cytokines in the formation, progression, and complications of atherosclerosis, in ischemic (acute myocardial infarction), and nonischemic injury to the myocardium (myocarditis) and the progression to heart failure. We also review the clinically available IL-1 inhibitors, although not currently approved for cardiovascular indications, and discuss other IL-1 inhibitors, not currently approved, as well as oral NLRP3 inflammasome inhibitors currently in clinical development. Canakinumab, IL-1β antibody, prevented the recurrence of ischemic events in patients with prior acute myocardial infarction in a large phase III clinical trial, including 10 061 patients world-wide. Phase II clinical trials show promising data with anakinra, recombit IL-1 receptor antagonist, in patients with ST-segment-elevation acute myocardial infarction or heart failure with reduced ejection fraction. Anakinra also improved outcomes in patients with pericarditis, and it is now considered standard of care as second-line treatment for patients with recurrent/refractory pericarditis. Rilonacept, a soluble IL-1 receptor chimeric fusion protein neutralizing IL-1α and IL-1β, has also shown promising results in a phase II study in recurrent/refractory pericarditis. In conclusion, there is overwhelming evidence linking the NLRP3 inflammasome and the IL-1 cytokines with the pathogenesis of cardiovascular diseases. The future will likely include targeted inhibitors to block the IL-1 isoforms, and possibly oral NLRP3 inflammasome inhibitors, across a wide spectrum of cardiovascular diseases. The progress in the understanding of the pathophysiology of rheumatic diseases provided a rational basis for the development of biologic disease‑modifying antirheumatic drugs (bDMARDs) and targeted synthetic DMARDs (tsDMARDs), which have completely revolutionized the treatment of inflammatory conditions. These agents differ in terms of their effectiveness for controlling specific rheumatic diseases depending on the pivotal cytokine driving the inflammatory process. Cytokine blockers were the first to be developed and rapidly expanded. They include agents that act against tumor necrosis factor α (TNF‑α) (etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol) and interleukin (IL) 6 (tocilizumab and sarilumab), IL‑1 (anakinra, canakinumab, and rilonacept), IL‑17 (secukinumab and ixekizumab), and IL-12/23 (ustekinumab) receptors. Lymphocyte‑targeting agents include rituximab and belimumab, which act against B cells by different mechanisms, and abatacept, which is a T cell costimulation modulator. tsDMARDs, also known as small‑molecule inhibitors, are oral drugs based on a novel strategy to treat inflammatory diseases. Janus kinase (JAK) inhibitors (tofacitinib, baricitinib, and upadacitinib) and phosphodiesterase 4 inhibitors (apremilast) form this group. The major concern with the use of bDMARDs and tsDMARDs is a higher risk of infections. Performance of blood tests as well as screening for tuberculosis and hepatitis viral infection are mandatory prior to biologic therapy initiation. Adherence to an immunization program is also recommended. Whenever possible, the choice of bDMARDs and tsDMARDs should be guided by the patient's comorbidities. There have been limited data on the use of these drugs during pregcy, but anti‑TNF‑α therapy, rituximab, and anakinra seem to be safe. Biologic agents are expensive, but biosimilars have emerged as a cost‑effective option with a potential to treat a greater number of patients. Recent advances have shown impressive results by anti-interleukin 1 (IL-1) agents in refractory idiopathic recurrent pericarditis. PURPOSE OF REVIEW: We critically discuss the current state of the art of therapy of relapsing pericarditis, with a focus on new pharmacological approaches and on specific clinical settings such as pregcy, pediatric patients, and secondary forms of relapsing pericarditis. RECENT FINDINGS: Antagonism of the IL-1 is highly effective in idiopathic recurrent pericarditis with autoinflammatory features. Currently, available anti-IL-1 agents are anakinra and canakinumab. Rilonacept is another IL-1 antagonist, currently studied in the phase-3 clinical trial RHAPSODY. Available data suggest similar efficacy and safety profiles of these three agents, although only anakinra has been tested in randomized clinical trials. These agents have slightly different pharmacological properties, being canakinumab a specific IL-1ß antagonist while anakinra and rilonacept are unselective IL-1α and IL-1ß blockers. To date, there is no evidence that specificity against IL-1ß affects safety and efficacy in patients with relapsing pericarditis, although it has been proposed that unspecific blockage might be useful in severe disease. Anakinra is the first anti-IL-1 agent with well-documented efficacy and safety in adult and pediatric patients with idiopathic relapsing pericarditis. Other anti-IL-1 agents are currently under study. Future research should clarify the optimal duration of therapy and tapering schedule of treatment with these agents. Moreover, biomarkers would be required to understand which patients will benefit from early administration of IL-1 blockers due to refractoriness to conventional therapy and which others will suffer from recurrences during the tapering of these agents. Lastly, future studies should focus on the subjects with the autoimmune or the pauci-inflammatory phenotype of idiopathic refractory pericarditis. BACKGROUND: Interleukin-1 has been implicated as a mediator of recurrent pericarditis. The efficacy and safety of rilonacept, an interleukin-1α and interleukin-1β cytokine trap, were studied previously in a phase 2 trial involving patients with recurrent pericarditis. METHODS: We conducted a phase 3 multicenter, double-blind, event-driven, randomized-withdrawal trial of rilonacept in patients with acute symptoms of recurrent pericarditis (as assessed on a patient-reported scale) and systemic inflammation (as shown by an elevated C-reactive protein [CRP] level). Patients presenting with pericarditis recurrence while receiving standard therapy were enrolled in a 12-week run-in period, during which rilonacept was initiated and background medications were discontinued. Patients who had a clinical response (i.e., met prespecified response criteria) were randomly assigned in a 1:1 ratio to receive continued rilonacept monotherapy or placebo, administered subcutaneously once weekly. The primary efficacy end point, assessed with a Cox proportional-hazards model, was the time to the first pericarditis recurrence. Safety was also assessed. RESULTS: A total of 86 patients with pericarditis pain and an elevated CRP level were enrolled in the run-in period. During the run-in period, the median time to resolution or near-resolution of pain was 5 days, and the median time to normalization of the CRP level was 7 days. A total of 61 patients underwent randomization. During the randomized-withdrawal period, there were too few recurrence events in the rilonacept group to allow for the median time to the first adjudicated recurrence to be calculated; the median time to the first adjudicated recurrence in the placebo group was 8.6 weeks (95% confidence interval [CI], 4.0 to 11.7; hazard ratio in a Cox proportional-hazards model, 0.04; 95% CI, 0.01 to 0.18; P<0.001 by the log-rank test). During this period, 2 of 30 patients (7%) in the rilonacept group had a pericarditis recurrence, as compared with 23 of 31 patients (74%) in the placebo group. In the run-in period, 4 patients had adverse events leading to the discontinuation of rilonacept therapy. The most common adverse events with rilonacept were injection-site reactions and upper respiratory tract infections. CONCLUSIONS: Among patients with recurrent pericarditis, rilonacept led to rapid resolution of recurrent pericarditis episodes and to a significantly lower risk of pericarditis recurrence than placebo. (Funded by Kiniksa Pharmaceuticals; RHAPSODY ClinicalTrials.gov number, NCT03737110.). OBJECTIVE: Recurrent pericarditis (RP) incurs significant morbidity. Rilonacept inhibits both interleukin-1 alpha (IL-1α) and IL-1β; these cytokines are thought to play a major role in RP. This phase II study evaluated rilonacept efficacy and safety in RP. METHODS: This multicentre, open-label study enrolled adult patients with idiopathic or postpericardiotomy RP, symptomatic (≥2 pericarditis recurrences) or corticosteroid (CS) dependent (≥2 recurrences prior).Patients received rilonacept 320 mg SC load/160 mg SC weekly maintece in a 6-week base treatment period (TP) followed by an optional 18-week on-treatment extension period (EP) (option to wean background therapy). RESULTS: Outcomes: pericarditis pain (numeric rating scale (NRS)) and inflammation (C reactive protein (CRP)) for symptomatic patients; disease activity after CS taper for CS-dependent patients. SECONDARY OUTCOMES: health-related quality of life (HRQOL), pericarditis manifestations and additional medications. 25 unique patients enrolled, while 23 completed the EP (seven colchicine failures and five CS failures). In symptomatic patients, NRS and CRP decreased; response was observed after first rilonacept dose. NRS decreased from 4.5 at baseline to 0.7, and CRP decreased from 4.62 mg/dL at baseline to 0.38 mg/dL at end of TP. Median time to CRP normalisation: 9 days. Pericarditis manifestations resolved. 13 patients on CS at baseline completed the EP; 11 (84.6%) discontinued CS, and 2 tapered; CRP and NRS remained low without recurrence. Mean HRQOL scores improved in symptomatic patients. One serious adverse event (SAE) resulted in discontinuation of rilonacept. CONCLUSIONS: Rilonacept led to rapid and sustained improvement in pain, inflammation (CRP and pericarditis manifestations) and HRQOL. CSs were successfully tapered or discontinued; safety was consistent with known rilonacept safety profile. TRIAL REGISTRATION NUMBER: NCT03980522.

Which deep learning framework has been developed for cancer molecular subtype classification?

Molecular subtyping of cancer is a critical step towards more individualized therapy and provides important biological insights into cancer heterogeneity. Although gene expression signature-based classification has been widely demonstrated to be an effective approach in the last decade, the widespread implementation has long been limited by platform differences, batch effects, and the difficulty to classify individual patient samples. DeepCC is a novel deep learning-based framework for cancer molecular subtype classification. It is platform independent, robust to missing data, and can be used for single sample prediction facilitating clinical implementation of cancer molecular subtyping.

What class of drugs have been given a black box warning for suicide?

In 2004, the European and American authorities released a black-box warning on antidepressants indicating an association with an increased risk of suicidality (suicidal ideation and behavior) in young people

OBJECTIVE: In 2002, 264 children and adolescents ages 5-14 died by suicide in the United States, the fifth leading cause of death. Of these suicides, 260 were in the 10-14 year age group, making suicide the third largest cause of death behind accidents and maligcy. Although 60% of suicides in the general population occur in the midst of a mood disorder, usually untreated, little is known about the relationship between treatment of mood disorders and youth suicide. The FDA recently linked adverse event reports of suicidal ideation among children and adolescents in randomized controlled trials to selective serotonin reuptake inhibitors (SSRIs) and consequently required a change in labeling that included a black box warning regarding SSRI use for all age groups. Given that the age-adjusted suicide rate is about six times higher in 15-19 year olds compared with 10-14 year olds, the risk-benefit ratio may be different in younger children. Therefore, this study examined the association between antidepressant medication prescription rate and suicide rate in children ages 5-14 prior to the FDA findings by analyzing associations at the county level across the United States. METHOD: National county-level suicide rate data among children ages 5-14 were broken down by sex, income, and race during the period 1996-1998. National county-level antidepressant prescription rate data were expressed as number of pills prescribed per person. The primary outcome measure was the suicide rate in each county expressed as number of suicides for a given population size. RESULTS: After adjustment for sex, race, income, access to mental health care, and county-to-county variability in suicide rates, higher SSRI prescription rates were associated with lower suicide rates in children and adolescents. CONCLUSIONS: The aggregate nature of these observational data precludes a direct causal interpretation of the results. More SSRI prescriptions are associated with lower suicide rates in children and may reflect antidepressant efficacy, treatment compliance, better quality mental health care, and low toxicity in the event of a suicide attempt by overdose. Regulatory agencies of different European countries and the United States have been critically examining the possible link between suicidality and antidepressant use in children and adults, which has resulted in an FDA directive to the manufacturers of all antidepressant medications to add a 'black box' warning. 'Black box' warning describes the increased risk of suicidality in persons who take antidepressants. Because the news media's coverage of the antidepressant-suicide controversy has increased dramatically in the last few years, serious concerns need to be performed. In this review, the possible risk-benefit ratio has been estimated according to the use of selective serotonin reuptake inhibitors (SSRIs) antidepressants due to relevant psychobiological, clinical and epidemiologic data. The risk of suicidal behavior associated with antidepressant treatment is an issue of debate and concern. The US FDA has required that antidepressants carry a black box warning that there may be a risk of suicidal ideations in depressed pediatric patients treated with these medications, and recently extended the warning to include individuals up age 24. However studies of antidepressant-induced suicidality in adults have yielded contradictory findings and conclusions. This article discusses investigations of this poorly understood phenomenon and the clinical implications of research findings and FDA warnings for clinicians treating adults with depression. Although antidepressant-induced suicidality apparently occurs only rarely, close monitoring and follow up care after the initiation of a new antidepressant is indicated. CONTEXT: On January 31, 2008, the Food and Drug Administration issued an alert regarding increased risk of suicidal thoughts and behavior related to use of antiepileptic drugs (AEDs). On July 10, 2008, a Food and Drug Administration scientific advisory committee voted that, yes, there was a significant positive association between AEDs and suicidality but voted against placing a black box warning on AEDs for suicidality. OBJECTIVE: To determine if AEDs increase the risk of suicide attempt in patients with bipolar disorder. DESIGN: A pharmacoepidemiologic study in which suicide attempt rates were compared before and after treatment and with a medication-free control group. Analyses were restricted to AED and lithium monotherapy. SETTING: We used the PharMetrics medical claims database to study the relationship between the 11 AEDs identified in the FDA alert, and lithium, to suicide attempts. MAIN OUTCOME MEASURE: Suicide attempts. Patients A cohort of 47 918 patients with bipolar disorder with a minimum 1-year window of information before and after the index date of their illness. RESULTS: Overall, there was no significant difference in suicide attempt rates for patients treated with an AED (13 per 1000 person-years [PY]) vs patients not treated with an AED or lithium (13 per 1000 PY). In AED-treated subjects, the rate of suicide attempts was significantly higher before treatment (72 per 1000 PY) than after (13 per 1000 PY). In patients receiving no concomitant treatment with an antidepressant, other AED, or antipsychotic, AEDs were significantly protective relative to no pharmacologic treatment (3 per 1000 vs 15 per 1000 PY). CONCLUSIONS: Despite Food and Drug Administration reports regarding increased risk of suicidality associated with AED treatment, the current study reveals that, as a class, AEDs do not increase risk of suicide attempts in patients with bipolar disorder relative to patients not treated with an AED or lithium. Use of AEDs reduces suicide attempt rates both relative to patients not receiving any psychotropic medication and relative to their pretreatment levels. Objective. This study evaluates changes in use of antidepressants in children and adolescents after the US Food and Drug Administration black box warning for increased risk of suicide.Method. A retrospective chart review was completed for children and adolescents (ages 4-17) who were diagnosed with depressive or anxiety disorders in an outpatient clinic and offered a trial of antidepressants between September 2003 and February 2004 (before the black box warning) and between January 2005 and June 2005 (after the black box warning). Statistical analyses were performed with the SPSS version 17 and R package version 2.9.1. Univariate analysis was conducted using the Fisher's Exact test.Results. The odds ratio calculated for the different groups suggests that in all the groups, the proportion of acceptance of antidepressant use was greater before the black box warning as compared to after the black box warning (odds ratio>1). It was also found that upon combining the age groups after the warning and comparing them, based on the diagnoses, there was a greater degree of refusal of antidepressant therapy when a diagnosis of anxiety disorder was made as compared to a diagnosis of depressive disorder (p=0.017).Conclusion. There has been a decrease in the use of antidepressant therapy in children and adolescents following the US Food and Drug Administration black box warning for risk of suicide. A limitation of this study is that reasons for refusal of antidepressent therapy by parents or guardians of children and adolescents were not collected; therefore, there is no certainty that the black box warning was the primary reason for refusal. OBJECTIVES: To study prescribing trends for antidepressants in Hawai'i following the FDA black box warning regarding the possible risk of suicide in children and adolescents. We also explored relationships between changes in prescribing trends and patient and provider characteristics. STUDY DESIGN: Analysis of an existing insurance data set of prescriptions to children and adolescents within the State of Hawai'i. STUDY POPULATION: Children and adolescents under 18-years-old insured through the largest (>60%) third-party insurance company in the state. RESULTS: Our results showed variations in changes in prescribing trends for different selective serotonin reuptake inhibitors (SSRIs) following the FDA black box warning. SSRIs with more evidence-based research supporting their safety and efficacy were least affected as were those that were less implicated by the FDA analysis of the possible link between SSRIs and Suicidality. Trends were apparent for all age groups examined and for both females and males. CONCLUSIONS: Changes in prescribing patterns of psychiatric medications for children and adolescents in Hawai'i were identified. Differing patterns have evolved since 2003 following the series of concerns raised regarding SSRIs and suicidality in children and adolescents. OBJECTIVE: Isotretinoin (13-cis-retinoic acid), approved by the US Food and Drug Administration for the treatment of acne, carries a black box warning related to the risk of depression, suicide, and psychosis. Retinoic acid, the active form of vitamin A, regulates gene expression in the brain, and isotretinoin is its 13-cis isomer. Retinoids represent a group of compounds derived from vitamin A that perform a large variety of functions in many systems, in particular the central nervous system, and abnormal retinoid levels can have neurologic effects. Although infrequent, proper recognition and treatment of psychiatric side effects in acne patients is critical given the risk of death and disability. This article reviews the evidence for isotretinoin's relationships with depression and suicidality. DATA SOURCES: The PsycINFO, MEDLINE, and PubMed searchable database indexes were searched for articles published in the English language from 1960 to June 2010 using the key words isotretinoin, retinoids, retinoic acid, depression, depressive disorders, and vitamin A. Evidence examined includes (1) case reports; (2) temporal association between onset of depression and exposure to the drug; (3) challenge-rechallenge cases; (4) class effect (other compounds in the same class, like vitamin A, having similar neuropsychiatric effects); (5) dose response; and (6) biologically plausible mechanisms. STUDY SELECTION: All articles in the literature related to isotretinoin, depression, and suicide were reviewed, as well as articles related to class effect, dose response, and biologic plausibility. DATA EXTRACTION: Information from individual articles in the literature was extracted, including number of episodes of depression, suicidality, suicide, psychosis, violence and aggression, past psychiatric history, time of onset in relation to isotretinoin usage, medication dosage, duration of treatment, and dechallenge and challenge history. RESULTS: The literature reviewed is consistent with associations of isotretinoin administration with depression and with suicide in a subgroup of vulnerable individuals. CONCLUSIONS: The relationship between isotretinoin and depression may have implications for a greater understanding of the neurobiology of affective disorders. Depending on symptom severity, psychopharmacological treatment can be a valuable option in the treatment of depressive disorders in childhood and adolescence. This review provides recommendations for clinical treatment, focusing on suicidality and treatment-resistant patients. The quality of studies regarding the psychopharmacological therapy of depressive disorders in childhood and adolescence has improved since the «black box» warning of the FDA concerning the occurrence of suicidality under treatment with selective serotonin reuptake inhibitors (SSRIs). In Germany, there is proof for a trend toward a more evidence-based psychopharmacological treatment approach within recent years. Depression represents a huge pharmaceutical market opportunity. There are approximately 350 million people worldwide with depression, and it is the leading cause of disability in the world. In the U.S., 9.1% of the population suffers from depression. Globally, fewer than half of depression sufferers receive treatment for their illness, and in some countries this figure falls to fewer than 1 in 10. The high incidence rate, combined with limited market penetration, makes depression a high potential market for pharmaceuticals. However, companies developing drugs for depression also face a number of serious challenges. Psychosocial treatment options remain the preferred first-line therapy ahead of medication-and when it comes to drug treatment, the abundance of generic options available has significantly contributed to halving the value of the branded antidepressant market over recent years. Another hurdle faced by new drugs is the requirement that all antidepressants carry a black-box warning regarding the increased risk of suicide in children, adolescents and young adults, which limits their use in this population. Switching between medications presents both an opportunity and a challenge, as a significant number of patients will switch away from their first medication within the first year of treatment. The lack of complete understanding of why depression occurs also makes this area a difficult one, although it opens the door for the development of drugs with novel mechanisms of action. Suicide is one of the major causes of morbidity and mortality amongst children and adolescents. In 2004 the Food and Drug Administration (FDA) issued a "black-box" warning for antidepressants in children and adolescents, stating that these drugs may increase suicidality, a term encompassing both suicidal thoughts and behavior, especially in the first few weeks of treatment. The warning was extended in 2007 to antidepressants prescribed to adults aged 25 and under. The evidence behind this decision stemmed from meta-analyses of antidepressant clinical trials that demonstrated a slight increase in suicidality in those receiving antidepressants versus those treated with a placebo. Due to methods of this pooled data compilation, the relationship between antidepressants and suicidality remains controversial. This report investigates a case where a 14 year old with major depressive disorder (MDD) developed suicidal ideation shortly after being prescribed a selective serotonin reuptake inhibitor (SSRI). Investigating the role antidepressants may play in suicidality suggests the need to explore the neurobiological mechanisms within the serotonin system. This case and its theoretical explanations attempt to bridge the gap between neurobiology and pharmacology in order to better delineate the etiology of this adverse effect. Epidemiological data suggests suicide is uncommon in childhood but becomes an extremely serious issue among adolescents.Several risk factors have been identified and include the presence of psychiatric illness, a previous suicide attempt, family factors, substance abuse, sexual and physical abuse, disorders in gender identity or bullying. Pediatricians have a primary role in searching for these risk factors, recognizing them and acting synergistically with other specialists to prevent and treat suicidal behavior.Pediatricians should also be able to identify the "warning signs" for suicide since their presence implies a need for immediate action, as attempted suicide may occur in a few hours or days.The use of antidepressant drugs and its association with suicidal risk in pediatric age is another topic of ongoing debate. Food and Drug Administration has recently introduced the so-called "black box" on antidepressants' packages with the aim of gaining attention to the possible risk of suicide among adolescents who are treated with antidepressants, with a warning that the risk of suicide is higher when starting a therapy or while adjusting its dosage. The decision made in the year 2004 by the U.S. Food and Drug Administration (FDA) to require a boxed warning on antidepressants regarding the risk of suicidality in young adults still represents a matter of controversy. The FDA warning was grounded on industry-sponsored trials carried one decade ago or earlier. However, within the past decade, an increasing number of reports have questioned the actual validity of the FDA warning, especially considering a decline in the prescription of the antidepressant drugs associated with an increase in the rate of suicidal events among people with severe depression. The present report provides an overview of the FDA black box warning, also documenting two Major Depressive Disorder patients whose refusal to undergo a pharmacological antidepressant treatment possibly led to an increased risk for suicidal behaviors. The concerns raised by the FDA black box warning need to be considered in real-world clinical practice, stating the associated clinical and public health implications. Publisher: Zusammenfassung. Die Diskussion über das Auftreten von suizidalen Symptomen bzw. Suizid unter einer Antidepressivatherapie ist bereits jahrzehntealt. Sowohl die Fachwelt als auch die Laienpresse setzte sich mit dem Thema immer wieder kritisch auseider. Die vorliegende Arbeit gibt einen Überblick über die aktuelle Evidenzlage: Zurzeit bestehen keine Hinweise für eine Erhöhung des Suizidrisikos. Hingegen wurde insbesondere bei Kindern, Jugendlichen und jungen Erwachsenen zu Beginn der Therapie eine höhere Rate an Suizidalitätssymptomen (Suizidgedanken, Suizidhandlungen) beobachtet. Diese Symptome waren gegenüber der Periode vor Einsetzen der Antidepressivatherapie nicht erhöht und nehmen im Verlauf der Therapie kontinuierlich ab. Zu Beginn der Behandlung ist deshalb eine besonders intensive Therapieführung nötig. Allgemein gilt, dass eine unterlassene Therapie einer Depression das grösste Risiko für einen Suizid darstellt. The United States Food and Drug Administration issued a Black Box warning in October 2004 after placebo-controlled trials of antidepressant medications found an increased risk of suicidal thoughts and behaviors among children and adolescents taking antidepressant medications relative to placebo. Subsequently, some researchers have concluded that the Black Box warning caused severe unintended consequences; specifically, they have argued that the warning led to reduced use of antidepressants among youth, which led to more suicides. In this paper, we critically examine research regarding the Black Box warning's alleged deleterious consequences. One study claimed that controlled trials did not actually find an increased risk of suicidality among youth taking fluoxetine relative to those taking placebo, but its measure of suicidality is likely invalid. We found that ecological time series studies claiming that decreasing antidepressant prescriptions are linked to higher rates of suicide attempts or actual suicides among youth were methodologically weak. These studies exhibited shortcomings including: selective use of time points, use of only a short-term time series, lack of performing statistical analysis, not examining level of severity/impairment among participants, inability to control confounding variables, and/or use of questionable measures of suicide attempts. Further, while some time-series studies claim that increased antidepressant prescriptions are related to fewer youth suicides, more recent data suggests that increasing antidepressant prescriptions are related to more youth suicide attempts and more completed suicides among American children and adolescents. We also note that case-control studies show increased risk of suicide attempts and suicide among youth taking antidepressants, even after controlling for some relevant confounds. As clinical trials have the greatest ability to control relevant confounds, it is important to remember such trials demonstrated increased risk of suicidality adverse events among youth taking antidepressants. The Black Box warning is firmly rooted in solid data whereas attempts to claim the warning has caused harm are based on quite weak evidence. In 2004, the US Food and Drug Administration (FDA) controversially issued a black box warning that antidepressants were associated with an increased risk of suicidal thoughts and behaviours in people aged under 18 years. In 2007, the warning was expanded to include young adults aged under 25 years. In 2005, the Australian Therapeutic Goods Administration responded to the FDA warning by requiring Product and Consumer Information leaflets to be updated to reflect the risk. However, there was considerable debate, and at times emotive backlash, in academic journals and the international media. Prominent US and Australian mental health organisations and psychiatrists challenged the FDA warning. They argued that, on balance, antidepressant use was likely to reduce the risk of suicide. Several ecological studies were cited misleadingly as evidence that decreasing antidepressant use increases suicide risk. From 2008 to 2018, Australian per-capita child, adolescent and young adult antidepressant dispensing (0-27 years of age) and suicide (0-24 years) rates have increased approximately 66% and 49%, respectively. In addition, there was a 98% increase in intentional poisonings among 5 to 19 year-olds in New South Wales and Victoria between 2006 and 2016, with substantial overlap between the most commonly dispensed psychotropics and the drugs most commonly used in self-poisoning. These results do not support claims that increased antidepressant use reduces youth suicide risk. They are more consistent with the FDA warning and the hypothesis that antidepressant use increases the risk of suicide and self-harm by young people. Causal relationships cannot be established with certainty until there is a vast improvement in post-marketing surveillance. However, there is clear evidence that more young Australians are taking antidepressants, and more young Australians are killing themselves and self-harming, often by intentionally overdosing on the very substances that are supposed to help them.

What nerve is affected in Carpel Tunnel syndrome?

Carpel tunnel syndrome (CTS) is a condition in which median nerve compression results in paresthesias and pain in the wrist and hand.

Sensory conduction of the median nerve at the carpal tunnel for eight consecutive 1 cm segments of the nerve was evaluated in 217 hands of 153 of our patients with carpal tunnel syndrome. Impairment was found to be highly focal and often confined to a single 1 cm segment of the nerve. The section of the nerve at or just distal to the distal margin of the carpal tunnel was affected most frequently, the section within the tunnel was affected less often, and the section proximal to the tunnel at the level of the mid-carpal and radio-carpal joints was affected least. The greatest contrast between frequencies of slowing at adjacent segments occurred at the proximal and distal margins of the carpal tunnel. The distribution of the nerve impairment was similar between the sexes; however, among the men the segment affected most frequently was located 1 cm distal to the segment affected most frequently among the women. The general pattern of slowing which we found does not substantiate some commonly-held opinions about the aetiology of carpal tunnel syndrome. Motor axons supplying lumbrical muscles are less severely affected than axons supplying thenar muscles in the carpal tunnel syndrome; sometimes lumbrical motor fibers are less affected than digit 2 sensory fibers. This pattern is consistent with compression of both the anterior and posterior aspects of the median nerve in the carpal tunnel because nerve fibers responsible for thenar, lumbrical, and digit 2 functions lie in an anterior-posterior gradient within the distal median nerve. Recognition of lumbrical sparing supports the electrodiagnosis of carpal tunnel syndrome when the distal latency to thenar muscles or the palm-to-wrist mixed median nerve conduction velocity is normal. The carpal tunnel, a narrow space closed distally by the anterior annular ligament of the wrist, and containing flexor tendons and the median nerve, is the most frequent site of tunnel syndromes, compression in the canal leading to medium nerve lesion. The carpal tunnel syndrome usually affects women aged between 40 and 60 years, and presents typically as parasthesia of the fingers, mainly at night, in the regions served by the median nerve, sometimes associated with hypoesthesia and difficulty in movements. Motor disorders, particularly affecting the thumb, occur during the advanced stages. Electrical tests may confirm diagnosis and enable assessment of severity. More than half the cases are idiopathic in nature, presenting as hypertrophy of the annular ligament and fibrous thickening in the canal, but other forms may be observed including those due to wrist injuries, anatomical anomalies, rheumatic affections, or tumours. Associated disorders may be Dupuytren's disease, cubital nerve compression in Guyon's canal, or nodular tendinitis leading to a trigger finger. Surgical treatment is simple and should be employed when medical measures fail. The nerve should therefore be liberated if parasthesia persists after two or three local corticoid infiltrations. After a wide exploratory incision, the nerve is freed along the total length of the canal and up to the distal extremity of the forearm. Results are excellent, 98 p. cent of patients being relieved of their pain. Persistent motor disorders require surgical intervention before amyotrophy and muscle weakness develops. The declining capacity for injured peripheral nerves to regenerate their axons with time and distance is accounted for, at least in part, by the chronic axotomy of the neurons and Schwann cell denervation prior to target reinnervation. A largely unrecognized site of delay is the surgical suture site where, in rats, 4 weeks is required for all neurons to regenerate their axons across the site. Low frequency stimulation for just 1 h after surgery accelerates this axon crossing in association with upregulation of neurotrophic factors in the neurons. We translated these findings to human patients by examining the number of reinnervated motor units in the median nerve-innervated thenar muscles before and after carpel tunnel release surgery in a randomized controlled trial. Motor unit number estimates (MUNE) in patients with moderate and severe carpal tunnel syndrome were significantly lower than normal. This number increased significantly by 6-8 months after surgery and reached normal values by 12 months in contrast to a non-significant increase in the control unstimulated group. Tests including the Purdue Pegboard Test verified the more rapid functional recovery after stimulation. The data indicate a feasible strategy to promote axonal regeneration in humans that has the potential to improve functional outcomes, especially in combination with strategies to sustain the regenerative capacity of neurons and the support of Schwann cells over distance and time. INTRODUCTION: Carpal tunnel syndrome is a neuropathy caused by compression of the median nerve within the carpal tunnel. However, the severity of symptoms and signs does not often correlate well with the extent of nerve damage. METHODS AND OUTCOMES: We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of drug treatments, non-drug treatments, surgical treatments, and postoperative treatments for carpal tunnel syndrome? We searched: Medline, Embase, The Cochrane Library and other important databases up to December 2006 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). RESULTS: We found 54 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. CONCLUSIONS: In this systematic review we present information relating to the effectiveness and safety of the following interventions: acupuncture; diuretics; carpal tunnel release surgery (open, and endoscopic); internal neurolysis; local and systemic corticosteroids; massage therapy; nerve and tendon gliding exercises; non-steroidal anti-inflammatory drugs; pyridoxine; therapeutic ultrasound; and wrist splints. Electrical stimulation (ES) of injured peripheral nerves accelerates axonal regeneration in laboratory animals. However, clinical applicability of this intervention has never been investigated in human subjects. The aim of this pilot study was to determine the effect of ES on axonal regeneration after surgery in patients with median nerve compression in the carpal tunnel causing marked motor axonal loss. A randomized control trial was conducted to provide proof of principle for ES-induced acceleration of axon regeneration in human patients. Carpel tunnel release surgery (CTRS) was performed and in the stimulation group of patients, stainless steel electrode wires placed alongside the median nerve proximal to the surgical decompression site for immediate 1 h 20 Hz bipolar ES. Subjects were followed for a year at regular intervals. Axonal regeneration was quantified using motor unit number estimation (MUNE) and sensory and motor nerve conduction studies. Purdue Pegboard Test, Semmes Weinstein Monofilaments, and Levine's Self-Assessment Questionnaire were used to assess functional recovery. The stimulation group had significant axonal regeneration 6-8 months after the CTRS when the MUNE increased to 290+/-140 (mean+/-SD) motor units (MU) from 150+/-62 MU at baseline (p<0.05). In comparison, MUNE did not significantly improve in the control group (p>0.2). Terminal motor latency significantly accelerated in the stimulation group but not the control group (p>0.1). Sensory nerve conduction values significantly improved in the stimulation group earlier than the controls. Other outcome measures showed a significant improvement in both patient groups. We conclude that brief low frequency ES accelerates axonal regeneration and target reinnervation in humans. OBJECTIVES: To provide a quantitative analysis of ultrasonographic measurements and possible pathophysiology of carpal tunnel syndrome by comparing cross-sectional areas of the median nerve, carpal tunnel, and nerve/tunnel index and the difference in ultrasonographic findings between affected and nonaffected hands and between sexes. DESIGN: Blinded comparison study. SETTING: Secondary referral and training hospital of institutional practice. PARTICIPANTS: Patients (N=51; 42 women, 9 men) with suspected carpal tunnel syndrome who underwent sonography within 1 week after the electrodiagnostic study. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Electrodiagnostic and ultrasonographic studies were conducted on both upper extremities. Cross-sectional areas of the median nerve and carpal tunnel were measured at 2 separate levels; proximal and distal cross-sectional areas of the carpal tunnel were each measured at the scaphoid-pisiform and trapezium-hamate levels, respectively. RESULTS: Comparison between normative (n=24) and abnormal hands (n=78) revealed the following: the mean proximal cross-sectional areas of the median nerve, carpal tunnel, and nerve/tunnel index of electrodiagnostically normative hands were 10.941mm(2), 192.43mm(2), and 5.635%, respectively, whereas those of abnormal hands were 13.74mm(2), 208.87mm(2), and 6.693%, respectively, showing statistically significant differences for all (P<.05). Distal measurements of the cross-sectional area of the median nerve, carpal tunnel, and nerve/tunnel index were 10.088mm(2), 150.4mm(2), and 6.762%, respectively, in normative hands, and 11.178mm(2), 149.6mm(2), and 7.493%, respectively, in abnormal hands, showing no statistically significant differences (P>.05). In women, proximal cross-sectional areas of the median nerve and nerve/tunnel index of abnormal hands showed statistically significant differences, but no ultrasonographic measurement with a statistically significant difference was observed in men. CONCLUSIONS: Compared with nonaffected hands, the proximal cross-sectional areas of the median nerve and carpal tunnel were greater, but the distal ultrasonographic measurements were not in affected hands. Ultrasonographic findings of carpal tunnel syndrome were different according to sex. Carpal tunnel syndrome is the most common entrapment neuropathy, affecting the median nerve at the wrist. Acupuncture is a minimally-invasive and conservative therapeutic option, and while rooted in a complex practice ritual, acupuncture overlaps significantly with many conventional peripherally-focused neuromodulatory therapies. However, the neurophysiological mechanisms by which acupuncture impacts accepted subjective/psychological and objective/physiological outcomes are not well understood. Eligible patients (n = 80, 65 female, age: 49.3 ± 8.6 years) were enrolled and randomized into three intervention arms: (i) verum electro-acupuncture 'local' to the more affected hand; (ii) verum electro-acupuncture at 'distal' body sites, near the ankle contralesional to the more affected hand; and (iii) local sham electro-acupuncture using non-penetrating placebo needles. Acupuncture therapy was provided for 16 sessions over 8 weeks. Boston Carpal Tunnel Syndrome Questionnaire assessed pain and paraesthesia symptoms at baseline, following therapy and at 3-month follow-up. Nerve conduction studies assessing median nerve sensory latency and brain imaging data were acquired at baseline and following therapy. Functional magnetic resoce imaging assessed somatotopy in the primary somatosensory cortex using vibrotactile stimulation over three digits (2, 3 and 5). While all three acupuncture interventions reduced symptom severity, verum (local and distal) acupuncture was superior to sham in producing improvements in neurophysiological outcomes, both local to the wrist (i.e. median sensory nerve conduction latency) and in the brain (i.e. digit 2/3 cortical separation distance). Moreover, greater improvement in second/third interdigit cortical separation distance following verum acupuncture predicted sustained improvements in symptom severity at 3-month follow-up. We further explored potential differential mechanisms of local versus distal acupuncture using diffusion tensor imaging of white matter microstructure adjacent to the primary somatosensory cortex. Compared to healthy adults (n = 34, 28 female, 49.7 ± 9.9 years old), patients with carpal tunnel syndrome demonstrated increased fractional anisotropy in several regions and, for these regions we found that improvement in median nerve latency was associated with reduction of fractional anisotropy near (i) contralesional hand area following verum, but not sham, acupuncture; (ii) ipsilesional hand area following local, but not distal or sham, acupuncture; and (iii) ipsilesional leg area following distal, but not local or sham, acupuncture. As these primary somatosensory cortex subregions are distinctly targeted by local versus distal acupuncture electrostimulation, acupuncture at local versus distal sites may improve median nerve function at the wrist by somatotopically distinct neuroplasticity in the primary somatosensory cortex following therapy. Our study further suggests that improvements in primary somatosensory cortex somatotopy can predict long-term clinical outcomes for carpal tunnel syndrome. Carpel tunnel syndrome (CTS) is a condition in which median nerve compression results in paresthesias and pain in the wrist and hand. We are going to report a rare case of topiramate-induced neuropathy which clinically resembles CTS. Discontinuation of topiramate resulted in spontaneous resolution of numbness, paresthesia and pain in a few days. High clinical suspicion is advised in patients who are on topiramate and present with signs of compressive neuropathy. Level of evidence: V. BACKGROUND: Carpal tunnel syndrome refers to a constellation of symptoms resulting from compression of the median nerve at the wrist. The characteristic symptoms include pain and numbness in the hands. AIM: To identify the risk factors responsible for carpal tunnel syndrome, to identify the symptom severity as well as functional status of patients with carpal tunnel syndrome, and to determine the relationship between symptom severity and functional status among patients with carpal tunnel syndrome. SETTINGS AND DESIGN: A non-experimental descriptive study was conducted. MATERIALS AND METHODS: A semi-structured questionnaire was administered to assess the risk factors. Standardized questionnaires included the symptom severity scale (SSS) and functional status scale (FSS). RESULTS: The risk factors assessed were the female gender (64%), premorbidities (75%), diabetes mellitus (53%), hypertension (25%), dyslipidemia (24%), osteoarthritis (8%), and impaired thyroid functions (10%). Fifty-one patients were overweight and 8 were obese. There was an association between symptom severity and presence of any of the premorbidities (χ2 = 5.80; P < 0.05). There was also an association between symptom severity and diabetes mellitus (χ2 = 13.62; P < 0.05). A positive correlation was also noted between the symptom severity and the functional status of patients with carpal tunnel syndrome (r = 0.705; P = 0.00). CONCLUSIONS: Prompt recognition, timely management, and avoidance of risk factors responsible for the manifestations of carpal tunnel syndrome have practical implications in the treatment of carpal tunnel syndrome. L’objectif de cette étude est d’évaluer l’utilité de l’échographie dans le diagnostic de syndrome du canal carpien par rapport à l’électroneuromyogramme (ENMG). Il s’agit d’une étude transversale incluant 50 patients sur une période de 6 mois. Tous les patients avaient bénéficié d’une échographie avec mesure de la surface du nerf médian à l’entrée du canal carpien et une étude électroneuromyographique des 2 poignets. La sensibilité et la spécificité de l’échographie ont été comparées à ceux de l’ENMG comme référence. L’âge moyen est de 49,6 ans avec une nette prédomice féminine dans 98%. La majorité des patientes étaient des femmes au foyer. Les paresthésies étaient le motif le plus fréquent de consultation dans 86%. La symptomatologie clinique était bilatérale dans 78% des cas. L’ENMG était pathologique chez 89 poignets (89%). L’échographie était anormale chez 63 poignets (63%) avec une médiane de la surface du nerf médian de 11 mm2. D’après cette étude, la sensibilité d’échographie était de 70%, et la spécificité était de 100% avec une valeur prédictive positive (VPP) de 100% et une valeur prédictive négative (VPN) de 29,7%. On a conclu que l’échographie n’est sensible que pour le syndrome de canal carpien avec une atteinte sévère à l’ENMG. The most common etiology of carpal tunnel syndrome (CTS) is idiopathic. However, secondary causes of CTS should be considered when symptoms are unilateral, or electrodiagnostic studies are discrepant with the clinical presentation. Imaging of the carpal tunnel should be performed when secondary causes of CTS are suspected. An ultrasound evaluation of the carpal tunnel can assess for pathologic changes of the median nerve, detect secondary causes of CTS, and aid in surgical planning.

What drug, used to treat rheumatoid arthritis, is an interleukin-1 receptor antagonist?

Anakinra is an oral interleukin-1 receptor antagonist that is used to treat rheumatoid arthritis.

Anakinra, a recombit human interleukin-1 (IL-1) receptor antagonist, is the first biological agent approved to block the pro-inflammatory effects of IL-1 in patients with rheumatoid arthritis. In a double-blind, randomised trial in 472 patients with active, severe or very severe rheumatoid arthritis, recipients of subcutaneous anakinra 150 mg/day achieved higher response rates [assessed using the American College of Rheumatology (ACR) composite score] and accumulated more mean productivity days after 6 months than placebo recipients. However, the response rates and accumulated productivity days of patients receiving subcutaneous anakinra 30 or 75 mg/day for 6 months were similar to those of placebo. With respect to the total Get radiographic scores, the same study showed that all anakinra treatment regimens slowed disease progression after 6 months to a greater extent than placebo. In double-blind, randomised trials in patients with rheumatoid arthritis, combined treatment with anakinra and methotrexate was associated with higher ACR 20, 50 and 70 response rates than with methotrexate alone. Anakinra, used alone or in combination with methotrexate, was generally well tolerated, with the most frequent adverse event being a mild injection-site reaction of transient duration. Infections requiring antibacterial therapy or hospitalisation occurred more commonly in anakinra recipients than in placebo recipients, but were a rare cause for discontinuation of anakinra therapy (approximately 1%) in clinical trials. PURPOSE: Interleukin -1 receptor antagonist ( IL-1Ra ) is a new option among biotherapies against rheumatoid arthritis ( RA ). THE AIM: of this review is to recall the rationale of use of IL-1Ra and to analyse the results available in the current literature. CURRENT KNOWLEDGE AND KEY POINTS: Pathophysiological data of RA give a specific position for IL-1 as a potential target for immunotherapy in this disease, confirmed in animal models. Phase II and III studies with IL-1Ra (Anakinra) demonstrated clinical efficacy versus placebo (42% responders in ACR 20 in Anakinra + methotrexate, and 23% in the placebo + methotrexate group at 24 weeks) and a structural effect (slowing of radiological progression at six months). Anakinra has obtained an European license and is indicated in RA not controlled by methotrexate, in daily subcutaneous administration (100 mg/day), in combination with methotrexate. Tolerance is fair; the most frequent side effect is represented by injection site reactions. FUTURE PROSPECTS AND PROJECTS: This ambulatory biotherapy offers new perspectives in combination with other slow acting drugs as well as biologic agents such as anti-TNF, currently under evaluation. Anakinra, a recombit human interleukin-1 receptor antagonist offers a new potent treatment for rheumatoid arthritis(RA). It is administered as a daily single subcutaneous injection. Recent randomized, double-blind, placebo-controlled trials revealed that anakinra significantly reduces the signs and symptoms of RA, reduces joint destruction, and is safe and tolerated. It was also revealed that anakinra is more potent when used in combination with methotrexate. Interleukin-1 (IL-1) is one of the main inflammatory mediators associated with development of rheumatoid arthritis (RA). Deficiency in secretion of the natural IL-1 antagonist was found in RA patients, and enhanced joint inflammation. This finding was confirmed by numerous studies using the animal model of the disease. Two randomized, multicenter clinical trials with anakinra, a recombit IL-1 receptor antagonist (rHHHuIL-1Ra), revealed that application of anakinra with or without methotrexate induces remission (ACR 20) in 38-71% of patients with RA. Induction of remission was dose and treatment-time-dependent. The present paper reviews theoretical application of rHuIL-1Ra in patients with RA and summarizes results of published clinical trials of the drug. New treatment strategies in rheumatoid arthritis are targeted to interfere with critical mediators of inflammation. Proinflammatory cytokines like IL-1 beta and TNFalpha play a crucial role in induction and maintece of synovitis, pannus formation and bone and cartilage destruction. Within a few years, these morphological changes may lead to joint destruction and consecutively to functional impairment. Since April 2002 a recombit human interleukin-1 receptor antagonist (Anakinra) is available in Germany for treatment of patients with rheumatoid arthritis. Anakinra (Kineret(R)) is approved for therapy in combination with methotrexate and should be applied according to guidelines established by the German Rheumatology Society for the use of biologicals in treatment of patients with rheumatoid arthritis. The approval of anakinra as a new therapeutic is based on data obtained in large multicenter, placebo-controlled, and randomised trials in comparison to placebo. Treatment of Anakinra as monotherapy or in combination with methotrexate lead to significant improvement of signs and symptoms of disease as measured by the ACR 20 (or more) response and was associated with a slower radiographic progression with regard to joint space narrowing and development of erosions. Anakinra showed a favourable safety profile with injection side reactions as the predomit side effect that occurs in 70% of patients usually after 10-12 days of treatment and that are mostly mild to moderate and self-limiting. Patients with previous pneumonia or other risk factors for pulmonary infections such as chronic obstructive lung disease seem to show a slightly increased risk of developing infectious complications of the bronchopulmonary system while being on anakinra and should be monitored appropriately. Combining IL-1ra treatment with the use of anti-TNF agents showed an increased risk of infectious complications in clinical studies and is not recommended at present. Studies are currently assessing the use of anakinra for treatment of other rheumatic diseases like psoriatic arthritis, juvenile arthritis or spondylarthropathy. Anakinra, the recombit form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret trade mark, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E2 production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1beta. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombit human IL-1beta either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1beta produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombit protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombit protein became progressively less effective. This was particularly evident under conditions of continuous IL-1beta synthesis. Anakinra is a recombit human interleukin-1 receptor antagonist (IL-1ra) recently approved by the FDA as a new therapy for patients with rheumatoid arthritis. Four clinical trials have been completed which have demonstrated that anakinra is an effective anti-rheumatic therapy either used alone or in combination with methotrexate. The most frequent adverse events reported in the clinical trials are injection-site reactions which are generally mild to moderate and rapidly resolve. A large, prospective safety study which allowed a wide-variety of comorbid conditions and concomitant medications demonstrated that anakinra therapy is a well-tolerated treatment for rheumatoid arthritis in the patient population seen by the practicing rheumatologist. Unlike therapies designed to affect TNF-alpha, there have not yet been reports of the development of tuberculosis or other fungal infections, demylinating syndromes or worsening of congestive heart failure. The safety profile of etanercept and infliximab were similar to that of anakinra in the phase I-phase III clinical trials. Unlike anakinra, these medications were not studied in the usual rheumatoid arthritis population which includes a number of patients with a wide variety of co-morbid disease and utilizing a number of concomitant anti-rheumatic medications. Post approval, several safety concerns, including patients at risk for serious infection and the emergence of latent tuberculosis and other opportunistic infections have emerged with the use of anti TNF therapy. OBJECTIVE: To assess the efficacy and safety of 100 mg daily anakinra (Kineret), a recombit form of the naturally occurring interleukin 1 receptor antagonist, plus methotrexate (MTX) in reducing the signs and symptoms of rheumatoid arthritis (RA). METHODS: Patients with active RA (n = 506) despite current treatment with MTX were enrolled in this multicentre, double blind, randomised, placebo controlled study. Patients received subcutaneous injections of anakinra 100 mg/day or placebo. They were assessed monthly for 6 months for improvement in signs and symptoms of RA and for adverse events. The primary efficacy measure was the percentage of patients attaining ACR20 response at week 24. RESULTS: Significantly greater proportions of patients treated with anakinra compared with placebo achieved ACR20 (38% v 22%; p<0.001), ACR50 (17% v 8%; p<0.01), and ACR70 (6% v 2%; p<0.05) responses. The response to anakinra was rapid; the proportion of patients with an ACR20 response at the first study assessment (4 weeks) was twice as high with anakinra as with placebo (p<0.005). Clinically meaningful and statistically significant responses were also seen in individual components of the ACR response (for example, Health Assessment Questionnaire, pain, C reactive protein levels, and erythrocyte sedimentation rate). Anakinra was well tolerated, with a safety profile, similar to that of placebo with one exception: mild to moderate injection site reactions were more common with anakinra than with placebo (65% v 24%). CONCLUSIONS: This study confirms previous observations from a dose-ranging study showing that anakinra, in combination with MTX, is an effective and safe treatment for patients with RA who have inadequate responses to MTX alone. BACKGROUND: The efficacy and safety of anakinra, a recombit human interleukin 1 (IL1) receptor antagonist used in rheumatoid arthritis, has been documented in five randomised controlled studies. However, long term post-marketing efficacy data are lacking. OBJECTIVE: To evaluate the efficacy, safety, and drug survival of anakinra in clinical practice. METHODS: All patients with rheumatoid arthritis who started anakinra in six hospitals between May 2002 and February 2004 were included in a two year prospective, in part retrospective, cohort study. Efficacy was assessed using the 28 joint disease activity score (DAS28) and the EULAR response criteria. Safety was evaluated using the common toxicity criteria. Drug survival and prognostic factors were analysed using Kaplan-Meier and Cox proportional hazard analyses. RESULTS: After three months, 55% of the patients (n = 146) showed a response (43% moderate, 12% good). A subset of patients continuing anakinra after 18 months had a sustained clinical response compared with patients who switched to other disease modifying antirheumatic drug treatment (DAS28 improvement, 2.46 v 1.79). Drug survival was 78%, 54%, and 14% after three, six, and 24 months, respectively. The reason for discontinuation was lack of efficacy in 78% and adverse events in 22%. Except for higher drug survival in women (odds ratio = 0.51, 95% confidence interval, 0.27 to 0.97), no prognostic factors were found. Adverse events were reported 206 times in 111 patients, the most common being injection site reactions (36%). Serious adverse events occurred in 12% of the patients, with one classified as related. CONCLUSIONS: The short term efficacy and safety profile of anakinra are comparable to those found in randomised clinical studies. However, the drug survival of anakinra after two years is low, mostly because of lack of efficacy. Anakinra is a specific receptor antagonist of interleukin-1 that differs from naturally occurring interleukin-1 receptor antagonist by the presence of a methionine group. It is administered by daily subcutaneous injection. Anakinra improves the clinical signs and symptoms of rheumatoid arthritis, slows radiographic progression and improves patient physical function. The most common adverse event is an injection site reaction. Anakinra has been associated with an increased incidence of serious infections and has an increased standardized incidence ratio for lymphoma. It has not been found to be associated with the development of opportunistic infections, worsening of congestive heart failure or the development of demyelinating disease. It appears to be effective in treating adult Stills disease, systemic-onset juvenile idiopathic arthritis and chronic infantile neurological cutaneous and articular syndrome (also known as neonatal-onset multisystem inflammatory disease syndrome). Impaired healing of the skin is a notable cause of patient morbidity and mortality. In diabetic individuals, dysregulated inflammation contributes to delayed wound healing. Specific immunomodulatory agents may have a role in the treatment of diabetic wounds. One of these molecules is interleukin-1 receptor antagonist (Anakinra; Amgen Corp.). Although interleukin-1 receptor antagonist (Anakinra; Amgen Corp.) is approved by the Food and Drug Administration (FDA) for the treatment of rheumatoid arthritis and neonatal-onset multisystem inflammatory disease, little is known about the local use this drug in cutaneous wound healing. Therefore, the aim of this study is to determine the effect of locally administered interleukin-1 receptor antagonist on delayed wound healing, specifically, in a diabetic mouse model. Two 6-mm full-thickness wounds were created on the dorsa of diabetic (db/db) mice and stented. One-hour postwounding, wound margins were subcutaneously injected with either (1) low-dose interleukin-1 receptor antagonist in a gelatin-transglutaminase gel vehicle or (2) the gel vehicle only. Wounds were imaged on days 0, 7, 14, and 21 postwounding, and wound area was determined. Wound biopsies were collected on day 21 and immunohistochemically stained for neutrophil and macrophage infiltration. Wounds treated with interleukin-1 receptor antagonist had significantly smaller wound area than nontreated wounds on day 7 and day 14 postwounding. Treated wounds also showed significantly less neutrophil and macrophage infiltration. These findings support the hypothesis that interleukin-1 receptor antagonist may have an important role in cutaneous wound healing, possibly by promoting successful resolution of acute inflammation and hence accelerating wound closure. Thereby, administration of IL-1Ra may be useful in the treatment of nonhealing wounds. BACKGROUND: Anakinra, an interleukin-1 receptor antagonist and tocilizumab, an interleukin-6 receptor blocker, are used for the treatment of rheumatoid arthritis. We investigated the differential effects of anakinra and tocilizumab on myocardial and vascular function in an atherosclerosis model of patients with rheumatoid arthritis. METHODS: 120 patients with rheumatoid arthritis were randomized to anakinra (n = 40), tocilizumab (n = 40) or prednisolone (n = 40) for 3 months. Primary outcome measure was the change of left ventricular longitudinal strain after 3 months of treatment. Additionally, we measured coronary flow reserve, flow-mediated dilatation of the brachial artery, carotid-femoral pulse wave velocity, malondialdehyde and protein carbonyls as oxidative stress markers and C-reactive protein blood levels at baseline and post-treatment. RESULTS: At baseline, patients among the three treatment arms had similar age, sex, disease activity score and atherosclerotic risk factors. Compared with baseline, all patients had improved longitudinal strain (- 16% vs. - 17.8%), coronary flow reserve (2.56 vs. 2.9), malondialdehyde (2.0 vs. 1.5 µM/L), protein carbonyls (0.0132 vs. 0.0115 nmol/mg), and C-reactive protein post-treatment. In all patients, the percent decrease of malondialdehyde was correlated with percent increase of longitudinal strain (p < 0.001). Compared with tocilizumab and prednisolone, anakinra treatment resulted in a greater improvement of longitudinal strain (18.7% vs. 9.7% vs. 6%) and coronary flow reserve (29% vs. 13% vs. 1%), while pulse wave velocity and brachial blood pressure were improved only after tocilizumab treatment (11 ± 3 vs. 10.3 ± 2 m/s p < 0.05 for all comparisons). CONCLUSIONS: Anakinra is associated with an improvement in cardiac function and tocilizumab with improvement in vascular function. CLINICAL TRIAL REGISTRATION: URL: https:// http://www.clinicaltrials.gov . Unique identifier: NCT03288584.

Herpes viruses have what type of genome?

The Herpesviridae are a family of viruses which have a large genome of linear, double-stranded DNA (> 120 kb)

Vectors derived from herpes simplex virus provide a means of gene delivery to postmitotic neurons. The virus is readily taken up at nerve terminals, passes by rapid retrograde and anterograde transport within neurons, and is selectively transferred across synapses, thus allowing it entry from the periphery into the brain. This virus can enter a state of latency in some neurons, where it exists as an episomal element in the nucleus and is transcriptionally active to a reduced extent. In this state, the virus is apparently benign and can effect stable expression of foreign genes. The large (150 kb) genome of this double-stranded virus has been completely sequenced. Many of its 70 genes can be replaced while still allowing the virus to replicate in at least some cultured cells. Some mutations in the viral genome can compromise the toxicity of the virus and reduce or eliminate its ability to replicate within neurons. Many uses for herpes vectors can be envisioned, including evaluation of neuronal promoter elements and functions of neural proteins in culture and in vivo, as well as therapeutic delivery of genes to modulate nerve function and for gene replacement therapy in vivo. Varicella-zoster virus (human herpesvirus 3; VZV) is one of eight herpes viruses that routinely infect humans. It is classified as a member of the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. Of the other human herpes viruses it is most closely related to the herpes simplex viruses (also members of the Alphalerpesvirinae). Like all herpes viruses, the virus has a large double-stranded DNA genome within an icosahedral nucleocapsid. This is surrounded by a proteinaceous tegument and a trilaminar membrane derived from host-cell membranes into which the viral glycoproteins are inserted. The structure of the virion is summarized in Fig. 1. Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 and pac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that pac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids. The Herpesviridae are a family of viruses which have a large genome of linear, double-stranded DNA (> 120 kb). It has been quite difficult to clone and modify herpesvirus genomes because of their large sizes. Recently, several groups of investigators demonstrated successful cloning of infectious herpesvirus genomes as bacterial artificial chromosomes (BACs). In this review, I describe the recent development of methods to modify the cloned viral genomes. The methods are: (i) homologous recombination in E. coli which allows targeted mutagenesis of any specific viral sequence, and (ii) mutagenesis which allows random modification of entire viral genomes. Both methods should facilitate both the study of herpes viruses and the development of herpesvirus-based vector system. Herpesviruses are large double stranded DNA animal viruses with the distinguishing ability to establish latent, life-long infections. To date, eight human herpesviruses that exhibit distinct biological and corresponding pathological/clinical properties have been identified. During their life cycles, herpesviruses execute an intricate chain of events geared towards optimizing their replication. This sets an interesting paradigm to study fundamental biological processes. This review summarizes recent developments in herpesvirus research with emphasis on genome transactions, particularly with respect to the prototypic herpes simplex virus type-1. For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral a sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes. BACKGROUND: Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of approximately 120-200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. METHODS AND RESULTS: Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but < 400 sites; 69(23.9%) in > 400 but < 700 sites; and the 9(3.1%) enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a oparticle for microbicide development and a "recombit lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII. CONCLUSION: Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission. Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) are doublestranded DNA viruses with a genome size of 152 kbp. The genome consists of two unique regions, U(L) (long) and U(s) (short), flanked by repeated sequences (Fig. 1; for review, see ref. 1). The two viruses are closely related, and both infect humans, producing mucocutaneous sores that are predomitly facial in the case of HSV-1 and genital in the case of HSV-2. To launch an effective antiviral immune response, cells must recognize the virus, activate a cytokine response, and initiate inflammatory processes. Herpes simplex virus 1 (HSV-1) and HSV-2 are nuclear-replicating viruses composed of a double-stranded DNA genome plus glycoproteins that are incorporated into a lipid bilayer envelope that surrounds an icosahedral capsid. Several novel receptors that mediate innate recognition of HSV and that activate the innate immune response have been identified in recent years. The host-virus interactions that lead to type I interferon (IFN), type III IFN, and cytokine production include cellular recognition of viral envelope and structural proteins, recognition of viral genomic DNA and recognition of virus-derived double-stranded RNAs. Such RNAs can interact with cellular pattern-recognition receptors, including Toll-like receptors and a number of cytoplasmic and nuclear receptors for virus DNA and virus-derived RNAs. In this review, I present a systematic overview of innate cellular recognition of HSV infection that leads to immune activation, and I discuss the implications of the known cell-host interactions. In addition, I discuss the use of innate stimulation to improve anti-HSV treatment and vaccine response and I discuss future research aims. The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

Is liraglutide effective for weight reduction?

Yes, liraglutide is effective and approved for weight reduction.

AIMS: Effects of the long acting GLP-1 analogue--liraglutide in subjects with type 2 diabetes. METHODS: 144 type 2 diabetic subjects on metformin treatment (1000 mg BID) were randomised to 5 weeks of treatment (double-blind) with metformin plus liraglutide, liraglutide or metformin, or metformin plus glimepiride (open label). The dose of liraglutide was increased weekly from 0.5 to 2 mg OD. RESULTS: Liraglutide added to metformin monotherapy was associated with a significant reduction in fasting serum glucose (FSG) (-3.9 mM -4.9; -2.9) (primary objective), and HbA1c levels (-0.8% -1.2; -0.4). Furthermore, liraglutide in combination with metformin vs. metformin plus glimepiride significantly reduced FSG (-1.2 mM -2.2; -0.2). In addition, body weight was significantly lower in the metformin plus liraglutide vs. the metformin plus glimepiride group (-2.9 kg -3.6; -2.1). There were no biochemically confirmed episodes of hypoglycaemia with liraglutide treatment. Nausea was the most frequently reported adverse event following liraglutide therapy, it was transient in nature, and led to withdrawal of only 4% of the subjects. CONCLUSIONS: Using a weekly dose-titration liraglutide is well tolerated up to 2 mg daily. While liraglutide caused transient gastrointestinal side effects, this rarely interfered with continuing treatment. An improvement in FSG over that in control groups was seen for liraglutide as an add-on to metformin. In the latter case, body weight was reduced in comparison to metformin plus glimepiride. Liraglutide is a promising drug for the treatment of type 2 diabetes. Liraglutide is a long-acting analog of GLP-1, being developed by Novo Nordisk and currently undergoing regulatory review for the treatment of type 2 diabetes. Upon injection, liraglutide binds non-covalently to albumin, giving it a pharmacokinetic profile suitable for once-daily administration. In clinical trials of up to 1 year duration, liraglutide has been demonstrated to have beneficial effects on islet cell function, leading to improvements in glycemic control. Both fasting and postprandial glucose concentrations are lowered, and are associated with lasting reductions in HbA1c levels. Liraglutide is effective as monotherapy and in combination therapy with oral antidiabetic drugs, and reduces HbA1c by up to approximately 1.5% from baseline (8.2%-8.4%). Because of the glucose-dependency of its action, there is a low incidence of hypoglycemia. Liraglutide is associated with body weight loss, and reductions in systolic blood pressure have been observed throughout the clinical trials. The most common adverse events reported with liraglutide are gastrointestinal (nausea, vomiting and diarrhea). These tend to be most pronounced during the initial period of therapy and decline with time. Further clinical experience with liraglutide will reveal its long-term durability, safety and efficacy. AIM: The effect on body composition of liraglutide, a once-daily human glucagon-like peptide-1 analogue, as monotherapy or added to metformin was examined in patients with type 2 diabetes (T2D). METHODS: These were randomized, double-blind, parallel-group trials of 26 [Liraglutide Effect and Action in Diabetes-2 (LEAD-2)] and 52 weeks (LEAD-3). Patients with T2D, aged 18-80 years, body mass index (BMI) < or =40 kg/m(2) (LEAD-2), < or =45 kg/m(2) (LEAD-3) and HbA1c 7.0-11.0% were included. Patients were randomized to liraglutide 1.8, 1.2 or 0.6 mg/day, placebo or glimepiride 4 mg/day, all combined with metformin 1.5-2 g/day in LEAD-2 and to liraglutide 1.8, 1.2 or glimepiride 8 mg/day in LEAD-3. LEAD-2/3: total lean body tissue, fat tissue and fat percentage were measured. LEAD-2: adipose tissue area and hepatic steatosis were assessed. RESULTS: LEAD-2: fat percentage with liraglutide 1.2 and 1.8 mg/metformin was significantly reduced vs. glimepiride/metformin (p < 0.05) but not vs. placebo. Visceral and subcutaneous adipose tissue areas were reduced from baseline in all liraglutide/metformin arms. Except with liraglutide 0.6 mg/metformin, reductions were significantly different vs. changes seen with glimepiride (p < 0.05) but not with placebo. Liver-to-spleen attenuation ratio increased with liraglutide 1.8 mg/metformin possibly indicating reduced hepatic steatosis. LEAD-3: reductions in fat mass and fat percentage with liraglutide monotherapy were significantly different vs. increases with glimepiride (p < 0.01). CONCLUSION: Liraglutide (monotherapy or added to metformin) significantly reduced fat mass and fat percentage vs. glimepiride in patients with T2D. Liraglutide is a glucagon-like peptide-1 (GLP-1) analog marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide also reduces body weight. It is not fully understood how liraglutide induces weight loss or to what degree liraglutide acts directly in the brain. Here, we determined that liraglutide does not activate GLP-1-producing neurons in the hindbrain, and liraglutide-dependent body weight reduction in rats was independent of GLP-1 receptors (GLP-1Rs) in the vagus nerve, area postrema, and paraventricular nucleus. Peripheral injection of fluorescently labeled liraglutide in mice revealed the presence of the drug in the circumventricular organs. Moreover, labeled liraglutide bound neurons within the arcuate nucleus (ARC) and other discrete sites in the hypothalamus. GLP-1R was necessary for liraglutide uptake in the brain, as liraglutide binding was not seen in Glp1r(-/-) mice. In the ARC, liraglutide was internalized in neurons expressing proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Electrophysiological measurements of murine brain slices revealed that GLP-1 directly stimulates POMC/CART neurons and indirectly inhibits neurotransmission in neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) via GABA-dependent signaling. Collectively, our findings indicate that the GLP-1R on POMC/CART-expressing ARC neurons likely mediates liraglutide-induced weight loss. BACKGROUND: Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, has been shown to possess pleiotropic effects including body weight reduction. However, long-term effect of liraglutide on body weight and glycemic control has not been elucidated in Japanese type 2 diabetes (T2D) subjects. Present study investigates whether liraglutide treatment maintains the body weight-decreasing and glucose-lowering effects for 2 years in Japanese T2D subjects. METHODS: The enrolled subjects were 86 T2D patients [age; 59.8 ± 12.8 years, duration of diabetes; 15.8 ± 9.5 years, glycated hemoglobin (HbA1c); 8.5 ± 1.5%, body mass index (BMI); 27.3 ± 5.4 kg/m(2) (15.8 - 46.5 kg/m(2)), mean ± SD]. Among 86 subjects, liraglutide was introduced in 25 inpatients and 61 outpatients, and 46 subjects were followed for 2 years. Clinical parameters were measured at baseline and 3, 6, 9, 12, and 24 months after liraglutide introduction. The increase in liraglutide dosage and the additional usage of glucose-lowering agents depended on each attending physician. RESULTS: At 1 year after liraglutide introduction, 69 patients (80.2%) decreased body weight and 58 patients (67.4%) improved glycemic control. Body mass index (BMI) was changed 27.3 ± 5.4 kg/m(2) to 25.9 ± 4.8 kg/m(2) and percent reduction of body weight was significant and maintained over 4% at 2 years after liraglutide introduction. HbA1c was significantly decreased from 8.5 ± 1.5% to 7.7 ± 1.2% for 2 years. Liraglutide treatment tended to ameliorate lipid profile and hepatic enzymes. Stepwise regression analysis demonstrated that baseline BMI and previous insulin dose were positively associated with body weight reduction and baseline HbA1c was positively associated with reduction of HbA1c at 2 years after liraglutide introduction. CONCLUSIONS: Long-term liraglutide treatment effectively maintained the reduction of body weight and the fair glycemic control, and also improved lipid profile and liver enzymes in Japanese T2D subjects. IMPORTANCE: Weight loss of 5% to 10% can improve type 2 diabetes and related comorbidities. Few safe, effective weight-management drugs are currently available. OBJECTIVE: To investigate efficacy and safety of liraglutide vs placebo for weight management in adults with overweight or obesity and type 2 diabetes. DESIGN, SETTING, AND PARTICIPANTS: Fifty-six-week randomized (2:1:1), double-blind, placebo-controlled, parallel-group trial with 12-week observational off-drug follow-up period. The study was conducted at 126 sites in 9 countries between June 2011 and January 2013. Of 1361 participants assessed for eligibility, 846 were randomized. Inclusion criteria were body mass index of 27.0 or greater, age 18 years or older, taking 0 to 3 oral hypoglycemic agents (metformin, thiazolidinedione, sulfonylurea) with stable body weight, and glycated hemoglobin level 7.0% to 10.0%. INTERVENTIONS: Once-daily, subcutaneous liraglutide (3.0 mg) (n = 423), liraglutide (1.8 mg) (n = 211), or placebo (n = 212), all as adjunct to 500 kcal/d dietary deficit and increased physical activity (≥150 min/wk). MAIN OUTCOMES AND MEASURES: Three coprimary end points: relative change in weight, proportion of participants losing 5% or more, or more than 10%, of baseline weight at week 56. RESULTS: Baseline weight was 105.7 kg with liraglutide (3.0-mg dose), 105.8 kg with liraglutide (1.8-mg dose), and 106.5 kg with placebo. Weight loss was 6.0% (6.4 kg) with liraglutide (3.0-mg dose), 4.7% (5.0 kg) with liraglutide (1.8-mg dose), and 2.0% (2.2 kg) with placebo (estimated difference for liraglutide [3.0 mg] vs placebo, -4.00% [95% CI, -5.10% to -2.90%]; liraglutide [1.8 mg] vs placebo, -2.71% [95% CI, -4.00% to -1.42%]; P < .001 for both). Weight loss of 5% or greater occurred in 54.3% with liraglutide (3.0 mg) and 40.4% with liraglutide (1.8 mg) vs 21.4% with placebo (estimated difference for liraglutide [3.0 mg] vs placebo, 32.9% [95% CI, 24.6% to 41.2%]; for liraglutide [1.8 mg] vs placebo, 19.0% [95% CI, 9.1% to 28.8%]; P < .001 for both). Weight loss greater than 10% occurred in 25.2% with liraglutide (3.0 mg) and 15.9% with liraglutide (1.8 mg) vs 6.7% with placebo (estimated difference for liraglutide [3.0 mg] vs placebo, 18.5% [95% CI, 12.7% to 24.4%], P < .001; for liraglutide [1.8 mg] vs placebo, 9.3% [95% CI, 2.7% to 15.8%], P = .006). More gastrointestinal disorders were reported with liraglutide (3.0 mg) vs liraglutide (1.8 mg) and placebo. No pancreatitis was reported. CONCLUSIONS AND RELEVANCE: Among overweight and obese participants with type 2 diabetes, use of subcutaneous liraglutide (3.0 mg) daily, compared with placebo, resulted in weight loss over 56 weeks. Further studies are needed to evaluate longer-term efficacy and safety. TRIAL REGISTRATION: clinicaltrials.gov Identifier:NCT01272232. Liraglutide (LIRA) treatment is associated with the dose-dependent reduction of weight. Higher doses are more effective than lower doses, although higher doses are also more poorly tolerated. Metformin may enhance the weight-lowering potential of LIRA via the stimulatory modulation of incretin in addition to its direct beneficial effects in PCOS. The aim of the present study was to evaluate whether metformin as an adjunct to low-dose LIRA affects body weight with increased efficacy compared with low-dose LIRA alone in obese patients with PCOS. In a 12-week study, 44 obese women with PCOS were randomly offered either combined treatment (COMBO) with 1,000 mg metformin twice a day and 1.2 mg LIRA once a day, or treatment with 1.2 mg LIRA alone. The primary outcome of treatment was an alteration in the levels of obesity. A total of 43 patients [aged 30.3±4.4 years; body mass index (BMI) 37.2±4.5 kg/m2; mean ± standard deviation] completed the study. The subjects treated with COMBO lost on average 6.2±2.4 kg compared with a 3.8±3.5 kg weight loss in the patients treated with LIRA alone (P=0.024). The BMI decreased by 2.2±0.8 kg/m2 in patients treated with COMBO and by 1.4±1.2 kg/m2 in patients treated with LIRA alone (P=0.024). A clinically significant ≥5% weight reduction was achieved in 59.1% of patients treated with COMBO and 42.9% of patients treated with LIRA alone. Reductions in glucose levels following oral glucose tolerance testing, as well as in androstenedione levels in the COMBO group were significantly greater compared with those in the LIRA group. The side effects were mild and transient in the two treatment groups. A combination of metformin and low-dose LIRA was more effective than low-dose LIRA alone in reducing body weight in obese patients with PCOS. Liraglutide, a glucagon-like peptide (GLP-1) receptor agonist, has showed favorable effects in the glycaemic control and weight reduction in patients with type 2 diabetes mellitus (T2DM). The meta-analysis was to compare the efficacy and safety of liraglutide added to metformin with other treatments in patients with T2DM. A systematic literature search on PubMed, Embase, Web of Science and the Cochrane library databases were performed. Eligible studies were randomized controlled trials (RCTs) of patients with T2DM who received the combination treatment of liraglutide and metformin. Pooled estimates were performed using a fixed-effects model or random-effects model. A total of nine RCTs met the inclusion criteria. Compared with control (placebo, sitagliptin, glimepiride, dulaglutide, insulin glargine, and NPH), liraglutide in combination with metformin resulted in significant reductions in HbA1c, bodyweight, FPG, and PPG, and similar reductions in SBP, and DBP. Moreover, liraglutide combined with metformin did not increase the risk of hypoglycemia, but induced a higher incidence of gastrointestinal disorders. In conclusion, this meta-analysis confirmed the use of liraglutide as add-on to metformin appeared to be effective and safe for patients with T2DM. However, considering the potential limitations in this study, more large-scale, well-conducted RCTs are needed to identify our findings. INTRODUCTION: For people with type 2 diabetes (T2DM) inadequately controlled with oral antidiabetic drugs (OADs), evidence from both randomized controlled trials (RCTs) and real-world studies has demonstrated that treatment intensification with liraglutide offers effective glycemic control, weight reduction, and a lower risk of hypoglycemia compared to treatment intensification with insulin or additional OADs. Sodium glucose cotransporter 2 (SGLT-2) inhibitors are a new class of OADs that have also been shown to be effective in T2DM patients inadequately controlled with OADs. Currently there are no head-to-head RCTs comparing these to liraglutide. METHODS: We aimed to evaluate the relative efficacy, using network meta-analysis (NMA), of treatment intensification with liraglutide and SGLT-2 inhibitors people with T2DM who have been treated with metformin (alone or in combination with SU, DPP-4, and TZD). We performed a systematic literature review to identify relevant RCTs comparing liraglutide (1.2 and 1.8 mg), canagliflozin (100 and 300 mg), empagliflozin (10 and 25 mg), or dapagliflozin (5 and 10 mg) to placebo. To strengthen the indirect evidence base, we also included non-placebo RCTs where sitagliptin (100 mg) was the active comparator. Bayesian NMA was performed on the following outcomes to assess the relative efficacy and safety of interventions: reduction (change) in HbA1c, weight, and fasting plasma glucose (FPG) as well as proportion reaching target HbA1c (<7%), and risk of hypoglycemia. Doses for each intervention were considered separately. RESULTS: A total of 16 RCTs were identified. All trials were similar with respect to important baseline characteristics and study design. Both doses of liraglutide were generally statistically significantly superior to the SGLT-2s with respect to change from baseline in HbA1c and FPG as well as odds of reaching target HbA1c <7%. For weight, canagliflozin 300 mg was superior to liraglutide 1.2 mg, and SGLT-2s were generally associated with larger change from baseline in weight. For risk of major or minor hypoglycemia, no differences were found between treatments. CONCLUSIONS: Compared to SGLT-2 inhibitors, liraglutide offers improvement in HbA1c and FPG. Reductions in weight are likely comparable between liraglutide and SGLT-2s. Liraglutide did not differ from SGLT-2s in terms of risk of hypoglycemia. Given the lack of head-to-head evidence, this analysis provides valuable insight into the comparative outcomes of liraglutide versus SGLT-2 inhibitors. BACKGROUND: Obesity represents the second preventable mortality cause worldwide, and is very often associated with type 2 Diabetes Mellitus (T2DM). The first line treatment is lifestyle modification to weight-loss, but for those who fail to achieve the goal or have difficulty in maintaining achieved results, pharmacological treatment is needed. Few drugs are available today, because of their side effects. OBJECTIVE: We aim to review actual pharmacological management of obese patients, highlighting differences between Food and Drug Administration - and European Medicine Agency-approved molecules, and pointing out self-medications readily obtainable and widely distributed. METHODS: Papers on obesity, weight loss, pharmacotherapy, self- medication and diet-aid products were selected using Medline. Research articles, systematic reviews, clinical trials and meta-analyses were screened. RESULTS: Anti-obesity drugs with central mechanisms, such as phentermine and lorcaserin, are available in USA, but not in Europe. Phentermine/topiramate and naltrexone/bupropion combinations are now available, even though the former is still under investigation from EMA. Orlistat, with peripheral mechanisms, represents the only drug approved for weight reduction in adolescents. Liraglutide has been approved at higher dose for obesity. Anti-obesity drugs, readily obtainable from the internet, include crude-drug products and supplements for which there is often a lack of compliance to national regulatory standards. CONCLUSIONS: Mechanisms of weight loss drugs include the reduction of energy intake or the increase in energy expenditure and sense of satiety as well as the decrease of hunger or the reduction in calories absorption. Few drugs are approved, and differences exist between USA and Europe. Moreover, herbal medicines and supplements often sold on the internet and widely used by obese patients, present a risk of adverse effects. BACKGROUND: A few Randomized Controlled Trials (RCTs) have evaluated the use of liraglutide in Type 1 Diabetes (T1D). Through the present systematic review and meta-analysis, we aim at critically appraising and summarizing those RCTs, providing precise effect estimates. METHODS: We searched major databases and grey literature from their inception to October 2018, for RCTs with a duration ≥ 12 weeks, comparing liraglutide with placebo or any other comparator as adjunct to insulin in patients with T1D, investigating major efficacy and safety endpoints. This review is reported in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement. RESULTS: We included 5 trials with 2,445 randomized participants. Liraglutide provided modest reductions in HbA1c, with liraglutide 1.8 mg producing the greatest decrease (MD = -0.24%, 95% CI -0.32 to -0.16, I2=0%). Significant weight reduction, up to 4.87 kg with liraglutide 1.8 mg was also observed (95% CI -5.31 to -4.43, I2=0%). Decrease in total daily insulin dose, primarily driven by a decrease in bolus insulin requirements, was demonstrated. Liraglutide decreased non-significantly the odds for severe hypoglycemia (OR=0.80, 95% CI 0.57-1.14, I2=0%), while it increased significantly the odds for gastrointestinal adverse events (for nausea, OR=4.70, 95% CI 3.68-6.00, I2=37%, and for vomiting, OR=2.50, 95% CI 1.54-4.72, I2=27%). A significant increase in heart rate was also demonstrated. No association with diabetic ketoacidosis or maligcies was identified. CONCLUSION: In patients with T1D, liraglutide might prove be an adjunct to insulin, improving glycemic control, inducing body weight loss and decreasing exogenous insulin requirements and severe hypoglycemia. Introduction: Obesity poses a significant increase in morbidity and mortality and thus five anti-obesity drugs have been approved currently by US FDA. Several phase 3 trials have shown a significant improvement in cardio-metabolic profile including significant weight reduction with these agents compared to placebo.Areas covered: We systematically searched the database of PubMed, Embase, The Cochrane Library and The ClinicalTrials.gov up to 30 September 2019 and retrieved all the randomized controlled trials (RCTs) that were conducted with these five drugs for ≥1 year and explicitly reported their efficacy versus placebo. Subsequently, we have conducted the meta-analysis to primarily study the effect of these anti-obesity drugs on weight reduction. We additionally reviewed the effect of these drugs on other cardio-metabolic parameters including key adverse events.Expert opinion: This meta-analysis finds a significant reduction in body weight with orlistat (N = 10,435; ∆ -3.07 Kg, 95% CI, -3.76 to -2.37), phentermine plus topiramate (N = 2985; ∆ -9.77 Kg; 95% CI, -11.73 to -7.81), lorcaserin (N = 16,856; ∆ -3.08 Kg; 95% CI, -3.49 to -2.66), naltrexone plus bupropion (N = 3239; ∆ -4.39 Kg; 95% CI, -5.05 to -3.72) and liraglutide (N = 4978; ∆ -5.25 Kg; 95% CI, -6.17 to -4.32), compared to placebo (all p < 0.00001). As a chronic and relapsing disease, obesity negatively impacts the health of men to a greater extent than that of women, with a higher risk of cardiovascular disease. Since lifestyle modifications alone are often challenging and limited for the maintece of weight reduction, pharmacotherapy should be considered in a timely manner for obese men or overweight patients with weight-related comorbidities. Recent advances in anti-obesity drugs have enabled the potential of achieving clinically significant weight loss. Increasing evidence has shown that behavior-based interventions with one of these medications can result in greater weight loss than that elicited by usual care conditions. Data from most recent meta-analyses showed that the overall placebo-subtracted weight reduction (%) with the use of anti-obesity drugs for at least 12 months ranges from 2.9% to 6.8%; phentermine/topiramate (-6.8%) liraglutide (-5.4%), naltrexone/bupropion (-4.0%), lorcaserin (-3.1%), and orlistat (-2.9%). However, they have a high cost and may cause adverse outcomes depending on the individual. Very recently, on February 13, 2020, the US Food and Drug Administration requested withdrawal of lorcaserin from the market because a safety clinical trial showed an increased occurrence of cancer. Therefore the decision to initiate drug therapy in obese individuals should be made after the benefits and risks are considered. Thereafter, treatment should be tailored to specific patient subpopulations depending on their chronic conditions, comorbidities, and preferences. Herein, we provide an overview of the latest developments in weight loss medications, which may serve as one of the strategies for long-term obesity control. BACKGROUND: The increase in global obesity rates over the past three decades has been remarkable, a true epidemic, both in developed and in developing countries. The projections, based on current trends, suggest an increase in the prevalence of obesity at 60% in adult men, 40% in adult women and 25% in children in 2050. Given the limitations of lifestyle and surgery interventions bariatric, drug therapy approaches for the treatment of obesity, therefore become important options. AIM: The purpose of this review is a review of the literature, based on research on MEDLINE until 2019, on the possible pharmacological options in the treatment of obesity. RESULTS: Currently, the FDA has approved several molecules for the treatment of obesity, both in monotherapy and in combination. Pharmacological monotherapies focus mainly on a single protein target and include orlistat, lorcaserin and liraglutide while the combination molecules propose a multitarget approach and include phentermine/topiramate and naltrexone/bupropion. All the approved drugs showed, in the different studies, a weight reduction of at least 5%, compared to placebo, in 52 weeks of observation. Phentermine-topiramate and liraglutide have been associated with the highest probability of at least 5% weight loss. Liraglutide and naltrexone-bupropion had the lowest rates of therapy discontinuation due to adverse events. CONCLUSION: The drugs, associated with the standard diet and/or exercise protocols, represent a good therapeutic opportunity to allow not only weight loss but also to reduce the risk of developing diseases caused by obesity, particularly cardiovascular diseases, and to maintain the set objectives over time. However, future research on the pharmacological treatment of obesity should encourage greater personalization of therapy, given the differences in safety, efficacy and response to therapy, in the different subpopulations of patients with obesity. AIMS/INTRODUCTION: Obesity and metabolic syndrome are well-known to be associated with multiple chronic diseases. Currently, high-dose liraglutide has been used for weight control in non-diabetic patients. Considering incretin-based therapy is more effective in Asian populations, the effect of low-dose liraglutide in weight control among these non-diabetic groups has not been well evaluated. Our study aimed to evaluate the efficacy of low-dose liraglutide in weight control among Taiwan patients. MATERIALS AND METHODS: From July 2017 to December 2018, 46 non-diabetic patients with metabolic syndrome were included. They had received low-dose liraglutide at 0.6 or 1.2 mg per day for weight reduction for 12 weeks. After then, changes in bodyweight, waist and metabolic factors were examined. Overt bodyweight reduction was defined as a decrease of >5% within 12 weeks. RESULTS: With 12 weeks of medication use, both groups showed statistical weight reduction. Higher doses of liraglutide had better efficacy, and 44.4% of patients in the liraglutide 1.2 mg group reached overt weight reduction, whereas just 32.1% in the 0.6 mg group had achieved this. Young age was found to be a predictor factor for a positive finding (odds ratio 0.941, P = 0.037). Early responders with decreased bodyweight of >4.2% within the first 4 weeks indicated a better chance to achieve measurable weight reduction. CONCLUSIONS: Low-dose liraglutide still has high efficacy in weight reduction in Taiwanese people, especially for those of younger age.

Is lorcaserin associated with increased cancer risk?

The US Food and Drug Administration (FDA) reported an increased risk of cancer with lorcaserin in the follow-up of the CAMELLIA-TIMI 61 trial. However, subsequent meta-analysis did not confirm the increased risk of cancer with lorcaserin but suggests a trend in this direction, with a greater incidence of some subtypes such as lung and pancreas.

Is Eflornithine and Sulindac are effective for prevention of progression in Familial Adenomatous Polyposis?

No. In a clinical trial, the incidence of progression in Familial Adenomatous Polyposis was not significantly lower with the combination of eflornithine and sulindac than with either drug alone.

BACKGROUND: The efficacy and safety of combination therapy with eflornithine and sulindac, as compared with either drug alone, in delaying disease progression in patients with familial adenomatous polyposis are unknown. METHODS: We evaluated the efficacy and safety of the combination of eflornithine and sulindac, as compared with either drug alone, in adults with familial adenomatous polyposis. The patients were stratified on the basis of anatomical site with the highest polyp burden and surgical status; the strata were precolectomy (shortest projected time to disease progression), rectal or ileal pouch polyposis after colectomy (longest projected time), and duodenal polyposis (intermediate projected time). The patients were then randomly assigned in a 1:1:1 ratio to receive 750 mg of eflornithine, 150 mg of sulindac, or both once daily for up to 48 months. The primary end point, assessed in a time-to-event analysis, was disease progression, defined as a composite of major surgery, endoscopic excision of advanced adenomas, diagnosis of high-grade dysplasia in the rectum or pouch, or progression of duodenal disease. RESULTS: A total of 171 patients underwent randomization. Disease progression occurred in 18 of 56 patients (32%) in the eflornithine-sulindac group, 22 of 58 (38%) in the sulindac group, and 23 of 57 (40%) in the eflornithine group, with a hazard ratio of 0.71 (95% confidence interval [CI], 0.39 to 1.32) for eflornithine-sulindac as compared with sulindac (P = 0.29) and 0.66 (95% CI, 0.36 to 1.24) for eflornithine-sulindac as compared with eflornithine. Among 37 precolectomy patients, the corresponding values in the treatment groups were 2 of 12 patients (17%), 6 of 13 (46%), and 5 of 12 (42%) (hazard ratios, 0.30 [95% CI, 0.07 to 1.32] and 0.20 [95% CI, 0.03 to 1.32]); among 34 patients with rectal or ileal pouch polyposis, the values were 4 of 11 patients (36%), 2 of 11 (18%), and 5 of 12 (42%) (hazard ratios, 2.03 [95% CI, 0.43 to 9.62] and 0.84 [95% CI, 0.24 to 2.90]); and among 100 patients with duodenal polyposis, the values were 12 of 33 patients (36%), 14 of 34 (41%), and 13 of 33 (39%) (hazard ratios, 0.73 [95% CI, 0.34 to 1.52] and 0.76 [95% CI, 0.35 to 1.64]). Adverse and serious adverse events were similar across the treatment groups. CONCLUSIONS: In this trial involving patients with familial adenomatous polyposis, the incidence of disease progression was not significantly lower with the combination of eflornithine and sulindac than with either drug alone. (Funded by Cancer Prevention Pharmaceuticals; ClinicalTrials.gov number, NCT01483144; EudraCT number, 2012-000427-41.).

Which cancer can be treated with Darolutamide?

Darolutamide is used for treatment of nonmetastatic castration-resistant prostate cancer.

Darolutamide (ODM-201) is a novel androgen receptor (AR) antagonist with a chemical structure distinctly different from currently approved AR antagonists that targets both wild-type and mutated ligand binding domain variants to inhibit AR nuclear translocation. Here, we evaluate the activity of darolutamide in enzalutamide-resistant castration resistant prostate cancer (CRPC) as well as in AR mutants detected in patients after treatment with enzalutamide, abiraterone, or bicalutamide. Darolutamide significantly inhibited cell growth and AR transcriptional activity in enzalutamide-resistant MR49F cells in vitro, and led to decreased tumor volume and serum prostate-specific antigen levels in vivo, prolonging survival in mice bearing enzalutamide-resistant MR49F xenografts. Moreover, darolutamide inhibited the transcriptional activity of AR mutants identified in the plasma of CRPC patients progressing on traditional therapies. In particular, darolutamide significantly inhibited the transcriptional activity of the F877L, H875Y/T878A, F877L/T878A, and the previously unreported T878G AR mutants, that transform enzalutamide into a partial agonist. In silico cheminformatics computer modeling provided atomic level insights confirming darolutamide antagonist effect in F877L and T878G AR mutants. In conclusion, our results provide a rationale for further clinical evaluation of darolutamide in enzalutamide-resistant CRPC, in particular in combination with circulating tumor DNA assays that detect AR mutants sensitive to darolutamide, in a precision oncology setting. PATIENT SUMMARY: In this study we evaluated the novel drug darolutamide in preclinical models of prostate cancer. We found that darolutamide delays growth of enzalutamide-resistant prostate cancer, in particular in cells with mutated forms of the androgen receptor after previous treatment. Our data supports further evaluation of darolutamide in clinical trials. The phase III ARAMIS study shows that the androgen-receptor antagonist darolutamide delays metastasis in men with castration-resistant prostate cancer by a median of 22 months compared with a placebo. Researchers also found that side effects associated with other drugs in this class were no more common in the placebo group than the darolutamide group. Darolutamide (NUBEQA™) is a structurally distinct non-steroidal androgen receptor antagonist being developed by Orion and Bayer as a treatment for prostate cancer. Based on positive results in the phase III ARAMIS trial, darolutamide was recently approved in the USA for the treatment of men with non-metastatic castration-resistant prostate cancer. This article summarizes the milestones in the development of darolutamide leading to this first approval. The antiandrogen therapeutics apalutamide and darolutamide entered the clinic in 2018 and 2019, respectively, for the treatment of castration-resistant prostate cancer (CRPC). Increased expression of the enzyme aldo-keto reductase 1C3 (AKR1C3) is phenotypic of CRPC. The enzyme acts to circumvent castration by producing potent androgens that drive proliferation. Furthermore, AKR1C3 mediates chemotherapeutic resistance to the standard of care, enzalutamide, a structural analogue of apalutamide. Resistance develops in almost all CRPC patients within three months of beginning treatment. Herein, we report that both apalutamide and the structurally distinct darolutamide induce AKR1C3 expression in in vitro models of prostate cancer and are susceptible to AKR1C3-mediated resistance. This effect is countered by pretreatment with a potent and highly selective AKR1C3 inhibitor, sensitizing high AKR1C3 expressing prostate cancer cell lines to the action of both chemotherapeutics with a concomitant reduction in expression of AKR1C3 and the biomarker prostate-specific antigen. BACKGROUND: Among men with high-risk non-metastatic castrate-resistant prostate cancer (nmCRPC), we used network meta-analysis to compare non-steroidal anti-androgens (NSAAs) and stratified class-level meta-analysis to identify subgroups with particular benefit from NSAAs with androgen deprivation therapy versus androgen deprivation therapy alone. MATERIALS AND METHODS: We performed a systematic review of phase III parallel-group randomized controlled trials in adult men with nmCRPC. Primary outcome was metastasis-free survival (MFS). Secondary outcomes included overall survival (OS), prostate-specific antigen (PSA) progression-free survival (PFS), and rates of grade 3 to 4 adverse events (AEs). We assessed class-level effects using random effects models; effect modification owing to subgroup effects using random-effects models to pool study-level differences; and comparative outcomes between agents using fixed-effect network models in a Bayesian framework. RESULTS: Three randomized controlled trials were identified. Pooled MFS, PSA-PFS, and OS were significantly greater with NSAA versus placebo (hazard ratio [HR], 0.32; 95% confidence interval [CI], 0.25-0.41; HR, 0.08; 95% CI, 0.05-0.13; and HR, 0.74; 95% CI, 0.61-0.90, respectively). Subgroup analysis demonstrated a greater benefit with NSAAs in men with Eastern Cooperative Oncology Group performance status 0 (HR, 0.30; 95% CI, 0.24-0.38) versus 1 (HR, 0.45; 95% CI, 0.36-0.56; P = .005), but no difference owing to PSA doubling time (P = .43) or use of osteoclast targeting therapy (P = .77). Bayesian analysis showed apalutamide and enzalutamide had a 56% and 44% likelihood of maximizing MFS, respectively, with subgroup analysis demonstrating these agents were preferred regardless of PSA doubling time and performance status. There was a 44%, 41%, and 15% likelihood that apalutamide, darolutamide and enzalutamide offered the greatest OS benefit, respectively. Grade 3 to 4 AEs were more common with NSAAs (odds ratio [OR], 1.47; 95% CI, 1.27-1.71) and there was a 61% chance that darolutamide was preferred. CONCLUSIONS: NSAAs improve survival in high-risk nmCRPC. Apalutamide and enzalutamide may result in improved oncologic outcomes. Darolutamide may result in fewer AEs. Phase IV data are needed to validate these findings. Darolutamide is a novel, nonsteroidal androgen receptor (AR)-signaling inhibitor. It serves as a second-generation antiandrogen and is currently indicated for the treatment of patients with nonmetastatic castration-resistant prostate cancer (nmCRPC). The product was approved by the United States Food and Drug Administration (FDA) in July 2019 and by the Japanese Ministry of Health, Labour and Welfare (MHLW) in January 2020 for the treatment of men with nmCRPC, and is awaiting approval in the E.U. for the same indication. This review will cover the background, preclinical development, safety, pharmacokinetics, pharmacodynamics and clinical studies that led to the approval of darolutamide. The key clinical data, ongoing trials and future directions for darolutamide are also discussed herein. The next-generation antiandrogen drugs, XTANDI (enzalutamide), ZYTIGA (abiraterone acetate), ERLEADA (apalutamide) and NUBEQA (darolutamide) extend survival times and improve quality of life in patients with advanced prostate cancer. Despite these advances, resistance occurs frequently and there is currently no definitive cure for castration-resistant prostate cancer. Our previous studies identified that similar mechanisms of resistance to enzalutamide or abiraterone occur following treatment and cross-resistance exists between these therapies in advanced prostate cancer. Here, we show that enzalutamide- and abiraterone-resistant prostate cancer cells are further cross-resistant to apalutamide and darolutamide. Mechanistically, we have determined that the AKR1C3/AR-V7 axis confers this cross-resistance. Knockdown of AR-V7 in enzalutamide-resistant cells resensitize cells to apalutamide and darolutamide treatment. Furthermore, targeting AKR1C3 resensitizes resistant cells to apalutamide and darolutamide treatment through AR-V7 inhibition. Chronic apalutamide treatment in C4-2B cells activates the steroid hormone biosynthesis pathway and increases AKR1C3 expression, which confers resistance to enzalutamide, abiraterone, and darolutamide. In conclusion, our results suggest that apalutamide and darolutamide share similar resistant mechanisms with enzalutamide and abiraterone. The AKR1C3/AR-V7 complex confers cross-resistance to second-generation androgen receptor-targeted therapies in advanced prostate cancer. BACKGROUND: Darolutamide is recently approved for the treatment of non-metastatic castrate resistance prostate cancer. Hitherto, no stereoselective pharmacokinetic data have been published pertaining to darolutamide and its diastereomers in animals or humans. The key aims of the experiment were to examine darolutamide, S,S-darolutamide and S,R-darolutamide with respect to (a) assessment of in vitro metabolic stability and protein binding and (b) characterization of in vivo oral and intravenous pharmacokinetics in mice. METHODS: In vitro (liver microsomes stability and protein binding) and in vivo experiments (oral/intravenous dosing to mice) were carried out using darolutamide, S,S-darolutamide and S,Rdarolutamide. Besides, tissue levels of darolutamide, S,S-darolutamide and S,R-darolutamide were measured following oral and intravenous dosing. Appropriate plasma/tissue samples served to determine the pharmacokinetics of various analytes in mice. Liquid chromatography in tandem with mass spectrometry procedures enabled the delineation of the plasma pharmacokinetics, in vitro and tissue uptake data of the various analytes. RESULTS: Chiral inversion was absent in the metabolic stability study. However, darolutamide showed profound stereoselectivity (S,S-darolutamide greater than S,R-darolutamide) after either intravenous or oral dosing. S,R-darolutamide but not S,S-darolutamide showed conversion to its antipode post oral and intravenous dosing to mice. Regardless of oral or intravenous dosing, active keto darolutamide formation was evident after administration of darolutamide, S,S-darolutamide or S,R- darolutamide. Tissue data supported the observations in plasma; however, tissue exposure of darolutamide, S,Sdarolutamide and S,R-darolutamide was much lower as compared to plasma. CONCLUSION: In lieu of the human pharmacokinetic data, although the administration of diastereomeric darolutamide was justified, it is proposed to delineate the clinical pharmacokinetics of S,Rdarolutamide and S,S-darolutamide relative to darolutamide in future clinical pharmacology studies. Enzalutamide is the first second-generation nonsteroidal androgen receptor (AR) antagonist with a strong binding affinity to AR. Most significantly, enzalutamide can prolong not only overall survival time and metastatic free survival time for patients with lethal castration-resistant prostate cancer (CRPC), but also castration-resistant free survival time for patients with castration-sensitive prostate cancer (CSPC). Enzalutamide has thus been approved by the US Food and Drug Administration (FDA) for the treatment of both metastatic (in 2012) and non-metastatic (in 2018) CRPC, as well as CSPC (2019). This is an inspiring drug discovery story created by an amazing interdisciplinary collaboration. Equally important, the successful clinical use of enzalutamide proves the notion that the second-generation AR antagonists can serve as hormonal therapeutics for three forms of advanced prostate cancer. This has been further verified by the recent FDA approval of the other two second-generation AR antagonists, apalutamide and darolutamide, for the treatment of prostate cancer. This review focuses on the rational design and discovery of these three second-generation AR antagonists, and then highlights their syntheses, clinical studies, and use. Strategies to overcome the resistance to the second-generation AR antagonists are also reviewed. Prostate cancer affects one in every nine men in the USA and is the second leading cause of cancer-related death. The treatment landscape of advanced prostate cancer is changing rapidly. Multiple agents including abiraterone, enzalutamide, apalutamide, darolutamide, docetaxel, cabazitaxel, radium-223, and sipuleucel-T have been approved for advanced prostate cancer. Appropriate drug selection remains crucial in this evolving landscape to derive maximum benefit for the patients. We summarize clinical trials leading to recent drug approvals and discuss optimal treatment selection. We also review recent advances in genomics including its evolving role in prognosis, in elucidating mechanisms of treatment resistance, and in guiding treatment decisions. Since 2018, apalutamide, darolutamide, and enzalutamide have been approved for the treatment of men with non-metastatic castration-resistant prostate cancer (M0CRPC). These approvals were based on the results of three separate randomized, placebo-controlled, phase III trials: SPARTAN (apalutamide), ARAMIS (darolutamide) and PROSPER (enzalutamide). These trials included men with M0CRPC and a short PSA doubling time (≤10 months). Results demonstrated a longer metastasis-free survival with these agents when used in conjunction with androgen deprivation therapy (ADT), compared to ADT alone. Updated results of these trials presented in the 2020 annual meeting of American Society of Oncology (ASCO) showed significantly improved overall survival with these agents. Based on these results, apalutamide, darolutamide, and enzalutamide can now be considered the standard of care treatment options for the treatment of men with M0CRPC. Collaborators: Metrebian S, Montes de Oca LF, Richardet M, Dowling A, Hovey E, Joshi A, Krieger L, Oliveira N, Parnis F, Meran J, Shariat S, Gedrevich Z, Polyakov S, Forget F, Waltregny D, Werbrouck P, Wynendaele W, Alves da Silva BS, Andrade L, Assed Bastos D, Beato CA, Borges GS, Cruz FJ, do Reis R, Eyll BM, Lago S, Lazaretti N, Luz M, Mavignier Carcano F, Murad AM, Paula AP, Pires LA, Ribeiro R, Rios L, Silveira GC, Zereu M, Mladenov D, Stamboliyski V, Chin J, Emmenegger U, Fleshner N, Gotto G, Jacobson A, Jansz KG, Ouellette P Jr, Rendon R, Siemens R, Correa JJ, Lobaton Ramirez JF, Salazar Rey M, Hanus T, Heracek J, Jandeisek J, Krolupper M, Matouskova M, Vseticka J, Pokker H, Hirvonen O, Marttila T, Rannikko A, Tammela T, Azria D, Azzouzi AR, Cathelineau X, Coloby P, Culine S, de la Taille A, Dourthe LM, Eymard JC, Fizazi K, Mouillet G, Haillot O, Helissey C, Lagneau E, Lebret T, Mahammedi H, Pfister C, Rigaud J, Rotarski M, Roubaud G, Ruffion A, Spaeth D, Tourani JM, Vincendeau S, Banek S, Binder M, Bogemann M, Carl S, Feyerabend S, Gleissner J, Heinemann V, Maier S, Niegisch G, Rudolph R, Schmitz-Drager B, Schneider T, Schostak M, Stenzl A, Stroelin P, Thomas C, Warnack W, Wiegl A, Wirth M, Ali T, Beli L, Csoszli T, Kocsis J, Nyirady P, Pajor L, Szabo A, Torzsok F, Toth Z, Grikshats E, Aglietta M, Damiano R, di Giorgi U, Facchini G, Gasparro D, Magna C, Natoli C, Sabbatini R, Scagliotti GV, Serretta V, Anai S, Asano K, Edamura K, Fukasawa S, Fukaya Y, Hirata A, Iinuma M, Kasuya Y, Kato M, Kawakita M, Kitamura H, Kobayashi K, Kondo Y, Masumori N, Matsubara N, Matsumoto H, Matsushima H, Miki K, Mizusawa H, Momma T, Motoyama D, Nagata M, Nakashima T, Namima T, Nishimatsu H, Nishimura K, Noguchi M, Nomura T, Okegawa T, Oya M, Sakai H, Suzuki K, Tabata K, Takamoto A, Tamada S, Tobe T, Tsurusaki T, Uemura H, Uemura H, Ukimura O, Yamaguchi S, Buldinskis G, Hublarovs O, Laukmanis A, Lietuvietis V, Litavniece D, Vjaters E, Auskalnis S, Jievaltas M, Ramonas H, Ulys A, Venckus R, Abrill Mendoza GV, Efrain Alarcon-Rozas A, Aleman Polanco DS, Philco Salas MJ, Badzio A, Borowka A, Kmieciak R, Majek A, Marcheluk A, Mruk A, Niezabitowski J, Senkus-Konefka E, Skoneczna IA, Wiechno P, Campos Pinheiro L, Faustino I, Fonseca J, Lima E, Mauricio J, Menezes N, Pina F, Prisco R, Ramos R, Rodrigues T, Bumbu G, Cauni V, Cebotaru CL, Calin Chibelean B, Dinu M, Doru Ghizdavescu G, Harza M, Jinga V, Lungulescu DS, Schenker M, Al-Shukri SH, Alekseev B, Alyasova A, Apolikhin O, Bljumberg B, Buiniakova A, Dykhno Y, Ivanov S, Khvorostenko D, Kogan M, Kopyltsov E, Lykov A, Pimenov I, Rodicheva N, Safina SZ, Semenov A, Shirinkin VB, Vladimirov V, Zaripov M, Colovic V, Stojanovic V, Breza J, Goncalves F, Sokol R, Coetzee L, Harris JK, Heyns C, Kraus P, Smit S, Choi HY, Chung BH, Hong SJ, Kang TW, Kim CS, Kim CI, Kim S, Kim WJ, Kwak C, Kwon TG, Yoon SJ, Alvarez-Ossorio J, Benejam JM, Carles J, Casas Nebra FJ, Garcio-Rojo D, Garde Noguera J, Gomez Veiga F, Gomez-Ferrer A, Gonzalez Del Alba A, Gonzalez Enguita C, Herdez Ferdez C, Juarez A, Jurado JM, Llorente Abarca C, Loizaga A, Medina Lopez RA, Mendez Vidal MJ, Ortiz M, Pinto Marin A, Puente Vazquez J, Ribal Caparros MJ, Rodriguez Antolin A, Saez Medina M, Suarez JF, Virizuela JA, Andren O, Damber JE, Haggman M, Hellstrom M, Huang SP, Ou YC, Pang ST, Pu YS, Wu TL, Esen A, Muezzinoglu T, Ozyurt C, Sozen TS, Yildirim A, Adamchuk H, Antonyan I, Bondarenko I, Grygorenko V, Ivashchenko P, Lyulko O, Rusyn A, Zaitsev V, Beesley S, Beresford M, Birtle A, Bryan N, Dixit S, Hussain S, Jones R, Kanaga Sundaram S, Koh P, Kynaston H, Madaan S, Ng Y, Rogers P, Vengalil S, Adams GW, Albany C, Aragon-Ching J, Aronson WJ, Bailen J, Belkoff L, Bernstein G, Bidair M, Bowles DW, Brown G, Cinman A, Clark RL, Clark W, Cohen T, Concepcion RS, Cookson M, Dumbadze I, Franks M, Gabrail NY, Garcia J, Gartrell B, Given RW, Kahnoski RJ, Karsh L, Koletsky A, Korman H, Matrana M, Merrick G, Millard FE, Mocharnuk R, Moss RA, Nordquist L, Patel K, Pieczonka C, Rivera I, Rosenberg SJ, Schwab WC, Sharifi R, Shevrin D, Shore N, Smith M, Taksey J, Tebyani N, Thara E, Tolia BM, Trainer AF, Tutrone RF, Ward E, Wertheim MS, Zorsky P. Author information: (1)Department of Urology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. (2)Department of Urology, The Jikei University School of Medicine, Tokyo, Japan. (3)Research Center for Evidence Based Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. (4)Deaprtment of Urology, PRES Centre Val de Loire, CHRU Tours, France, Université François Rabelais de Tours, Tours, France. (5)Department of Urology, King Fahad Specialist Hospital, Dammam, Saudi Arabia. (6)Institute for Urology and Reproductive Health, Sechenov University, Moscow, Russia. (7)Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. (8)Division of Urology, Department of Special Surgery, The University of Jordan, Amman, Jordan. (9)Cancer Prognostics and Health Outcomes Unit, University of Montreal Health Centre, Montreal, Canada. (10)Department of Urology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. [email protected]. (11)Institute for Urology and Reproductive Health, Sechenov University, Moscow, Russia. [email protected]. (12)Division of Urology, Department of Special Surgery, The University of Jordan, Amman, Jordan. [email protected]. (13)Department of Urology, Weill Cornell Medical College, New York, NY, USA. [email protected]. (14)Department of Urology, University of Texas Southwestern, Dallas, TX, USA. [email protected]. (15)Karl Landsteiner Institute of Urology and Andrology, Vienna, Austria. [email protected]. (16)Department of Urology, Second Faculty of Medicine, Charles University, Prague, Czech Republic. [email protected]. (17)European Association of Urology Research Foundation, Arnhem, Netherlands. [email protected]. Author information: (1)Department of Urology and Renal Transplantation, Yokohama City University Medical Center, 4-57 Urafune-cho, Minami-ku, Yokohama, 232-0024, Japan. [email protected]. (2)Department of Urology, Tokyo Metropolitan Police Hospital, 4-22-1 Nakano, Nakano-ku, 164-8541, Japan. (3)Department of Urology, Yokosuka Kyosai Hospital, 1-16 Yonegahamadori, Yokosuka, 238-8558, Japan. (4)Department of Urology, National Hospital Organization, Shinshu Ueda Medical Center, 1-27-21 Midorigaoka, Ueda, 386-8610, Japan. (5)Department of Urology, The Fraternity Memorial Hospital, 2-1-11 Yokoami, Sumida-ku, 130-8587, Japan. (6)Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805, Villejuif Cedex, France. (7)Massachusetts General Hospital Cancer Center, 55 Fruit Street, Boston, MA, 02114, USA. (8)Carolina Urologic Research Center, 823 82nd Parkway, Myrtle Beach, SC, 29572, USA. (9)Tampere University Hospital, Urologian poliklinikka, PL 2000, Teiskontie 35, 33521, Tampere, Finland. (10)Department of Urology, Kitasato University Hospital, 1-15-1 Kitazato Minami-ku, Sagamihara, 252-0375, Japan. (11)Department of Breast and Medical Oncology, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, 277-8577, Japan. (12)Department of Urology, National Hospital Organization, Mito Medical Center, 280 Sakuranosato Ibarakimachi, Higashiibaraki, 311-3193, Japan. (13)Department of Urology, Kindai University, 377-2, Onohigashi, Osakasayama, 589-8511, Japan. (14)Department of Urology, Keio University, 35 Shio-machi, Shinjuku-ku, 160-8582, Japan. (15)Department of Urology, National Hospital Organization, Saitama National Hospital, 2-1 Suwa, Wako, 351-0102, Japan. (16)Department of Urology, Kobe City Medical Center General Hospital, 2-1-1 Minatojimaminamimachi Chuo-ku, Kobe, 650-0047, Japan. (17)Division of Urology, Chiba Cancer Center, 666-2, Nitona-cho, Chuo-ku, Chiba, 260-8717, Japan. (18)Department of Urology, Fukui Prefectural Hospital, 2-8-1 Yotsui, Fukui, 910-8526, Japan. (19)Clinical Statistics, Bayer AG, Building P300, B342, 13342, Berlin, Germany. (20)Bayer Healthcare SAS, 220 Avenue de la Recherche, 59120, Loos, France. (21)Orion Corporation Orion Pharma, Orionintie 1, P.O. Box 65, FI-02101, Espoo, Finland. (22)PCI Biotech, Ullernchausseen 64, 0379, Oslo, Norway. (23)Department of Urology, Gunma University, 3-39-15 Showa-machi, Maebashi, 371-8511, Japan. Oral darolutamide (Nubeqa™) is a novel second-generation, nonsteroidal, selective androgen receptor (AR) inhibitor indicated for the treatment of non-metastatic castration-resistant prostate cancer (nmCRPC). In the pivotal multinational, phase 3 ARAMIS trial in men with nmCRPC, relative to placebo plus ongoing androgen deprivation therapy (ADT), darolutamide (+ ADT) significantly prolonged metastasis-free survival (MFS) at the time of the primary analysis and overall survival (OS) at the time of the final OS analysis and was generally well tolerated in extended follow-up. Albeit long-term data from the real-world setting are required to fully define the safety profile of darolutamide, current evidence from the final ARAMIS analysis indicates that darolutamide has a low propensity for CNS-related adverse events (AEs) associated with other currently approved second-generation AR inhibitors. Given the efficacy and safety evidence from the final ARAMIS analysis and the key role of second-generation AR inhibitors in the management of nmCRPC, darolutamide + ADT represents an important emerging option for the treatment of men with nmCRPC who are at high risk of developing metastatic prostate cancer.

Which cancers can be treated with Selpercatinib?

Selpercatinib was recently approved by the US FDA for the treatment of RET fusion-positive non-small-cell lung cancer, RET fusion-positive thyroid cancer and RET-mutant medullary thyroid cancer.

INTRODUCTION: Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described. METHODS: Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays. RESULTS: After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs. CONCLUSIONS: RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors. Selpercatinib (RETEVMO™) is a receptor tyrosine kinase RET (rearranged during transfection) inhibitor being developed by Loxo Oncology for the treatment of cancers harbouring RET alterations. Based on results from the phase I/II LIBRETTO-001 trial, selpercatinib was recently approved by the US FDA for the treatment of RET fusion-positive non-small-cell lung cancer, RET fusion-positive thyroid cancer and RET-mutant medullary thyroid cancer. This article summarizes the milestones in the development of selpercatinib leading to this first approval. Author information: (1)From Memorial Sloan Kettering Cancer Center and Weill Cornell Medical College (A.D., E.R.) and New York University Langone Medical Center (V.V.), New York, and Roswell Park Comprehensive Cancer Center, Buffalo (G.K.D.) - all in New York; Dana-Farber Cancer Institute (G.R.O.) and Massachusetts General Hospital (J.G.), Boston; National Cancer Centre Singapore, Singapore (D.S.W.T.); the Chinese University of Hong Kong, Hong Kong, China (H.H.F.L.); Sarah Cannon Research Institute, Nashville (M.J.); University of California, San Francisco-Helen Diller Family Comprehensive Cancer Center, San Francisco (C.E.M.), University of California, San Diego, Moores Cancer Center, La Jolla (L.B.), and City of Hope Comprehensive Cancer Center, Duarte (K.L.R.) - all in California; University of Bern, Bern, and Cantonal Hospital of Lucerne, Lucerne - both in Switzerland (O.G.); Institut Gustave Roussy, Villejuif (B.B.), Hospital La Timone, Marseille (F.B.), and Centre Léon Bérard, Lyon (P.A.C.) - all in France; Severance Hospital, Yonsei University Health System (B.C.C.), and Samsung Medical Center, Sungkyunkwan University School of Medicine (K.P.), Seoul, and Seoul National University Bundang Hospital, Seongnam (Y.J.K.) - all in South Korea; Soroka University Medical Center, Beer-Sheva, Israel (N.P.); University of North Carolina-Chapel Hill, Chapel Hill (J. Weiss); National Cancer Center Hospital (Y.O.) and Cancer Institute Hospital of the Japanese Foundation for Cancer Research (M. Nishio), Tokyo, National Hospital Organization Kyushu Cancer Center, Fukuoka (T. Seto), Tottori University Hospital, Tottori (T. Sakamoto), Hyogo Cancer Center, Akashi (M.S.), Okayama University Hospital, Okayama (K.O.), and National Cancer Center Hospital East, Chiba (K.G.) - all in Japan; University of Chicago, Chicago (J.P.); the Ohio State University Comprehensive Cancer Center, Columbus (M.H.S.), and Cleveland Clinic, Cleveland (N.A.P.); Istituto Nazionale Tumori-National Cancer Institute, Università degli Studi di Milano, Milan (F.D.B.); Vall d'Hebron Institute of Oncology, Hospital Universitario Vall d'Hebron, Barcelona (E.G.); Center for Integrated Oncology, University Hospital of Cologne, Cologne, Germany (J. Wolf); Peter MacCallum Cancer Institute, Melbourne, VIC, Australia (B.S.); Sarah Cannon Research Institute at HealthONE, Denver (G.F.); Loxo Oncology, Stamford, CT (K.E., M. Nguyen, B.N., E.Y.Z., L.Y., X.H., E.O., S.M.R.); and the University of Texas M.D. Anderson Cancer Center, Houston (V.S.). Author information: (1)From Massachusetts General Hospital (L.J.W.) and Dana-Farber Cancer Institute (J. Lorch), Boston; Memorial Sloan Kettering Cancer Center, New York (E.S., A.D.); Royal North Shore Hospital, St. Leonards, NSW (B.R.), and Peter MacCallum Cancer Institute, Melbourne, VIC (B.S.) - both in Australia; University of California, San Francisco-Helen Diller Family Comprehensive Cancer Center, San Francisco (H.K.), David Geffen School of Medicine at UCLA, Los Angeles (J.W.G.), and Chao Family Comprehensive Cancer Center, University of California Irvine, Orange (V.W.Z.) - all in California; University of Michigan, Ann Arbor (F.W.), and START Midwest, Grand Rapids (N.L.) - both in Michigan; University of Pennsylvania, Philadelphia (M.B.); University of Chicago, Chicago (J.P.); Gustave Roussy, Villejuif (S.L.), Institut Bergonié, Bordeaux (Y.G.), Aix Marseille University, Centre National de la Recherche Scientifique, INSERM, Centre de Recherche en Cancérologie de Marseille, Assistance Publique-Hôpitaux de Marseille, Early Phase Cancer Trial Center CLIP2, Hospital La Timone, Marseille (F.B.), Centre Léon Bérard, Lyon (C.D.L.F.), and Hôpital Européen Georges-Pompidou, Faculté de Médecine Paris-Descartes, Paris (J.M.) - all in France; Mayo Clinic-Rochester, Rochester, MN (J.C.M.); Winship Cancer Institute of Emory University, Atlanta (T.K.O.); National Cancer Center Singapore, Singapore (D.S.W.T.); University of Bern, Bern, and Cantonal Hospital of Lucerne, Lucerne - both in Switzerland (O.G.); University of North Carolina-Chapel Hill, Chapel Hill (J.W.); University of Wisconsin-Carbone Cancer Center, Madison (M.E.B.); British Columbia Cancer Agency, Vancouver, Canada (J. Laskin); Oregon Health and Science University, Portland (M.H.T.); Universitätsklinikum Würzburg, Department of Internal Medicine I, Division of Endocrinology and Diabetology, Würzburg, Germany (M.K.); Sarah Cannon Research Institute-Tennessee Oncology, Nashville (T.M.B.); Johns Hopkins Kimmel Cancer Center, Washington, DC (B.L.); Fundación Jimenez Diaz, START-Madrid, Madrid (V.M.); Loxo Oncology, Stamford, CT (K.E., M.N., D.H., E.Y.Z., X.H., L.Y., J.K., S.M.R.); University of Texas M.D. Anderson Cancer Center, Houston (V.S., M.E.C.); and Ohio State University Comprehensive Cancer Center, Columbus (M.H.S.). Conflict of interest statement: Conflicts of interest disclosure statement: E.Y.R. reports research support from Bayer. M.L.J. reports research funds to her institution from Loxo Oncology, Inc., a wholly owned subsidiary of Eli Lilly and Company (hereafter referred to as Loxo Oncology); AbbVie; Acerta; Adaptimmune; Apexigen; Array BioPharma; AstraZeneca; Atreca; BeiGene; Birdie; Boehringer Ingelheim; Checkpoint Therapeutics; Corvus Pharmaceuticals; CytomX; Daiichi Sankyo; Dynavax; Eli Lilly and Company; EMD Serono; Genentech/Roche; Genmab; Genocea Biosciences; GlaxoSmithKline; Gritstone Oncology; Guardant Health; Hengrui Therapeutics; Immunocore; Incyte; Janssen; Jounce Therapeutics; Kadmon Pharmaceuticals; Lycera; Merck; Mirati Therapeutics; Neovia Oncology; Novartis; OncoMed Pharmaceuticals; Pfizer; Regeneron Pharmaceuticals; Sanofi; Shattuck Labs; Stem CentRx; Syndax Pharmaceuticals; Takeda Pharmaceuticals; Tarveda; University of Michigan; WindMIL; TCR2 Therapeutics; Arcus Biosciences; Ribon Therapeutics; and Amgen. M.L.J. also reports consulting/advisory roles through her institution for AbbVie, Achilles Therapeutics, AstraZeneca, Atreca, Boehringer Ingelheim, Calithera Biosciences, Genentech, GlaxoSmithKline, Gritstone Oncology, Guardant Health, Incyte, Janssen, Eli Lilly and Company, Loxo Oncology, Merck, Mirati Therapeutics, Novartis, Pfizer, Ribon Therapeutics, Sanofi, and Association of Community Cancer Centers; fees for food/beverage/travel from Abbvie, Astellas, AstraZeneca, Boehringer Ingelheim, Clovis, Daiichi Sankyo, EMD Serono, Bristol Myers Squibb, Exelixis, Genentech/Roche, Incyte, Merck, Pfizer, Sysmex Inostics, Vapotherm, Janssen, Eli Lilly and Company, Novartis, and Sanofi; and Contract Lobbyist roles of her spouse for Astellas and Otsuka Pharmaceuticals. S.E.C. reports no conflicts of interest. R.S. reports research funding from Loxo Oncology, Helsinn Healthcare, Merus, and Elevation Oncology. J.F.K. is an employee of Loxo Oncology. J.S., A.A.B., M.A.D., and E.G. report no conflicts of interest. E.V.I. reports being an inventor on a pending patent related to manipulating, culturing, and evaluating tumor organotypic spheroids in 3D microfluidic devices. D.N.H. is an employee of Loxo Oncology. E.M.K., M. Lin, and M.S.D.M. report no conflicts of interest. B.C.N. and E.A.O. are employees of Loxo Oncology. J.E.S. and M.V. report no conflicts of interest. K.E. is an employee and stockholder of Loxo Oncology. J.F.H. reports a research grant from Eli Lilly and Company and an honorarium from Illumina. B.T.L. reports two pending institutional patents at Memorial Sloan Kettering Cancer Center (US62/685,057, US62/514,661); consultant/advisory board member roles for Roche/Genentech, Biosceptre International, Thermo Fisher Scientific, Mersana Therapeutics, Hengrui Therapeutics, Guardant Health, Eli Lilly and Company; and research funds to his institution from Roche/Genentech, Daiichi Sankyo, Hengrui Therapeutics, Illumina, Guardant Health, BioMed Valley Discoveries, AstraZeneca, GRAIL, MORE Health, Amgen, and Eli Lilly and Company. L.M.S. reports consulting fees from EMD Serono, scientific advisory board roles for Loxo Oncology and Foghorn Therapeutics, and honorarium from Astra Zeneca. B.S.T. reports honoraria and research funding from Genentech and discovery board activities for Boehringer Ingelheim and Loxo Oncology; all stated activities were outside of the work described herein. M. Ladanyi reports ad hoc advisory board roles for Eli Lilly and Company and Loxo Oncology, and research support from Helsinn Healthcare and Loxo Oncology. P.A.J. reports consulting fees from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche/Genentech, Takeda Oncology, ACEA Biosciences, Eli Lilly and Company, Araxes Pharma, Ignyta, Mirati Therapeutics, Novartis, Loxo Oncology, Daiichi Sankyo, Sanofi Oncology, Voronoi, SFJ Pharmaceuticals and Biocartis; receiving post-marketing royalties from DFCI owned intellectual property on EGFR mutations licensed to Lab Corp; sponsored research agreements with AstraZeneca, Daichi-Sankyo, PUMA, Boehringer Ingelheim, Eli Lilly and Company, Revolution Medicines and Astellas Pharmaceuticals; and stock ownership in Loxo Oncology and Gatekeeper Pharmaceuticals. S.M.R., a former employee of Loxo Oncology, was employed by Loxo Oncology at the time of this work. A.E.D. reports consulting/advisory roles for Ignyta, Loxo Oncology, TP Therapeutics, AstraZeneca, Pfizer, Blueprint Medicines, Genentech/Roche, Helsinn Therapeutics, BeiGene, Hengrui Therapeutics, Exelixis, Bayer, Tyra Biosciences, Verastem, Takeda/Millennium, BerGenBio, MORE Health, Eli Lilly and Company, and Verastem; royalties for Pocket Oncology; honoraria from Medscape, OncLive, PeerVoice, Physician’s Education Resource, Targeted Oncology, MORE Health, Research to Practice, Foundation Medicine, and Peerview; and research funding from Foundation Medicine. G.R.O. reports consulting fees from Abbvie, AstraZeneca, Blueprint, Dropworks, GRAIL, Janssen, Illumina, Inivata, Loxo Oncology, Sysmex, and Takeda; and honoraria from Foundation Medicine and Guardant. BACKGROUND: Targeted kinase inhibitors have been increasingly utilized in the treatment of advanced medullary thyroid cancer (MTC) over the last decade. Recently, highly potent next generation selective RET inhibitors have been clinically validated, and selpercatinib was recently Food and Drug Administration (FDA)-approved for advanced MTC. The advent of highly selective, potent RET inhibitors is broadening the treatment options for patients with RET-mutated cancers. METHODS: We report the first published case of neoadjuvant selpercatinib followed by surgery for a patient with initially unresectable, widely metastatic, RET-mutated MTC who was treated on a single patient protocol. RESULTS: After greater than 50% RECIST response, the patient underwent complete surgical resection followed by selpercatinib resumption. He remains locoregionally disease-free 21 months after starting therapy with stable metastatic disease (after initial partial response); and calcitonin/CEA continue to decline. CONCLUSION: This novel treatment strategy for locoregionally advanced RET-mutated MTC warrants further study in clinical trials. On May 8, 2020, the FDA granted accelerated approval to selpercatinib for (i) adult patients with metastatic RET fusion-positive non-small cell lung cancer (NSCLC), (ii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET-mutant medullary thyroid cancer who require systemic therapy, and (iii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET fusion-positive thyroid cancer who require systemic therapy and who are radioactive iodine refractory (if radioactive iodine is appropriate). Approval was granted on the basis of the clinically important effects on the overall response rate (ORR) with prolonged duration of responses observed in a multicenter, open-label, multicohort clinical trial (LIBRETTO-001, NCT03157128) in patients whose tumors had RET alterations. ORRs within the approved patient populations ranged from 64% [95% confidence interval (CI), 54-73] in prior platinum-treated RET fusion-positive NSCLC to 100% (95% CI, 63-100) in systemic therapy-naïve RET fusion-positive thyroid cancer, with the majority of responders across indications demonstrating responses of at least 6 months. The product label includes warnings and precautions for hepatotoxicity, hypertension, QT interval prolongation, hemorrhagic events, hypersensitivity, risk of impaired wound healing, and embryo-fetal toxicity. This is the first approval of a drug specifically for patients with RET alterations globally.

What is the target of Volanesorsen?

Volanesorsen is a second-generation antisense oligonucleotide inhibiting apoC-III (apolipoprotein C-III) transcription/translation that has been recently approved in Europe for Familial Chylomicronemia Syndrome (FCS) treatment.

OBJECTIVE: To determine the effects of volanesorsen (ISIS 304801), a second-generation 2'-O-methoxyethyl chimeric antisense inhibitor of apolipoprotein (apo)C-III, on triglyceride (TG) levels and insulin resistance in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: A randomized, double-blind, placebo-controlled trial was performed in 15 adult patients with type 2 diabetes (HbA1c >7.5% [58 mmol/mol]) and hypertriglyceridemia (TG >200 and <500 mg/dL). Patients were randomized 2:1 to receive volanesorsen 300 mg or placebo for a total of 15 subcutaneous weekly doses. Glucose handling and insulin sensitivity were measured before and after treatment using a two-step hyperinsulinemic-euglycemic clamp procedure. RESULTS: Treatment with volanesorsen significantly reduced plasma apoC-III (-88%, P = 0.02) and TG (-69%, P = 0.02) levels and raised HDL cholesterol (HDL-C) (42%, P = 0.03) compared with placebo. These changes were accompanied by a 57% improvement in whole-body insulin sensitivity (P < 0.001). Importantly, we found a strong relationship between enhanced insulin sensitivity and both plasma apoC-III (r = -0.61, P = 0.03) and TG (r = -0.68, P = 0.01) suppression. Improved insulin sensitivity was sufficient to significantly lower glycated albumin (-1.7%, P = 0.034) and fructosamine (-38.7 μmol/L, P = 0.045) at the end of dosing and HbA1c (-0.44% [-4.9 mmol/mol], P = 0.025) 3 months postdosing. CONCLUSIONS: Volanesorsen reduced plasma apoC-III and TG while raising HDL-C levels. Importantly, glucose disposal, insulin sensitivity, and integrative markers of diabetes also improved in these patients after short-term treatment. Elevated triglyceride levels (higher than ~1000 mg/dL) are associated with an increased risk for pancreatitis. Apolipoprotein-CIII (apoC-III) plays a key role in the metabolism of triglycerides and triglyceride-rich lipoproteins. Loss of function mutations in the gene encoding apoC-III (APOC3) is associated with low triglyceride levels and a decreased risk for cardiovascular disease (CVD) while overexpression of APOC3 is associated with hypertriglyceridemia. Although many drugs such as fibrates, statins and omega-3 fatty acids modestly decrease triglyceride levels (and apoC-III concentrations), there are many patients who still have severe hypertriglyceridemia and are at increased risk for pancreatitis and potentially for CVD. The antisense oligonucleotide (ASO) against APOC3 mRNA volanesorsen (previously called ISIS 304801, ISIS-ApoCIIIRx and IONIS-ApoCIIIRx) robustly decreases both, apoC-III production and triglyceride concentrations and is being currently evaluated in phase 3 trials. In this narrative review, we present the currently available clinical evidence on the efficacy and safety of volanesorsen for the treatment of hypertriglyceridemia. PURPOSE OF REVIEW: Apolipoprotein CIII (ApoCIII) is now recognized as a key regulator in severe hypertriglyceridemia, chylomicronemia, and conditions of triglyceride-rich lipoprotein (TRL) remt excess due to its inhibition of lipoprotein lipase (LPL) and hepatic lipase, leading to decreased hepatic reuptake of TRLs, as well as enhanced synthesis and secretion of VLDL from the liver. ApoCIII gain-of-function mutations are associated with atherosclerosis and coronary heart disease (CHD), and contribute to the development of cardiometabolic syndrome, hypertriglyceridemia, and type 2 diabetes mellitus. Conversely, loss-of-function mutations in ApoCIII are associated with lower levels of plasma triglycerides (TG), attenuation of vascular inflammatory processes such as monocyte adhesion and endothelial dysfunction, and potentially, a reduction in the incidence and progression of atherosclerosis and cardioprotection. RECENT FINDINGS: Evidence is now emerging that volanesorsen, a second-generation antisense oligonucleotide drug targeting ApoCIII messenger RNA resulting in decreases in TG in patients with familial chylomicronemia syndrome, severe hypertriglyceridemia, and metabolic dyslipidemia with type 2 diabetes giving support to the hypothesis that ApoCIII is a powerful inhibitor of LPL, and when reduced, endogenous clearance of TRLs can result in substantial reductions in TG levels. Discovery of the ApoCIII inhibitor volanesorsen opens a new era of lipid-lowering drugs for reduction in TG and potentially for reduction in LDL-C. Herein, this review will provide an update on the pathophysiology of ApoCIII-linked atherosclerosis and the development of the first drug to target ApoCIII, volanesorsen, as a promising lipid-lowering agent. BACKGROUND: Volanesorsen, an investigational inhibitor of apoC-III synthesis, significantly reduced triglyceride levels in clinical trials in patients with familial chylomicronemia syndrome (FCS), a rare genetic disorder characterized by marked chylomicronemia leading to a spectrum of symptoms, including recurrent abdominal pain and episodes of potentially fatal acute pancreatitis (AP). OBJECTIVE: To determine the effect of volanesorsen on burden of disease on patients with FCS Methods: ReFOCUS was a retrospective global web-based survey open to patients with FCS who received volanesorsen for ≥3 months in an open-label extension study. The survey included questions about patients' experiences before and after volanesorsen treatment. RESULTS: Twenty-two respondents had received volanesorsen for a median of 222 days. Volanesorsen significantly reduced the number of symptoms per patient across physical, emotional, and cognitive domains. Significant reductions from baseline were reported for steatorrhea, pancreatic pain, and constant worry about an attack of pain/AP. Respondents reported that volanesorsen improved overall management of symptoms and reduced interference of FCS with work/school responsibilities. Reductions in the negative impact of FCS on personal, social, and professional life were also reported. CONCLUSIONS: Treatment with volanesorsen has the potential to reduce disease burden in patients with FCS through modulation of multiple symptom domains. Familial chylomicronemia syndrome (FCS) is a rare autosomal recessive disorder typically caused by mutations in genes for lipoprotein lipase (LPL), apolipoprotein C-II (Apo-CII), apolipoprotein A-V (Apo-AV), lipase maturation factor 1 (LMF1) and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPI-HBP1). FCS is associated with severe morbidity that includes recurrent pancreatitis and other problems. Effective treatment to reliably prevent complications has been unavailable, so there is a quest to identify novel interventions to achieve sustained triglyceride lowering and prevention of pancreatitis. Apolipoprotein C-III (Apo-CIII) interferes with triglyceride clearance by blocking LPL and alternative pathways. Volanesorsen is an experimental antisense oligonucleotide that inhibits translation of Apo-CIII mRNA, thereby substantially lowering plasma levels of Apo-CIII and triglycerides. It is being developed for treatment of patients with FCS and refractory hypertriglyceridemia. Data from a variety of clinical trials have been very encouraging, with documentation of excellent triglyceride-lowering efficacy, but there have been concerns about the risk of drug-related thrombocytopenia and bleeding that contributed to the recent decision by the Food and Drug Administration (FDA) to not approve the drug for clinical use. Clinical trials testing the safety and efficacy of volanesorsen are ongoing, so there is hope that the drug ultimately will be approved and available for treatment of high-risk patients with FCS. BACKGROUND: Familial chylomicronemia syndrome is a rare genetic disorder that is caused by loss of lipoprotein lipase activity and characterized by chylomicronemia and recurrent episodes of pancreatitis. There are no effective therapies. In an open-label study of three patients with this syndrome, antisense-mediated inhibition of hepatic APOC3 mRNA with volanesorsen led to decreased plasma apolipoprotein C-III and triglyceride levels. METHODS: We conducted a phase 3, double-blind, randomized 52-week trial to evaluate the safety and effectiveness of volanesorsen in 66 patients with familial chylomicronemia syndrome. Patients were randomly assigned, in a 1:1 ratio, to receive volanesorsen or placebo. The primary end point was the percentage change in fasting triglyceride levels from baseline to 3 months. RESULTS: Patients receiving volanesorsen had a decrease in mean plasma apolipoprotein C-III levels from baseline of 25.7 mg per deciliter, corresponding to an 84% decrease at 3 months, whereas patients receiving placebo had an increase in mean plasma apolipoprotein C-III levels from baseline of 1.9 mg per deciliter, corresponding to a 6.1% increase (P<0.001). Patients receiving volanesorsen had a 77% decrease in mean triglyceride levels, corresponding to a mean decrease of 1712 mg per deciliter (19.3 mmol per liter) (95% confidence interval [CI], 1330 to 2094 mg per deciliter [15.0 to 23.6 mmol per liter]), whereas patients receiving placebo had an 18% increase in mean triglyceride levels, corresponding to an increase of 92.0 mg per deciliter (1.0 mmol per liter) (95% CI, -301.0 to 486 mg per deciliter [-3.4 to 5.5 mmol per liter]) (P<0.001). At 3 months, 77% of the patients in the volanesorsen group, as compared with 10% of patients in the placebo group, had triglyceride levels of less than 750 mg per deciliter (8.5 mmol per liter). A total of 20 of 33 patients who received volanesorsen had injection-site reactions, whereas none of the patients who received placebo had such reactions. No patients in the placebo group had platelet counts below 100,000 per microliter, whereas 15 of 33 patients in the volanesorsen group had such levels, including 2 who had levels below 25,000 per microliter. No patient had platelet counts below 50,000 per microliter after enhanced platelet-monitoring began. CONCLUSIONS: Volanesorsen lowered triglyceride levels to less than 750 mg per deciliter in 77% of patients with familial chylomicronemia syndrome. Thrombocytopenia and injection-site reactions were common adverse events. (Funded by Ionis Pharmaceuticals and Akcea Therapeutics; APPROACH Clinical Trials.gov number, NCT02211209.). OBJECTIVE: ApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients. CONCLUSIONS: Our results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels. INTRODUCTION: The prevalence of hypertriglyceridemia (HTG) is increasing. Elevated triglyceride (TG) levels are associated with an increased cardiovascular disease (CVD) risk. Moreover, severe HTG results in an elevated risk of pancreatitis, especially in severe HTG with an up to 350-fold increased risk. Both problems emphasize the clinical need for effective TG lowering. AREAS COVERED: The purpose of this review is to discuss the currently available therapies and to elaborate the most promising novel therapeutics for TG lowering. EXPERT OPINION: Conventional lipid lowering strategies do not efficiently lower plasma TG levels, leaving a residual CVD and pancreatitis risk. Both apolipoprotein C-III (apoC-III) and angiopoietin-like 3 (ANGPTL3) are important regulators in TG-rich lipoprotein (TRL) metabolism. Several novel agents targeting these linchpins have ended phase II/III trials. Volanesorsen targeting apoC-III has shown reductions in plasma TG levels up to 90%. Multiple ANGPLT3 inhibitors (evinacumab, IONIS-ANGPTL3-LRx, ARO-ANG3) effectuate TG reductions up to 70% with concomitant potent reduction in all other apoB containing lipoprotein fractions. We expect these therapeutics to become players in the treatment for (especially) severe HTG in the near future. The aim of this study was to assess the effect of volanesorsen on the corrected QT (QTc) interval. This thorough QT study enrolled 52 healthy male and female subjects who were randomized at a single site in a four-way crossover study. Subjects were randomly assigned to 1 of 12 treatment sequences and crossed over into four treatment periods over the course of which each subject was to receive a single therapeutic dose of volanesorsen as a 300 mg subcutaneous (SC) injection, a single supratherapeutic dose of volanesorsen as 300 mg intravenous (IV) infusion, a single oral (PO) dose of moxifloxacin (positive control), and placebo dose. The study demonstrated that volanesorsen 300 mg SC and 300 mg IV did not have a clinically relevant effect on ΔΔQTcF exceeding 10 ms. The largest mean effect at any postdose time point was 3.0 ms (90% confidence interval [CI]: 0.8-5.2) after SC dosing and 1.8 ms (90% CI -0.4 to 4.0) after IV dosing. Volanesorsen, at the studied therapeutic and supratherapeutic doses, does not have a clinically meaningful effect on the QTc. INTRODUCTION: Severe hypertriglyceridemia (sHTG) is a complex disorder of lipid metabolism characterized by plasma levels of triglyceride (TG) greater than 885 mg/dl (>10 mmol/L). The treatment of sHTG syndromes is challenging because conventional treatments are often ineffective in reducing TG under the threshold to prevent acute pancreatitis (AP). The inhibition of APOC3, which encodes a protein involved in triglyceride (TG)-rich lipoproteins (TGRLs) removal, has been reported to be a novel target for the treatment of sHTG. Volanesorsen is a second-generation antisense oligonucleotide inhibiting apoC-III transcription/translation that has been recently approved in Europe for Familial Chylomicronemia Syndrome (FCS) treatment. AREAS COVERED: This review summarizes the evidences on the efficacy and safety of volanesorsen for the treatment of sHTG syndromes. EXPERT OPINION: Volanesorsen effectively reduces TG in sHTG through a mechanism that is mainly LPL-independent, potentially decreasing the risk of AP. Some safety concerns have been raised with the use of volanesorsen, mainly represented by the occurrence of thrombocytopenia. Due to the potential severity of side effects, some caution is needed before affirming the long-term utility of this drug. Despite this, volanesorsen currently remains the only drug that has been demonstrated effective in FCS, which otherwise remains an untreatable disease.

Roflumilast Cream is effective for which disease?

Roflumilast Cream has been shown to be effective for psoriasis.

BACKGROUND: Oral phosphodiesterase (PDE)4 inhibitors have shown efficacy in chronic obstructive pulmonary disease and psoriasis. OBJECTIVES: To assess the effectiveness, local safety and tolerability, and systemic pharmacokinetics of two topical PDE4 inhibitors, roflumilast and TAK-084, in plaque psoriasis. METHODS: An intraindividual comparison of six topical products was made in 15 patients aged 18-65 years with stable chronic plaque psoriasis in an investigator-blinded, within-subject randomized study. The products evaluated were calcipotriol 0·005% cream; betamethasone valerate 0·1% (both in their marketed formulations); investigational cream formulations of roflumilast 0·5% and TAK-084 0·5% and 5%; and a vehicle cream formulation as a control. Each treatment was applied daily to different test sites located on psoriasis plaques for 3 weeks. RESULTS: The primary end point of (mean) change from baseline in skin infiltrate thickness after 3 weeks of treatment showed statistically significant improvements for all treatments: betamethasone valerate cream (-286·9 μm), the selective PDE4 inhibitors roflumilast 0·5% (-237·1 μm) and TAK-084 (0·5% cream, -153·6 μm; 5% cream, -216·7 μm) and calcipotriol 0·005% (-187·7 μm) when compared with vehicle cream control (all P < 0·001). Both the TAK-084 5% and roflumilast 0·5% formulations performed well overall compared with the potent corticosteroid, betamethasone, and were ranked better than the vitamin D analogue calcipotriol. All adverse events were mild or moderate and none was serious. CONCLUSIONS: Topical treatment with cream formulations of the PDE4 inhibitors roflumilast and TAK-084 reduced inflammation, measured as a change in skin infiltrate thickness, and reduced psoriasis severity. Corticosteroid treatments have known systemic and cutaneous side-effects; PDE4 inhibitors could offer an alternative to these and deserve further study. BACKGROUND: Systemic oral phosphodiesterase type 4 (PDE-4) inhibitors have been effective in the treatment of psoriasis. Roflumilast cream contains a PDE-4 inhibitor that is being investigated for the topical treatment of psoriasis. METHODS: In this phase 2b, double-blind trial, we randomly assigned adults with plaque psoriasis in a 1:1:1 ratio to use roflumilast 0.3% cream, roflumilast 0.15% cream, or vehicle (placebo) cream once daily for 12 weeks. The primary efficacy outcome was the investigator's global assessment (IGA) of a status of clear or almost clear at week 6 (assessed on a 5-point scale of plaque thickening, scaling, and erythema; a score of 0 indicates clear, 1 almost clear, and 4 severe). Secondary outcomes included an IGA score indicating clear or almost clear plus a 2-grade improvement in the IGA score for the intertriginous area and the change in the Psoriasis Area and Severity Index (PASI) score (range, 0 to 72, with higher scores indicating worse disease). Safety was also assessed. RESULTS: Among 331 patients who underwent randomization, 109 were assigned to roflumilast 0.3% cream, 113 to roflumilast 0.15% cream, and 109 to vehicle cream. An IGA score indicating clear or almost clear at week 6 was observed in 28% of the patients in the roflumilast 0.3% group, in 23% in the roflumilast 0.15% group, and in 8% in the vehicle group (P<0.001 and P = 0.004 vs. vehicle for roflumilast 0.3% and 0.15%, respectively). Among the approximately 15% of patients overall who had baseline intertriginous psoriasis of at least mild severity, an IGA score at week 6 indicating clear or almost clear plus a 2-grade improvement in the intertriginous-area IGA score occurred in 73% of the patients in the roflumilast 0.3% group, 44% of those in the roflumilast 0.15% group, and 29% of those in the vehicle group. The mean baseline PASI scores were 7.7 in the roflumilast 0.3% group, 8.0 in the roflumilast 0.15% group, and 7.6 in the vehicle group; the mean change from baseline at week 6 was -50.0%, -49.0%, and -17.8%, respectively. Application-site reactions occurred with similar frequency in the roflumilast groups and the vehicle group. CONCLUSIONS: Roflumilast cream administered once daily to affected areas of psoriasis was superior to vehicle cream in leading to a state of clear or almost clear at 6 weeks. Longer and larger trials are needed to determine the durability and safety of roflumilast in psoriasis. (Funded by Arcutis Biotherapeutics; ARQ-151 201 ClinicalTrials.gov number, NCT03638258.). Background: Roflumilast cream (ARQ-151) is a highly potent, selective phosphodiesterase-4 inhibitor in development for once-daily topical treatment of chronic plaque psoriasis. Objectives: To assess the safety and efficacy of once-daily roflumilast cream 0.5% and 0.15% in patients with chronic plaque psoriasis. Methods: This phase 1/2a study enrolled a single-dose, open-label cohort (Cohort 1: 0.5% cream applied to 25 cm² psoriatic plaques), and a 28-day, double-blinded cohort (Cohort 2: 1:1:1 randomization to roflumilast cream 0.5%, 0.15%, or vehicle). Patients had chronic plaque psoriasis of >6 months' duration with ≤5% body surface area involvement. Outcomes included safety (adverse events) and efficacy (percentage change in the Target Plaque Severity Score [TPSS] × Target Plaque Area [TPA]) at week 4. Results: For Cohorts 1 (n=8) and 2 (n=89), adverse events (all mild/moderate; none severe or serious) were similar between active arms and vehicle. Treatment-related events were confined to the application site, without differences between drug and vehicle. No patient discontinued treatment due to adverse events. The primary efficacy endpoint was met for both roflumilast cream doses: TPSS×TPA improvement at week 4 was statistically significant for roflumilast 0.5% (P=0.0007) and 0.15% (P=0.0011) versus vehicle; significance was reached as early as 2 weeks. For both roflumilast cream doses, 66%-67% improvement from baseline was observed at week 4, without reaching a plateau, versus 38% improvement for vehicle. Conclusion: Roflumilast cream was safe and highly effective at doses of 0.5% and 0.15% and represents a potential novel once-daily topical therapy for the treatment of chronic plaque psoriasis. ClinicalTrials.gov NCT03392168. J Drugs Dermatol. 2020;19(8): doi:10.36849/JDD.2020.5370. Author information: (1)St John's Institute of Dermatology, Guy's and St Thomas' NHS Foundation Trust, and Kings College London, London, England, UK (S.K.M., C.H.S.).

Describe the mechanism of action of Givosiran.

Givosiran is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the siRNA to hepatocytes. This results in downregulation of ALAS1 mRNA and prevents accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks.

BACKGROUND: Induction of delta aminolevulinic acid synthase 1 ( ALAS1) gene expression and accumulation of neurotoxic intermediates result in neurovisceral attacks and disease manifestations in patients with acute intermittent porphyria, a rare inherited disease of heme biosynthesis. Givosiran is an investigational RNA interference therapeutic agent that inhibits hepatic ALAS1 synthesis. METHODS: We conducted a phase 1 trial of givosiran in patients with acute intermittent porphyria. In part A of the trial, patients without recent porphyria attacks (i.e., no attacks in the 6 months before baseline) were randomly assigned to receive a single subcutaneous injection of one of five ascending doses of givosiran (0.035, 0.10, 0.35, 1.0, or 2.5 mg per kilogram of body weight) or placebo. In part B, patients without recent attacks were randomly assigned to receive once-monthly injections of one of two doses of givosiran (0.35 or 1.0 mg per kilogram) or placebo (total of two injections 28 days apart). In part C, patients who had recurrent attacks were randomly assigned to receive injections of one of two doses of givosiran (2.5 or 5.0 mg per kilogram) or placebo once monthly (total of four injections) or once quarterly (total of two injections) during a 12-week period, starting on day 0. Safety, pharmacokinetic, pharmacodynamic, and exploratory efficacy outcomes were evaluated. RESULTS: A total of 23 patients in parts A and B and 17 patients in part C underwent randomization. Common adverse events included nasopharyngitis, abdominal pain, and diarrhea. Serious adverse events occurred in 6 patients who received givosiran in parts A through C combined. In part C, all 6 patients who were assigned to receive once-monthly injections of givosiran had sustained reductions in ALAS1 messenger RNA (mRNA), delta aminolevulinic acid, and porphobilinogen levels to near normal. These reductions were associated with a 79% lower mean annualized attack rate than that observed with placebo (exploratory efficacy end point). CONCLUSIONS: Once-monthly injections of givosiran in patients who had recurrent porphyria attacks resulted in mainly low-grade adverse events, reductions in induced ALAS1 mRNA levels, nearly normalized levels of the neurotoxic intermediates delta aminolevulinic acid and porphobilinogen, and a lower attack rate than that observed with placebo. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02452372 .). Recent advances in pathophysiological and genetic mechanisms of some neuromuscular diseases and a rapid progress in new pharmacological technologies led to an accelerated development of innovative treatments, generating an unexpected therapeutic revolution. In part 1, we report already commercially available drugs, just approved drugs and new therapeutic promises in the treatment of peripheral neuropathies. Hereditary transthyretin amyloidosis (hATTR) is a devastating disease due to amyloid accumulation in peripheral nerves, heart and autonomic system. The first specific drug approved for hATTR was tafamidis, a TTR tetramer stabilizer. In 2018, the positive results of two phase 3 trials have been reported leading to start of regulatory approval route for inotersen, an antisense oligonucleotide and patisiran, the first-ever RNA interference (RNAi) therapeutic. System biology targeting approach has indicated baclofen, naltrexone and sorbitol in combination (PXT3003) as candidate drugs for Charcot-Marie-Tooth disease type 1A. This hypothesis was confirmed in experimental models and in phase 2 and 3 clinical trials. Givosiran, another RNAi therapeutic, targeting 5-aminolevulinic acid synthase, has been positively tested in acute intermittent porphyria in phase 1/2 and ongoing phase 3 trials. Although allogenic hematopoietic stem cell transplantation resulted recently a long-term therapy in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a new strategy is liver transplantation which is able to revert the severe biochemical and clinical imbalance of the disease. Recently, a gene therapy has been tested in a MNGIE murine model, indicating that it may become a new therapeutic option. In November 2019 givosiran became the second small interfering RNA (siRNA)-based drug to receive US Food and Drug Administration (FDA) approval, it has been developed for the treatment of acute intermittent porphyria (AIP), a disorder characterized by life-threatening acute neurovisceral attacks. The porphyrias are a group of disorders in which enzymatic deficiencies in heme production lead to toxic accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG), which are involved in the neurovisceral attacks. Givosiran acts as a conventional siRNA to trigger RNA interference (RNAi)-mediated gene silencing on delta-ALA synthase 1 (ALAS1), thus returning ALA and PBG metabolites to the physiological level to attenuate further neurotoxicity. Givosiran makes use of a new hepatic-delivery system that conjugates three GalNac (N-acetylgalactosamine) molecules to the siRNA passenger strand. GalNac binds to the liver asialoglycoprotein receptor, favoring the internalization of these GalNac-conjugated siRNAs into the hepatic cells. In a phase I study, subcutaneous monthly administration of givosiran 2.5 mg/kg reduced > 90% of ALA and PBG content. This siRNA is being analyzed in ENVISION (NCT03338816), a phase III, multicenter, placebo-controlled randomized controlled trial. In preliminary results, givosiran achieved clinical endpoints for AIP, reducing urinary ALA levels, and presented a safety profile that enabled further drug development. The clinical performance of givosiran revealed that suppression of ALAS1 by GalNac-decorated siRNAs represents an additional approach for the treatment of patients with AIP that manifests recurrent acute neurovisceral attacks. Givosiran is a small interfering ribonucleic acid agent that was recently approved in the United States for the treatment of acute hepatic porphyria (AHP). This phase I study evaluated the safety, pharmacokinetic, and pharmacodynamic profile of subcutaneously (SC) administered givosiran in patients with acute intermittent porphyria, the most common AHP type. Givosiran was rapidly absorbed from the SC injection site with peak plasma concentrations achieved within 0.5-5 hours followed by elimination with a short half-life of 4-10 hours. Plasma exposures of AS(N-1)3' givosiran, an active metabolite with equal potency as givosiran, was 35%-75%. Givosiran treatment resulted in a rapid and dose-dependent reduction in urinary aminolevulinic acid (ALA) and porphobilinogen (PBG) towards the upper limit of normal (ULN) in AHP patients. Greater and more sustained reductions in ALA and PBG were achieved with once monthly dosing compared with once quarterly dosing. After monthly dosing, trough ALA levels were reduced to below the ULN, approximately 95% reduction from baseline, at both the 2.5 and 5.0 mg/kg doses. Givosiran (Givlaari™) is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the siRNA to hepatocytes. This results in downregulation of ALAS1 mRNA and prevents accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks. Givosiran is being developed by Alnylam Pharmaceuticals for the treatment of acute hepatic porphyria (AHP). In November 2019, givosiran was approved in the USA for the treatment of adults with AHP based on the positive results from the multinational, phase III ENVISION trial. In the EU, givosiran received a positive opinion in January 2020 for the treatment of AHP in adults and adolescents aged 12 years and older. This article summarizes the milestones in the development of givosiran leading to this first approval for the treatment of adults with AHP. State-of-the-art small interfering RNA (siRNA) therapeutics such as givosiran and fitusiran are constructed from three variable components: a fully-modified RNA core that conveys metabolic stability, a targeting moiety that mediates target-cell uptake, and a linker. This structural complexity poses challenges for metabolite characterization and risk assessment after long-term patient exposure. In this study, we show that basic phosphorothioate modification of a siRNA targeting the oncoprotein Lin28B provides a useful increase in metabolic stability, without greatly compromising potency. We found that its stability in vitro matched that of oparticle-free patisiran in serum and surpassed it in liver tritosome extracts, although it did not reach the stability of the fitusiran siRNA core structure. Liver and kidney were the main sites of accumulation after its subcutaneous administration in mice. Despite the lack of a delivery agent-free antitumor effect, we anticipate our study to be a starting point to develop alternative siRNA scaffolds that can be degraded into naturally-occurring metabolites and help alleviate the aforementioned challenges. Furthermore, Lin28B is a promising target for cancers, and the development of such simplified siRNA analogs, possibly together with novel targeting units, holds potential. BACKGROUND: Up-regulation of hepatic delta-aminolevulinic acid synthase 1 (ALAS1), with resultant accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen, is central to the pathogenesis of acute attacks and chronic symptoms in acute hepatic porphyria. Givosiran, an RNA interference therapy, inhibits ALAS1 expression. METHODS: In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned symptomatic patients with acute hepatic porphyria to receive either subcutaneous givosiran (2.5 mg per kilogram of body weight) or placebo monthly for 6 months. The primary end point was the annualized rate of composite porphyria attacks among patients with acute intermittent porphyria, the most common subtype of acute hepatic porphyria. (Composite porphyria attacks resulted in hospitalization, an urgent health care visit, or intravenous administration of hemin at home.) Key secondary end points were levels of ALA and porphobilinogen and the annualized attack rate among patients with acute hepatic porphyria, along with hemin use and daily worst pain scores in patients with acute intermittent porphyria. RESULTS: A total of 94 patients underwent randomization (48 in the givosiran group and 46 in the placebo group). Among the 89 patients with acute intermittent porphyria, the mean annualized attack rate was 3.2 in the givosiran group and 12.5 in the placebo group, representing a 74% lower rate in the givosiran group (P<0.001); the results were similar among the 94 patients with acute hepatic porphyria. Among the patients with acute intermittent porphyria, givosiran led to lower levels of urinary ALA and porphobilinogen, fewer days of hemin use, and better daily scores for pain than placebo. Key adverse events that were observed more frequently in the givosiran group were elevations in serum aminotransferase levels, changes in serum creatinine levels and the estimated glomerular filtration rate, and injection-site reactions. CONCLUSIONS: Among patients with acute intermittent porphyria, those who received givosiran had a significantly lower rate of porphyria attacks and better results for multiple other disease manifestations than those who received placebo. The increased efficacy was accompanied by a higher frequency of hepatic and renal adverse events. (Funded by Alnylam Pharmaceuticals; ENVISION ClinicalTrials.gov number, NCT03338816.). RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO® (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)-siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field. RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Is Olaparib effective for prostate cancer?

Yes, olaparib was shown to be effective for treatment of prostate cancer. Olaparib led to stable disease or tumor regressions of prostate cancer patients.

The evolving field of personalised medicine is playing an increasingly important role in cancer prevention, diagnosis, prognosis and therapeutics. Its importance in clinical management is demonstrated by the recent introduction into routine clinical practice of various individualised, molecularly targeted therapies with increased efficacy and/or reduced toxicity. The identification of cancer predisposition genes, such as the BRCA genes in breast cancer, permits screening programmes to identify patients "at-risk" of developing cancer and helps them make decisions on individual risk-modification behaviours. Personalised medicine also plays an increasingly important role in cancer treatment. It is increasingly clear that there are molecularly distinct subtypes of various common cancers, with different therapeutic approaches required for each subtype, for example, the use of the monoclonal antibodies (trastuzumab and cetuximab) in HER2-positive breast cancer and wild-type KRAS colorectal cancer; tyrosine kinase inhibitors (imatinib, gefitinib, erlotinib and crizotinib) in chronic myeloid leukaemia, gastrointestinal stromal tumours and non-small-cell lung cancer and intracellular agents (vemurafenib and olaparib) in metastatic maligt melanoma and ovarian, breast and prostate cancer. The efficacy of various targeted therapies in such disparate tumours suggests that we are entering an era in which treatment decisions will be based on tumour molecular abnormality profile or "signature," rather than tumour tissue type or anatomical site of origin, improving patient prognosis and quality of life. This mini review focuses on the role of personalised medicine in cancer prevention and treatment as well as its future direction in oncology. Author information: (1)Bella Kaufman and Ronnie Shapira-Frommer, Sheba Medical Center, Tel Hashomer; Georgeta Fried, Institute of Oncology, Rambam Health Care Campus; Mariana Steiner, Linn Medical Centre, Haifa; Salomon M. Stemmer, Rabin Medical Center, Petah Tikva; Ayala Hubert, Hadassah-Hebrew University Hospital, Sharett Institute of Oncology; Ora Rosengarten, Shaare Zedek Medical Centre, Jerusalem, Israel; Rita K. Schmutzler, Center for Familial Breast and Ovarian Cancer and Center of Integrated Oncology, Cologne, Germany; M. William Audeh, Samuel Oschin Cancer Institute, Los Angeles, CA; Michael Friedlander, Prince of Wales Clinical School, University of New South Wales, Sydney, New South Wales; Gillian Mitchell, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia; Judith Balmaña, Vall d'Hebron Institute of Oncology, Barcelona, Spain; Niklas Loman, Skånes Universitetssjuk Lund, Lund, Sweden; Karin Bowen and Anitra Fielding, AstraZeneca, Macclesfield, United Kingdom; and Susan M. Domchek, Basser Research Center and Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA. (2)Bella Kaufman and Ronnie Shapira-Frommer, Sheba Medical Center, Tel Hashomer; Georgeta Fried, Institute of Oncology, Rambam Health Care Campus; Mariana Steiner, Linn Medical Centre, Haifa; Salomon M. Stemmer, Rabin Medical Center, Petah Tikva; Ayala Hubert, Hadassah-Hebrew University Hospital, Sharett Institute of Oncology; Ora Rosengarten, Shaare Zedek Medical Centre, Jerusalem, Israel; Rita K. Schmutzler, Center for Familial Breast and Ovarian Cancer and Center of Integrated Oncology, Cologne, Germany; M. William Audeh, Samuel Oschin Cancer Institute, Los Angeles, CA; Michael Friedlander, Prince of Wales Clinical School, University of New South Wales, Sydney, New South Wales; Gillian Mitchell, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia; Judith Balmaña, Vall d'Hebron Institute of Oncology, Barcelona, Spain; Niklas Loman, Skånes Universitetssjuk Lund, Lund, Sweden; Karin Bowen and Anitra Fielding, AstraZeneca, Macclesfield, United Kingdom; and Susan M. Domchek, Basser Research Center and Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA. [email protected]. In a phase II study, researchers found that the PARP inhibitor olaparib led to stable disease or tumor regressions in patients with advanced breast, ovarian, pancreatic, and prostate cancers who had germline mutations in BRCA1 or BRCA2. Olaparib (Lynparza™) is an oral, small molecule, poly (ADP-ribose) polymerase inhibitor being developed by AstraZeneca for the treatment of solid tumours. The primary indication that olaparib is being developed for is BRCA mutation-positive ovarian cancer. A capsule formulation of the drug has received approval for use in this setting in the EU and USA, and a tablet formulation is in global phase III trials (including in the USA, EU, Australia, Brazil, Canada, China, Israel, Japan, Russia and South Korea). In addition, phase III trials in breast, gastric and pancreatic cancer are underway/planned, and phase I/II investigation is being conducted in other maligcies, including prostate cancer, non-small cell lung cancer, Ewing's sarcoma and advanced cancer. This article summarizes the milestones in the development of olaparib leading to this first approval for ovarian cancer. BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.). In the phase II TOPARP-A clinical trial, patients with metastatic castrate-resistant prostate cancer who were treated with the PARP inhibitor olaparib lived nearly three times longer without their cancer worsening if their tumors had mutations in at least one of 12 DNA repair genes. However, physicians say that a larger trial is needed to confirm olaparib's effectiveness against the disease before they start routinely sequencing tumors and prescribing the drug. : Advances in DNA sequencing technology have created a wealth of information regarding the genomic landscape of prostate cancer. It had been thought that BRCA1 and BRCA2 mutations were associated with only a small fraction of prostate cancer cases. However, recent genomic analysis has revealed that germline or somatic inactivating mutations in BRCA1 or BRCA2, or other genes involved in the homologous recombination (HR) pathway of DNA repair collectively occur in as much as 20%-25% of advanced prostate cancers. A synthetic lethal therapeutic approach using poly(ADP-ribose) polymerase inhibitor therapy has been developed for BRCA mutant- and HR deficient-related cancers (those with "BRCAness") and is being studied in multiple clinical trials. This article discusses the current understanding of the genomic landscape of prostate cancer, focusing on the occurrence of DNA repair mutations and the therapeutic opportunities that this presents. IMPLICATIONS FOR PRACTICE: This review aims to update oncologists about the increased understanding of the genomes of prostate cancers and, in particular, the prevalence of mutations in DNA repair genes. These observations provide potential new therapeutic opportunities for the use of poly(ADP-ribose) polymerase inhibitors and other therapies, especially in advanced forms of the disease. Of note is the recent U.S. Food and Drug Administration breakthrough therapy designation of olaparib for the treatment of BRCA1/2- or ATM-mutated metastatic castration-resistant prostate cancer. The implications of this new knowledge for clinical practice now and in the future are discussed. Olaparib is an FDA-approved PARP inhibitor (PARPi) that has shown promise as a synthetic lethal treatment approach for BRCA-mutant castration-resistant prostate cancer (CRPC) in clinical use. However, emerging data have also shown that even BRCA-mutant cells may be resistant to PARPi. The mechanistic basis for these drug resistances is poorly understood. Polo-like kinase 1 (Plk1), a critical regulator of many cell-cycle events, is significantly elevated upon castration of mice carrying xenograft prostate tumors. Herein, by combination with Plk1 inhibitor BI2536, we show a robust sensitization of olaparib in 22RV1, a BRCA1-deficient CRPC cell line, as well as in CRPC xenograft tumors. Mechanistically, monotherapy with olaparib results in an override of the G1-S checkpoint, leading to high expression of Plk1, which attenuates olaparib's overall efficacy. In BRCA1 wild-type C4-2 cells, Plk1 inhibition also significantly increases the efficacy of olaparib in the presence of p53 inhibitor. Collectively, our findings not only implicate the critical role of Plk1 in PARPi resistance in BRCA-mutant CRPC cells, but also shed new light on the treatment of non-BRCA-mutant patient subgroups who might also respond favorably to PARPi. Mol Cancer Ther; 16(3); 469-79. ©2017 AACR. Although mortality from prostate cancer has declined over the past 20 years as a result of early detection and treatment, the 5-year survival rate for men with prostate cancer who develop metastatic disease is only 29%. Current treatment options for metastatic castration-recurrent prostate cancer (mCRPC) are associated with toxicity and a limited durable response; therefore, additional lines of efficacious and minimally toxic therapy are needed. Olaparib, a poly(adenosine 5'-diphosphate) ribose polymerase (PARP) inhibitor, received a U.S. Food and Drug Administration breakthrough therapy designation in January 2016 for the treatment of patients with BRCA1/2 or ATM gene-mutated mCRPC based on results of a compelling phase II trial of olaparib in patients with advanced castration-resistant prostate cancer (TOPARP-A). This study found that men with mCRPC and genetic mutations in DNA damage repair genes had an overall response rate of nearly 90% with olaparib treatment. In this review, we describe current therapies for mCRPC, the rationale for anti-PARP therapies, the pharmacology of olaparib for prostate cancer, clinical trials of olaparib for mCRPC, our clinical experience with olaparib for prostate cancer at a comprehensive cancer center, and future directions of olaparib for the treatment of mCRPC. Olaparib may constitute a promising treatment to prolong survival in patients with mCRPC, with an acceptable adverse effect profile. As the role of PARP inhibition in prostate cancer and other maligcies becomes further elucidated, olaparib may be shown to be beneficial for other patient populations. Castration-resistant prostate cancer remains as an incurable disease. Exploiting DNA damage repair defects via inhibition of poly (ADP-ribose) polymerase (PARP) is becoming an attractive therapeutic option. The TOPARP-A clinical trial demonstrated that the PARP inhibitor olaparib may be an effective strategy for treating prostate cancer. However, several uswered questions regarding the use of olaparib remain: 1) How do we best stratify patients for olaparib treatment? 2) Where do we place olaparib in the treatment sequence paradigm? 3) Is there cross-resistance between olaparib and currently used therapies? Here, we tested putative cross-resistance between current therapies and olaparib in treatment-resistant castration-resistant prostate cancer models. Docetaxel-resistant cells exhibited robust resistance to olaparib which could be attributed to blunted PARP trapping in response to olaparib treatment. Upregulated ABCB1 mediates cross-resistance between taxanes and olaparib, which can be overcome through decreasing ABCB1 expression or inhibiting ABCB1 using elacridar or enzalutamide. We also show that combining olaparib with enzalutamide is more effective in olaparib-sensitive cells than either single agent. Our results demonstrate that cross-resistance between olaparib and other therapies could blunt response to treatment and highlight the need to develop strategies to maximize olaparib efficacy. The approval of upfront abiraterone for castration-sensitive prostate cancer and the approval of enzalutamide and apalutamide for non-metastatic castration-resistant prostate cancer have led to early utilization of potent androgen receptor (AR) signaling inhibitors in treating advanced prostate cancer. There is an unmet need to develop novel therapies beyond targeting AR signaling for metastatic castration-resistant prostate cancer (mCRPC). Poly (ADP-ribose) polymerase inhibitors (PARPi) belong to a class of targeted agents being developed for the treatment of homologous recombination repair (HRR) deficient tumors. Olaparib, rucaparib, niraparib, veliparib, and talazoparib were evaluated in early phase trials as a monotherapy for HRR-deficient mCRPC. Among them, olaparib and rucaparib have breakthrough designations for BRCA1/2-mutated mCRPC. Phase II studies also reported clinical activity of the PARPi and abiraterone combination and the PARPi checkpoint inhibitor combination in HRR-intact mCRPC. Ongoing phase III trials are testing these combinations as frontline or later line treatments for mCRPC. This review summarizes the critical clinical data as well as ongoing clinical trials for developing PARPi in treating mCRPC. Conflict of interest statement: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. BENEDITO A. CARNEIRO: Consulting or Advisory Role: Bristol-Myers Squibb, Bayer HealthCare Pharmaceuticals KATHARINE ANN COLLIER: No relationship to disclose REBECCA J. NAGY: Employment: Guardant Health Stock and Other Ownership Interests: Guardant Health SAHITHI PAMARTHY: No relationship to disclose VINAY SAGAR: No relationship to disclose STEPHEN FAIRCLOUGH: Employment: Guardant Health Stock and Other Ownership Interests: Guardant Health Research Funding: Guardant Health Patents, Royalties, Other Intellectual Property: Guardant Health Travel, Accommodations, Expenses: Guardant Health JUSTIN ODEGAARD: Employment: Guardant Health Stock and Other Ownership Interests: Guardant Health RICHARD B. LANMAN: Employment: Guardant Health, Veracyte Leadership: Guardant Health Stock and Other Ownership Interests: Guardant Health Research Funding: Guardant Health RICARDO COSTA: No relationship to disclose TIMOTHY TAXTER: Employment: Tempus Stock and Other Ownership Interests: Tempus TIMOTHY M. KUZEL: Honoraria: Genentech/Roche, Celgene, Eisai, Argos Therapeutics, Amgen, Astellas Pharma, Bristol-Myers Squibb, Medivation, Exelixis, AbbVie, Merck Consulting or Advisory Role: CVS, Kyowa Hakko Kirin, Stemline Therapeutics Speakers’ Bureau: Celgene, Genentech/Roche, Astellas Pharma, Medivation Research Funding: Genentech/Roche (Inst), Eisai (Inst), Bristol-Myers Squibb (Inst) Travel, Accommodations, Expenses: Genentech, Celgene, Astellas Pharma, Argos Therapeutics, Medivation, Amgen, Kyowa Hakko Kirin, Merck, Stemline Therapeutics ALICE FAN: Stock and Other Ownership Interests: Molecular Decisions Consulting or Advisory Role: Verily Research Funding: Calithera Biosciences Patents, Royalties, Other Intellectual Property: Stanford Patent has been licensed YOUNG KWANG CHAE: Consulting or Advisory Role: Foundation Medicine, Boehringer Ingelheim, Biodesix, Counsyl, AstraZeneca, Guardant Health, Speakers’ Bureau: Merck, Genentech/Roche Travel, Accommodations, Expenses: Hanmi MASSIMO CRISTOFANILLI: Honoraria: Dompé Farmaceutici, Pfizer Consulting or Advisory Role: Dompé Farmaceutici, Newomics, Vortex Biosciences MAHA H. HUSSAIN: Honoraria: Onclive, Sanofi Research Funding: Genentech (Inst), Pfizer (Inst), PCCTC (Inst), AstraZeneca (Inst) Patents, Royalties, Other Intellectual Property: Title: Systems and methods for tissue imaging, 3676 our file, serial No. UM-14437/US-1/PRO 60/923,385UM-14437/US-2/ORD 12/101,753US 8,185,186 (US patent No.), Systems and methods for tissue imaging (issued patent) EP 08745653.9 (EP application No.), Systems and methods for tissue imaging (pending) CA 2683805 (Canadian application No.), Systems and methods for tissue imaging (pending) US 13/362,500 (US application No.), Systems and methods for tissue imaging (continuation application of US 8,185,186); Method of treating cancer, docket No., serial No. 224990/10-016P2/311733 61/481/671, application filed on February 5, 2011; Dual inhibition of MET and VEGF for the treatment of castration resistant prostate cancer and osteoblastic bone metastases, applicant/proprietor: Exelexis, application No./patent No. 11764665.4- 1464, application No./patent No. 11764656.2-1464, application filed on September 26, 2011 Travel, Accommodations, Expenses: Sanofi SARKI A. ABDULKADIR: Stock and Other Ownership Interests: Vortex Therapeutics Patents, Royalties, Other Intellectual Property: Patent pending through Northwestern University for new MYC inhibitors FRANCIS J. GILES: Employment: Actuate Leadership: Actuate Honoraria: Novartis Consulting or Advisory Role: Novartis Travel, Accommodations, Expenses: MedImmune, Novartis, Foundation Medicine BACKGROUND: Metastatic castration-resistant prostate cancer is enriched in DNA damage response (DDR) gene aberrations. The TOPARP-B trial aims to prospectively validate the association between DDR gene aberrations and response to olaparib in metastatic castration-resistant prostate cancer. METHODS: In this open-label, investigator-initiated, randomised phase 2 trial following a selection (or pick-the-winner) design, we recruited participants from 17 UK hospitals. Men aged 18 years or older with progressing metastatic castration-resistant prostate cancer previously treated with one or two taxane chemotherapy regimens and with an Eastern Cooperative Oncology Group performance status of 2 or less had tumour biopsies tested with targeted sequencing. Patients with DDR gene aberrations were randomly assigned (1:1) by a computer-generated minimisation method, with balancing for circulating tumour cell count at screening, to receive 400 mg or 300 mg olaparib twice daily, given continuously in 4-week cycles until disease progression or unacceptable toxicity. Neither participants nor investigators were masked to dose allocation. The primary endpoint of confirmed response was defined as a composite of all patients presenting with any of the following outcomes: radiological objective response (as assessed by Response Evaluation Criteria in Solid Tumors 1.1), a decrease in prostate-specific antigen (PSA) of 50% or more (PSA50) from baseline, or conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to <5 cells per 7·5 mL blood). A confirmed response in a consecutive assessment after at least 4 weeks was required for each component. The primary analysis was done in the evaluable population. If at least 19 (43%) of 44 evaluable patients in a dose cohort responded, then the dose cohort would be considered successful. Safety was assessed in all patients who received at least one dose of olaparib. This trial is registered at ClinicalTrials.gov, NCT01682772. Recruitment for the trial has completed and follow-up is ongoing. FINDINGS: 711 patients consented for targeted screening between April 1, 2015, and Aug 30, 2018. 161 patients had DDR gene aberrations, 98 of whom were randomly assigned and treated (49 patients for each olaparib dose), with 92 evaluable for the primary endpoint (46 patients for each olaparib dose). Median follow-up was 24·8 months (IQR 16·7-35·9). Confirmed composite response was achieved in 25 (54·3%; 95% CI 39·0-69·1) of 46 evaluable patients in the 400 mg cohort, and 18 (39·1%; 25·1-54·6) of 46 evaluable patients in the 300 mg cohort. Radiological response was achieved in eight (24·2%; 11·1-42·3) of 33 evaluable patients in the 400 mg cohort and six (16·2%; 6·2-32·0) of 37 in the 300 mg cohort; PSA50 response was achieved in 17 (37·0%; 23·2-52·5) of 46 and 13 (30·2%; 17·2-46·1) of 43; and circulating tumour cell count conversion was achieved in 15 (53·6%; 33·9-72·5) of 28 and 13 (48·1%; 28·7-68·1) of 27. The most common grade 3-4 adverse event in both cohorts was anaemia (15 [31%] of 49 patients in the 300 mg cohort and 18 [37%] of 49 in the 400 mg cohort). 19 serious adverse reactions were reported in 13 patients. One death possibly related to treatment (myocardial infarction) occurred after 11 days of treatment in the 300 mg cohort. INTERPRETATION: Olaparib has antitumour activity against metastatic castration-resistant prostate cancer with DDR gene aberrations, supporting the implementation of genomic stratification of metastatic castration-resistant prostate cancer in clinical practice. FUNDING: Cancer Research UK, AstraZeneca, Prostate Cancer UK, the Prostate Cancer Foundation, the Experimental Cancer Medicine Centres Network, and the National Institute for Health Research Biomedical Research Centres. KEY POINTS • The prognosis for patients with mCRPC has improved over the last few years due to the introduction of novel agents. • The optimal sequence of administering these therapeutic agents remains as a moving target and is not well established. Decisions are usually made according to patients' clinical conditions and disease characteristics, and the safety profile and availability of new drugs. • Recently, cabazitaxel improved outcomes in the third-line setting after docetaxel and an ARTA. Olaparib is an additional option for second- and third-line treatment in those with alterations in BRCA1, BRCA2, and ATM. • Understanding the mechanisms of resistance may provide a rationale for suggesting specific strategies. • A subset of patients may benefit from molecularly targeted therapies, which highlights the importance of genomic testing in the castration-resistant setting. • Immunotherapy may provide benefit to some subsets of patients, such as those with MSI-high tumors. Studies regarding combination therapy with immune checkpoint inhibitors are ongoing. CONTEXT: The goal of precision oncology is to use the underlying genomic characteristics of the patient and the cancer to select the optimal treatment at a given time. The recent Food and Drug Administration (FDA) approval of the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and rucaparib for the treatment of advanced prostate cancer heralds the onset of precision medicine for this disease. OBJECTIVE: To discuss the emerging role that PARP inhibitors may play as a personalised future treatment option in patients with prostate cancer, with a focus on patients with metastatic castration-resistant prostate cancer (mCRPC) whose tumour cells harbour mutations resulting from deficient homologous recombination repair (HRR). EVIDENCE ACQUISITION: To identify publications relevant to this review, a systematic literature search of PubMed was conducted for articles and proceedings of relevant major congresses, published between January 2010 and March 2020, reporting the use of PARP inhibitors in the treatment of cancers. EVIDENCE SYNTHESIS: A total of 168 publications were identified, and 18 of these met the criteria for subsequent review. In addition, 15 phase 2 or on-going phase 3 (mCRPC) studies evaluating PARP inhibitors as monotherapy or in combination, which had not yet reported data, were identified through ClinicalTrials.gov. Emerging data suggest that the greatest efficacy with single-agent PARP inhibitors is seen in mCRPC patients with germline or somatic BRCA1/2 alterations (especially BRCA2 or biallelic mutations), with potential efficacy also observed in men with PALB2 and FANCA mutations. CONCLUSIONS: PARP inhibitors have demonstrated efficacy in mCRPC, and similar to ovarian and breast cancers, the greatest effect is observed in patients with HRR deficiency. The PARP inhibitors olaparib and rucaparib are now FDA approved for mCRPC patients with HRR mutations and BRCA1/2 mutations, respectively. Furthermore, when PARP inhibition is combined with novel hormonal therapies, a treatment benefit may be observed regardless of the HRR deficiency status. Gaps in the knowledge and understanding around PARP inhibitor use in prostate cancer, including the most appropriate diagnostic testing method for identifying an HRR mutation, remain to be resolved. PATIENT SUMMARY: The poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and rucaparib are now approved by the Food and Drug Administration for the treatment of advanced prostate cancer. Here, we reviewed the literature and proceedings from meeting presentations and published papers relevant to the use of PARP inhibitors in the treatment of prostate cancer. Testing methods for detecting homologous recombination repair gene mutations, as diagnostic tools to help identify patients most likely to benefit from PARP inhibitor treatment, are also discussed. BACKGROUND: We previously reported that olaparib led to significantly longer imaging-based progression-free survival than the physician's choice of enzalutamide or abiraterone among men with metastatic castration-resistant prostate cancer who had qualifying alterations in homologous recombination repair genes and whose disease had progressed during previous treatment with a next-generation hormonal agent. The results of the final analysis of overall survival have not yet been reported. METHODS: In an open-label, phase 3 trial, we randomly assigned patients in a 2:1 ratio to receive olaparib (256 patients) or the physician's choice of enzalutamide or abiraterone plus prednisone as the control therapy (131 patients). Cohort A included 245 patients with at least one alteration in BRCA1, BRCA2, or ATM, and cohort B included 142 patients with at least one alteration in any of the other 12 prespecified genes. Crossover to olaparib was allowed after imaging-based disease progression for patients who met certain criteria. Overall survival in cohort A, a key secondary end point, was analyzed with the use of an alpha-controlled, stratified log-rank test at a data maturity of approximately 60%. The primary and other key secondary end points were reported previously. RESULTS: The median duration of overall survival in cohort A was 19.1 months with olaparib and 14.7 months with control therapy (hazard ratio for death, 0.69; 95% confidence interval [CI], 0.50 to 0.97; P = 0.02). In cohort B, the median duration of overall survival was 14.1 months with olaparib and 11.5 months with control therapy. In the overall population (cohorts A and B), the corresponding durations were 17.3 months and 14.0 months. Overall, 86 of 131 patients (66%) in the control group crossed over to receive olaparib (56 of 83 patients [67%] in cohort A). A sensitivity analysis that adjusted for crossover to olaparib showed hazard ratios for death of 0.42 (95% CI, 0.19 to 0.91) in cohort A, 0.83 (95% CI, 0.11 to 5.98) in cohort B, and 0.55 (95% CI, 0.29 to 1.06) in the overall population. CONCLUSIONS: Among men with metastatic castration-resistant prostate cancer who had tumors with at least one alteration in BRCA1, BRCA2, or ATM and whose disease had progressed during previous treatment with a next-generation hormonal agent, those who were initially assigned to receive olaparib had a significantly longer duration of overall survival than those who were assigned to receive enzalutamide or abiraterone plus prednisone as the control therapy, despite substantial crossover from control therapy to olaparib. (Funded by AstraZeneca and Merck Sharp and Dohme; PROfound ClinicalTrials.gov number, NCT02987543.). Poly (ADP-ribose) polymerase inhibitors (PARPi) are a unique class of antineoplastic agents that function by inducing synthetic lethality. Synthetic lethality occurs when PARPi and either another agent or an underlying genetic alteration together lead to overwhelming DNA damage and ultimately cell death. PARPi first showed promise as a cancer therapy in patients with BRCA1/2 mutations and have become part of standard treatment for breast and ovarian cancer. In prostate cancer, two PARPi, rucaparib and olaparib, have been FDA approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC). While both agents are approved for tumors with BRCA1/2 alterations, for olaparib the indication is also expanded to patients with 12 other homologous recombination deficiency (HRD) gene alterations including ATM and PALB2. PARPi differ in their pharmacokinetics and pharmacodynamics, and additional studies are being conducted with niraparib, veliparib, and talazoparib in prostate cancer. While PARPi are fairly well tolerated, common toxicities include hematologic (anemia/thrombocytopenia) and gastrointestinal effects (nausea/vomiting). Ongoing studies are being conducted combining PARPi with other agents in patients with and without HRD alterations. Early data are promising for the combination of PARPi with second-generation antiandrogens and with immunotherapy. As additional trials are developed and reported, the hope is that the patient population who may benefit from PARPi will continue to expand. Genomic instability is one of the hallmarks of cancer. The incidence of genetic alterations in homologous recombination repair genes increases during cancer progression, and 20% of prostate cancers (PCas) have defects in DNA repair genes. Several somatic and germline gene alterations drive prostate cancer tumorigenesis, and the most important of these are BRCA2, BRCA1, ATM and CHEK2. There is a group of BRCAness tumours that share phenotypic and genotypic properties with classical BRCA-mutated tumours. Poly(ADP-ribose) polymerase inhibitors (PARPis) show synthetic lethality in cancer cells with impaired homologous recombination genes, and patients with these alterations are candidates for PARPi therapy. Androgen deprivation therapy is the mainstay of PCa therapy. PARPis decrease androgen signalling by interaction with molecular mechanisms of the androgen nuclear complex. The PROFOUND phase III trial, comparing olaparib with enzalutamide/abiraterone therapy, revealed increased radiological progression-free survival (rPFS) and overall survival (OS) among patients with metastatic castration-resistant prostate cancer (mCRPC) with BRCA1, BRCA2 or ATM mutations. The clinical efficacy of PARPis has been confirmed in ovarian, breast, pancreatic and recently also in a subset of PCa. There is growing evidence that molecular tumour boards are the future of the oncological therapeutic approach in prostate cancer. In this review, we summarise the data concerning the molecular mechanisms and preclinical and clinical data of PARPis in PCa.

What is Corkscrew Esophagus?

Corkscrew esophagus is a classic finding of diffuse esophageal spasm in barium studies reflecting abnormal contractions, leading to compartmentalization and curling of the esophagus, ultimately giving an appearance similar to a corkscrew or rosary beads.

"Corkscrew oesophagus" is characterised on the basis of two case reports and attention is drawn to thoracic pain of oesophageal origin. Corkscrew oesophagus is a radiological diagnosis and is characterised by twisted segments in the distal third of the oesophagus. The condition can sometimes be demonstrated endoscopically and it is due to a basic disturbance in the motility of the oesophagus. Painful conditions in the oesophagus are most frequently caused by gastro-oesophageal reflux or disturbances in motility and the latter is frequently complicated by reflux oesophagitis. Pain of oesophageal origin is frequently a diagnosis by exclusion and requires exclusion of ischaemic heart disease. The initial treatment should be directed to the reflux oesophagitis. The diagnosis and information about the origin of the pain and the benign course of the condition will calm the majority of the patients and remove their fear of a possible fatal heart disease. Nonpropulsive esophageal contractions radiologically described as tertiary contractions or "corkscrew" esophagus suggest the presence of an underlying motility disorder and may lead to impaired acid clearance. The goals of this study were to determine the prevalence and role of gastroesophageal reflux (GER) in patients with tertiary contractions. Thirty-five consecutive patients with spontaneous, repetitive, nonpropulsive esophageal contractions noted on esophagography were studied with endoscopy, infusion esophageal manometry, and 24-h ambulatory pH monitoring. All patients had esophageal symptoms, mainly dysphagia, heartburn, and chest pain, but only three were found to have esophagitis by endoscopy and biopsy. Nineteen patients had repetitive, nonlumen-obliterating, nonperistaltic (tertiary) contractions, six had corkscrew esophagus, and 10 had forceful, lumen-obliterating simultaneous contractions (rosary bead esophagus). Twenty patients (58%) had GER by pH criteria with mean values: % time pH less than 4, 40.9; %upright pH less than 4, 41; %supine pH less than 4, 44.3%; number of episodes with greater than 5 min of pH less than 4, 12. Esophageal motility revealed "nutcracker" esophagus in eight, low LESP in two, and nonspecific esophageal motility disorder in 10. Symptoms or severity of nonperistaltic contractions did not correlate with GER. Radiologically demonstrable free reflux or the presence of heartburn did not predict GER. We conclude that 1) GER occurs in up to 58% of patients with nonpropulsive (tertiary) esophageal contractions on esophagography, and may play a role in the induction of abnormal peristaltic activity of the esophageal body; 2) GER is usually not associated with endoscopic evidence of esophagitis or characteristic symptoms, and is recognized by 24-h pH monitoring. We speculate that detection and treatment of GER may improve the symptomatic management of patients with nonpropulsive esophageal contractions. The aim of this paper is to describe and discuss, on the basis of the available literature, the case of an old female patient, admitted to our university hospital because of a severe dysphagia for solid foods, in whom laboratory data showed a marked hypomagnesemia. She reported a long history (20 years) of allergic bronchial asthma treated with theophylline. Esophagography evidenced a disorder of esophagus motility with diffuse multiple spasm, reminiscent of the 'corkscrew esophagus'. A link with the severe hypomagnesemia (Mg 1.1 mEq/l, normal range 1.6-2.1) was suspected, and a therapy with oral pidolate of Mg (1.5 g/twice a day) was started and continued for 4 months. This was associated with a slow progressive normalization of the Mg plasma level and reverted radiographic esophageal findings with disappearance of dysphagia. Mg is an important element for health and disease, and today Mg deficiency in man has become an accepted medical problem which might complicate many diseases. Neuromuscular disorders, as laryngeal spasm, are recognized complications of hypomagnesemia, but until now only 1 case of motor esophageal disorder associated with a low Mg plasma level was briefly reported in the literature, even if dysphagia is generally included in the symptomatological pattern of hypomagnesemia. Our observation of a severe form of esophageal spasm, associated with hypomagnesemia, in an aged female patient underlines the pathophysiological meaning of the plasma Mg level and suggests the need for routine Mg determination in the clinical setting. Corkscrew esophagus (also referred as rosary bead esophagus) is a classic finding of diffuse esophageal spasm (DES) in barium studies reflecting abnormal contractions, leading to compartmentalization and curling of the esophagus, ultimately giving an appearance similar to a corkscrew or rosary beads. We review the pathophysiology of this finding, correlating it to corkscrew and rosary images that originated this classic description.

List 3 conventional synthetic DMARDs.

Three conventional synthetic (cs) DMARDs include methotrexate (MTX), leflunomide, and sulfasalazine.

In light of the recent emergence of new therapeutics for rheumatoid arthritis, such as kinase inhibitors and biosimilars, a new nomenclature for disease-modifying antirheumatic drugs (DMARDs), which are currently often classified as synthetic (or chemical) DMARDs (sDMARDS) and biological DMARDs (bDMARDs), may be needed. We propose to divide the latter into biological original and biosimilar DMARDs (boDMARDs and bsDMARDs, respectively, such as abatacept, adalimumab, anakinra, certolizumab pegol, etanercept, golimumab, infliximab, rituximab or tocilizumab, but also emerging ones like clazakizumab, ixekizumab, sarilumab, secukinumab or sirukumab) and the former into conventional synthetic and targeted synthetic DMARDs (csDMARDs and tsDMARDs, respectively). tsDMARDs would then constitute only those that were specifically developed to target a particular molecular structure (such as tofacitinib, fostamatinib, baricitinib or apremilast, or agents not focused primarily on rheumatic diseases, such as imatinib or ibrutinib), while csDMARDs would comprise the traditional drugs (such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine, gold salts and others). The proposed nomenclature may provide means to group and distinguish the different types of DMARDs in clinical studies and review articles. Disease-modifying antirheumatic drugs (DMARDs) have become essential treatments in the management of patients with inflammatory rheumatic diseases. Their use has resulted in a marked improvement of disease control and a limitation of joint damage, although some patients still require subsequent corrective or joint replacement surgery. Due to their immunosuppressive effects, some DMARDs are associated with an increased risk of infection. The aim of this review is to discuss the available literature on the management of DMARDs during the perioperative period, particularly in the case of orthopaedic surgery. Conventional synthetic DMARDs such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine appear to be safe during the perioperative period. Conflicting results on biological DMARDs, mainly tumour necrosis factor antagonists, are reported in the literature, including both increased and unchanged risk of superimposed infections after surgery. Taking into account the available literature, we included some propositions for the management of patients who will undergo surgical interventions.

Is isradipine effective for Parkinson's disease?

No. Long-term treatment with immediate-release isradipine did not slow the clinical progression of early-stage Parkinson's disease.

Is MK-1602 a CGRP antagonist?

Yes, MK-1602 is a CGRP antagonist.

AIM: The aim of this trial was to evaluate the efficacy and tolerability of ubrogepant (MK-1602), a calcitonin gene-related peptide receptor antagonist (CGRP-RA), for the acute treatment of migraine. METHODS: This double-blind, placebo-controlled study randomized 834 participants to treat one migraine attack with ubrogepant 1 mg, 10 mg, 25 mg, 50 mg, 100 mg, or placebo in a 1:1 ratio. The co-primary endpoints were pain freedom and headache response at two hours. The first primary hypothesis tested the dose-response trend for two-hour pain freedom using a logistic regression model. Subsequent hypotheses tested the effects of each dose on the co-primary endpoints, using a closed sequential testing procedure to control for multiplicity. RESULTS: A total of 527 participants received ubrogepant and 113 received placebo. A positive response trend in the proportion of participants achieving two-hour pain freedom was demonstrated (p < 0.001). Ubrogepant 100 mg was significantly superior to placebo for two-hour pain freedom (25.5% vs 8.9%) but not for two-hour headache response. Per the prespecified multiplicity strategy, this nonsignificant result precluded further formal hypothesis testing, although the 50 mg and 25 mg doses demonstrated nominal significance over placebo for two-hour pain freedom (unadjusted p < 0.05). Overall, adverse events were similar between ubrogepant and placebo. CONCLUSION: This trial supports ubrogepant's efficacy and provides further evidence that CGRP-RAs are viable options for the acute treatment of migraine.

Which company developed eptinezumab?

Eptinezumab was developed by Lundbeck Seattle BioPharmaceuticals.

Eptinezumab-jjmr (referred to as eptinezumab hereafter; Vyepti™) is a humanised monoclonal antibody that binds to calcitonin gene-related peptide (CGRP) and blocks its binding to the receptor. CGRP is believed to play a major role in the pathophysiology of migraine. Eptinezumab, delivered by intravenous (IV) administration, is being developed by Lundbeck Seattle BioPharmaceuticals for the prevention of migraine. In February 2020, eptinezumab was approved in the USA for the preventive treatment of migraine in adults. This article summarizes the milestones in the development of eptinezumab leading to this first approval.

What are the features of the AESOP syndrome?

Adenopathy and Extensive Skin Patch Overlying a Plasmacytoma is defined as the AESOP Syndrome.

In our manuscript we describe the cutaneous manifestations of a rare condition termed Adenopathy and Extensive Skin Patch Overlying Plasmacytoma (AESOP) syndrome. We emphasize the importance of clinically following and subsequently removing the osteolytic tumor to make the diagnosis. POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein and skin signs) and AESOP (adenopathy and extensive skin patch overlying a plasmacytoma) syndromes are rare paraneoplastic conditions due to an underlying plasma cell dyscrasia. We report a 70-year-old patient with the rare coexistence of POEMS and AESOP syndromes and in whom skin signs, that differ both clinically and histologically, were the clues to the diagnosis of a plasma cell disorder. Vascular endothelial growth factor-A overexpression seems to be the common pathogenetic link of the different clinicopathological presentations of the skin lesions. A 56-year-old previously healthy man presented to the dermatology clinic with a 2-year history of an expanding, violaceous, infiltrated plaque on the right flank. Biopsy revealed a diffuse dermal vascular proliferation of bland, capillary-sized vessels admixed with conspicuous fibrohistiocytic cells including scattered multinucleated floret cells. Further workup revealed a monoclonal gammopathy, an osteolytic chest wall plasmacytoma underlying the plaque, and regional lymphadenopathy leading to a diagnosis of adenopathy and extensive skin patch overlying a plasmacytoma (AESOP). Biopsy of an enlarged lymph node revealed Castleman disease. The patient subsequently developed polyneuropathy and peripheral edema, which supported an additional diagnosis of polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes (POEMS) syndrome. Herein, we discuss the unique findings of our patient, the potential pathogenesis of AESOP, and the link between these three rare paraneoplastic entities along with review of the literature.

Which drugs are included in the VIFUP regimen for breast cancer?

ViFuP includes vinorelbine, cisplatin and continuous infusion of 5-fluorouracil.

OBJECTIVE: We undertook a prospective phase II study to assess the feasibility and activity of a new induction chemotherapy regimen followed by hyperfractionated irradiation in locally advanced squamous cell head and neck cancer. METHODS: 25 patients with locally advanced head and neck cancer were treated with 4 cycles of vinorelbine (20 mg i.v. day 1, 3), cisplatin (60 mg/m(2) i.v. day 1) and 5-fluorouracil (200 mg/m(2) continuous i.v. infusion day 1-21) (ViFuP regimen) followed by bifractionated radiotherapy (bidRT) up to 74.4 Gy in 62 fractions of 1.2 Gy twice daily. RESULTS: Chemotherapy was well tolerated, 6 patients developed grade 3 and one patient grade 4 neutropenia. Response to chemotherapy was observed in 19 patients (76%) including three complete responses and 16 partial responses. Planned bidRT was completed in 25 patients and all but one received planned bidRT dose without interruptions. Radiotherapy was well tolerated, mucositis was the most common side effect (grade 3-12 patients, grade 4-1 patient). At evaluation after the completion of bidRT, 13 patients had complete response (52%), 7 partial response (28%), 2 stable disease and 3 tumor progression. At the median follow-up of 18.2 months, 11 patients were alive and free of disease, and 14 patients had died (12 of tumor). Late xerostomy was observed in all but one 3-month survivors. Late mandibular necrosis was seen in 1 patient. CONCLUSIONS: bidRT preceded by ViFuP seems a feasible and active combination in locally advanced head and neck cancer. Good patient compliance did not compromise the delivery of planned dose of bidRT. However, short median duration of response (14.6 months) and moderate median overall survival (18.7 months) indicate the need for more intensive therapeutic strategies. On the basis of these results, modifications of our treatment schedule (shortening the overall treatment time by reduction of chemotherapy cycles and the use of chemotherapy concomitantly with irradiation) are planned for the future study. BACKGROUND: Experimental data on perioperative chemotherapy (PeCT) indicate that its initiation might be most useful if administered as close as possible to the time of first 'disturbance of the tumour'. Regimens including 5-fluorouracil (5-FU) as continuous infusion are commonly used in the preoperative setting, especially for large tumours and locally advanced disease. We therefore evaluated the role of PeCT with 5-FU as continuous infusion after preoperative chemotherapy (PreCT), covering the surgical phase and acute wound healing period, in patients with breast cancer too large to attempt breast-conserving surgery upon diagnosis. PATIENTS AND METHODS: Breast cancer patients, clinical stages T2-T3, N0-N2, M0, and Ki-67 labelling index >/= 20%, were treated every 3 weeks with a maximum of six courses of vinorelbine 20 mg total dose intravenously (i.v.) on days 1 and 3, cisplatin 60 mg/ m(2) i.v. on day 1 and 5-FU 200 mg/m(2)/day as a continuous infusion (ViFuP regimen). Patients who achieved a clinical and radiological objective remission with PreCT were also treated with perioperative 5-FU that was continued until 30 min before, and restarted immediately after surgery, prolonging infusion until 15 days after surgery. RESULTS: Following preoperative treatment, 39 of 49 evaluable patients [80%; 95% confidence interval (CI) 70% to 90%] had an objective response. Pathological complete remission (pCR) was achieved in 14 (29%) patients. No relevant clinical or haematological toxicity due to PeCT was observed. In 36 patients submitted to PeCT the rate of pCR was 33% (95% CI 18% to 48%). The highest response of the primary tumour to PreCT and PeCT was observed in women with tumours not expressing estrogen and progesterone receptors (pCR 46%; 95% CI 19% to 73%). CONCLUSIONS: Preoperative therapy can be protracted into the surgical (and wound healing) period without significant additional short-term toxicity. Proper selection of patients according to biological features might improve the therapeutic yield of preoperative therapies.

Which drugs are included in the EE-4A regimen for Wilm's tumor?

EE-4A regimen includes dactinomycin and vincristine.

Does inactivation of CYLD help in colorectal cancer?

Νο. Inactivation of CYLD in intestinal epithelial cells exacerbates colitis-associated colorectal carcinogenesis.

PURPOSE: CYLD is a tumor suppressor that has been linked to the development of various human maligcies, including colon cancer. The tumor-suppressing function of CYLD is associated with its deubiquitinating activity, which maps to the carboxyl-terminal region of the protein. In the present study we evaluated the role of intestinal epithelial CYLD in colitis-associated cancer using a conditional mouse CYLD inactivation model. METHODS: In order to evaluate the role of CYLD in intestinal epithelial carcinogenesis, mice (IEC-Cyld (Δ9) mice) that carry a mutation that eliminates the deubiquitinating domain of CYLD in intestinal epithelial cells (IEC) were generated by crossing Villin-Cre transgenic mice to previously generated mice carrying a loxP-flanked Cyld exon 9 (Cyld (flx9) mice). RESULTS: We found that IEC-Cyld (Δ9) mice did not present spontaneous intestinal abnormalities up to one year of age. However, upon challenge with a combination of genotoxic (AOM) and pro-inflammatory (DSS) agents we found that the number of adenomas in the IEC-Cyld (Δ9) mice was dramatically increased compared to the control mice. Inactivation of CYLD in intestinal epithelial cells did not affect the classical nuclear factor-kappaB (NF-κB) and c-Jun kinase (JNK) activation pathways under physiological conditions, suggesting that these pathways do not predispose CYLD-deficient intestinal epithelia to colorectal cancer development before the onset of genotoxic and/or pro-inflammatory stress. CONCLUSIONS: Our findings underscore a critical tumor-suppressing role for functional intestinal epithelial CYLD in colitis-associated carcinogenesis. CYLD expression and its associated pathways in intestinal tumors may be exploited for future prognostic and therapeutic purposes.

Which network analysis method can you use for prioritization of metabolic disease genes?

metPropagate is a network-guided propagation of metabolomic information for prioritization of metabolic disease genes. metPropagate was able to prioritize at least one causative gene in the top 20th percentile of candidate genes for 92% of patients with known IEMs.

Many inborn errors of metabolism (IEMs) are amenable to treatment, therefore early diagnosis is imperative. Whole-exome sequencing (WES) variant prioritization coupled with phenotype-guided clinical and bioinformatics expertise is typically used to identify disease-causing variants; however, it can be challenging to identify the causal candidate gene when a large number of rare and potentially pathogenic variants are detected. Here, we present a network-based approach, metPropagate, that uses untargeted metabolomics (UM) data from a single patient and a group of controls to prioritize candidate genes in patients with suspected IEMs. We validate metPropagate on 107 patients with IEMs diagnosed in Miller et al. (2015) and 11 patients with both CNS and metabolic abnormalities. The metPropagate method ranks candidate genes by label propagation, a graph-smoothing algorithm that considers each gene's metabolic perturbation in addition to the network of interactions between neighbors. metPropagate was able to prioritize at least one causative gene in the top 20th percentile of candidate genes for 92% of patients with known IEMs. Applied to patients with suspected neurometabolic disease, metPropagate placed at least one causative gene in the top 20th percentile in 9/11 patients, and ranked the causative gene more highly than Exomiser's phenotype-based ranking in 6/11 patients. Interestingly, ranking by a weighted combination of metPropagate and Exomiser scores resulted in improved prioritization. The results of this study indicate that network-based analysis of UM data can provide an additional mode of evidence to prioritize causal genes in patients with suspected IEMs.

Which drugs are included in the IROX regimen for colorectal cancer?

IROX regimen for colorectal cancer includes irinotecan and oxaliplatin.

BACKGROUND: Fluorouracil (5-FU), oxaliplatin and irinotecan combinations improve time to tumor progression (TTP), objective response and overall survival (OS) in patients with metastatic colorectal cancer (MCRC). Here we identify and describe patients treated on Intergroup study N9741 who initially had inoperable MCRC, but who obtained sufficient chemotherapeutic benefit to allow removal of their metastatic disease. PATIENTS AND METHODS: Patient research records in study arms (A) irinotecan/5-FU/leucovorin (LV) (IFL, n = 264), (F) oxaliplatin/5-FU/LV (FOLFOX4, n = 267) and (G) oxaliplatin/irinotecan (IROX, n = 265) were reviewed. TTP and median OS were calculated. RESULTS: Twenty-four (3.3%) of 795 randomized patients underwent curative metastatic disease resection [hepatectomy, 16; radiofrequency-ablation (RFA), six; lung resection, two]. Twenty-two out of 24 (92%) resected patients received an oxaliplatin-based regimen (FOLFOX4, 11; IROX, 11). Seven patients (29.2%) remain disease-free; relapses occurred mainly in the resected organ. Median OS in resected patients is 42.4 months, and median TTP is 18.4 months. All six patients treated with RFA have recurred. Four out of five (80%) patients who received chemotherapy following resection are disease-free. CONCLUSIONS: Resection of metastatic disease after chemotherapy is possible in a small but important subset of patients with MCRC, particularly after receiving an oxaliplatin-based chemotherapy regimen, with encouraging OS and TTP observed in these highly selected patients. BACKGROUND: Cancer of unknown primary (CUP) lacks established therapy although it affects 3% of cancer patients. We evaluated the irinotecan-oxaliplatin combination (IROX regimen) in previously untreated patients with non-favorable subsets of unknown primary carcinomas. METHODS: This was a multicenter phase-II trial. Protocol treatment consisted of oxaliplatin 80 mg/m(2) followed by irinotecan 160 mg/m(2) administered every 3 weeks. The primary end points were response rate and toxicity, and secondary end points were time to progression and survival. RESULTS: Forty-seven patients with liver, bone or multiple visceral metastases entered into the trial and received a median 6 chemotherapy cycles (1-11). The regimen was very well tolerated with one febrile neutropenia case and six cases with diarrhea grade 3 (16%). In intent-to-treat analysis the tumor response rate was 13% (95% CI = 4.8-25.7%) and 12 patients (27%, 95%CI 13.9-40.4%) had at least 4 months' duration of disease stabilization. The median time to progression was 2.7 months and the median survival was 9.5 months, with 40% of patients alive at 1 year. CONCLUSIONS: The IROX regimen demonstrated similar efficacy and a favorable toxicity profile compared to other more toxic chemotherapy combinations in patients with poor-prognosis CUP. PURPOSE To determine whether irinotecan plus oxaliplatin (IROX) is superior to irinotecan alone in patients with metastatic colorectal cancer (CRC) previously treated with single-agent fluoropyrimidines. PATIENTS AND METHODS A phase III, randomized, open-label, multicenter study of patients with metastatic or recurrent CRC that had progressed or recurred during or after adjuvant or first-line fluoropyrimidines (fluorouracil/leucovorin or capecitabine, the latter only for metastatic CRC). Patients received IROX (irinotecan 200 mg/m(2) plus oxaliplatin 85 mg/m(2)) or irinotecan alone (350 mg/m(2)) every 3 weeks. RESULTS: At the data cutoff (when 447 of 628 randomly assigned patients had died), median overall survival was 13.4 months (95% CI, 12.4 to 14.7 months) and 11.1 month (95% CI, 10.0 to 12.7 months) in the IROX and irinotecan groups, respectively (hazard ratio = 0.78; 95% CI, 0.65 to 0.94; P = .0072). Overall response rate (22% v 7%, respectively; P < .0001), median time to progression (5.3 v 2.8 months, respectively; P < .0001), and improvement in tumor-related symptoms (32% v 19%, respectively; P = .0072) were also improved with IROX as compared with irinotecan. With the exception of granulocytopenia (25% v 13%), diarrhea (28% v 23%), and sensory disturbances (5% v 0%), grade 3 to 4 toxicities were comparable between the IROX and irinotecan groups, respectively. CONCLUSION IROX is an effective treatment for metastatic CRC that has progressed after first-line fluoropyrimidine therapy. IROX improves efficacy compared with irinotecan alone, providing an additional option in the postadjuvant or second-line treatment setting for patients who experience treatment failure with single-agent fluoropyrimidine therapy. BACKGROUND: Gemcitabine- and 5-fluorouracil (5-FU)- based chemotherapy is a commonly used adjuvant or palliative treatment for patients with pancreatic cancer. However, a standard chemotherapy regimen has yet to be developed for patients refractory to gemcitabine and 5-FU treatment. We attempted to evaluate the efficacy and safety of a combination of irinotecan and oxaliplatin (IROX) as a salvage treatment for patients with gemcitabine- and 5-FU- refractory pancreatic cancer. PATIENTS AND METHODS: Patients with advanced pancreatic cancer who were refractory to prior gemcitabine- and 5-FU- based chemotherapy were enrolled in this study. IROX chemotherapy was administered as follows: Irinotecan, 150 mg/m(2) on day 1; and oxaliplatin, 85 mg/m(2) on day 1 over 90 min every 2 weeks. RESULT: From Mar. 2006 to Dec. 2008, a total of 14 patients were administered 50 cycles of chemotherapy. The male-to-female ratio of the patient group was 11:3. These patients ranged in age from 48 to 73 years (median 65.5 years old). 3 patients (21.4%) evidenced partial responses. four patients (28.6%) exhibited stable disease. The median time to progression and overall survival time were 1.4 (95% CI: 1.2-1.6) months and 4.1 (95% CI: 2.0-6.2) months, respectively. Major hematologic toxicities included grade 1-2 anemia (88%), neutropenia (36%), thrombocytopenia (30%), and grade 3-4 neutropenia (10%). The most frequently detected non-hematological toxicities were grade 3 diarrheas (14%). CONCLUSION: The IROX regimen appears to constitute a feasible and tolerable salvage therapy in patients with advanced pancreatic cancer who have been previously treated with gemcitabine- and 5-FU-based chemotherapy. 5-Fluorouracil (5-FU) is an antimetabolite that acts during the S phase of the cell cycle. The active metabolite, 5-fluorodeoxyuridine monophosphate inhibits thymidylate synthase (TS), thus preventing DNA synthesis, which leads to imbalanced cell growth and ultimately cell death. 5-FU and its oral prodrug capecitabine are used in the treatment of a number of solid tumors, including colorectal, breast, gastric, pancreatic, prostate, and bladder cancers. Common side effects include leukopenia, diarrhea, stomatitis, nausea, vomiting, and alopecia. Hand-foot syndrome (HFS) is a relatively common side effect of cytotoxic chemotherapy. It is more frequently associated with 5-FU, capecitabine, and cytarabine. This article reports on the case of a 55-year-old black man with metastatic colorectal carcinoma that was refractory to recommended treatment measures who developed grade 3 HFS after treatment with modified FOLFOX6 (leucovorin [LV]/5-FU/oxaliplatin) and bFOL (bolus 5-FU/LV/oxaliplatin) regimens. Treatment was discontinued despite excellent response to chemotherapy. The patient had progression of disease on IROX (irinotecan/oxaliplatin) and irinotecan/cetuximab regimens. He was started on gemcitabine/capecitabine and developed HFS again, which was controlled with aggressive skin care and vitamin B6 treatment. Full sequencing of the dihydropyrimidine dehydrogenase (DPYD) gene and analysis of the human TS gene (TYMS) promoter region was performed. Pharmacogenetic testing revealed 2R/2R genotype of TYMS gene, which is associated with up to a 2.5-fold risk of toxicity to 5-FU therapy. Hand-foot syndrome has proven to be a dose-limiting toxicity of 5-FU, especially of capecitabine, leading to significant morbidity. Hand-foot syndrome seems to be dose dependent, and both peak drug concentration and total cumulative dose determine its occurrence. Genetic variations such as polymorphic abnormality of TYMS are potential causative factors for a significant portion of serious adverse reactions to 5-FU-based therapy. PURPOSE: With three available chemotherapy drugs for advanced colorectal cancer (CRC), response rate (RR) and survival outcomes have improved with associated morbidity, accentuating the need for tools to select optimal individualized treatment. Pharmacogenetics identifies the likelihood of adverse events or response based on variants in genes involved in drug transport, metabolism, and cellular targets. PATIENTS AND METHODS: Germline DNA was extracted from 520 patients on the North American Gastrointestinal Intergroup N9741 study. Three study arms were evaluated: IFL (fluorouracil [FU] + irinotecan [IRN]), FOLFOX (FU + oxaliplatin), and IROX (IRN + oxaliplatin). Information on adverse events, response, and disease-free survival was available. Thirty-four variants in 15 candidate genes for analysis based on previous associations with adverse events or outcome were assessed. Genotyping was performed using pyrosequencing. RESULTS: All variants were polymorphic. The homozygous UGT1A1*28 allele observed in 9% of patients was associated with risk of grade 4 neutropenia in patients on IROX (55% v 15%; P = .002). Deletion in GSTM1 was associated with grade 4 neutropenia after FOLFOX (28% v 16%; P = .02). Patients with a homozygous variant genotype for GSTP1 were more likely to discontinue FOLFOX because of neurotoxicity (24% v 10%; P = .01). The presence of a CYP3A5 variant was significantly associated with RR on IFL (29% v 60%; P = .0074). Most previously published genotype-toxicity or -efficacy relationships were not validated in this study. CONCLUSION: This study provides a platform to evaluate pharmacogenetic predictors of response or severe adverse events in advanced CRC. Pharmacogenetic studies can be conducted in multicenter trials, and our findings demonstrate that with continued research, clinical application is practical. PURPOSE: The optimal end point for randomized phase II trials of anticancer therapies remains controversial. We simulated phase II trials by resampling patients from N9741, a randomized phase III trial of chemotherapy regimens for metastatic colorectal cancer, and compared the power of various end points to detect the superior therapy (FOLFOX [infusional fluorouracil, leucovorin, and oxaliplatin] had longer overall survival than both IROX [irinotecan plus oxaliplatin] and IFL [irinotecan and bolus fluorouracil plus leucovorin]). METHODS: Tumor measurements and progression-free survival (PFS) data were obtained for 1,471 patients; 1,002 had consistently measured tumors and were resampled (5,000 replicates) to simulate two-arm, randomized phase II trials with α = 0.10 (one sided) and 20 to 80 patients per arm. End points included log ratio of tumor size at 6, 12, and 18 weeks relative to baseline; time to tumor growth (TTG), estimated using a nonlinear mixed-effects model; and PFS. Arms were compared using rank sum tests for log ratio and TTG and a log-rank test for PFS. RESULTS: For FOLFOX versus IFL, TTG and PFS had similar power, with both exceeding the power of log ratio at 18 weeks; for FOLFOX versus IROX, TTG and log ratio at 18 weeks had similar power, with both exceeding the power of PFS. The best end points exhibited > 80% power with 60 to 80 patients per arm. CONCLUSION: TTG is a powerful end point for randomized phase II trials of cytotoxic therapies in metastatic colorectal cancer; it was either comparable or superior to PFS and log ratio at 18 weeks. Additional studies will be needed to clarify the potential of TTG as a phase II end point. This network meta-analysis compared the short-term and long-term efficacies of first-line chemotherapy regimens in patients with advanced colorectal cancer (CRC). The 10 regimens included folinic acid + 5-fluorouracil + oxaliplatin (FOLFOX), folinic acid + 5-fluorouracil + irinotecan (FOLFIRI), folinic acid + 5-fluorouracil + gemcitabine (FFG), folinic acid + 5-fluorouracil + trimetrexate (FFT), folinic acid + 5-fluorouracil (FF), irinotecan + oxaliplatin (IROX), raltitrexed + oxaliplatin (TOMOX), folinic acid + tegafur-uracil (FTU), raltitrexed, and capecitabine. Electronic searches were performed in the Cochrane Library, PubMed and Embase databases from inception to June 2017. Network meta-analysis combined direct and indirect evidence to obtain odds ratios (ORs) and surface under the cumulative ranking curves (SUCRA) of different chemotherapy regimens for advanced CRC. Fourteen randomized controlled trails (RCTs) covering 4,383 patients with advanced CRC were included. The results revealed that FOLFOX, FOLFIRI, IROX, and TOMOX all showed higher overall response rates (ORRs) than FF or raltitrexed. Compared with raltitrexed, the aforementioned four regimens also had higher 1-year progression-free survival (PFS) rates. In addition, FOLFOX and FOLFIRI exhibited higher disease control rates (DCRs) and 1-year PFS rates than FF or raltitrexed. Cluster analysis revealed that FOLFOX, FOLFIRI, and TOMOX had better short-term and long-term efficacies. These findings suggest FOLFOX, FOLFIRI, and TOMOX are superior to other regimens for advanced CRC. These three regimens are therefore recommended for clinical treatment of advanced CRC.

What are the targets of pemigatinib?

Pemigatinib is a small molecule inhibitor of fibroblast growth factor receptor (FGFR) 1, FGFR2 and FGFR3, received accelerated approval for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test.

BACKGROUND: Fibroblast growth factor receptor (FGFR) 2 gene alterations are involved in the pathogenesis of cholangiocarcinoma. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. This study evaluated the safety and antitumour activity of pemigatinib in patients with previously treated, locally advanced or metastatic cholangiocarcinoma with and without FGFR2 fusions or rearrangements. METHODS: In this multicentre, open-label, single-arm, multicohort, phase 2 study (FIGHT-202), patients aged 18 years or older with disease progression following at least one previous treatment and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 recruited from 146 academic or community-based sites in the USA, Europe, the Middle East, and Asia were assigned to one of three cohorts: patients with FGFR2 fusions or rearrangements, patients with other FGF/FGFR alterations, or patients with no FGF/FGFR alterations. All enrolled patients received a starting dose of 13·5 mg oral pemigatinib once daily (21-day cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, withdrawal of consent, or physician decision. The primary endpoint was the proportion of patients who achieved an objective response among those with FGFR2 fusions or rearrangements, assessed centrally in all patients who received at least one dose of pemigatinib. This study is registered with ClinicalTrials.gov, NCT02924376, and enrolment is completed. FINDINGS: Between Jan 17, 2017, and March 22, 2019, 146 patients were enrolled: 107 with FGFR2 fusions or rearrangements, 20 with other FGF/FGFR alterations, 18 with no FGF/FGFR alterations, and one with an undetermined FGF/FGFR alteration. The median follow-up was 17·8 months (IQR 11·6-21·3). 38 (35·5% [95% CI 26·5-45·4]) patients with FGFR2 fusions or rearrangements achieved an objective response (three complete responses and 35 partial responses). Overall, hyperphosphataemia was the most common all-grade adverse event irrespective of cause (88 [60%] of 146 patients). 93 (64%) patients had a grade 3 or worse adverse event (irrespective of cause); the most frequent were hypophosphataemia (18 [12%]), arthralgia (nine [6%]), stomatitis (eight [5%]), hyponatraemia (eight [5%]), abdominal pain (seven [5%]), and fatigue (seven [5%]). 65 (45%) patients had serious adverse events; the most frequent were abdominal pain (seven [5%]), pyrexia (seven [5%]), cholangitis (five [3%]), and pleural effusion (five [3%]). Overall, 71 (49%) patients died during the study, most frequently because of disease progression (61 [42%]); no deaths were deemed to be treatment related. INTERPRETATION: These data support the therapeutic potential of pemigatinib in previously treated patients with cholangiocarcinoma who have FGFR2 fusions or rearrangements. FUNDING: Incyte Corporation. Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombit human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations. Author information: (1)Division of Hematology/Oncology, Department of Internal Medicine, Mayo Clinic, Phoenix, AZ 85054, USA. (2)Division of Cancer Sciences, University of Manchester & Department of Medical Oncology, The Christie Hospital NHS Foundation Trust, The University of Manchester, Manchester, UK. (3)Department of Oncology, University of Leuven, Leuven, Belgium. (4)Department of Oncology and Hematology, Humanitas Clinical and Research Center-IRCCS, Rozzano, Milan, Italy. (5)Department of Biomedical Sciences, Humanitas University, Pieve Emanuele, Milan, Italy. (6)Department of Medical Oncology, Kyorin University, Tokyo, Japan. (7)Department of Cancer Survey and Gastrointestinal Oncology, Osaka International Cancer Institute, Osaka, Japan. (8)Department of Medicine, University of Verona, Verona, Italy. (9)Medical Oncology Department, Vall d'Hebron University Hospital & Vall d'Hebron Institute of Oncology, Barcelona, Spain. (10)Research Department of Oncology, UCL Cancer Institute, University College London, London, UK. (11)Department of Medical Oncology, Hammersmith Hospital, Imperial College Health Care Trust, London, UK. (12)Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (13)Department of Medicine, Weill Medical College, Cornell University, New York, NY, USA. (14)Incyte Corporation, Wilmington, DE, USA. (15)Incyte Biosciences International Sàrl, Morges, Switzerland. (16)Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany. BACKGROUND: Prognosis of patients affected by metastatic esophageal-gastric junction (EGJ) or gastric cancer (GC) remains dismal. Trastuzumab, an anti-HER2 monoclonal antibody, is the only targeted agent approved for the first-line treatment of patients with HER2-overexpressing advanced EGJ or GC in combination with chemotherapy. However, patients invariably become resistant during this treatment. We recently identified the overexpression of fibroblast growth factor (FGF) receptor 3 (FGFR3) as a molecular mechanism responsible for trastuzumab resistance in GC models, providing the rationale for the inhibition of this receptor as a potential second-line strategy in this disease. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. METHODS: The FiGhTeR trial is a phase II, single-arm, open-label study to assess safety and activity of the FGFR inhibitor pemigatinib as second-line treatment strategy in metastatic EGJ/GC patients progressing under trastuzumab-containing therapies. The primary endpoint is the 12-week progression-free survival rate. Plasma and tumor tissue samples will be collected for translational research analyses at baseline, during treatment, and at progression on pemigatinib. DISCUSSION: Co-alterations in genes coding for different tyrosine-kinase receptors are emerging as relevant mechanisms of acquired resistance to anti-HER2 therapeutic strategies in GC. In particular, our group has recently identified that in GC models the overexpression of FGFR3 sustains the acquired resistance to trastuzumab. This trial aims to assess the safety, tolerability and activity of the FGFR inhibitor pemigatinib as a second-line treatment in metastatic EGJ/GC patients refractory to first-line trastuzumab-containing therapies. Furthermore, this study offers the opportunity to prospectively study mechanisms and pathways involved in trastuzumab resistance. PROTOCOL NUMBER: CRC2017_02. EUDRACT NUMBER: 2017-004522-14.

Is there high nucleotide diversity in the Drosophila suzukii species?

Native to Asia, the soft-skinned fruit pest Drosophila suzukii has recently invaded the United States and Europe. The eastern United States represents the most recent expansion of their range, and presents an opportunity to test alternative models of colonization history. There are high levels of nucleotide diversity in this species and research suggests that the recent invasions of Europe and the continental United States are independent demographic events.

Which tools have been developed for identifying and visualising ncRNA promoters?

Epd, ncpro-ml and ucsc genome browser are tools that have been developed for identifying and visualising ncRNA promoters.

What is the mode of administration of AZD8601?

AZD8601 is administered intradermally.

Author information: (1)Drug Metabolism and Pharmacokinetics, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. (2)Fraunhofer-Chalmers Centre, Chalmers Science Park, Gothenburg, Sweden. (3)Clinical Pharmacology and Quantitative Pharmacology, Clinical Pharmacology and Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. (4)Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA. (5)Bioscience Cardiovascular, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. (6)Integrative Systems Biology, Department of Biomedical Engineering, Linköping University, Linköping, Sweden. (7)Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. (8)Integrated Cardiometabolic Center, Karolinska Institute, Huddinge, Sweden. (9)Department of Cell and Molecular Biology and Medicine, Karolinska Institute, Stockholm, Sweden.

What is SAR425899?

SAR425899 ia a dual glucagon-like peptide-1 receptor/glucagon receptor agonist.

AIM: To evaluate the change in insulin sensitivity, β-cell function and glucose absorption after 28 days of treatment with high and low doses of SAR425899, a novel dual glucagon-like peptide-1 receptor/glucagon receptor agonist, versus placebo. MATERIALS AND METHODS: Thirty-six overweight to obese subjects with type 2 diabetes were randomized to receive daily subcutaneous administrations of low-dose SAR425899 (0.03, 0.06 and 0.09 mg) and high-dose SAR425899 (0.06, 0.12 and 0.18 mg) or placebo for 28 days; dose escalation occurred after days 7 and 14. Mixed meal tolerance tests were conducted before treatment (day -1) and on days 1 and 28. Oral glucose and C-peptide minimal models were used to quantify metabolic indices of insulin sensitivity, β-cell responsiveness and glucose absorption. RESULTS: With low-dose SAR425899, high-dose SAR425899 and placebo, β-cell function from day -1 to day 28 increased by 163%, 95% and 23%, respectively. The change in area under the curve for the rate of meal glucose appearance between 0 and 120 minutes was -32%, -20% and 8%, respectively. CONCLUSIONS: After 28 days of treatment, SAR425899 improved postprandial glucose control by significantly enhancing β-cell function and slowing glucose absorption rate.

Which class of disorders are caused by AMPA receptor GluA2 subunit defects?

Mutations in the AMPA receptor GluA2 subunit cause a variety of neurodevelopmental disorders including autism spectrum disorder.

Is Hunter's disease is associated with the X Chromosome?

Yes, Hunter's disease is associated with the X Chromosome.

We report the occurrence of Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl who was one of identical twins. Molecular studies showed nonrandom X-inactivation in both her fibroblasts and lymphocytes, while her normal twin showed equal usage of both X chromosomes. In view of previous reports of 7 pairs of identical female twins in which one had Duchenne muscular dystrophy, it seems that twinning may be strongly associated with nonrandom X-inactivation, and is not specific to the properties of the disease causing gene. We report the results of studies on the characterization of the mutation associated with marked unbalanced expression of the mutant X chromosome in a karyotypically normal girl with Hunter disease (mucopolysaccharidosis type II). Southern analysis of DNA extracted from somatic cell hybrids containing only the mutant X chromosome showed deletion of the Xq27.3-q28 loci: DXS297 (VK23AC), DXS293 (VK16), FRAXA (pfxa3), DXS296 (VK21A), and the 3' end of the iduronatesulfatase (IDS) gene. The flanking loci--DXS52 (St14-1), DXS304 (U6.2), and DXS369 (RN1)--were intact. On the basis of these results, we concluded that the mutation was a simple deletion extending a maximum of 3-5 cM to the centromeric side of the IDS gene. Both Southern analysis of DNA from somatic cell hybrids, using short segments of IDS cDNA, and PCR of reverse-transcribed RNA from cultured skin fibroblasts indicated that the telomeric terminus of the deletion was localized to a region near the middle of the coding sequences of the gene. Segregation analysis on five samples of families with Hunter's syndrome (158 cases overall) shows that the mutant allele segregates in agreement with Mendelian expectations for an X linked recessive disease, but the proportion of sporadic cases is significantly lower than expected under mutation-selection equilibrium. Heterogeneity among the samples is apparent, but it is caused entirely by a sample of Ashkenazi families, whose segregation pattern has previously been interpreted as supporting the hypothesis of prenatal selection in favour of the pathological allele. Conversely, our joint analysis of the five samples by a maximum likelihood approach does not suggest segregation distortion. Possible reasons for the apparent lack of sporadic cases include the effect of ascertainment bias. Cytogenetic re-evaluation of a fibroblast cell line from a female Hunter's syndrome case with a balanced X;autosome translocation, which had previously been reported to have a breakpoint in Xq26 to Xq27, showed the breakpoint to be either between Xq27 and Xq28 or within Xq28. The normal X chromosome was preferentially inactivated, supporting the view that the translocation had disrupted the Hunter gene. The new localisation is now in full agreement with our previous linkage work and other published data. Results of further linkage studies using probes defining the loci DXS86, DXS144, DXS100, DXS102, DXS105, F8C, and DXS134 are also consistent with our original conclusion that the Hunter locus lies within the distal region of the X chromosome long arm. A 2.5-year-old girl who presented with abdominal distension, hepatomegaly, coarse facies, hirsutism and contraction deformities was investigated for mucopolysaccharidoses. Urinary excretion showed increased total glycosaminoglycans (105 mg/mmol creatinine; normal for age 9-20 mg/mmol) with marked increases of dermatan and heparan sulphates. A number of lysosomal enzyme activities were measured on leucocytes, serum and cultured fibroblasts. Normal or high activities were found for alpha-iduronidase, N-acetylgalactosamine-6-sulphatase, beta-galactosidase, arylsulphatase B and beta-glucuronidase. However a marked deficiency of iduronate sulphate sulphatase activity was observed, consistent with a diagnosis of Hunter's disease. Activities were reduced to less than 2% of mean control values in the patient's leucocytes, serum and cultured fibroblasts. Normal activities were measured in samples from the father and younger sister but a partial deficiency (43% of control serum) was found in the mother. Chromosome studies on the patient revealed a partial deletion of the long arm of one X-chromosome, most probably of band Xq25, which was not inherited from either parent. Studies using BrdU indicated that the deleted X chromosome was consistently late replicating, and as a result the Hunter gene was fully expressed on the other X chromosome. Hunter disease or mucopolysaccharidosis type II is an X-linked disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS gene (24 kb) contains nine exons and has been completely sequenced. A pseudogene (IDS-2 locus) distal to the functional IDS gene has recently been identified. This work reports the characterization of IDS gene alterations in two severely affected patients. Patient 1 has a partial deletion that removes exons I to VI and extends about 200 kb upstream of the IDS gene. Patient 2 has an internal deletion of exons IV, V, VI, and VII, which results from an IDS gene-pseudogene exchange between highly homologous regions. In the rearranged gene, the junction intron contains pseudogene intron 3- and intron 7-related sequences. An interchromosomal recombination is probably the cause of this rearranged X chromosome. Hunter disease is an X-linked recessive mucopolysaccharide storage disorder caused by iduronate-2-sulfatase deficiency and is rare in females. We describe here findings in a girl with Hunter disease of the severe type. She had a normal karyotype but a marked deficiency of iduronate-2-sulfatase activity in lymphocytes and cultured fibroblasts. In a sequence analysis of the iduronate-2-sulfatase gene, evidence was obtained for the R468Q (G1403 to A) mutation, a common one in Hunter disease. RT-PCR showed her cDNA to represent only the R468Q allele, although at the genomic level she was a heterozygote with one normal allele. Her brother had the R468Q mutation, and their mother was a carrier of this mutation. The fusion products of CHO (TG(R),Neo(R)) with patient's fibroblasts cultured in HAT/G418 selective medium, carried only the maternal allele. However, in genomic DNA from the patient's fibroblasts, only the paternal allele of the androgen receptor gene, a gene subjected to differential methylation of the inactive X-chromosome, was methylated. These findings strongly suggest that the severe form of Hunter disease in this girl was the result of selective expression of the maternal allele carrying the missense mutation R468Q, which in turn resulted from skewed X inactivation of the paternal nonmutant X chromosome. The utility of polymerase chain reaction (PCR) amplification of amelogenin gene as a reliable and rapid means of determination of sex chromosomes was tested in 20 patients of X-linked disorders (Duchenne muscular dystrophy, haemophilia and Wiscott-Aldrich and Hunter's syndromes), 12 of intersex (testicular feminization syndrome, male pseudohermaphrodites, true hermaphrodites) and 21 of congenital adrenal hyperplasia. Of these, 26 (49%) cases were for prenatal diagnosis of X-linked diseases and congenital adrenal hyperplasia (CAH). The presence of X and Y chromosomes was determined within 24 h of receiving the samples. The results were in conformity with cytogenetic studies in all instances. The analysis of amelogenin gene proved helpful in the diagnosis and management of these patients. Hunter disease is an X-linked recessive disorder caused by a deficiency of iduronate-2-sulfatase activity. We describe a pair of brother/sister siblings with a typical feature of Hunter disease (mucopolysaccharidosis type II). They had normal karyotypes but a marked deficiency of iduronate-2-sulfatase activity in both lymphocytes and fibroblasts. The molecular analysis of the iduronate-2-sulfatase gene revealed the R468L(G1403-->T) substitution in their genes. Although the sister's genomic DNA was heterozygous for the mutant allele, the sister's cDNA was found to be homogeneous for this mutation. The mother was found to be a heterozygote. The analysis of X chromosome inactivation by comparison of the methylation patterns of the androgen-receptor (AR) gene which was isolated from the sister's fibroblasts and leucocytes revealed a skewed X chromosome inactivation of the paternal allele. These findings indicate that a skewed X chromosome inactivation of the paternal gene and a point mutation in the maternal gene were responsible for the lack of iduronate-2-sulfatase activity in the sister. INTRODUCTION: Hunter syndrome, or mucopolysaccharidosis type II, is an inherited disease linked to the X chromosome that is caused by a deficit of the enzyme iduronate-2-sulfatase and its main symptoms affect the bones, neurological system and the viscera. In order to further our knowledge of its natural history, a registry containing data about patients' clinical histories was compiled. The purpose of this review is to describe how the registry works and to present the initial data concerning Spanish patients with Hunter syndrome included in it. DEVELOPMENT: The Hunter Outcome Survey (HOS) registry is a multi-centre, world-wide, observational, long-term follow-up study that is open to all patients diagnosed with the disease. The registry includes clinical data and information from the complementary examinations that are commonly performed as usual practice while attending these patients. Its main aims are to describe the population of patients with the disease, to further our knowledge of its natural history, to keep a check on the safety and effectiveness of enzyme replacement therapy in patients who are candidates for such treatment and to create a database that makes it possible to draw up a set of guidelines for clinical practice. CONCLUSIONS: Specific registries of low-prevalence diseases, such as the HOS registry for patients with Hunter syndrome, are important to improve the follow-up of patients and to determine the impact of new treatments. The Spanish HOS registry is an important step forward in furthering our knowledge of the current situation of the patients registered throughout the country. Familial X-chromosome inactivation (XCI) skewing was investigated in a family in which a female mucopolysaccharidosis type II (MPS II) (Hunter syndrome, an X-linked genetic disease) occurred. Among eight related females aged under 60 years from three generations who were tested, four revealed a non-random pattern of XCI. Detailed genetic analysis failed to find mutations in genes that were previously reported as important for the XCI process. Haplotype analysis excluded linkage of non-random XCI with genes localized on the X-chromosome. We propose that analysis of the XCI pattern should be taken into consideration when assessing risk factors for X-linked recessive genetic disorders. Mucopolysaccharidosis type II (MPS II, Hunter disease) is an X chromosome-linked inherited metabolic disease caused by mutations resulting in deficiency of activity of iduronate-2-sulfatase (IDS) and accumulation of undegraded glycosaminoglycans (GAGs), heparan sulfate, and dermatan sulfate. Previous experiments with cell cultures and studies on animal model of MPS II suggested that gene expression-targeted isoflavone therapy (GET IT), based on genistein-mediated reduction of efficiency of GAG synthesis, might be a suitable therapy for this disease. In this report, we demonstrate efficacy of GET IT in connective tissue elasticity, particularly in improving the range of joint motion in seven patients with MPS II after 26 weeks of treatment with an isoflavone extract at the dose corresponding to 5 mg/kg/day of genistein. BACKGROUND: Global developmental delay and mental retardation are associated with X-linked disorders including Hunter syndrome (mucopolysaccharidosis type II) and Fragile X syndrome (FXS). Single nucleotide mutations in the iduronate 2-sulfatase (IDS) gene at Xq28 most commonly cause Hunter syndrome while a CGG expansion in the FMR1 gene at Xq27.3 is associated with Fragile X syndrome. Gene deletions of the Xq27-28 region are less frequently found in either condition with rare reports in females. Additionally, an association between Xq27-28 deletions and skewed X-inactivation of the normal X chromosome observed in previous studies suggested a primary role of the Xq27-28 region in X-inactivation. CASE PRESENTATION: We describe the clinical, molecular and biochemical evaluations of a four year-old female patient with global developmental delay and a hemizygous deletion of Xq27.3q28 (144,270,614-154,845,961 bp), a 10.6 Mb region that contains >100 genes including IDS and FMR1. A literature review revealed rare cases with similar deletions that included IDS and FMR1 in females with developmental delay, variable features of Hunter syndrome, and skewed X-inactivation of the normal X chromosome. In contrast, our patient exhibited skewed X-inactivation of the deleted X chromosome and tested negative for Hunter syndrome. CONCLUSIONS: This is a report of a female with a 10.6 Mb Xq27-28 deletion with skewed inactivation of the deleted X chromosome. Contrary to previous reports, our observations do not support a primary role of the Xq27-28 region in X-inactivation. A review of the genes in the deletion region revealed several potential genes that may contribute to the patient's developmental delays, and sequencing of the active X chromosome may provide insight into the etiology of this clinical presentation. Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a rare X-linked disorder caused by deficient activity of the lysosomal enzyme, iduronate-2-sulfatase (IDS). Phenotypic expression of MPS II in female patients rarely occurs and may be the result of (i) structural abnormalities of the X chromosome, (ii) homozygosity for disease-causing mutations, or (iii) skewed X-chromosome inactivation, in which the normal IDS allele is preferentially inactivated and the abnormal IDS allele is active. We report here on a female patient with clinical MPS II manifestations, deficiency of IDS enzyme activity and a de novo balanced reciprocal X;9 translocation. As our patient has a skewed XCI pattern, but neither genomic IDS mutations nor abnormal IDS transcripts were detected, we speculate about the possible role of the chromosomal rearrangement in reducing the IDS translation efficiency.

Where are integrins localized in a cell?

Integrins are transmembrane glycoproteins that are broadly distributed in living organisms.

The extracellular domain of plasma membrane integrin αvβ3 contains a cell surface receptor for thyroid hormone analogues. The receptor is largely expressed and activated in tumor cells and rapidly dividing endothelial cells. The principal ligand for this receptor is l-thyroxine (T4), usually regarded only as a prohormone for 3,5,3'-triiodo-l-thyronine (T3), the hormone analogue that expresses thyroid hormone in the cell nucleus via nuclear receptors that are unrelated structurally to integrin αvβ3. At the integrin receptor for thyroid hormone, T4 regulates cancer and endothelial cell division, tumor cell defense pathways (such as anti-apoptosis), and angiogenesis and supports metastasis, radioresistance, and chemoresistance. The molecular mechanisms involve signal transduction via mitogen-activated protein kinase and phosphatidylinositol 3-kinase, differential expression of multiple genes related to the listed cell processes, and regulation of activities of other cell surface proteins, such as vascular growth factor receptors. Tetraiodothyroacetic acid (tetrac) is derived from T4 and competes with binding of T4 to the integrin. In the absence of T4, tetrac and chemically modified tetrac also have anticancer effects that culminate in altered gene transcription. Tumor xenografts are arrested by unmodified and chemically modified tetrac. The receptor requires further characterization in terms of contributions to nonmaligt cells, such as platelets and phagocytes. The integrin αvβ3 receptor for thyroid hormone offers a large panel of cellular actions that are relevant to cancer biology and that may be regulated by tetrac derivatives. Author information: (1)State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing, 400716, China; Cancer Center, Medical Research Institute, Southwest University, Chongqing, 400716, China; Chongqing Engineering and Technology Research Centre for Silk Biomaterials and Regenerative Medicine, Chongqing, 400716, China; Engineering Research Center for Cancer Biomedical and Translational Medicine, Southwest University, Chongqing, 400716, China. (2)State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing, 400716, China; Cancer Center, Medical Research Institute, Southwest University, Chongqing, 400716, China; Chongqing Engineering and Technology Research Centre for Silk Biomaterials and Regenerative Medicine, Chongqing, 400716, China; Engineering Research Center for Cancer Biomedical and Translational Medicine, Southwest University, Chongqing, 400716, China. Electronic address: [email protected]. (3)State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing, 400716, China; Cancer Center, Medical Research Institute, Southwest University, Chongqing, 400716, China; Chongqing Engineering and Technology Research Centre for Silk Biomaterials and Regenerative Medicine, Chongqing, 400716, China; Engineering Research Center for Cancer Biomedical and Translational Medicine, Southwest University, Chongqing, 400716, China. Electronic address: [email protected].

What is a bacteriocin?

Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. One such strategy involves using narrow-spectrum protein antibiotics (so-called bacteriocins), which diverse bacteria use to compete against closely related species.

Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. However, bacteriocins display diverse mechanisms of action, a narrow spectrum of activity, and inherent challenges in natural product isolation making in vitro verification of putative bacteriocins difficult. A subset of bacteriocins exert their antimicrobial effects through favorable biophysical interactions with the bacterial membrane mediated by the charge, hydrophobicity, and conformation of the peptide. We have developed a pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using machine learning and a simple biophysical trait filter to generate 20 amino acid peptides that can be synthesized and evaluated for activity. We generated 28,895 total 20-mer candidate peptides and scored them for charge, α-helicity, and hydrophobic moment. Of those, we selected 16 sequences for synthesis and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. Peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination to create an original approach to swiftly discover bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use. Many enterococcal strains produce bacteriocins, which could be useful as natural food preservatives through inhibition of pathogenic and spoilage microorganisms. There is little knowledge of the distribution and spectrum of bacteriocin activity and the distribution of bacteriocin-encoding genes in enterococci isolated from dogs. Therefore, we subjected 160 enterococcal isolates (E. faecium n=92, E. faecalis n=35, E. hirae n=28, E. casseliflavus n=3, E. mundtii n=2) from 105 samples of dog faeces to polymerase chain reaction (PCR) detection of genes for enterocin A, P, B, L50A, L50B, AS-48, and bac31 and to screening for bacteriocin activity. The results showed the presence of at least one of the tested genes in 54/160 isolates, with E. faecium the most common gene-possessing species. The most frequently occurring gene for production of enterocin A was observed in combination with enterocin P and B. Bacteriocin activity was observed in 76/160 isolates against at least one of 5 indicator bacteria from the genus Listeria, Enterococcus, Streptococcus and Staphylococcus. Four selected strains (IK25, Bri, I/Dz, P10) were active mostly against different species of Enterococcus (in the range 400-25 600 AU/mL) and Listeria sp. (800-12 800 AU/mL) but no Gram-negative bacteria were inhibited. Protein character, thermostability (up to 121°C) and stability at different pH values (3.0-10.0) were confirmed for crude bacteriocins of these four strains. The antimicrobial substance of E. faecium IK25 strain was identified as enterocin B using molecular weight detection and the presence of genes.

What protein complex is altered in "Coffin-Siris syndrome"?

he genes causative of CSS mainly encode the SWI/SNF complex, which contributes to chromatin remodeling and regulates the access of transcriptional factors to specific gene sites.

Author information: (1)West of Scotland Regional Genetics Service, Laboratory Medicine Building, Queen Elizabeth University Hospital, Glasgow, United Kingdom. Electronic address: [email protected]. (2)Yorkshire Regional Genetics Service, Department of Clinical Genetics, Chapel Allerton Hospital, Leeds, United Kingdom. (3)Bristol Clinical Genetics Service, St Michael's Hospital, Bristol, United Kingdom. (4)Clinical Genetics Department, Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom. (5)Department of Clinical Genetics, Addenbrookes Hospital, Cambridge, United Kingdom. (6)South West Thames Regional Genetics Service, St. George's Hospital, London, United Kingdom. (7)Academic Unit of Medical Genetics and Clinical Pathology, School of Medicine, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom. (8)Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom. (9)West of Scotland Regional Genetics Service, Laboratory Medicine Building, Queen Elizabeth University Hospital, Glasgow, United Kingdom. Electronic address: [email protected]. PURPOSE: SMARCB1 encodes a subunit of the SWI/SNF complex involved in chromatin remodeling. Pathogenic variants (PV) in this gene can give rise to three conditions. Heterozygous loss-of-function germline PV cause rhabdoid tumor predisposition syndrome and schwannomatosis. Missense PV and small in-frame deletions in exons 8 and 9 result in Coffin-Siris syndrome, which is characterized by intellectual disability (ID), coarse facial features, and fifth digit anomalies. METHODS: By a gene matching approach, individuals with a similar SMARCB1 PV were identified. Informed consent was obtained and patient data were collected to further establish genotype-phenotype relationship. RESULTS: A recurrent de novo missense PV (c.110G>A;p.Arg37His) in exon 2 of SMARCB1, encoding the DNA-binding domain, was identified in four individuals from different genetic centers. They shared a distinct phenotype consisting of profound ID and hydrocephalus due to choroid plexus hyperplasia. Other shared features include severe neonatal feeding difficulties; congenital heart, kidney, and eye anomalies; obstructive sleep apnea; and anemia. CONCLUSION: The p.Arg37His PV in the DNA-binding domain of SMARCB1 causes a distinctive syndrome, likely through a gain-of-function or domit-negative effect, which is characterized by severe ID and hydrocephalus resulting from choroid plexus hyperplasia. This report broadens the phenotypic spectrum associated with PV in SMARCB1. ARID2 loss-of-function is associated with a rare genetic disorder characterized in 14 reported patients to date. ARID2 encodes a member of the SWItch/sucrose non-fermentable chromatin remodeling complex. Other genes encoding subunits of this complex, such as ARID1A, ARID1B, and SMARCA2, are mutated in association with Coffin-Siris syndrome (CSS) and Nicolaides Baraitser syndrome (NCBRS) phenotypes. Previously reported ARID2 mutations manifested clinically with a CSS-like phenotype including intellectual disability, coarsened facial features, fifth toenail hypoplasia, and other recognizable dysmorphisms. However, heterogeneity exists between previously reported patients with some patients showing more overlapping features with NCBRS. Herein, we present a patient with a novel disease-causing ARID2 loss-of-function mutation. His clinical features included intellectual disability, coarse and dysmorphic facial features, toenail hypoplasia, ADHD, short stature, and delayed development consistent with prior reports. Our patient also presented with previously unreported clinical findings including ophthalmologic involvement, persistent fetal fingertip and toetip pads, and diffuse hyperpigmentary and hypopigmentary changes sparing his face, palms, and soles. The anomalous skin findings are particularly of interest given prior literature outlining the role of ARID2 in melanocyte homeostasis and melanoma. This clinical report and review of the literature is further affirming of the characteristic symptoms and expands the phenotype of this newly described and rare syndrome. Author information: (1)Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada. (2)Centre Hospitalier Universitaire Sainte-Justine Research Center, University of Montreal, Montreal, QC H3T 1C5, Canada. (3)Department of Biology, Faculty of Science, Western University, London, ON N6A 5B7, Canada; Division of Genetics and Development, Children's Health Research Institute, London, ON N6C 2V5, Canada. (4)Department of Biology, Faculty of Science, Western University, London, ON N6A 5B7, Canada. (5)Department of Pediatrics, Central Hospital, Aichi Human Service Center, Kasugai 480-0392, Japan. (6)Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. (7)Faculty of Medicine, University of Southampton, Southampton SO17 1BJ, UK. (8)Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK. (9)Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. (10)APHP, Département de Génétique, Centre de Référence Déficiences Intellectuelles de Causes Rares, Groupe Hospitalier Pitié Salpêtrière et GHUEP Hôpital Trousseau, Paris, France. (11)Institut National de la Santé et de la Recherche Médicale (INSERM), U 1127, CNRS UMR 7225, Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Univ Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle Épinière, 75013 Paris, France; Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, UMR 7104, INSERM U964, Université de Strasbourg, 67400 Illkirch, France; Institute of Human Genetics, University Hospital Essen, University of Duisburg, Essen, 45147 Essen, Germany. (12)Birmingham Women's and Children's National Health Service Foundation Trust, Mindelsohn Way, Birmingham B15 2TG, UK. (13)Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada; Department of Biology, Faculty of Science, Western University, London, ON N6A 5B7, Canada; Division of Genetics and Development, Children's Health Research Institute, London, ON N6C 2V5, Canada. Electronic address: [email protected]. (14)Centre Hospitalier Universitaire Sainte-Justine Research Center, University of Montreal, Montreal, QC H3T 1C5, Canada; Department of Pediatrics, University of Montreal, Montreal, QC H4A 3J1, Canada. Electronic address: [email protected]. Coffin-Siris Syndrome (CSS) is a rare neurodevelopmental disorder characterized by intellectual disability, coarse facial features, hypoplastic digits/nails, and hypertrichosis. The genes causative of CSS mainly encode the SWI/SNF complex, which contributes to chromatin remodeling and regulates the access of transcriptional factors to specific gene sites. While ARID1B mutations account for a third of all CSS cases, the condition's phenotypic features vary widely. We document the case of a girl with CSS who presented with a variant facial appearance, global developmental delay with speech impairment, agenesis of the corpus callosum, funnel chest, and bilateral renal stones without hypertrichosis or hypoplasia of the fifth fingernail. Genetic analysis revealed that the patient had a novel heterozygous frameshift mutation c.2201dupG (p.Ser736Ilefs*27) on the ARID1B gene.

What is Aortitis?

Aortitis is the inflammation of the aorta due to various causes, such as the manifestation of an underlying infectious or noninfectious disease process.

Aortitis is defined as an inflammatory process that involves one or more layers of the aortic wall (internal elastic lamina, tunica media, and adventitia) and can be caused by multiple mechanisms. Clinical features are usually nonspecific and a high index of suspicion is required for making the diagnosis. Although noninvasive imaging studies are being increasingly used in evaluating patients for diagnosis, angiography remains the gold standard for confirming aortic involvement. When tissue is available, pathologic examination can aid in clarifying the diagnosis. Aortitis, irrespective of the underlying cause, frequently displays similar clinical, pathologic, and arteriographic features and therefore understandably presents a therapeutic and diagnostic dilemma. Whatever the cause, early identification and aggressive treatment is required in order to avert the potentially life-threatening sequelae. The treatment of aortitis is determined by the underlying cause. If diagnosed early, infectious aortitis responds to appropriate antimicrobial therapy, whereas noninfectious, immune-mediated aortitis requires immunosuppressive therapy. However, in many instances, primarily because of the delay in diagnosis, surgical intervention is necessary to treat the associated anatomic and physiologic sequelae. Less definitive and more controversial is the role of inflammation in both primary and secondary accelerated atherosclerosis of the aorta and its modality of treatment. Aortitis is typically a chronic, progressive disease manifestation associated with large vessel vasculitidies, most notably giant cell, Takayasu arteritis, and a newly described entity, isolated aortitis. The aortitis may lead to aneurysm formation and symptoms associated with branch vessel occlusion in these diseases, but aortic dissection is rare and usually a late complication of smoldering, incompletely treated disease. We present a case of aortitis in a previously healthy 39-year-old man who succumbed to aortic dissection hours after the onset of symptoms. No aneurysm or fibrosis was found on postmortem examination. The inflammation was characterized by disruption of the media with patchy transmural chronic and focally acute inflammatory infiltrate. We review case reports of other individuals with aortitis, who initially or very early in their course presented with aortic dissection in the absence of known rheumatic disease and most without evidence of aneurysm formation. We believe that this represents a process characterized by an aggressive vasculitis of the aorta with its own clinical features, a fulmit variety of isolated aortitis. 1. Syphilitic aortitis is a productive inflammatory process, the earliest and most constant feature of which is a perivascular round cell infiltration in the adventitia. 2. The typical gross picture of luetic aortitis is often obscured by a superimposed, diffuse atherosclerosis. In the early cases the aorta appears fairly normal, presenting only the characteristic histological changes. 3. A pure aortic insufficient valve, with the exception of an infectious endocarditis, is always luetic. 4. Cardiac hypertrophy is not a complication of luetic aortitis. When present it is usually associated with a nephritis. 5. The demonstration of Spirochoeta pallida, even in advanced specimens of syphilitic aortas, is doubtful. 6. An antigen prepared from alcoholic extract of guinea pig heart with the original Wassermann technique should be preferred in diagnosing luetic aortitis. 7. Positive complement fixations in patients suffering with syphilis for a period of about fifteen years or longer suggest the probability, at least, of histological luetic changes in the aorta in 80 to 90 per cent of the cases. 60 per cent of these die from aortitis. 8. About 94 per cent of patients suffering with aortitis give positive Wassermann reactions. Aortitis is the all-encompassing pathological term ascribed to inflammation of the aorta. Regardless of the etiology, it frequently results in aortic root dilatation and aortic insufficiency rather than aortic stenosis. The rare case of aortitis such as isolated idiopathic aortitis may occur without evidence of systenic inflammatory disease or infection, and usually has subclinical nature. Even though the goals of therapy include immediate treatment of aortic inflammation or infection, the optimal management of isolated idiopathic aortitis is uncertain. We report a rare case of isolated idiopathic aortitis mimicking acute severe aortic stenosis, which was improved after steroid therapy. Aortitis is a general term that refers to a broad category of infectious or noninfectious conditions in which there is abnormal inflammation of the aortic wall. These inflammatory conditions have different clinical and morphologic features and variable prognoses. The clinical manifestations are usually vague and nonspecific and may include pain, fever, vascular insufficiency, and elevated levels of acute phase reactants, as well as other systemic manifestations. As a result, aortitis is often overlooked during the initial work-up of patients with constitutional symptoms and systemic disorders. A multimodality imaging approach is often required for assessment of both the aortic wall and aortic lumen, as well as for surveillance of disease activity and treatment planning. Noninvasive cross-sectional imaging modalities such as magnetic resoce (MR) imaging, MR angiography, and computed tomographic angiography play a critical role in initial evaluation and further assessment of aortitis. Radiologists should be familiar with the clinical features and imaging findings of the different types of aortitis. Aortitis is a general term denoting inflammation of the aortic wall. Various infectious and non-infectious diseases can be complicated by aortitis; in addition, isolated idiopathic aortitis has also been described. In a 12-year nationwide Danish population-based study, the prevalence of aortitis among 1,210 resected thoracic aorta samples was 6.1%, with nearly three-quarters of cases being idiopathic. Identified risk factors for aortitis included advanced age, a history of connective tissue disease, diabetes mellitus, and heart valve pathology. As in virtually all pathological studies, this study has a bias toward reporting the most severe cases of aortitis requiring surgical repair. "Aortitis" is a pathologic term that refers to an abnormal inflammation of the aortic wall. A wide spectrum of infectious, inflammatory, and idiopathic conditions may result in the development of aortitis. Infectious aortitis may be secondary to bacterial, tubercular, syphilitic, and viral pathogens. Although Takayasu arteritis and giant cell arteritis are the most common rheumatologic causes of aortitis, the other systemic diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Behçet disease, and Cogan syndrome, may also be associated with aortitis. In addition, aortitis may also occur without any systemic diseases or infectious causes (idiopathic). Clinical features of aortitis are nonspecific and may include fever, abdominal or chest pain, and vascular insufficiency. Patients may have elevated serum levels of acute phase reactants. A high index of clinical suspicion is always needed for the diagnosis of aortitis in a timely manner. Cross-sectional imaging techniques, such as computed tomographic angiography, magnetic resoce imaging, magnetic resoce angiography, and positron emission tomography, are extremely helpful in diagnosis, assessing disease activity, treatment planning, and post-treatment follow-up. Many of the patients with aortitis may require a multimodality imaging approach for appropriate diagnosis. Knowledge of the clinical features and cross-sectional imaging findings of different types of aortitis permit optimal patient management. BACKGROUND: Aortitis is a subtype of the more general term "vasculitis", an inflammatory condition of infectious or noninfectious origin involving the vessel wall. The term "vasculitis" refers to a broad spectrum of diseases with different aetiologies, pathophysiologies, clinical presentations and prognoses. The clinical manifestations are nonspecific, as are the laboratory findings such as pain, fever, weight loss, vascular insufficiency and elevated levels of acute phase reactants, as well as other systemic manifestations, and sometimes may mimic other entities. Thus, if not suspected as part of the initial differential diagnosis, aortitis can be overlooked during the workup of patients with constitutional symptoms and systemic disorders. METHODS: Imaging is rarely used for the primary diagnosis, but imaging findings, although nonspecific, can help in the assessment of these patients and is often required for making the final diagnosis. Imaging can be critical in the initiation of appropriate management and therapy. RESULTS: Noninvasive cross-sectional imaging modalities such as contrast-enhanced CT, magnetic resoce (MR) imaging, nuclear medicine and in particular positron emission tomography (PET) are the leading modalities in modern diagnostic imaging of aortitis for both the initial diagnosis and follow-up. CONCLUSION: This review focusses on the most common manifestations of aortitis with which radiologists should be familiar. TEACHING POINTS : • Aortitis is an inflammatory condition of infectious/noninfectious origin involving the vessel wall. • Imaging findings can help in the assessment of aortitis and are often crucial for the final diagnosis. • Contrast-enhanced CT, MRI and PET-CT are used for both the initial diagnosis and follow-up of aortitis. Aortitis is a pathological term designating inflammation of the aortic wall, regardless of its cause. The clinical presentation of aortitis is nonspecific and variable. Symptoms include abdominal pain, fever, and weight loss; acute phase reactants may also be elevated. Aortitis can be caused by a wide spectrum of entities, including from infectious processes to autoimmune diseases (Takayasu arteritis and giant cell arteritis are among the most common of these causing aortitis), and the prognosis and treatment of these entities vary widely. Various imaging techniques can be used to evaluate the lumen and wall of the aorta (such as multidetector computed tomography, magnetic resoce imaging, angiography, or PET-CT). This review focuses on the most common diseases that cause aortitis and on the clinical and radiological findings that are most useful for diagnosing and treating this condition appropriately. BACKGROUND Inflammation of the aortic wall, known as aortitis, is a rare clinical entity which is frequently asymptomatic, or identified when the patient presents with an aortic aneurysm or dissection. It is most often caused by infection or autoimmune vasculitides such as giant cell or Takayasu's arteritis. CASE REPORT The case presented is that of a 55-year-old man with symptomatic occlusion of the right coronary artery caused by a plasmacytic aortitis suggestive of IgG4 disease, which was successfully treated with coronary artery bypass grafting and an ascending aortic graft. CONCLUSIONS A review of the current literature emphasizes how poorly the etiology and natural history of plasmacytic aortitis is understood. IgG4 related thoracic aortitis is a recent addition to the differential diagnosis for inflammatory aortic disease - a condition which is often underappreciated until complications arise such as aneurysmal formation or aortic dissection. Currently, IgG4 aortitis remains a post-surgical diagnosis reliant on positive immunohistochemistry findings. Management is guided by the extent of disease involvement, which can be gauged by serum IgG4 levels and radiological findings. Options include surgical resection, corticosteroid therapy and steroid-sparing agents to prevent relapses. INTRODUCTION: Takayasu aortitis is a well known yet rare form of large vessel vasculitis. Also known as pulseless disease, occlusive thromboaortopathy, and Martorell syndrome, is a chronic inflammatory aortitis. Vessel inflammation leads to wall thickening, fibrosis, stenosis, and thrombus formation. METHODS: A 64-year-old woman was referred to emergency for lack of pulse in the upper and lower limbs and changes in heart rate. AngioRMN revealed dissection of the ascending aorta while in PET, intense uptake of FDG-F18 involving ascending, crossa, descending thoracic and abdominal segments of the aorta, was evident urgent surgical correction occurred. An aorta ring segment with 2.5cm length, showed whitish and smooth intimate, with linear transversal laceration, with regular borders. Dissection 1cm long of the medial tunica was occupied by a clot in continuity with a thrombus occupying the neoformed lumen. RESULTS: Microscopy examination confirmed hyalinization of the tunica media with impregnation of fibrin / thrombus with blood cell elements. Endothelial inflammatory characteristics together with vasa vasorum and vascular trajectory of the periphery of the tunica media with inflammatory cells involvement allowed the diagnosis of Takayasu aortitis. CONCLUSION: Takayasu aortitis is rare in the presented age group, with early non-specific symptoms. The diagnosis of aortic dissection was crucial, constituting a medical emergency. Heather L-Gomik (2008) supports the hyaline structural alteration of the tunica media. The disease has been recognized for more than 100 years, and patients with Takayasu aortitis remain relatively poor and treatment is suboptimal. Key areas for improvement include the need for increase disease awareness and earlier diagnosis, and improved means for monitoring disease activity. The demonstration of diferential expression of Toll-like receptors in arteries, is particularly intriguing and worthy of further investigation. Aortitis can be the manifestation of an underlying infectious or noninfectious disease process. An autoimmune cause is suggested in a large proportion of noninfectious causes. Similar to other autoimmune diseases, the pathophysiology of aortitis has been investigated in detail, but the etiology remains unknown. Most cases of aortitis often go undetected for a long time and are often identified at late stages of the disease. Recent advances in imaging techniques have significantly improved the diagnosis of aortitis. However, significant challenges associated with the imaging techniques limit their use. Several routine inflammation-based markers, such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and inflammatory cytokines, are nonspecific and, therefore, have limited use in the diagnosis of aortitis. The search for more specific serum biomarkers, which can facilitate detection and progression is under progress. Several autoantibodies have been identified, but assigning their role in the pathogenesis as well as their specificity remains a challenge. The current review addresses some of these issues in detail. KEY MESSAGES: • Noninfectious aortitis is an autoimmune disease. • Several biomarkers, including cytokines and autoantibodies, are increased in aortitis. • Imaging techniques, commonly used to detect aortitis, are associated with the high cost and technical challenges. • There is a need to develop low-cost biomarker-based detection tools. • The knowledge of biomarkers in aortitis detection is discussed. Aortitis includes conditions with infectious or non-infectious etiology, characterized by inflammatory changes in one or more layers in aortic wall. Age at onset, geographic predilections, distribution and pattern of involvement in aorta, its branches and pulmonary arteries, and systemic associations provide a clue to etiology. Clinical presentations are often non-specific. An integrated approach including clinical, laboratory and imaging assessment is essential to confirm diagnosis and plan treatment. Assessment of disease activity is the key as it influences timing and outcome of treatment. Markers of activity include clinical, laboratory and imaging. Medical management remains the first-line therapy. Revascularization is indicated in the presence of hemodynamically significant stenosis and inactive disease. In the presence of flash pulmonary edema, left ventricular dysfunction or hypertensive encephalopathy, revascularization is performed irrespective of disease activity. Endovascular management is favored over surgery due to its high success and low restenosis rates. Symptomatic aneurysmal disease is usually managed by surgery. Aortitis is a pathological term that refers to the inflammation of one or more layers of the aortic wall. It is associated with a wide spectrum of inflammatory diseases of infectious and non-infectious origins, and often present with vague clinical findings and non-specific laboratory results that can model other entities. As a result, aortitis may not form part of the initial workup and appropriate treatment can be delayed or missed. Therefore, imaging modalities are required to assess for inflammation and structural changes in the aorta to support or exclude the diagnosis of aortitis. This review presents current literature on the imaging modalities utilized in the diagnosis and management of aortitis, which surgeons and physicians should be familiar with for providing optimal patient care. PURPOSE OF REVIEW: Aortitis is the inflammation of the aorta due to various causes. Clinical presentations vary as well as the imaging findings. Exact pathogenetic mechanisms or triggering factors, as well as the best diagnostic and monitoring modalities and treatment strategies, are yet to be elucidated. We reviewed recent studies in aortitis and associated diseases. RECENT FINDINGS: Multiple cohort studies reporting long-term outcomes in patients with noninfectious aortitis were recently published. Comparative features of isolated aortitis were described. Six angiographic clusters for giant cell arteritis and Takayasu have been identified. New classification criteria have been proposed for IgG4-related disease by a data-driven method. The ultrasonographic slope sign and a halo score were described as specific imaging parameters in giant cell arteritis. The promising role of PET-computed tomography, not only in the diagnosis of aortitis but also in monitoring disease activity, has been noted. Results of in-vitro studies on Janus kinase (JAK)/signal transducers and activators of transcription and mammalian target of rapamycin (mTOR) pathways, comparative studies with leflunomide as an induction therapy, and a long-term follow-up study with tocilizumab may contribute to the management of Takayasu arteritis. SUMMARY: An impressive number of studies have addressed aortitis in recent years. However, there still is a lack of robust data on causes, monitoring disease activity by imaging and biomarkers, and drugs providing steroid-free remission in noninfectious aortitis.

Please list the syndromes that are part of Castleman's disease AKA TAFRO

The syndromes that are part of Castleman's disease AKA TAFRO are:1) organomegaly, 2) anasarca, 3) myelofibrosis, 4) thrombocytopenia and 5) reticulin fibrosis.

Recently, a unique clinicopathologic variant of multicentric Castleman's disease (MCD) has been identified in Japan. This disease is characterized by a constellation of symptoms, as listed in the title, and multiple lymphadenopathy of mild degree with a pathologic diagnosis of atypical CD, often posing diagnostic and therapeutic problems for pathologists and hematologists, respectively. These findings suggest that this disease represents a novel clinical entity belonging to systemic inflammatory disorders with a background of immunological abnormality beyond the ordinal spectrum of MCD. To define this disorder more clearly, Japanese participants presented clinicopathologic data at the Fukushima and Nagoya meetings. Many of the patients presented by the participants were significantly accompanied by a combination of thrombocytopenia, ascites (anasarca), pleural effusions, microcytic anemia, fever, myelofibrosis, renal dysfunction, and organomegaly (TAFRO). Multiple lymphadenopathies were generally of mild degree, less than 1.5 cm in diameter, and consistently featured the histopathology of mixed- or less hyaline vascular-type CD. Autoantibodies were often detected. However, this disease did not fulfill the diagnostic criteria for well-known autoimmune diseases including systemic lupus erythematosus. Castleman-Kojima disease and TAFRO syndrome (the favored clinical term) were proposed for this disease. The patients were sensitive to steroid and anti-interleukin-6 receptor antibody (tocilizumab), but some exhibited a deteriorated clinical course despite the treatment. The participants proposed a future nationwide survey and a Japanese consortium to facilitate further clinical and therapeutic studies of this novel disease. [J Clin Exp Hematop 53(1): 57-61, 2013]. Multicentric Castleman's disease (MCD) is a polyclonal lymphoproliferative disorder that manifests as marked hyper-γ-globulinemia, severe inflammation, anemia, and thrombocytosis. Recently, Takai et al. reported a new disease concept, TAFRO syndrome, named from thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly. Furthermore, Kojima et al. reported Japanese MCD cases with effusion and thrombocytopenia (Castleman-Kojima disease). Here, we report two cases of MCD associated with marked pleural effusion, ascites, and thrombocytopenia, and discuss the independence of the TAFRO syndrome (Castleman-Kojima disease). Case 1: A 57-year-old woman had fever, anemia, anasarca, and some small cervical lymphadenopathy. Although she had been administered steroid therapy, and full-coverage antibiotics, her general condition, including fever, systemic inflammation, and anasarca, deteriorated steadily. We administered chemotherapy [CHOEP (cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisolone) regimen], but despite a transient improvement, she died due to septic shock. Case 2: A 73-year-old man with a history of aplastic anemia and remission presented with fever, severe inflammation, and anasarca. Prednisolone was administered (15 mg daily), and his hyperinflammation once improved. Nevertheless, his general condition, including pleural effusion and ascites, worsened, and C-reactive protein and interleukin-6 levels showed marked increases. The patient died due to multiorgan failure. Cases of TAFRO syndrome (Castleman-Kojima disease) are still rare. Therefore, it is necessary to conduct multicenter clinical surveys including similar cases, such as ours, to reach a consensus regarding diagnostic criteria, therapeutic strategy, and pathophysiological etiology for this syndrome. Multicentric Castleman's disease is a systemic inflammatory disorder characterized by lymphadenopathy and excessive interleukin-6 production. A unique clinicopathologic variant of multicentric Castleman's disease, TAFRO (i.e., thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly) syndrome, was recently proposed in Japan. Despite the successful use of anti-interleukin-6 therapy in some patients with TAFRO syndrome, not all patients achieve remission. The pathophysiological etiology of and suitable therapeutic strategies for this variant have not been established. Here, we present our experience of a unique case of TAFRO syndrome in a 78-year-old woman whose symptoms responded differently to several therapies. Tocilizumab, an anti-interleukin-6 receptor antibody, successfully induced remission of fever and lymphadenopathy. However, severe thrombocytopenia persisted and she developed anasarca, ascites, and pleural effusion shortly thereafter. Rituximab, an anti-CD20 antibody, and glucocorticoid therapy provided no symptom relief. In contrast, cyclosporine A, an immunosuppressive agent that blocks T cell function by inhibiting interleukin-2, yielded immediate improvements in systemic fluid retention and a gradual increase in platelet count, with complete resolution of disease symptoms. Excessive serum interleukin-2, when used as an anti-cancer agent, has been reported to cause side effects such as fluid retention, thrombocytopenia, and renal failure. Our case was unique because the anti-interleukin-2 therapy successfully improved symptoms that were not relieved with anti-interleukin-6 therapy. The present report therefore provides insight into the possible role of interleukin-2, in addition to interleukin-6, in TAFRO syndrome. This report will certainly help to clarify the pathogenesis of and optimal treatment strategies for TAFRO syndrome. Thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome is a unique clinicopathologic variant of multicentric Castleman's disease that has recently been identified in Japan. Previous reports have shown that affected patients typically respond to immunosuppressive therapy, such as prednisolone and tocilizumab. However, the optimal treatment for refractory TAFRO syndrome, which can be fatal, remains unclear. We herein report a case of tocilizumab-resistant TAFRO syndrome successfully treated with cyclosporin A, indicating that cyclosporine A may be an alternative therapy for refractory TAFRO syndrome. Author information: (1)Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and, Pharmaceutical Sciences, Okayama, Japan. (2)Department of Cellular Transplantation Biology (Hematology/Oncology and Respiratory Medicine), Division of Cancer Medicine, Graduate School of Medical Sciences Kanazawa University, Kanazawa, Japan. (3)Department of Medicine, Division of Hematology & Oncology, Raymond & Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (4)Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and, Pharmaceutical Sciences, Okayama, Japan. (5)Division of Rheumatology, Kanazawa University Hospital, Kanazawa, Japan. (6)Department of Hematology, Chugoku Central Hospital, Fukuyama, Japan. (7)Department of Hematologic Oncology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Japan. (8)Division of Clinical Infection Control and Prevention, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. (9)Division of Hematology, Niigata City General Hospital, Niigata, Japan. (10)Division of Hematology and Oncology, Department of Medicine, Kameda Medical Center, Kamogawa, Japan. (11)Department of Hematology, National Hospital Organization Hokkaido Cancer Center, Sapporo, Japan. (12)Department of Hematology, Eiju General Hospital, Tokyo, Japan. (13)Department of Hematology, National Hospital Organization Nishigunma National Hospital, Shibukawa, Japan. (14)Department of Hematology and Medical Oncology, Fujita Health University School of Medicine, Toyoake, Japan. (15)Division of Pathophysiology, Okayama University Graduate School of Health Sciences, Okayama, Japan. (16)Department of Pathology, Tokai University School of Medicine, Kanagawa, Japan. (17)Cellular Transplantation Biology, Division of Medicine, Kanazawa University Institutes of Medical, Pharmaceutical, and Health Sciences, Kanazawa, Japan. Thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome is considered as a unique clinicopathologic variant of multicentric Castleman's disease and is recently reported in Japan. This entity represents a severe inflammatory state leading to organ failures such as severe liver dysfunction seen in our case, and can be treated by immunosuppressive agents, steroids, and cyclosporine shown in several case reports. A systematic review and our case suggest the potential utility of tocilizumab as a treatment for TAFRO syndrome. Multicentric Castleman's disease is a polyclonal lymphoproliferative disorder. Recently, a new variant of the disease was reported and named TAFRO syndrome, an acronym for thrombocytopenia, ascites, myelofibrosis, renal dysfunction, and organomegaly. A 55-year-old woman presented to our hospital with dyspnea on exertion and high fever. Laboratory tests revealed anemia, thrombocytopenia, and proteinuria. Computed tomography (CT) revealed a large anterior mediastinal mass, mild splenomegaly, bilateral pleural effusion, pericardial effusion, and mild systemic lymphadenopathy. A CT-guided biopsy was unable to establish a definitive diagnosis, so we resected the mediastinal mass for diagnostic and therapeutic purposes. Pathological findings were consistent with the hyaline vascular type of Castleman's disease (CD), and she was diagnosed with TAFRO syndrome. There has been no description of a patient with TAFRO syndrome with a large mass, and this is the first case of TAFRO syndrome treated with debulking surgery. Thrombocytopenia, anasarca, myelofibrosis, renal dysfunction and organomegaly (TAFRO) syndrome is a variant of Castleman's disease recently identified in Japan. A 73-year-old man was diagnosed with TAFRO syndrome according to clinical findings, and his symptoms improved after corticosteroid therapy. Ten months later, lymphadenopathy worsened during tapering of corticosteroids. Histological findings of abdominal lymph nodes showed diffuse large B-cell lymphoma. After 6 cycles of R-CHOP therapy, he has remained in sustained complete remission. This is a rare case of the development of maligt lymphoma during the treatment of TAFRO syndrome, which suggests an association between diffuse large B-cell lymphoma and TAFRO syndrome. Castleman's disease is a rare disorder, yet a rarer newly described syndrome called TAFRO syndrome was discovered to accompany it. TAFRO represents the constellation of symptoms (Thrombocytopenia, Anasarca, MyeloFibrosis, Renal failure, Organomegaly). Most cases were described in Japan. We present the first case of TAFRO syndrome in Syria. A 58-year-old Caucasian male with no relevant history presented with fatigue, oliguria, decreased platelets, increased creatinine level, hepatosplenomegaly, ascites, pitting edema and lymph node enlargement. Possible differential diagnoses were excluded by laboratory, radiologic and cytologic tests including TB, maligcy and autoimmune diseases. A biopsy of a supraclavicular lymph node confirmed Castleman disease. Our patient had Catleman's disease, and presented with only four diagnostic criteria for TAFRO syndrome (Myelofibrosis was absent) in addition to other minor characteristics (microcytic anemia, negative HIV and HHV-8 infections.) which make the presentation consistent with TAFRO syndrome described in the Japanese cases. The criteria for diagnosing TAFRO syndrome are still changing, and the pathophysiology behind it is unclear. We recommend further research to understand this syndrome taking into account that its prevalence might be worldwide. TAFRO syndrome is a newly recognized variant of idiopathic multicentric Castleman disease (iMCD) that involves a constellation of syndromes: thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Thrombocytopenia and severe anasarca accompanied by relatively low serum immunoglobulin levels are characteristic clinical findings of TAFRO syndrome that are not present in iMCD-not otherwise specified (iMCD-NOS). Lymph node biopsy is recommended to exclude other diseases and to diagnose TAFRO syndrome, which reveals characteristic histopathological findings similar to hyaline vascular-type CD. TAFRO syndrome follows a more aggressive course, compared with iMCD-NOS, and there is no standard treatment. BACKGROUND: Although thrombocytopenia, anasarca, fever, reticulin fibrosis and organomegaly (TAFRO) syndrome was first described as a variant of idiopathic multicentric Castleman disease (CD), patients with TAFRO syndrome demonstrate more aggressive clinical features. Because these patients may present with fever of unknown origin, general physicians need to recognise its characteristic laboratory data and clinical features during hospitalisation. AIMS: to describe the features, symptoms and characteristics of TAFRO syndrome and to compare them to those of idiopathic CD. METHODS: This was a retrospective study of patients with histopathologically confirmed TAFRO syndrome and idiopathic multicentric CD who were diagnosed and managed between April 2012 and June 2018 in a Japanese university hospital's General Medicine Department. RESULTS: We found that the hospitalisations were significantly longer among patients with TAFRO syndrome compared to those with idiopathic CD (median: 87 days; range: 34-236 days vs median: 30 days; range: 13-59 days; P < 0.01). Patients with TAFRO syndrome were more likely to present with fever, abdominal pain and elevated inflammatory markers and be misdiagnosed with an infectious disease during the first hospital visit. Approximately 40% of patients with TAFRO syndrome had no radiographically enlarged lymph nodes. CONCLUSIONS: TAFRO syndrome may present as an infectious disease with an aggressive clinical course. Our study highlights the importance of giving significance to chief complaints and laboratory data. Physicians need to recognise the clinical and laboratory features of this disease to avoid missing this potentially fatal disorder. Castleman disease (CD) is a rare lymphoproliferative disorder that can be unicentric or multicentric. Multicentric CD (MCD) is further subdivided into human herpesvirus type-8-associated, POEMS syndrome-associated, and idiopathic (iMCD). TAFRO syndrome is a newly identified disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. The TAFRO syndrome is sometimes regarded as a subtype of iMCD (TAFRO-iMCD), whereas iMCD without TAFRO syndrome is considered "not otherwise specified" (iMCD-NOS). However, a proportion of patients with TAFRO syndrome have been diagnosed without lymph node biopsies (TAFRO syndrome without proven iMCD; TAFRO-w/op-iMCD). To clarify the clinical features of iMCD-NOS, TAFRO-iMCD, and TAFRO-w/op-iMCD, we retrospectively analyzed 220 patients extracted from the database of the Multicenter Collaborative Retrospective Study for Establishing the Concept of TAFRO Syndrome. The patients included 87 with iMCD-NOS, 63 with TAFRO-iMCD, and 19 with TAFRO-w/op-iMCD. Patients in all three groups exhibited anemia, hypoalbuminemia, and elevated serum C-reactive protein and interleukin-6 levels. No significant differences in clinical, laboratory, and prognostic features were noted between the TAFRO-iMCD, and TAFRO-w/op-iMCD groups. However, the iMCD-NOS group exhibited polyclonal hyper-γ-globulinemia. The five-year survival rates of patients in the iMCD-NOS and TAFRO-involved groups were 100% and 66.5%, respectively (dropping markedly during the first few months in the latter). The iMCD-NOS and the TAFRO-iMCD samples typically showed plasma cell and mixed-type histologies, respectively. Thus, iMCD can be classified into two distinct subtypes, iMCD-NOS and TAFRO-iMCD. As such, TAFRO-iMCD and TAFRO-w/op-iMCD may be considered the same entity, requiring prompt diagnosis and intensive care. Castleman-Kojima disease, also known as idiopathic multicentric Castleman disease with TAFRO syndrome (iMCD-TAFRO), is a recently recognized systemic inflammatory disorder with a characteristic series of clinical symptoms, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Patients with iMCD-TAFRO often develop severe abdominal pain, elevated alkaline phosphatase levels, and systemic inflammation, but the etiological factors are unknown. To investigate the potential role of bacterial infection in the pathogenesis of iMCD-TAFRO, we performed polymerase chain reaction (PCR) for the bacterial 16S rRNA gene with DNA extracted from liver specimens of three patients with iMCD-TAFRO, four patients with amyotrophic lateral sclerosis, and seven patients with inflammatory conditions. Sequencing of the PCR product showed 99% DNA sequence identity with Campylobacter jejuni in all three patients with iMCD-TAFRO and in two patients with inflammatory conditions. Immunohistochemical and electron microscopy analyses could not identify C. jejuni in patients with iMCD-TAFRO. The findings indicated that C. jejuni infection is not the pathological cause of iMCD-TAFRO; however, this ubiquitous bacterium may play a role in uncontrolled systemic hypercytokinemia, possibly through the development of cross-reactive autoantibodies. INTRODUCTION: Thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome is a severe subtype of idiopathic multicentric Castleman's disease, characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, and organomegaly. Renal complication of this disease can be life-threatening and sometimes requires hemodialysis, but it has not been elucidated in detail. METHODS: Case-series was designed to evaluate the renal histology of patients with TAFRO syndrome treated at our hospital. RESULTS: Seven patients were eligible to the criteria. All of them had severe diuretic-resistant anasarca and 6 of 7 had mild proteinuria (<1 g daily). On light microscopy, all patients showed glomerular endotheliopathy characterized by endothelial cell swelling and a double contour of the glomerular basement membrane with mesangiolysis or mesangial loosening. Immunofluorescent staining and electron microscopy did not detect immune deposits in any patient. Electron microscopy revealed endothelial cell swelling with diffuse expansion of the subendothelial space, loss of mesangial architecture, and loss of endothelial cell fenestrations. Treatment with glucocorticoids and molecular-targeting agents, including tocilizumab and rituximab, improved renal dysfunction and anasarca. In 4 of 7 patients with persistent thrombocytopenia, hemorrhagic events occurred despite platelet transfusion or thrombopoietin receptor antagonist therapy. CONCLUSION: Severe diuretic-resistant anasarca with mild proteinuria and severe glomerular endotheliopathy were common characteristics of renal dysfunction due to TAFRO syndrome. In addition, endothelial changes mediated via interleukin (IL)-6 and vascular endothelial growth factor (VEGF) that lead to vascular hyperpermeability and water leakage might contribute to anasarca, because molecular-targeting therapy directed against IL-6 or VEGF improved renal dysfunction and severe endothelial damage.

What is known about the protein Curli?

A major component of bacterial biofilms is curli amyloid fibrils secreted by the curli biogenesis system. Curli is a bacterial α-synuclein (αSyn) which is deposited first in the enteric nervous system and amyloid deposits are propagated in a prion like manner to the central nervous system. curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities.

A major component of bacterial biofilms is curli amyloid fibrils secreted by the curli biogenesis system. Understanding the curli biogenesis mechanism is critical for developing therapeutic agents for biofilm-related infections. Here we report a systematic study of the curli biogenesis system, highlighted by structural, biochemical and functional analysis of the secretion channel complexes (CsgF-CsgG) with and without the curli substrate. The dual-pore architecture of the CsgF-CsgG complex was observed and used to develop an approach to inhibit the curli secretion by physically reducing the size of the CsgF pore. We further elucidated the assembly of the CsgFG complex with curli components (CsgA and CsgB) and curli-cell association through CsgF. Importantly, the recognition of the CsgA substrate by CsgG was uncovered. Nine crevices outside of the CsgG channel provide specific and highly-conserved recognition sites for CsgA N-terminus. Together with analysis of CsgE, our study provides comprehensive insights into curli biogenesis. Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate αSyn aggregation and disease in the gut and the brain.

List the core lung matrisome proteins.

LGALS7, ASPN, HSP90AA1, HSP90AB1, COL1A1, SCGB1A1, TAGLN, PSEN2, TSPAN1, CTSB, AGR2, CSPG2, SERPINB3, fibronectin, emilin-1, versican, decorin

Can Freund's complete adjuvant induce arthritis?

Yes, Rheumatoid arthritis (RA) was induced by Freund's Complete Adjuvant (FCA; 1 mg/0.1 ml paraffin oil), injected subcutaneously on days 0, 30 and 40

Rheumatoid arthritis (RA) is a chronic and accelerated autoimmune illness with proliferative and damaging synovitis, resulting in joint death and cartilage and bone erosion. This study focused on the potential therapeutic effect of wogonin on complete Freund's adjuvant (CFA) induced RA in rats and the underlying mechanisms. Arthritis was experimentally caused in rats by subcutaneously injecting 0.1 mL of CFA into the subplantar area of the left hind paw under moderate anesthesia on day zero. The regular oral doses of indomethacin/wogonin began on day zero and proceeded after injection to day 35. Wogonin reduced arthritic score considerably, enhanced body weight, and reduced paw thickness. Wogonin also boosted red blood cell considerably along with hemoglobin and reduced white blood cell count and erythrocyte sedimentation rate. Wogonin substantially improved an altered level of oxidative stress markers, antioxidant proteins, and inflammatory cytokines in a dose-dependent way. Wogonin inhibited p38 phosphorylation triggered by CFA and p65 nuclear translocation. BACKGROUND: In the present study, we aimed to understand the expression and methylation levels of the suppressor of cytokine signaling 3 (SOCS3) in rheumatoid arthritis (RA) synovial fibroblasts. METHOD: The RA model was established using Freund's complete adjuvant, and then the synovial fibroblasts were isolated and cultured. Next, RNA extraction and reverse transcription were performed. The SOCS3 transcription level was detected using qPCR, and SOCS3 protein expression was detected using western blotting (WB). Lastly, methylation-specific PCR (MSP) was performed. RESULTS: The RA model was successfully demonstrated. SOCS3 gene (p < .01) and protein expression levels were significantly increased in the RA rat group compared to in the wild type (WT) group. However, no significant difference was observed in the MSP products between the RA and WT groups. CONCLUSION: The increased expression of the SOCS3 can be correlated with the development of RA. BACKGROUND: Rheumatoid arthritis (RA) is associated with joint damage. Effectiveness of embelin has been established in a wide variety of inflammatory disorders, but its utility as a therapeutic agent is limited by its poor absorption, rapid metabolism, and fast systemic elimination. To apprehend these limitations, we propose to use highly bioavailable embelin-loaded chitosan oparticles (CS-embelin NPs) for the treatment of RA. METHODS: The rats were made arthritic using a subcutaneous injection with 0.1 ml complete Freund's adjuvant (CFA) into the footpad of the left hind paw. CS-embelin NPs (25 and 50 mg/kg) was administered from day 15 to day 28 after adjuvant injection. After the experimental period, the animals were sacrificed and various biochemical markers were assessed. RESULTS: Arthritic score and paw swelling were significantly reduced after treatment with CS-embelin NPs. Arthritis-induced rats showed a significant increase in malondialdehyde (MDA) and nitric oxide (NO) with a concomitant reduction of antioxidants in the paw tissue. CS-embelin NPs (25 and 50 mg/kg) reduced MDA and NO levels and restored antioxidant levels to normalcy by mitigating oxidative stress. The arthritic rats exhibited elevated tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1β) serum concentrations, upregulated TNF- α and IL-6 protein levels and upregulated nuclear factor-kB (NF-kB) mRNA expression in paw tissues. Treatment with CS-embelin NPs (25 and 50 mg/kg) significantly reduced serum levels and down-regulated inflammatory markers to normalcy, dose-dependently. CONCLUSION: The results suggest that CS-embelin NPs displayed a protective effect against adjuvant-induced arthritis in rats mediated through antioxidant and anti-inflammatory effects. BACKGROUND: There are conflicting data regarding angiotensin receptor blockers (ARBs) induced anemia and its beneficial anti-inflammatory effect in rheumatoid arthritis. The aim of the present study was to investigate the effect of telmisartan administration either alone or in combination with etanercept on anemia of chronic inflammatory diseases in a model of rheumatoid arthritis in rats. METHODS: Rheumatoid arthritis (RA) was induced by Freund's Complete Adjuvant (FCA; 1 mg/0.1 ml paraffin oil), injected subcutaneously on days 0, 30 and 40. Rats with RA received dimethyl sulfoxide (DMSO), etanercept (0.3 mg/kg 3 times/week; sc), telmisartan (1.5 mg/kg/day; orally) or combination of etanercept and telmisartan. Arthritis parameters (footpad circumference change and paw volume change), erythrocyte indices (hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin level changes), iron profile (serum iron and serum ferritin), serum levels of erythropoietin (EPO), hepcidin, tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 were evaluated, along with measuring serum urea and creatinine levels. RESULTS: All treated groups showed improvement of the measured parameters in comparison to RA-control subgroup. Telmisartan either alone or in combination with etanercept significantly improved arthritis and erythrocyte indices. Telmisartan showed significant increase in EPO and decrease in hepcidin compared to etanercept. Combination group showed significant improvement in serum iron, ferritin, EPO, hepcidin, TNF-α, IL-6, urea and creatinine, compared to etanercept. Telmisartan either alone or in combination, but not etanercept alone, significantly decreased creatinine level. CONCLUSION: Telmisartan improved anemia and arthritis parameters and showed anti-inflammatory and reno-protective effects, in a rat model of rheumatoid arthritis.

Can saponins be used as adjuvant?

Yes, saponin is an ideal adjuvant candidate.

The purified active fraction of Albizia julibrissin saponin (AJSAF) is an ideal adjuvant candidate that improves antigen-specific both cellular and humoral immune responses and elicits mixed Th1/Th2 responses, but its mechanisms remain unclear. The key features of action of AJSAF were investigated in mice immunized with Newcastle disease virus-based recombit influenza vaccine (rL-H5) and AJSAF at the same leg (AJSAF+rL-H5) or different legs (AJSAF/rL-H5). The adjuvant activity of AJSAF on rL-H5 is strictly dependent on their spatial colocalization. Serum H5 antigen (H5Ag)-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in AJSAF+rL-H5 group were significantly higher than those in AJSAF/rL-H5 group. The mechanisms of selectivity of Th1 or Th2 in mice induced by AJSAF was explored by the transcriptomic and proteomic profiles of H5Ag-stimulated splenocytes from the immunized mice using gene microarray and two-dimensional difference gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Compared to rL-H5 alone, AJSAF/rL-H5 induced more differentially expressed genes (DEGs) than AJSAF+rL-H5, whereas AJSAF+rL-H5 upregulated higher mRNA expression of Th1 (T-bet, IFN-γ, TNF-α, IL-12β, and IL-12Rβ1) and Th2 (IL-10 and AICDA) immune response genes. The neutrophil response and its derived S100A8 and S100A9 might be involved in the AJSAF-mediated Th1 response. Meanwhile, AJSAF might induce the adaptive immune responses by improving a local innate immune microenvironment. These findings expanded the current knowledge on the mechanisms of action of saponin-based adjuvants, and provided new insights into how adjuvants shape adaptive immune responses. Author information: (1)Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. (2)Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas/NUPEB, Departamento de Ciências Biológicas, Insituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil. (3)Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma 88806-000, Santa Catarina, Brazil. (4)Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, Minas Gerais, Brazil. (5)Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. (6)Fundação Hospitalar do Estado de Minas Gerais, Hospital Eduardo de Menezes, Belo Horizonte 30622-020, Minas Gerais, Brazil. (7)Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, Minas Gerais, Brazil. (8)Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Neuropsychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA. (9)Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, Minas Gerais, Brazil. Electronic address: [email protected].

Is erabutoxin b usually found in plants?

Erabutoxin b is a short-chain neurotoxic peptide purified from the venom of the sea snake Laticauda semifasciata.

The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map. Erabutoxin b is one of a family of snake venom neurotoxins, all low-molecular-weight proteins, which block neuromuscular transmission at the postsynaptic membrane. They specifically inhibit the acetylcholine receptor. The molecular shape is that of a shallow elongated saucer with a footed stand formed by the six-membered ring at the COOH-terminal end. The central core of the molecule is an assembly of four disulfide bridges. Three long chain loops emerge as broad fronds from the core region. Approximately 40% of the main chain is organized into a twisted antiparallel beta-pleated sheet of five short strands. In 28 snake venom neurotoxins of established sequence which inhibit the acetylcholine receptor, the four disulfide bridges and seven other residues remain invariant. Three substitution positions conserve residue type. In one wing of the molecule, there is a broad shallow depression which may characterize the reactive site. It is populated by the sevent invariant residues and two of the three type conserved residues. This region is "anchored" on the undersurface of the molecule by the hydroxyl group of Ser-9, the remaining conservatively substituted residue. alpha-neurotoxins from elapid snake venoms and alpha-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an alpha-neurotoxin from the false sea snake Laticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller alpha-conotoxins (from the marine snails Conus magus and Conus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake alpha-neurotoxins and their homologues. Six proteins unrelated to erabutoxin b were compared to the alpha-conotoxins to show that the extent of the erabutoxin b/alpha-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely alpha-cobratoxin from Naja naja siamensis and cytotoxin VII4 from Naja mossambica mossambica, were also analyzed. The extent of the matching with the alpha-conotoxins decreased in the series erabutoxin b greater than alpha-cobratoxin greater than cytotoxin VII4, and this also relates the order of similarity to the pharmacological properties of the alpha-conotoxins. The alpha-conotoxin-like area of the snake alpha-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake alpha-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity. The crystal structure of the protein postsynaptic neurotoxin, erabutoxin b, has been refined at 0.140-nm resolution (R = 0.22) by restrained least-squares and interactive computer graphics. The study has established complete structural identity of the two sea-snake venom toxins, erabutoxin b and neurotoxin b, isolated from Laticauda semifasciata snakes taken in different Pacific Ocean waters. Two chemical-sequence inversion errors in erabutoxin b have been discovered during refinement, corrected and subsequently confirmed in both erabutoxin b and erabutoxin a by chemical analysis. The correct sequences are His6-Gln7, hitherto unsuspected, and Ser18-Pro19. The sequence correction His6-Gln7 explains the anomalous results of 1H NMR solution studies and those of early chemical modification experiments, which were in conflict with the previously published three-dimensional structure of erabutoxin b. On refinement, the five-stranded beta sheet described earlier is now shown to be discontinuous, split into a two-stranded beta loop and a three-stranded beta sheet. Unique features of the Pro44-Gly49 peripheral segment have now been identified. 51 water molecule positions have been located. Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23-9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13mug/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b. 1. The toxic principles in the venom of the sea-snake Laticauda semifasciata were separated into two components by CM-cellulose chromatography and obtained in crystalline forms. They were named ;erabutoxins a and b'. 2. The homogeneity of each toxin was shown by rechromatography, by disk electrophoresis, by ultracentrifuging, by toxicity measurements before and after repeated crystallizations and by N-terminal analysis. 3. They had molecular weights of about 7000. Both of them contained 61 (or 62) amino acid residues/molecule. The only difference between erabutoxins a and b was that one of the aspartic acid (or asparagine) residues in erabutoxin a was replaced by a histidine residue in erabutoxin b. 4. Both of the toxins had LD(50) values of 0.15mug./g. body wt. for mice and 0.07mug./g. for rats. It was shown with frog-muscle preparations that they acted on postsynaptic membrane to block neuromuscular transmission. The fluorescence increase on the deuterium oxide addition to the solvent medium was studied in various tryptophan- (or indole-) and/or tyrosine-containing model compounds. It was shown how the rates of the deuteration at the indole NH group of tryptophan and at the OH group of tyrosine could be followed independently of each other. The method was applied to a study of erabutoxin b molecule, a neurotoxic protein from a sea snake, to analyze the microenvironments of its single tryptophan and tyrosine residues. It was shown that the "functionally invariant" single tryptophan residue was exposed to the solvent in the surface of the molecule and that the "structurally conserved" single tyrosine residue was buried in the molecule. The rate of deuteration of the tyrosine residue (80 s(-1) at pH 6.3 and 33 degrees C) was 1/20 of that of an exposed tyrosine. It was also found that the amino group of Lys-27 quenched the fluorescence of Trp-29 but its deuteration had no effect on the fluorescence. Structural features associated with the ability of a monoclonal antibody (mAb) to discriminate between protein variants are identified and engineered. The variants are the curaremimetic toxin alpha from Naja nigricollis and erabutoxin a or b from Laticauda semifasciata, which differ from each other by 16 substitutions and one insertion. The neutralizing mAb M alpha 1 recognizes with high affinity a topographical epitope on the surface of toxin alpha, but fails to recognize the erabutoxins although they possess most of the residues forming the presumed epitope. Examinations of the toxin alpha and erabutoxin 3-D structures and molecular dynamics simulations reveal several differences between the variants. In particular, the region involving the beta-turn 17-24 is organized differently. Analysis of the differences found in this region suggest that the insertion (or deletion) at position 18 of the variant amino acid sequences is particularly important in determining the differential cross-reactivity. To test this proposal, residue 18 was deleted in one erabutoxin using site-directed mutagenesis, and the biological properties of the resulting mutant were examined. We found that full antigenicity was restored in the previously unrecognized variant. The implications of this finding are discussed. The three-dimensional structure of erabutoxin b, a short-chain neurotoxic peptide purified from the venom of the sea snake Laticauda semifasciata, was determined in aqueous solution by two-dimensional proton nuclear magnetic resoce and simulated annealing-based calculations. On the basis of 883 assigned nuclear Overhauser effect (NOE) connectivities, 676 final distance constraints were derived and used together with 38 torsion angle (phi, chi 1) constraints, four distance constraints derived from disulfide bridges and 30 distance constraints derived from hydrogen bonds. A total of 14 converged structures were obtained from 50 runs of calculations. The atomic root-mean-square difference about the mean coordinate positions (excluding the residues 18 to 22) is 0.60 A for backbone atoms (N, C alpha and C'). The protein consists of a core region from which three finger-like loops emerge outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 2 to 5 and 13 to 16, a three-stranded antiparallel beta-sheet involving residues 23 to 30, 35 to 41 and 50 to 56, and four disulfide bridges in the core region. Comparison with two crystal structures of erabutoxin b at 1.4 A and 1.7 A resolution indicated that the solution and the crystal structures were very similar, but less defined regions were observed at the localized region of the tip of the central loop and the outside of the third loop in solution. Other short-chain alpha-neurotoxins showed structural characteristics similar to those of erabutoxin b. Here we examine the actions of six snake neurotoxins (alpha-cobratoxin from Naja naja siamensis, erabutoxin-a and b from Laticauda semifasciata; CM12 from N. haje annulifera, toxin III 4 from Notechis scutatus and a long toxin from N. haje) on nicotinic acetylcholine receptors in the cercal afferent, giant interneuron 2 synapse of the cockroach, Periplaneta americana. All toxins tested reduced responses to directly-applied ACh as well as EPSPs evoked by electrical stimulation of nerve XI with similar time courses, suggesting that their action is postsynaptic. Thus, these nicotinic receptors in a well-characterized insect synapse are sensitive to both long and short chain neurotoxins. This considerably expands the range of snake toxins that block insect nicotinic acetylcholine receptors and may enable further pharmacological distinctions between nAChR subtypes. Erabutoxins a and b are neurotoxins isolated from venom of a sea snake Laticauda semifasciata (erabu-umihebi). Amino acid sequences of the toxins indicated that the toxins are members of a superfamily consisting of short and long neurotoxins and cytotoxins found in sea snakes and terrestrial snakes. The short neurotoxins to which erabutoxins belong act by blocking the nicotinic acetylcholine receptor on the post synaptic membrane in a manner similar to that of curare. X-ray crystallography and NMR analyses showed that the toxins have a three-finger structure, in which three fingers made of three loops emerging from a dense core make a gently concave surface of the protein. The sequence comparison and the location of essential residues on the protein suggested the mechanism of binding of the toxin to the acetylcholine receptor. Classification of snakes by means of sequence comparison and that based on different morphological features were inconsistent, which led the authors to propose a hypothesis "Evolution without divergence."