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Are synonymous sites in primates and rodents functionally constrained?

No. Synonymous sites in primates exhibited evidence for higher selective constraint that those in rodents. In primates up to 24% of synonymous sites could be under purifying selection, while in rodents synonymous sites evolved neutrally.

It has been claimed that synonymous sites in mammals are under selective constraint. Furthermore, in many studies the selective constraint at such sites in primates was claimed to be more stringent than that in rodents. Given the larger effective population sizes in rodents than in primates, the theoretical expectation is that selection in rodents would be more effective than that in primates. To resolve this contradiction between expectations and observations, we used processed pseudogenes as a model for strict neutral evolution, and estimated selective constraint on synonymous sites using the rate of substitution at pseudosynonymous and pseudononsynonymous sites in pseudogenes as the neutral expectation. After controlling for the effects of GC content, our results were similar to those from previous studies, i.e., synonymous sites in primates exhibited evidence for higher selective constraint that those in rodents. Specifically, our results indicated that in primates up to 24% of synonymous sites could be under purifying selection, while in rodents synonymous sites evolved neutrally. To further control for shifts in GC content, we estimated selective constraint at fourfold degenerate sites using a maximum parsimony approach. This allowed us to estimate selective constraint using mutational patterns that cause a shift in GC content (GT ↔ TG, CT ↔ TC, GA ↔ AG, and CA ↔ AC) and ones that do not (AT ↔ TA and CG ↔ GC). Using this approach, we found that synonymous sites evolve neutrally in both primates and rodents. Apparent deviations from neutrality were caused by a higher rate of C → A and C → T mutations in pseudogenes. Such differences are most likely caused by the shift in GC content experienced by pseudogenes. We conclude that previous estimates according to which 20-40% of synonymous sites in primates were under selective constraint were most likely artifacts of the biased pattern of mutation.

Which animal bite can cause Capnocytophaga canimorsus infection?

Capnocytophaga canimorsus infection is typically associated with dog bites, especially in asplenic or immunocompromised patients, and typically manifest as sepsis and/or bacteremia.

CDC group DF-2 is the vernacular name given to a slow-growing gram-negative bacterium that causes septicemia and meningitis in humans. Infections frequently (one-third of cases) occur following dog bites or close contact with dogs or occasionally with cats. Splenectomy and alcoholism appear to be strong predisposing factors for DF-2 infection. In addition to 150 DF-2 strains received for identification, we received 9 DF-2-like strains; 6 were isolated from wound or eye infections, 3 of which were associated with dog bites and 1 of which was associated with a cat scratch, and 3 were isolated from dog mouths. The major characteristics of DF-2 include production of acid but no gas from lactose and maltose and usually D-glucose; positive reactions for oxidase, catalase, arginine dihydrolase, gliding motility, and o-nitrophenyl-beta-D-galactopyranoside; growth enhanced by serum and by incubation in a candle jar atmosphere; and negative reactions for sucrose, raffinose, inulin, melibiose, nitrate reduction, indole, and growth on MacConkey agar. DF-2-like strains had the same characteristics, except that acid was formed from sucrose, raffinose, inulin, and melibiose. By the hydroxyapatite method, DNAs from 12 DF-2 strains were 88% related in 60 degrees C reactions and 84% related in 75 degrees C reactions. Related sequences contained 0.5 to 1.5% unpaired bases (divergence). Three DF-2-like strains were 73 to 80% related at 60 degrees C (with 2.0 to 2.5% divergence) and 68 to 75% related at 75 degrees C. The relatedness of DF-2 and DF-2-like strains was 19 to 31% at 60 degrees Celsius and 13 to 19% at 75 degrees Celsius. The relatedness of DF-2 and DF-2-like strains to Capnocytophaga species was 4 to 7%. The DNA relatedness date indicate that eh DF-2 and the DF-2-like strains are separate, previously undescribed species. Both groups are phenotypically and genetically distinct from Capnocytophaga species, although they do share several characteristics with Capnocytophaga species, including cellular morphology, gliding motility, cellular fatty acid composition, enhancement of growth in a candle jar atmosphere, and G+C content. The new species differ from Capnocytophaga species by their positive oxidase and catalase reactions. We chose to avoid creating a new genus and proposed the names Capnocytophaga canimorsus sp. nov. for group DF-2 and C. cynodegmi sp. nov. for the DF-2-like strains. Capnocytophaga canimorsus causes dog-bite wound induced sepsis in adults, but infection may follow mucous membrane exposure. Systemic infection in children is extremely rare. A neonate with frequent exposure to a family dog and no cutaneous infection developed C. canimorsus meningitis. Suspicion of this pathogen requires laboratory consultation. Parental counseling can limit the risk of pet acquired infections. Capnocytophaga canimorsus, a commensal bacterium from dogs' mouths, can cause septicemia or meningitis in humans through bites or scratches. Here, we describe and characterize the inflammatory response of human and mouse macrophages on C. canimorsus infection. Macrophages infected with 10 different strains failed to release tumor necrosis factor (TNF)- alpha and interleukin (IL)-1 alpha . Macrophages infected with live and heat-killed (HK) C. canimorsus 5 (Cc5), a strain isolated from a patient with fatal septicemia, did not release IL-6, IL-8, interferon- gamma , macrophage inflammatory protein-1 beta , and nitric oxide (NO). This absence of a proinflammatory response was characterized by the inability of Toll-like receptor (TLR) 4 to respond to Cc5. Moreover, live but not HK Cc5 blocked the release of TNF- alpha and NO induced by HK Yersinia enterocolitica. In addition, live Cc5 down-regulated the expression of TLR4 and dephosphorylated p38 mitogen-activated protein kinase. These results highlight passive and active mechanisms of immune evasion by C. canimorsus, which may explain its capacity to escape from the host immune system. Capnocytophaga canimorsus has been recognized as an opportunistic pathogen causing systemic infections in immunocompromised individuals. It is part of the normal oral flora of the dog, and can be responsible for localized wound infections in humans in consequence of bites. This microorganism causes also septicemia, meningitis, endocarditis, ocular infections and rarely brain abscess. We describe the case of an immunocompetent 28-year-old male with temporal brain abscess from Capnocytophaga canimorsus secondary to dog's bite. Capnocytophaga canimorsus is a commensal bacterium from the canine oral flora, which can cause septicemia or meningitis in humans upon bite wound infections. C. canimorsus 5 (Cc5), a strain isolated from a patient with fatal septicemia, was used to investigate the interaction between C. canimorsus and J774.1 mouse macrophages. J774.1 cells infected at high multiplicity with Cc5 did not phagocytose nor kill Cc5 within 120 min of infection, unless the bacteria were opsonized with specific antibodies. Opsonization with complement, however, did not increase phagocytosis. Moreover, infection of J774.1 cells with live Cc5 led to the release of a soluble factor, which interfered with the ability of macrophages to kill other phagocytosed bacteria. These results provide an example of how C. canimorsus neutralizes the innate immune system. BACKGROUND: Dog bites are the most common animal bite injuries occurring in the United States. Estimated infection rates range between 15% and 20%. Polymicrobial infections are most common. Capnocytophaga canimorsus (C. canimorsus) is a Gram-negative rod strongly associated with dog bites, and is known to cause life-threatening infection in humans. OBJECTIVES: 1) Outline epidemiology of dog bites in the United States; 2) Identify host factors associated with infection, and common pathogens; 3) Discuss microbiology of C. canimorsus; 4) Discuss common clinical manifestations of C. canimorsus infection; 5) Outline treatment options. CASE REPORT: A 42-year-old woman with a remote history of Hodgkin's lymphoma (treated with irradiation) and thyroid carcinoma, both of which were in remission, presented to the Emergency Department with fever, abdominal pain, and diarrhea. She was found to be in septic shock. She was aggressively resuscitated and administered broad-spectrum antibiotics. Blood cultures grew C. canimorsus in 2/4 bottles. The patient recalled being bitten by the family dog 48 h before her initial presentation. She made an uneventful recovery. She was felt to be "functionally hyposplenic" due to her prior irradiation. CONCLUSIONS: C. canimorsus is a rare pathogen strongly associated with dog bites. By eliciting a history of animal bite, clinicians may be able to alert the laboratory of suspected C. canimorsus infection. Prolonged laboratory incubation times may be necessary as the organism is fastidious. Predisposing conditions include, among others, prior splenectomy and alcoholism. The mortality rate from C. canimorsus sepsis is high, so treatment should be promptly initiated. Sudden and unexpected nontraumatic death in individuals with asplenia or hyposplenia is usually due to fulmit bacterial sepsis, most often involving Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae. We report a case of a previously well 40-year-old man who died 5 hours after hospital admission. At autopsy Waterhouse-Friderichsen syndrome was identified and Capnocytophaga canimorsus was subsequently isolated on antemortem blood cultures. Infection of humans with this organism is most often due to dog bite or contact. Upon specific inquiry it was ascertained that 2 days before admission the deceased had suffered a superficial bite to his hand by his pet Staffordshire Bullterrier dog. His relevant history included a previous splenectomy following blunt abdominal trauma. Asplenia and hyposplenia at autopsy should prompt microbiological testing with consideration of unusual organisms such as C. canimorsus. Although histories of animal contact or injury are often not available at the time of autopsy, this should also be considered in cases of apparent fulmit sepsis. In individuals with asplenia or hyposplenia, dog bites do not have to involve excessive tissue trauma, vascular compromise, or blood loss to be lethal. Described in this study is the case of a 53-year-old woman who developed a life-threatening infection caused by the bacterium Capnocytophaga canimorsus (C. canimorsus), subsequent to being bitten by a dog. The patient presented to an Emergency Department with a 24-h history of diarrhoea and vomiting with dehydration but within 36 h of presentation developed an overwhelming severe sepsis with septic shock, disseminated intravascular coagulation, acute renal failure, metabolic acidosis and threatened acute respiratory failure requiring urgent intensive care intervention. At subsequent questioning her husband volunteered that she had been bitten on the wrist by the family dog 24h prior to the onset of symptoms; this bite had been extremely minor, requiring no treatment at the time and leaving only a very superficial wound. The causative organism was finally identified two weeks later as C. canimorsus, a common commensal in the oral flora of dogs. C. canimorsus has been reported as a rare cause of severe infection in susceptible individuals; however this case is of particular interest as there were no apparent predisposing factors conferring risk of severe infection. This case also raised significant practice issues for the treating hospital. Capnocytophaga canimorsus is a gram-negative bacterial species hosted in the oral cavity of dogs. C. canimorsus can cause sepsis, meningitis and endocarditis. Penicillin is the drug of choice. However, the species is a slow-grower and sometimes missed in blood cultures. Patients with a history of alcoholism, splenectomy or immunodeficiency are at an increased risk of contracting serious infections with C. canimorsus following dog bites. We report a case story of C. canimorsus meningitis contracted after a dog bite. BACKGROUND: Animal bites are typically harmless, but in rare cases infections introduced by such bites can be fatal. Capnocytophaga canimorsus, found in the normal oral flora of dogs, has the potential to cause conditions ranging from minor cellulitis to fatal sepsis. The tendency of C. canimorsus infections to present with varied symptoms, the organism's fastidious nature, and difficulty of culturing make this a challenging diagnosis. Rarely, bacterial cytotoxins such as those produced by C. canimorsus may act as causative agents of TTP, further complicating the diagnosis. Early recognition is crucial for survival, and the variability of presentation must be appreciated. We present the first known case of C. canimorsus infection resulting in TTP that initially presented as splenic infarction. CASE PRESENTATION: 72-year-old Caucasian male presented with a four-day history of abdominal pain, nausea, vomiting, diarrhea, and intermittent confusion. On presentation, vital signs were stable and the patient was afebrile. Physical examination was unremarkable apart from petechiae on the inner left thigh, and extreme diffuse abdominal pain to palpation and percussion along with positive rebound tenderness. Initial investigations revealed leukocytosis with left shift and thrombocytopenia, but normal liver enzymes, cardiac enzymes, lipase, INR and PTT. Abdominal CT demonstrated a non-enhancing spleen and hemoperitoneum, suggesting complete splenic infarction. Although the patient remained afebrile, he continued deteriorating over the next two days with worsening thrombocytopenia. After becoming febrile, he developed microangiopathic hemolytic anemia and hemodynamic instability, and soon after was intubated due to hypoxic respiratory failure and decreased consciousness. Plasma exchange was initiated but subsequently stopped when positive blood cultures grew a gram-negative organism. The patient progressively improved following therapy with piperacillin-tazobactam, which was switched to imipenem, then meropenem when Capnocytophaga was identified. CONCLUSIONS: There is a common misconception amongst practitioners that the presence of systemic infection excludes the possibility of TTP and vice versa. This case emphasizes that TTP may occur secondary to a systemic infection, thereby allowing the two processes to coexist. It is important to maintain a wide differential when considering the diagnosis of either TTP or C. canimorsus infection since delays in treatment may have fatal consequences. Capnocytophaga canimorsus is part of normal gingival flora of dogs and cats. The organism can cause septicemia, meningitis, and endocarditis in humans after contact with dogs or cats. In spite of the frequency of gastrointestinal symptoms in C. canimorsus infection patients, specific gastrointestinal disease or clinical images have not been reported. We report a case of C. canimorsus bacteremia presenting with acute cholecystitis in elderly woman. She suffered from general fatigue and right upper abdominal pain. She had leukocytosis and abnormal liver function tests. She showed abnormal findings of the gallbladder by abdominal computed tomography and ultrasonography. She was diagnosed with acute cholecystitis without gallstones and was administered with antibiotics. C. canimorsus was isolated from blood cultures. A history of an insignificant wound secondary to a dog bite was elicited. She recovered completely with antibiotic treatment. This case revealed that C. canimorsus bacteremia can be presented with acute cholecystitis, suggesting that C. canimorsus could cause cholecystitis. And this cholecystitis can be treated with antibiotics without operation. Physicians seeing patients with acute cholecysitis should ask questions regarding animal contact. Newly named in 1989, Capnocytophaga canimorsus is a bacterial pathogen found in the saliva of healthy dogs and cats, and is transmitted to humans principally by dog bites. This review compiled all laboratory-confirmed cases, animal sources, and virulence attributes to describe its epidemiology, clinical features, and pathogenesis. An estimated 484 patients with a median age of 55 years were reported, two-thirds of which were male. The case-fatality rate was about 26%. Its clinical presentations included severe sepsis and fatal septic shock, gangrene of the digits or extremities, high-grade bacteremia, meningitis, endocarditis, and eye infections. Predispositions were prior splenectomy in 59 patients and alcoholism in 58 patients. Dog bites before illness occurred in 60%; additionally, in 27%, there were scratches, licking, or other contact with dogs or cats. Patients with meningitis showed more advanced ages, higher male preponderance, lower mortality, and longer incubation periods after dog bites than patients with sepsis (p < 0.05). Patients with prior splenectomy presented more frequently with high-grade bacteremia than patients with intact spleens (p < 0.05). The organism possesses virulence attributes of catalase and sialidase production, gliding motility, cytotoxin production, and resistance to killing by serum complement due to its unique lipopolysaccharide. Penicillin is the drug of choice, but some practitioners prefer third-generation cephalosporins or beta-lactamase inhibitor combinations. C. canimorsus has emerged as a leading cause of sepsis, particularly post-splenectomy sepsis, and meningitis after dog bites. Animal bites represent a significant global health problem and account for approximately 1-2% of all visits to the emergency department. The vast majority of animal bite injuries are inflicted by dogs (80-90%,) and cats (5-15%). The most common complication following an animal bite is a wound infection, which tends to be polymicrobial and include both aerobic and anaerobic bacteria mainly of oropharyngeal origin. The likelihood of a cat bite becoming infected is double of that of a dog bite. Pasteurella spp. predominates in infected dog and cat bites. Dog bite injuries can be also associated with Capnocytophaga canimorsus, an aggressive organism which can cause disseminated infections (sepsis) and death, particularly in immunocompromised individuals. Early aggressive local wound cleansing is the most important therapy to prevent infection after animal bites. Due to the polymicrobial etiology of infected bite wounds, broad-spectrum antibiotics, covering both aerobic and anerobic bacteria, are often recommended as empiric treatment of animal bites. We describe a case of a life-threatening septicemia resulting from a previous dog bite wound. The isolated bacterium was Capnocytophaga canimorsus, a slow-growing Gram-negative bacillus commonly found in dog saliva. Known risk factors for invasive C. canimorsus infections are alcohol abuse, cigarette smoking, splenectomy or other forms of immunosuppression. Any clinician seeing patients with a history of a dog bite should consider this pathogen as a causative agent and take detailed history regarding exposure to animals. C. canimorsus and C. cynodegmi are dog and cat commensals which can be transmitted to humans via bites or scratches and can cause sepsis, meningitis, endocarditis, and eye- or wound infections. Recently an additional Capnocytophaga species was identified as part of the oral flora of healthy dogs and was given the name "C. canis". We previously identified a Capnocytophaga isolate that could not be typed with available diagnostic tests including MALDI-TOF, 16S rRNA sequencing or species-specific PCR. This strain and 21 other Capnocytophaga spp isolated in Sweden from clinical blood- or wound-cultures were subjected to whole genome sequencing using the Illumina platform. Phylogenetic analysis revealed that the previously non-typable isolate belongs to the putative new species "C. canis". Since this strain was isolated from a wound it also shows that members of "C. canis" have the potential to be pathogenic. In addition, our phylogenetic analysis uncovered an additional species of Capnocytophaga, which can be transmitted from dogs and cats to humans, suggesting a speciation within the Capnocytophaga family that has not been observed before. We propose the name of "C. stomatis" for this putative novel species. Capnocytophaga canimorsus is a gram-negative, capnophilic rod constituting normal bacterial flora of the oral cavity of dogs and cats. It is also considered to be an etiological factor of infections in human that may lead to multiple complications, i.a. sepsis, endocarditis and meningitis. C. canimorsus poses a serious threat, especially to patients with asplenia, cirrhosis or alcohol abuse. In most cases, infection occurs after a dog bite. Isolation and identification of the bacteria from the biological material is difficult and often delayed because of slow growth of the bacteria on microbiological media. Gold standard for bacteriological identification of C. canimorsus is polymerase chain reaction method. Amoxicillin with clavulanic acid is considered the drug of choice used in prophylaxis of C. canimorsus infections. Based on the data available from the literature, the authors present the epidemiology, risk factors, clinical picture, diagnostic methods and treatment of the C. canimorsus infection. BACKGROUND: Capnocytophaga canimorsus is a bacterium of the normal oral flora of dogs and cats. Human infection is caused by animal bite but is rarely observed, mainly in immunocompromised patients. We present 2 cases of C. canimorsus infection that occurred in immunocompetent patients and caused multiorgan failure and in both cases severe neurologic involvement. CASE REPORT: In the first case, we present a 69-year-old immunocompetent woman with septic shock derived from skin and soft tissue infection after a dog's bite. She developed ischemic necrosis evolving to gangrene of both forefeet and hands, infective aortic endocarditis, and neurologic involvement caused by large hemispheric hypodense lesions compatible with ischemic septical lesions. In the second case, we present a 65-year-old immunocompetent man with meningitis after a dog's bite. Despite antibiotic therapy, he developed neurologic clinical deterioration, with right sensitive hemisyndrome associated with lack of strength and motor skills of the right hand. Radiologic findings were consistent with the diagnosis of cerebritis. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Clinicians should always be aware of this pathogen, both in immunocompromised and immunocompetent patients, and consider prophylactic antibiotics after exposure. INTRODUCTION: Capnocytophaga canimorsus infections are associated with dog bites, especially in asplenic or immunocompromised patients, and typically manifest as sepsis and/or bacteremia. Meningitis has been rarely described, and its diagnosis may be delayed due to poor or slow growth using traditional culture techniques. We provide our experience using polymerase chain reaction (PCR) to establish the diagnosis and perform a comprehensive review of C. canimorsus meningitis cases to provide summary data on the clinical manifestations, diagnosis, and outcomes of this unusual infection. METHODS: A systematic review of the peer-reviewed English literature (PubMed, Embase, Ovid Medline) from January 1966 to March 2018 was conducted to identify cases of C. canimorsus meningitis. Data collected included demographics, risk factors, cerebrospinal fluid (CSF) findings, PCR results, treatments, and outcomes. Descriptive statistics are presented as numbers (percentages) and medians (ranges). RESULTS: A total of 37 patients were reviewed with a median age of 63 years (12 days to 83 years) with a male predomice (76%). A relatively low proportion had an immunocompromised state (16% splenectomy and 5% steroid use); the most common risk factor was alcoholism (19%). Fifty-nine percent reported a dog bite (all within ≤ 14 days prior to presentation), while 22% reported a non-bite dog exposure, 3% reported cat bite, and 3% reported both dog and cat exposures; 11% reported no animal contact. CSF parameters included a median white count of 1024 cells/mm3, 81% had neutrophilic predomice, median protein of 190 mg/dl, and median glucose CSF/serum ratio 0.23. In 54% of cases, blood cultures were positive for C. canimorsus (median, 4 days) and 70% had positive CSF cultures (median, 5 days). PCR established the diagnosis in eight (22%) cases. Antibiotic therapy was given for a median of 15 days (range, 7 to 42 days). Prognosis was overall favorable with only one (3%) death reported and adverse neurologic and/or physical sequelae in 19% of the survivors. CONCLUSION: C. canimorsus meningitis is a rare but increasingly important clinical entity occurring in patients of all ages, typically after dog exposure. While classically considered an infection among immunocompromised patients, most cases have occurred in previously healthy, immunocompetent persons. Diagnosis may be rapidly established by PCR, and this test should be considered in culture-negative cases with associated exposures. Outcome was generally favorable after a median antibiotic duration of 15 days. Bite infections caused by Capnocytophaga canimorsus are rare. Severe and fatal infections are more frequently reported in patients with immunodeficiency, splenectomy or alcohol abuse. We describe the case of a 63-year-old man who developed flu-like symptoms and presented after some delay with severe sepsis and purpura fulmis. He was found to be infected with C. canimorsus without a bite injury and did not demonstrate immunodeficiency or any other typical predisposition. Despite extensive intensive care, his conditions deteriorated and he died from multiorgan failure. LEARNING POINTS: Pet owners with banal, for instance flu-like, symptoms should urgently seek medical advice when symptoms are unusual.Capnocytophaga canimorsus infection should be considered and empirical antibiotic therapy immediately started or adjusted in the presence of purpura fulmis in the absence of animal bites or immunodeficiency. Capnocytophaga species are gram-negative bacilli that inhabit mammalian oral surfaces and can cause opportunistic infection, especially in asplenic patients. The species Capnocytophaga canimorsus is particularly associated with dog bites and is known to cause endocarditis, meningitis, and sepsis in the general population. In pregt patients, infections tied to Capnocytophaga species from human flora have been associated with preterm labor, chorioamnionitis, and neonatal septicemia. There is little known about the effects of zoonotically-acquired Capnocytophaga infection in pregt patients. In this case report, we present a patient with Capnocytophaga bacteremia acquired after a dog bite associated with profound thrombocytopenia and preterm labor. Dog bites are common in the United States, and we present basic recommendations for management of dog bites in pregt patients in order to avoid morbidity associated with delay in time to antibiotic treatment of infection as described in this case. Author information: (1)Department of Emergency and Critical Care Medicine, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (2)Department of Emergency and Critical Care Medicine, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (3)Department of Emergency and Critical Care Medicine, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (4)Department of Emergency and Critical Care Medicine, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (5)Department of Emergency and Critical Care Medicine, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (6)Department of Plastic and Reconstructive Surgery, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (7)Department of Plastic and Reconstructive Surgery, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (8)Department of Emergency and Critical Care Medicine, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (9)Department of Hematology, Kansai Medical University Medical Center, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan. Electronic address: [email protected]. (10)Department of Emergency and Critical Care Medicine, Kansai Medical University Hospital, 2-3-1 Shinmachi, Hirakata, Osaka 573-1191, Japan. Electronic address: [email protected].

What is the function of lysozyme?

Lysozymes are an ancient group of antimicrobial enzymes of the innate immune system. Lysozyme activity is a marker of Paneth cell function.

The ICChI is a 35-kDa, glycosylated protein isolated from the latex of the weed Ipomoea carnea. It displays chitinase and lysozyme activity, which could be important for the defense against pathogenic fungi, insects and bacteria. The ICChI enzyme was crystallized, and a diffraction data set was collected from a single crystal to 1.42 Å resolution. The crystals belong to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 57.9, c = 172.0 Å, and α = β = γ = 90°. The structure was elucidated by molecular replacement method using a mixed model of three homologous structures from the N-terminal sequence of ICChI. The refined model consists of 272 amino acid residues and has a Rfactor of 18.93% and Rfree of 22.42%. The protein consists of a single globular domain with a (α/β)8 triosephosphate isomerase barrel fold. Three of the consensus sites for N-glycosylation viz., Asn45, Asn172, and Asn194 containing carbohydrate moieties N-Acetylglucosamine (NAG), mannose, fucose, and xylose. The putative catalytic residues are Asp125, Glu127, and Tyr184. The crystal structure may provide fundamental information of GH18 family chitinases. The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin-covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (~2.4-fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine-liver cultures can be used as a platform for future investigations.

Explain amniotic band syndrome.

Amniotic band syndrome (ABS) is a rare congenital disease with variable manifestations ranging from simple constriction rings at the extremities to major defects such as exencephaly.

The amniotic band syndrome (ABS) refers to the infrequent occurrence of congenital deformities presumably due to fetal entanglement in strands of ruptured amniotic sac. The most commonly associated anomalies include amputations, constriction bands, syndactyly, craniofacial defects, club feet, and cleft lip. We present a typical case and short literature review of ABS. The infant reported also had a connective tissue nevus and a cutaneous striated muscle hamartoma. The amniotic band syndrome is a collection of fetal malformations associated with fibrous bands that appear to entangle or entrap various fetal parts in utero, leading to deformation, malformation, or disruption. This syndrome is given many names yet follows a clearly defined clinical pattern. Misdiagnosis and inappropriate family counseling are chronic features. This article reviews the clinical features of the amniotic band syndrome, its epidemiology, and the status of prenatal and neonatal diagnosis. The spectrum of malformations associated with amniotic bands is summarized and illustrated. Major etiologic theories are examined. It is recommended that the clinician involved in the delivery of any infant manifesting elements of this unusual combination of defects seek specialized consultation in the pursuit of an accurate and precise diagnosis and appropriate genetic counseling. The amniotic band syndrome is a collection of congenital deformities presumably due to rupture of amniotic sac. It appears to cause fetal injury through three basic mechanisms including malformation, disruption, and deformation. The associated anomalies vary from minor digital defect to major craniofacial and visceral defects. They can be categorized as neural tube-like defects, craniofacial anomalies, limb anomalies, abdominal and thoracic wall defects, visceral anomalies, and constriction bands. We present two autopsy cases and discuss the diagnostic features. Our findings support Torpin's theory that the fibrous constriction bands generated from early rupture of the amnion. An accurate diagnosis may be achieved by looking for the major features of amniotic band syndrome and a routine chromosome study and placental examination in cases with multiple congenital deformities. Amniotic band syndrome is a sporadic condition that occurs in approximately 1:1200 to 1:15,000 live births and that may result in amputations, constrictions and other deformities of the fetus. Although some cases present with congenital anomalies that are beyond surgical repair, a selected group of fetuses may show isolated limb constriction. It has been speculated that, without treatment, amputation or severe dysfunction of the limb may occur. Despite these potential complications, surgical treatment for this selected group of fetuses has not been previously performed. We report two cases that were successfully treated using novel minimally invasive surgical techniques. The cases involved fetuses with amniotic band syndrome with associated limb constriction in which the amniotic band was surgically interrupted to avoid spontaneous amputation of the extremity. Adequate blood flow distal to the obstruction was preserved and significant functional improvement of the extremity occurred in both cases, preserving the limbs. These cases represent the first prenatal surgical intervention successfully used to treat constricting amniotic bands in humans. In addition, these cases represent the first time that a non-lethal fetal entity has been surgically treated in utero. The results of this innovative therapy will encourage the efforts to continue developing minimally invasive techniques for the correction of birth defects. BACKGROUND: The amniotic band syndrome is a collection of fetal malformations associated with fibrous bands that appear to entrap or entangle various fetal parts in utero and can affect any organ or system and cause a single or multiple anomalies. The anomaly, acrania, is characterized by partial or complete absence of the calvarium with abnormal brain tissue development. Literature reports association of amniotic band syndrome and acrania postnatally, but not diagnosed prenatally by ultrasound. CASE: A young woman, gravida 1, para 0, presented for an initial prenatal visit at 35 weeks' gestation and had a first ultrasound that showed a single intrauterine pregcy at 36 weeks' gestation. This ultrasound also showed polyhydramnios, absence of or a very small cerebrum with either anencephaly or acrania. A targeted ultrasound scan was performed on the following day, which confirmed acrania in view of the fact that we did see an absence of the flat bones of the skull with a substantial amount of abnormal brain tissue present surrounded by a fetal membrane. The patient was counseled, and labor induction was scheduled with a male infant delivered weighing 1763 g after a spontaneous vaginal delivery. The infant was diagnosed with acrania, given supportive care, and died 11 hours later. CONCLUSION: Diagnosis of cranial bone defects can be established by ultrasound in the first trimester of pregcy. The prenatal diagnosis of acrania associated with amniotic bands by transvaginal ultrasound was visualized in the third trimester in this case; therefore, appropriate counseling and treatment options were offered. Amniotic band syndrome is an uncommon congenital pathological condition that may lead to malformations and foetal-infant death. We report an autoptic case. The patient was a male preterm infant. At 14 weeks of gestation, a routine ultrasonography showed severe craniofacial anomalies and a close contiguity of the foetal head with the amnios. The neonate survived three days, after which an autopsy was carried out. The infant had a frontoparietal meningoencephalocele; a fibrous band was attached to the skin, close to the meningoencephalocele base. Cleft lip and palate, nose deformation and agenesis of the right eye were also present. At the opening of the cranial cavity, occipital hyperostosis was observed. The herniated brain showed anatomical abnormalities that made identification of normal structures difficult. Microscopically, the nervous parenchyma had architectural disorganization and immaturity, and the fibrous band consisted of amniotic membranes. As evident from this case report, amniotic band syndrome may cause severe malformations and foetal-infant death. Ethmocephaly is the rarest form of holoprosencephaly, which occurs due to an incomplete cleavage of the forebrain. Clinically, the disease presents with a proboscis, hypotelorism, microphthalmos and malformed ears. Amniotic band syndrome is another rare congenital malformation with ring-like constriction bands in the limbs, head, face or trunk. We present a case of ethmocephaly with amniotic band syndrome, which is likely the first of its kind, published in the literature. Amniotic band syndrome (ABS) is a fetal congenital malformation, affecting mainly the limbs, but also the craniofacial area and internal organs. Two mains pathogenic mechanisms are proposed in its genesis. Firstly the early amnion rupture (exogenous theory) leading to fibrous bands, which wrap up the fetal body; secondly, the endogenous theory privileges vascular origin, mesoblastic strings not being a causal agent. The authors believe that the second theory explain the occurrence of ABS. The outcome of the disease during pregcy depends on the gravity of the malformations. Interruption of the pregcy is usually proposed when diagnosis of severe craniofacial and visceral abnormalities is confirmed. Whereas minor limb defects can be repaired with postnatal surgery. In case of an isolated amniotic band with a constricted limb, in utero lysis of the band can be considered to avoid a natural amputation. In an African country, such treatment is not possible as far as the antenatal diagnosis. Amniotic band syndrome is a group of sporadic congenital anomalies that involve the limbs, craniofacial regions and trunk, ranging from simple digital band constriction to complex craniofacial and central nervous system abnormalities. Placento-cranial adhesions in amniotic band syndrome are extremely rare, and severe conditions are associated with high morbidity and mortality rates. In this study, we pooled placento-cranial adhesion case reports that were published in the medical literature and added an unpublished case from our institution. The purpose of this article was to review and discuss the clinical features and outcomes of placento-cranial adhesions in amniotic band syndrome. Amniotic band syndrome is a rare congenital disorder caused by entrapment of fetal parts (usually a limb or digits) in fibrous amniotic bands while in utero that presents with complex multisystem anomalies. The authors report 2 children with amniotic band syndrome who presented to the ophthalmic unit of the authors' pediatric hospital. One of them presented with telecanthus, syndactyly, amputated toes, and unilateral epiphora diagnosed as congenital nasolacrimal duct obstruction. She was managed conservatively with lacrimal sac massage and provided with refractive correction while she simultaneously underwent multiple surgeries for correction of clubfoot and craniosynostosis. The second patient presented with cleft lip, cleft palate, multiple constriction bands in upper limbs and fingers with unilateral microphthalmos, microcornea, typical iris coloboma, and retinochoroidal coloboma, very similar to a case reported in literature. These 2 cases provide an overview of the clinical spectrum of ophthalmic manifestations along with their staged optimum rehabilitation. INTRODUCTION: Amniotic band syndrome is a rare congenital disorder with clinical presentation of constricting bands in different parts of extremities or whole extremities. Conservative or surgical treatment is provided depending on the type and severity of the anomaly. CASE OUTLINE: The paper presents the case of a neonate patient with constriction bands localized on the left leg. During the second week of life, a surgery was indicated, and a single-stage multiple Z-plasty was performed to correct the anomalies on the left lower leg. Postoperative edema in the distal part of the lower leg was easily managed by incisions and drainage. Two months later, the correction of the stricture of the left thigh was managed using the same procedure. The postoperative course was uneventful and the outcome was satisfactory after a two-year follow-up. CONCLUSION: Evaluation of a patient with amniotic band syndrome, as well as diagnosis, monitoring, treatment and postoperative care, should always be multidisciplinary. A single-stage correction approach provided satisfactory both functional and aesthetic results. Given many morphological variations of the syndrome, a decision on the strategy of treatment should be made individually for each patient. Amniotic band syndrome (ABS) is a rare congenital disease with variable manifestations ranging from simple constriction rings at the extremities to major defects such as exencephaly. Here we report the case of a female baby born full term (39 weeks) from a 35-year-old primiparous mother by cesarean section. In addition to the constriction rings at the extremities (fingers), the newborn presented facial malformations and a cranial anomaly suggestive of exencephaly. Supportive treatment was chosen because of the poor prognosis, and the child died 5 months later. Depending on the anomaly associated with ABS and its complexity, as in our case, genetic studies should be performed whenever possible, and the parents should be informed about the possibility of recurrences and incompatibility with life. Amniotic band sequence (ABS) is an uncommon and heterogeneous congenital disorder caused by entrapment of fetal parts by fibrous amniotic bands, causing distinctive structural abnormalities involving limbs, trunk, and craniofacial regions. The incidence ranges between 1/1200 and 1/15,000 live births, but is higher in stillbirths and previable fetuses. The intrinsic theory attributes the constriction band syndrome as an inherent development defect of embryogenesis while the extrinsic theory proposes that an early amnion rupture is responsible for the adherent bands. It is also suggested that amputations and constriction rings might be due to vascular disturbances. Anomalies resulting from amniotic bands are quite variable and sometimes may simulate chromosomal abnormalities. The authors report a case of a 36-week-gestation male neonate who lived for 29 hours after a vaginal delivery with an Apgar score of 8/9/9. The mother was primipara, and the prenatal was uneventful except for two episodes of urinary tract infections. The newborn examination depicted multiple anomalies characterized by exencephaly, bilateral labial cleft with distorted nostrils and palate cleft. There was also facial skin tag band, exophthalmos with hypoplasia of the eyelids. The limbs showed distal amputation of the fingers in both hands and feet, oligodactyly associated with syndactyly in the left foot, ring constriction in the right leg, the presence of right hyperextension, and clubfoot. The upper limbs showed length discrepancies. Karyotype analysis was normal at 46 XY. The authors conclude that the recognition of the malformations secondary to ABS is important in genetic counseling to prevent misdiagnosis between chromosomal and secondary disruption disorders. BACKGROUND: Postprocedural amniotic band disruption sequence is a condition that is associated with intrauterine interventions, and it is characterized by a constriction of the limbs or umbilical cord by fibrous strands, leading to edema, amputation, and/or fetal demise. OBJECTIVE: To evaluate the prevalence of, risk factors for, and the outcome of postprocedural amniotic band disruption sequence after fetoscopic laser surgery in twin-twin transfusion syndrome cases. STUDY DESIGN: All consecutive cases of twin-twin transfusion syndrome treated with fetoscopic laser coagulation of the vascular anastomoses at our center between January 2002 and March 2019 were included in the study. The occurrence of postprocedural amniotic band disruption sequence in these cases was recorded, and the potential risk factors were analyzed. RESULTS: Postprocedural amniotic band disruption sequence was detected, at birth, in 2.2% (15/672) of twin-twin transfusion syndrome cases treated with fetoscopic laser surgery, in both the recipients (10/15, 67%) and the donors (5/15, 33%). Postprocedural amniotic band disruption sequence primarily affected the lower extremities (11/15, 73%) and, less frequently, the upper extremities (2/15, 13%), both the upper and lower extremities (1/15, 7%), or the umbilical cord (1/15, 7%). Postprocedural amniotic band disruption sequence led to the amputation of toes in 5 of 15 cases (33%) and resulted in fetal demise because of constriction of the umbilical cord in 1 case (7%). The independent risk factors identified for postprocedural amniotic band disruption sequence were lower gestational age at laser surgery (odds ratio per week, 1.43; 95% confidence interval, 1.12-1.79; P=.003) and the presence of postprocedural chorioamniotic membrane separation on antenatal ultrasound examination (odds ratio, 41.66; 95% confidence interval, 5.44-319.25; P<.001). CONCLUSION: The prevalence of postprocedural amniotic band disruption sequence is low, but, when present, it may lead to severe consequences, with amputation of extremities or fetal demise occurring in more than one-third of the cases. Lower gestational age at the time of laser therapy and chorioamniotic membrane separation are independent risk factors for the postprocedural amniotic band disruption sequence.

Which are the main advantages of kallisto against similar methodologies?

Kallisto is a pseudo-alignment algorithm, which is a way of quantifying RNA-sequencing. It's used in RNA-seq because it's much faster and more efficient than other methodologies.

Author information: (1)Innovative Genomics Initiative, University of California, Berkeley, California, USA. (2)Department of Computer Science, University of California, Berkeley, California, USA. (3)Faculty of Industrial Engineering, Mechanical Engineering and Computer Science, University of Iceland, Reykjavik, Iceland. (4)Department of Mathematics, University of California, Berkeley, California, USA. (5)Department of Molecular &Cell Biology, University of California, Berkeley, California, USA. 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: Deconvolution is a mathematical process of resolving an observed function into its constituent elements. In the field of biomedical research, deconvolution analysis is applied to obtain single cell-type or tissue specific signatures from a mixed signal and most of them follow the linearity assumption. Although recent development of next generation sequencing technology suggests RNA-seq as a fast and accurate method for obtaining transcriptomic profiles, few studies have been conducted to investigate best RNA-seq quantification methods that yield the optimum linear space for deconvolution analysis. RESULTS: Using a benchmark RNA-seq dataset, we investigated the linearity of abundance estimated from seven most popular RNA-seq quantification methods both at the gene and isoform levels. Linearity is evaluated through parameter estimation, concordance analysis and residual analysis based on a multiple linear regression model. Results show that count data gives poor parameter estimations, large intercepts and high inter-sample variability; while TPM value from Kallisto and Salmon shows high linearity in all analyses. CONCLUSIONS: Salmon and Kallisto TPM data gives the best fit to the linear model studied. This suggests that TPM values estimated from Salmon and Kallisto are the ideal RNA-seq measurements for deconvolution studies. RNA-sequencing has become the gold standard for whole-transcriptome gene expression quantification. Multiple algorithms have been developed to derive gene counts from sequencing reads. While a number of benchmarking studies have been conducted, the question remains how individual methods perform at accurately quantifying gene expression levels from RNA-sequencing reads. We performed an independent benchmarking study using RNA-sequencing data from the well established MAQCA and MAQCB reference samples. RNA-sequencing reads were processed using five workflows (Tophat-HTSeq, Tophat-Cufflinks, STAR-HTSeq, Kallisto and Salmon) and resulting gene expression measurements were compared to expression data generated by wet-lab validated qPCR assays for all protein coding genes. All methods showed high gene expression correlations with qPCR data. When comparing gene expression fold changes between MAQCA and MAQCB samples, about 85% of the genes showed consistent results between RNA-sequencing and qPCR data. Of note, each method revealed a small but specific gene set with inconsistent expression measurements. A significant proportion of these method-specific inconsistent genes were reproducibly identified in independent datasets. These genes were typically smaller, had fewer exons, and were lower expressed compared to genes with consistent expression measurements. We propose that careful validation is warranted when evaluating RNA-seq based expression profiles for this specific gene set. The recently introduced Kallisto pseudoaligner has radically simplified the quantification of transcripts in RNA-sequencing experiments.  We offer cloud-scale RNAseq pipelines Arkas-Quantification, and Arkas-Analysis available within Illumina's BaseSpace cloud application platform which expedites Kallisto preparatory routines, reliably calculates differential expression, and performs gene-set enrichment of REACTOME pathways .  Due to inherit inefficiencies of scale, Illumina's BaseSpace computing platform offers a massively parallel distributive environment improving data management services and data importing.   Arkas-Quantification deploys Kallisto for parallel cloud computations and is conveniently integrated downstream from the BaseSpace Sequence Read Archive (SRA) import/conversion application titled SRA Import.  Arkas-Analysis annotates the Kallisto results by extracting structured information directly from source FASTA files with per-contig metadata, calculates the differential expression and gene-set enrichment analysis on both coding genes and transcripts. The Arkas cloud pipeline supports ENSEMBL transcriptomes and can be used downstream from the SRA Import facilitating raw sequencing importing, SRA FASTQ conversion, RNA quantification and analysis steps. RNA-seq is a vital method for understanding gene structure and expression patterns. Typical RNA-seq analysis protocols use sequencing reads of length 50 to 150 nucleotides for alignment to the reference genome and assembly of transcripts. The resultant transcripts are quantified and used for differential expression and visualization. Existing tools and protocols for RNA-seq are vast and diverse; given their differences in performance, it is critical to select an analysis protocol that is scalable, accurate, and easy to use. Tuxedo, a popular alignment-based protocol for RNA-seq analysis, has been updated with HISAT2, StringTie, StringTie-merge, and Ballgown, and the updated protocol outperforms its predecessor. Similarly, new pseudo-alignment-based protocols like Kallisto and Sleuth reduce runtime and improve performance. However, these tools are challenging for researchers lacking command-line experience. Here, we describe two new RNA-seq analysis protocols, in which all tools are deployed on CyVerse Cyberinfrastructure with user-friendly graphical user interfaces, and validate their performance using plant RNA-seq data. © 2018 by John Wiley & Sons, Inc. Rainbow smelt, Osmerus mordax, have an impressive ability to acclimate to very cold water. Rainbow smelt exposed to cold (<5 °C) for an extended period of time have faster sustained swimming speeds and increased contraction kinetics in their myotomal muscle compared to warm acclimated fish. We used RNA Sequencing reactions (RNA-Seq) to explore how gene expression underlies thermal acclimation by muscle in these fish. Transcriptome analysis is limited in species that lack an annotated genome, such as rainbow smelt. The Trinity software package permits the de novo assembly of a rainbow smelt transcriptome with a modest learning curve. The transcriptome was then analyzed with Kallisto to quantify the abundance of each transcript represented in the full transcriptome and Sleuth to analyze the resulting RNA-seq datasets. Subsequently qPCR was used to explore patterns of thermal acclimation and gene expression for genes of metabolic and muscle contractile function. These methodologies revealed shifts in both muscle and metabolic gene expression that contribute to the thermal acclimation response in rainbow smelt. In fast-twitch, anaerobic white muscle, slow isoforms of myosin heavy and light chain tended to be down-regulated with exposure to cold in myotomal muscle, while fast isoforms were unchanged. Genes associated with protein turnover and aerobic metabolism were up-regulated in the white muscle, while those associated with anaerobic metabolism and the cell cycle were down-regulated. Collectively the results suggest that thermal acclimation to cold is complex process of apparent shifts in gene expression.

What methodology does the HercepTest use?

The HercepTest is immunohistochemistry based.

BACKGROUND: HER2 status is a predictive biomarker of response to trastuzumab in advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. However, there is relatively little known about the role of HER2 in resected gastric or GEJ adenocarcinoma in the Western population. METHODS: Retrospective, observational, single centre study of patients with gastric or GEJ adenocarcinoma undergoing surgery with curative intent between January 2007 and June 2014 in the University Hospital Complex of Santiago de Compostela. The expression of HER2 was determined by immunohistochemistry (IHC) using DAKO-HercepTest™ and gene amplification with DuoCISH using a DAKO-DuoCISH kit. The study of HER2 expression and amplification was carried out in all the patients and it was correlated with classic clinicopathological parameters, survival and recurrence pattern. RESULTS: 106 patients were included. HER2 expression was as follows: 71.7% HER2 negative, 21.7% HER2 equivocal and 6.6% HER2 positive, or with HER2 overexpression. 13.2% of patients (14/106) had HER2 amplification by DuoCISH. A significant association was seen between overexpression and amplification of HER2 (p < 0.001).HER2 positivity was associated with the intestinal subtype (p = 0.010) and a low grade of differentiation (p = 0.018). Likewise, HER2 was significantly associated with a worse prognosis: overall survival (OS) 32.3 months HER2 positive versus 93.9 months HER2 negative (HR 0.42; confidence interval 95% 0.18-0.93; p = 0.028); and the presence of distant metastasis without accompanying locoregional recurrence (p = 0.048). CONCLUSION: HER2 status defines a subgroup with differentiated clinicopathological characteristics, worse prognosis and distant dissemination, without accompanying locoregional recurrence, in patients with resected gastric or GEJ adenocarcinoma operated on in a Western population.

Which R/Bioconductor package has been developed for gene expression signature searching?

SignatureSearch is an R/Bioconductor package that integrates a suite of existing and novel algorithms into an analysis environment for gene expression signature (ESE) searching combined with functional enrichment analysis (FEA) and visualization methods to facilitate the interpretation of the search results.

signatureSearch is an R/Bioconductor package that integrates a suite of existing and novel algorithms into an analysis environment for gene expression signature (GES) searching combined with functional enrichment analysis (FEA) and visualization methods to facilitate the interpretation of the search results. In a typical GES search (GESS), a query GES is searched against a database of GESs obtained from large numbers of measurements, such as different genetic backgrounds, disease states and drug perturbations. Database matches sharing correlated signatures with the query indicate related cellular responses frequently governed by connected mechanisms, such as drugs mimicking the expression responses of a disease. To identify which processes are predomitly modulated in the GESS results, we developed specialized FEA methods combined with drug-target network visualization tools. The provided analysis tools are useful for studying the effects of genetic, chemical and environmental perturbations on biological systems, as well as searching single cell GES databases to identify novel network connections or cell types. The signatureSearch software is unique in that it provides access to an integrated environment for GESS/FEA routines that includes several novel search and enrichment methods, efficient data structures, and access to pre-built GES databases, and allowing users to work with custom databases.

Which molecule is targeted by Upadacitinib?

Upadacitinib is a Janus kinase 1 inhibitor developed for treatment of moderate to severe rheumatoid arthritis.

AIMS: Upadacitinib (ABT-494) is a selective Janus kinase 1 inhibitor being developed for treatment of auto-immune inflammatory disorders. This work evaluated effects of high-fat meal, cytochrome P450 (CYP) 3A inhibition, CYP induction, and organic anion transporting polypeptide (OATP) 1B inhibition on upadacitinib pharmacokinetics. METHODS: Two Phase 1 evaluations were conducted, each in 12 healthy subjects. In Study 1, using a randomized, two-sequence crossover design, a 3 mg dose of upadacitinib (immediate-release capsules) was administered alone under fasting conditions, after high-fat meal, or on Day 4 of a 6-day regimen of 400 mg once-daily ketoconazole. In Study 2, a 12 mg upadacitinib dose was administered alone, with the first, and with the eighth dose of a 9-day regimen of rifampin 600 mg once daily. Upadacitinib plasma concentrations were characterized. RESULTS: Administration of upadacitinib immediate-release capsules after a high-fat meal decreased upadacitinib Cmax by 23% and had no impact on upadacitinib AUC relative to the fasting conditions. Ketoconazole (strong CYP3A inhibitor) increased upadacitinib Cmax and AUC by 70% and 75%, respectively. Multiple doses of rifampin (broad CYP inducer) decreased upadacitinib Cmax and AUC by approximately 50% and 60%, respectively. A single dose of rifampin (also an OATP1B inhibitor) had no effect on upadacitinib AUC. Upadacitinib was well tolerated when co-administered with ketoconazole, rifampin, or after a high-fat meal. CONCLUSIONS: Strong CYP3A inhibition and broad CYP induction result in a weak and moderate effect, respectively, on upadacitinib exposures. OATP1B inhibition and administration of upadacitinib immediate-release formulation with food does not impact upadacitinib exposure. Exposure-response analyses of QT data from early-stage clinical studies represent a valuable tool to assess the QT prolongation potential for drugs in development in lieu of standalone thorough QT (TQT) studies. However, demonstrating adequate electrocardiogram assay sensitivity can be challenging in the absence of a positive pharmacological control. Upadacitinib is a Janus kinase 1 inhibitor currently being evaluated in phase III rheumatoid arthritis trials. Exposure-response analyses to evaluate the QT prolongation potential for upadacitinib from phase I trials and the utility of the effect of food on QTcF to demonstrate ECG assay sensitivity are presented. The analyses demonstrated no effect of upadacitinib on QT interval and confirmed the sensitivity of the ECG assay to detect the small QT shortening effect caused by food. Lack of bias from manual ECG adjudication was also demonstrated. These analyses supported requesting a waiver for the regulatory requirement for a dedicated thorough QT study for upadacitinib. The past three decades have witnessed remarkable advances in our ability to target specific elements of the immune and inflammatory response, fuelled by advances in both biotechnology and disease knowledge. As well as providing superior treatments for immune-mediated inflammatory diseases (IMIDs), such therapies also offer unrivalled opportunities to study the underlying immunopathological basis of these conditions.In this review, we explore recent approaches to the treatment of IMIDs and the insights to pathobiology that they provide. We review novel biologic agents targeting the T-helper 17 axis, including therapies directed towards interleukin (IL)-17 (secukinumab, ixekizumab, bimekizumab), IL-17R (brodalumab), IL-12/23p40 (ustekinumab, briakinumab) and IL-23p19 (guselkumab, tildrakizumab, brazikumab, risankizumab, mirikizumab). We also present an overview of biologics active against type I and II interferons, including sifalumumab, rontalizumab, anifrolumab and fontolizumab. Emerging strategies to interfere with cellular adhesion processes involved in lymphocyte recruitment are discussed, including both integrin blockade (natalizumab, vedolizumab, etrolizumab) and sphingosine-1-phosphate receptor inhibition (fingolimod, ozanimod). We summarise the development and recent application of Janus kinase (JAK) inhibitors in the treatment of IMIDs, including first-generation pan-JAK inhibitors (tofacitinib, baricitinib, ruxolitinib, peficitinib) and second-generation selective JAK inhibitors (decernotinib, filgotinib, upadacitinib). New biologics targeting B-cells (including ocrelizumab, veltuzumab, tabalumab and atacicept) and the development of novel strategies for regulatory T-cell modulation (including low-dose IL-2 therapy and Tregitopes) are also discussed. Finally, we explore recent biotechnological advances such as the development of bispecific antibodies (ABT-122, COVA322), and their application to the treatment of IMIDs. BACKGROUND: There is a great unmet clinical need for efficacious, tolerable, economical and orally administrated drugs for the treatment of inflammatory bowel disease (IBD). New therapeutic avenues have become possible including the development of medications that target specific genetic pathways found to be relevant in other immune mediated diseases. AIMS: To provide an overview of recent clinical trials for new generation oral targeted medications that may have a future role in IBD management. METHODS: Pubmed and Medline searches were performed up to 1 March 2018 using keywords: "IBD", "UC", "CD", "inflammatory bowel disease" "ulcerative colitis", "Crohn's disease" in combination with "phase", "study", "trial" and "oral". A manual search of the clinical trial register, article reference lists, abstracts from meetings of Digestive Disease Week, United European Gastroenterology Week and ECCO congress were also conducted. RESULTS: In randomised controlled trials primary efficacy endpoints were met for tofacitinib (JAK 1/3 inhibitor-phase III), upadacitinib (JAK 1 inhibitor-phase II) and AJM300 (α4-integrin antagonist-phase II) in ulcerative colitis. Ozanimod (S1P receptor agonist-phase II) also demonstrated clinical remission. For Crohn's disease, filgotinib (JAK1 inhibitor-phase II) met primary endpoints and laquinimod (quinolone-3-carboxide small molecule-phase II) was also efficacious. Trials using mongersen (SMAD7 inhibitor) and vidofludimus (dihydroorotate dehydrogenase inhibitor) have been halted. CONCLUSIONS: This is potentially the start of an exciting new era in which multiple therapeutic options are at the disposal of physicians to treat IBD on an individualised basis. Head-to-head studies with existing treatments and longer term safety data are needed for this to be possible. Upadacitinib is a Janus kinase 1 inhibitor under development for the treatment of several inflammatory disorders including rheumatoid arthritis (RA). Upadacitinib was administered in the phase 2 RA trials primarily as twice-daily regimens of an immediate-release (IR) formulation. The upadacitinib extended-release (ER) formulation was developed to enable once-daily dosing. In the present study, upadacitinib pharmacokinetics were characterized after the administration of single and multiple once-daily doses of the ER formulation in healthy subjects relative to single and multiple twice-daily doses of the IR formulation. Increase in upadacitinib exposure was dose-proportional over the evaluated 15- to 30-mg ER dose range. Single 15- and 30-mg ER doses provided equivalent AUC0-inf compared with single 12- and 24-mg IR doses, respectively. A high-fat breakfast increased upadacitinib ER Cmax and AUC0-inf by only 20% and 17%, respectively, relative to fasting conditions. The median time to peak plasma concentrations was 2 to 4 hours for the ER formulation, and steady state was achieved by day 4 of once-daily dosing. Doses of 15 and 30 mg once daily using the ER formulation provided equivalent AUC0-24 , comparable Cmax and Cmin , and a fluctuation index over a 24-hour period at steady state similar to 6 and 12 mg twice daily, respectively, using the IR formulation. These results supported the use of upadacitinib 15- and 30-mg doses of the ER formulation in the phase 3 trials in RA. BACKGROUND: Upadacitinib is a selective inhibitor of Janus kinase 1 and was efficacious in phase 2 studies in patients with moderate-to-severe rheumatoid arthritis. We aimed to assess the efficacy of upadacitinib in patients with inadequate response to conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs). METHODS: This study is a double-blind, placebo-controlled trial at 150 sites in 35 countries. We enrolled patients aged 18 years or older with active rheumatoid arthritis for 3 months or longer, who had received csDMARDs for at least 3 months with a stable dose for at least 4 weeks before study entry, and had an inadequate response to at least one of the following csDMARDs: methotrexate, sulfasalazine, or leflunomide. Using interactive response technology, we randomly assigned patients receiving stable background csDMARDs (2:2:1:1) to receive a once-daily extended-release formulation of upadacitinib 15 mg or 30 mg, or placebo, for 12 weeks. Patients, investigators, and the funder were masked to allocation. After 12 weeks, patients taking placebo received 15 mg or 30 mg of upadacitinib once daily, according to the prespecified randomisation assignment. The primary endpoints were the proportion of patients at week 12 who achieved 20% improvement in American College of Rheumatology criteria (ACR20), and a 28-joint disease activity score using C-reactive protein (DAS28[CRP]) of 3·2 or less. We did efficacy analyses in the full analysis set of all randomly assigned patients who received at least one dose of study drug, and used non-responder imputation for assessment of the primary outcomes. This study is registered with ClinicalTrials.gov, number NCT02675426. FINDINGS: Between Dec 17, 2015, and Dec 22, 2016, 1083 patients were assessed for eligibility, of whom 661 were recruited and randomly assigned to receive upadacitinib 15 mg (n=221), upadacitinib 30 mg (n=219), or placebo (n=221). All patients received at least one dose of study drug, and 618 (93%) completed 12 weeks of treatment. At week 12, ACR20 was achieved by 141 (64%; 95% CI 58-70) of 221 patients receiving upadacitinib 15 mg and 145 (66%; 60-73) of 219 patients receiving upadacitinib 30 mg, compared with 79 (36%; 29-42) of 221 patients receiving placebo (p<0·0001 for each dose vs placebo). DAS28(CRP) of 3·2 or less was met by 107 (48%; 95% CI 42-55) patients receiving upadacitinib 15 mg and 105 (48%; 41-55) patients receiving upadacitinib 30 mg, compared with 38 (17%; 12-22) patients receiving placebo (p<0·0001 for each dose vs placebo). Adverse events were reported in 125 (57%) of 221 patients receiving upadacitinib 15 mg, 118 (54%) of 219 patients receiving upadacitinib 30 mg, and 108 (49%) of 221 patients receiving placebo. The most frequently reported adverse events (≥5% of patients in any group) were nausea (16 [7%] of 221 in the upadacitinib 15 mg group; three [1%] of 219 in the upadacitinib 30 mg group; and seven [3%] of 221 in the placebo group), nasopharyngitis (12 [5%]; 13 [6%]; and nine [4%]), upper respiratory tract infection (12 [5%]; 12 [5%]; and nine [4%]), and headache (nine [4%]; seven [3%]; and 12 [5%]). More infections were reported for upadacitinib (64 [29%] of 221 patients receiving 15 mg and 69 [32%] of 219 patients receiving 30 mg) versus placebo (47 [21%] of 221 patients). There were three herpes zoster infections (one [<1%] in the placebo group, one [<1%] in the upadacitinib 15 mg group, and one [<1%] in the upadacitinib 30 mg group) and one primary varicella zoster virus infection (one [<1%] in the upadacitinib 30 mg group), two maligcies (both in the upadacitinib 30 mg group), one adjudicated major adverse cardiovascular event (in the upadacitinib 30 mg group), and five serious infections (one [<1%] in the placebo group, one [<1%] in the upadacitinib 15 mg group, three [1%] in the upadacitinib 30 mg group). No deaths were reported during the trial. INTERPRETATION: Patients with moderately to severely active rheumatoid arthritis who received upadacitinib (15 mg or 30 mg) in combination with csDMARDs showed significant improvements in clinical signs and symptoms. FUNDING: AbbVie Inc. BACKGROUND: Phase 2 studies with upadacitinib, a selective Janus kinase 1 (JAK1) inhibitor, have shown safety and efficacy in the treatment of patients with active rheumatoid arthritis. We did this study to further assess the safety and efficacy of upadacitinib in patients with an inadequate response to biologic disease-modifying anti-rheumatic drugs (bDMARDs). METHODS: We did this double-blind, randomised controlled phase 3 trial at 153 sites in 26 countries. Patients were aged 18 years or older, had active rheumatoid arthritis and previous inadequate response or intolerance to bDMARDs, and were receiving concomitant background conventional synthetic DMARDS (csDMARDs). We randomly assigned patients (2:2:1:1) by interactive response technology to receive once-daily oral extended-release upadacitinib 15 mg or 30 mg or placebo for 12 weeks, followed by upadacitinib 15 mg or 30 mg from week 12 onwards. The two separate primary endpoints were the proportions of patients achieving a 20% improvement in American College of Rheumatology criteria (ACR20) at week 12 and the proportion of patients achieving a 28-joint disease activity score using C-reactive protein (DAS28[CRP]) of 3·2 or less at week 12. Efficacy and safety analyses were done in the modified intention-to-treat population of all patients who received at least one dose of study drug. Data are presented up to week 24 of this ongoing study. The trial is registered with ClinicalTrials.gov (NCT02706847). FINDINGS: Between March 15, 2016, and Jan 10, 2017, 499 patients were randomly assigned (n=165 upadacitinib 15 mg; n=165 upadacitinib 30 mg; n=85 placebo then upadacitinib 15 mg; and n=84 placebo then upadacitinib 30 mg) and one patient was withdrawn from the 15 mg upadacitinib group before the start of study treatment. Mean disease duration was 13·2 years (SD 9·5); 235 (47%) of 498 patients had received one previous bDMARD, 137 (28%) had received two, and 125 (25%) had received at least three; 451 (91%) patients completed treatment up to week 12 and 419 (84%) patients completed treatment up to week 24. At week 12, ACR20 was achieved by 106 (65%; 95% CI 57-72) of 164 patients receiving upadacitinib 15 mg and 93 (56%; 49-64) of 165 patients receiving upadacitinib 30 mg compared with 48 (28%; 22-35) of 169 patients receiving placebo (p<0·0001 for each dose vs placebo). DAS28(CRP) of 3·2 or less was achieved by 71 (43%; 95% CI 36-51) of 164 patients receiving upadacitinib 15 mg and 70 (42%; 35-50) of 165 patients receiving upadacitinib 30 mg versus 24 (14%; 9-20) of 169 patients receiving placebo (p<0·0001 for each dose vs placebo). Up to week 12, overall numbers of patients with adverse events were similar for the placebo group (95 [56%] of 169) and the upadacitinib 15 mg group (91 [55%] of 164), but higher in the upadacitinib 30 mg group (111 [67%] of 165). At week 12, the most common adverse events occurring in at least 5% of patients in any treatment group were upper respiratory tract infection (13 [8%] of 169 in the placebo group; 13 [8%] of 164 in the upadacitinib 15 mg group; ten [6%] of 165 in the upadacitinib 30 mg group), nasopharyngitis (11 [7%]; seven [4%]; nine [5%]), urinary tract infection (ten [6%]; 15 [9%]; nine [5%]), and worsening of rheumatoid arthritis (ten [6%]; four [2%]; six [4%]). The number of patients with serious adverse events was higher in the upadacitinib 30 mg group (12 [7%]) than in the upadacitinib 15 mg group (eight [5%]); no serious adverse events were reported in patients receiving placebo. More patients in the upadacitinib 30 mg group had serious infections, herpes zoster, and adverse events leading to discontinuation than in the upadacitinib 15 mg and placebo groups. During the placebo-controlled phase of the study, one case of pulmonary embolism, three maligcies, one major adverse cardiovascular event, and one death were reported in patients receiving upadacitinib; none were reported in patients receiving placebo. INTERPRETATION: Both doses of upadacitinib led to rapid and significant improvements compared with placebo over 12 weeks in patients with refractory rheumatoid arthritis. FUNDING: AbbVie Inc. Upadacitinib is a novel selective oral Janus kinase 1 (JAK) inhibitor being developed for treatment of several inflammatory diseases. Oral contraceptives are anticipated to be a common concomitant medication in the target patient populations. This study was designed to evaluate the effect of multiple doses of upadacitinib on the pharmacokinetics of ethinylestradiol and levonorgestrel in healthy female subjects. This phase I, single-center, open-label, 2-period crossover study evaluated the effect of multiple doses of 30 mg once daily extended-release upadacitinib on the pharmacokinetics of a single oral dose of ethinylestradiol/levonorgestrel (0.03/0.15 mg; administered alone in period 1 and on day 12 of a 14-day regimen of upadacitinib in period 2) in 22 healthy female subjects. The ratios (90% confidence intervals) for maximum plasma concentration and area under the plasma drug concentration-time curve from time zero to infinity following administration of ethinylestradiol/levonorgestrel with upadacitinib compared with administration of ethinylestradiol/ levonorgestrel alone were 0.96 (0.89-1.02) and 1.1 (1.04-1.19), respectively, for ethinylestradiol, and 0.96 (0.87-1.06) and 0.96 (0.85-1.07), respectively, for levonorgestrel. The harmonic mean terminal half-life for ethinylestradiol (7.7 vs 7.0 hours) and levonorgestrel (37.1 vs 33.1 hours) was similar in the presence and absence of upadacitinib. Ethinylestradiol and levonorgestrel were bioequivalent in the presence and absence of upadacitinib. Therefore, upadacitinib can be administered concomitantly with oral contraceptives containing ethinylestradiol or levonorgestrel. OBJECTIVES: We assessed the relative efficacy and safety of once-daily administration of 15 and 30 mg upadacitinib (a JAK1-selective inhibitor) in patients with active rheumatoid arthritis (RA). METHODS: We conducted a Bayesian network meta-analysis to combine the direct and indirect evidence from randomized controlled trials (RCTs) that examined the efficacy and safety of upadacitinib in patients with active RA. RESULTS: Five RCTs involving 4381 patients met the inclusion criteria. There were 15 pairwise comparisons, including eight direct comparisons and six interventions. The ACR20 response rate was significantly higher in the upadacitinib 15 and 30 mg + MTX (methotrexate) groups than in the MTX group (OR: 4.98, 95% CrI: 2.66-10.10; OR: 4.73, 95% CrI: 2.25-10.98). Adalimumab 40 mg + MTX, upadacitinib 30 mg, and upadacitinib 15 mg groups showed a significantly higher ACR20 response rate than did the MTX group. Ranking probability based on the surface under the cumulative ranking curve (SUCRA) indicated that upadacitinib 15 mg + MTX was likely to achieve the best ACR20 response rate (SUCRA = 0.838), followed by upadacitinib 30 mg + MTX, adalimumab 40 mg + MTX, upadacitinib 30 mg, upadacitinib 15 mg, and MTX (SUCRA = 0.784, 0.495, 0.471, 0.404, and 0.008, respectively). The safety based on the number of serious adverse events (SAEs) did not differ significantly among the six interventions. CONCLUSIONS: Upadacitinib 15 and 30 mg administration once daily in combination with MTX was the most efficacious intervention for active RA, with no significant risk for SAEs. BACKGROUND: Anti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib. METHODS: Structure-based hypotheses were used to design the JAK1 selective inhibitor upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines. RESULTS: Here, we show the structural basis for the JAK1 selectivity of upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting. CONCLUSIONS: The data presented here highlight the JAK1 selectivity of upadacinitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile. Upadacitinib is a selective Janus kinase 1 inhibitor being developed for the treatment of several inflammatory autoimmune diseases, including rheumatoid arthritis. Upadacitinib is a nonsensitive substrate for metabolism by cytochrome P450 3A enzymes. This open-label, single-dose, multicenter study assessed the pharmacokinetics of upadacitinib following oral administration of a single 15-mg dose of the upadacitinib extended-release formulation in subjects with mild (n = 6) and moderate (n = 6) hepatic impairment relative to demographically matched healthy subjects (n = 6). Subjects were assigned to 1 of the 3 groups according to the Child-Pugh classification. Relative to subjects with normal hepatic function, the ratios (90% confidence intervals) of upadacitinib area under the plasma concentration-versus-time profile from time 0 to infinity (AUCinf ) for subjects with mild and moderate hepatic impairment were 1.28 (0.91-1.79) and 1.24 (0.87-1.76), respectively. The central ratios of upadacitinib maximum observed concentration (Cmax ) were 1.04 (0.77-1.39) and 1.43 (1.05-1.95) in subjects with mild and moderate hepatic impairment, respectively, compared with subjects with normal hepatic function. No clinically significant changes in vital signs or hematology measurements were observed, and no new safety events were identified in this study. These results indicate that mild and moderate hepatic impairment has no clinically relevant effect on upadacitinib pharmacokinetics. BACKGROUND: Upadacitinib, an oral Janus kinase (JAK)1-selective inhibitor, showed efficacy in combination with stable background conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) in patients with rheumatoid arthritis who had an inadequate response to DMARDs. We aimed to evaluate the safety and efficacy of upadacitinib monotherapy after switching from methotrexate versus continuing methotrexate in patients with inadequate response to methotrexate. METHODS: SELECT-MONOTHERAPY was conducted at 138 sites in 24 countries. The study enrolled adults (≥18 years) who fulfilled the 2010 American College of Rheumatology (ACR)-European League Against Rheumatism (EULAR) classification criteria for rheumatoid arthritis. Patients with active rheumatoid arthritis despite stable methotrexate were randomly assigned 2:2:1:1 to switch to once-daily monotherapy of of upadacitinib or to continue methotrexate at their existing dose as blinded study drug; starting from week 14, patients assigned to continue methotrexate were switched to 15 mg or 30 mg once-daily upadacitinib per prespecified random assignment at baseline. The primary endpoints in this report are proportion of patients achieving 20% improvement in the ACR criteria (ACR20) at week 14, and proportion achieving low disease activity defined as 28-joint Disease Activity Score using C-reactive protein (DAS28[CRP]) of 3·2 or lower, both with non-responder imputation at week 14. Outcomes were assessed in patients who received at least one dose of study drug. This study is active but not recruiting and is registered with ClinicalTrials.gov, number NCT02706951. FINDINGS: Patients were screened between Feb 23, 2016, and May 19, 2017 and 648 were randomly assigned to treatment. 598 (92%) completed week 14. At week 14, an ACR20 response was achieved by 89 (41%) of 216 patients (95% CI 35-48) in the continued methotrexate group, 147 (68%) of 217 patients (62-74) receiving upadacitinib 15 mg, and 153 (71%) of 215 patients (65-77) receiving upadacitinib 30 mg (p<0·0001 for both doses vs continued methotrexate). DAS28(CRP) 3·2 or lower was met by 42 (19%) of 216 (95% CI 14-25) in the continued methotrexate group, 97 (45%) of 217 (38-51) receiving upadacitinib 15 mg, and 114 (53%) of 215 (46-60) receiving upadacitinib 30 mg (p<0·0001 for both doses vs continued methotrexate). Adverse events were reported in 102 patients (47%) on continued methotrexate, 103 (47%) on upadacitinib 15 mg, and 105 (49%) on upadacitinib 30 mg. Herpes zoster was reported by one (<1%) patient on continued methotrexate, three (1%) on upadacitinib 15 mg, and six (3%) on upadacitinib 30 mg. Three maligcies (one [<1%] on continued methotrexate, two [1%] on upadacitinib 15 mg), three adjudicated major adverse cardiovascular events (one [<1%] on upadacitinib 15 mg, two [<1%] on upadacitinib 30 mg), one adjudicated pulmonary embolism (<1%; upadacitinib 15 mg), and one death (<1%; upadacitinib 15 mg, haemorrhagic stroke [ruptured aneurysm]) were reported in the study. INTERPRETATION: Upadacitinib monotherapy showed statistically significant improvements in clinical and functional outcomes versus continuing methotrexate in this methotrexate inadequate-responder population. Safety observations were similar to those in previous upadacitinib rheumatoid arthritis studies. FUNDING: AbbVie Inc, USA. Orally bioavailable inhibitors of the tyrosine kinases (TYKs), also referred to as Janus kinases (JAKs), are being evaluated for the treatment of patients with Crohn's disease (CD), ulcerative colitis (UC), and other chronic inflammatory disorders. To date, three JAK inhibitors have been tested in patients with moderate-to-severe CD: tofacitinib (pan-JAK inhibitor), filgotinib (JAK1 inhibitor) and upadacitinib (JAK1 inhibitor). Clinical development of tofacitinib was discontinued in CD because the primary endpoint of clinical remission in the phase II induction and maintece trials was not met, although outcomes may have been influenced by trial design flaws and a high placebo rate was noted. In contrast, filgotinib did meet its primary endpoint of clinical remission at week 10 in the phase II FITZROY trial, in addition to several other clinically important secondary outcomes, spurring a subsequent larger phase III trial. Following promising results for upadacitinib in its phase II trial, larger phase III trials were also initiated to corroborate the efficacy results. Although JAK inhibitors appear to have an acceptable safety profile, higher rates of infections compared to placebo were noted. Overall, JAK inhibitors constitute a new promising class of drugs, given the efficacy signals observed in pivotal clinical trials in several chronic inflammatory diseases. Here we review the existing evidence on the pharmacology, safety and efficacy of JAK-STAT inhibitors that are currently under investigation for the treatment of patients with CD. The aim of this study was to characterize the effects of upadacitinib, a Janus kinase 1 inhibitor, on in vivo activity of different cytochrome P450 (CYP) enzymes using a cocktail approach. Healthy subjects (n = 20) received single oral doses of the modified Cooperstown 5+1 cocktail drugs (midazolam [CYP3A], caffeine [CYP1A2], warfarin + vitamin K [CYP2C9], omeprazole [CYP2C19], and dextromethorphan [CYP2D6]) without upadacitinib and on day 11 (midazolam) or 12 (all other probes) of a 15-day regimen of upadacitinib 30 mg once daily (extended-release formulation). Serial blood samples and 12-hour urine samples were collected for assays of the probe substrates and select metabolites. The ratio (90%CI) of area under the plasma concentration-time curve from time 0 to infinity (AUCinf ) central values when the cocktail drugs were administered with upadacitinib relative to when administered alone were 0.74 (0.68-0.80) for midazolam, 1.22 (1.15-1.29) for caffeine, 1.11 (1.07-1.15) for S-warfarin, 1.07 (0.95-1.22) for dextromethorphan, and 0.82 (0.72-0.94) for omeprazole. The ratio (90%CI) was 1.09 (1.00-1.19) for 5-hydroxy-omeprazole to omeprazole AUCinf ratio and 1.17 (0.97-1.41) for dextromethorphan to dextrorphan 12-hour molar urinary ratio. Upadacitinib 30 mg once daily (a dose that is twice the optimal dose in rheumatoid arthritis based on phase 3 results) has a limited effect on CYP3A activity (26% decrease in exposure of midazolam, a sensitive CYP3A substrate) and no relevant effects on CYP1A2, CYP2C9, CYP2C19, or CYP2D6 activity in vivo. No clinically relevant changes in plasma exposures are expected for drugs that are substrates for the evaluated CYP enzymes when coadministered with upadacitinib. The past decade has witnessed an explosion in trial data on JAK inhibitors (JAKi). These small molecules target the Janus kinase - signal transducer and activator of transcription (JAK-STAT) pathway, blocking crucial cytokines across a septum of rheumatic diseases. As a class, JAKi are beginning to demonstrate efficacy on par, if not superior to biologics. Two first generation JAKi are licensed for use in inflammatory arthritis; tofacitinib and baricitinib. Next-generation JAKi have been designed with selective affinity for one JAK enzymes, the aim to reduce unwanted adverse effects without declining clinical efficacy. Emerging data with selective JAK1 inhibitors upadacitinib and filgotinib looks very promising. Despite differences in selectivity between JAKi, an overlap exists in their safety profiles. Across the class, a characteristic safety signal is emerging with viral opportunistic infections, particularly herpes zoster. Post marketing drug surveillance will be essential in evaluating the long-term risk with these agents. Upadacitinib is a selective Janus Kinase 1 inhibitor which is being developed for the treatment of several inflammatory diseases including rheumatoid arthritis. Upadacitinib was evaluated in Phase 3 studies as an oral extended-release (ER) formulation administered once daily. The purpose of this study was to develop a level A in vitro-in vivo correlation (IVIVC) for upadacitinib ER formulation. The pharmacokinetics of four upadacitinib extended-release formulations with different in vitro release characteristics and an immediate-release capsule formulation of upadacitinib were evaluated in 20 healthy subjects in a single-dose, randomized, crossover study. In vivo pharmacokinetic data and in vitro dissolution data (USP Dissolution Apparatus 1; pH 6.8; 100 rpm) were used to establish a level A IVIVC. Three formulations were used to establish the IVIVC, and the fourth formulation was used for external validation. A non-linear IVIVC best described the relationship between upadacitinib in vitro dissolution and in vivo absorption profiles. The absolute percent prediction errors (%PE) for upadacitinib Cmax and AUC were less than 10% for all three formulations used to establish the IVIVC, as well as for the %PE for the external validation formulation and the overall mean internal validation. Model was cross-validated using the leave-one-out approach; all evaluated cross-validation runs met the regulatory acceptance criteria. A level A IVIVC was successfully developed and validated for upadacitinib ER formulation, which meets the FDA and EMA regulatory validation criteria and can be used as surrogate for in vivo bioequivalence. Inhibition of Janus kinases [JAKs] in Crohn's disease [CD] patients has shown conflicting results in clinical trials. Tofacitinib, a pan-JAK inhibitor, showed efficacy in ulcerative colitis [UC] and has been approved for the treatment of patients with moderate to severe UC. In contrast, studies in CD patients were disappointing and the primary end point of clinical remission could not be met in the respective phase II induction and maintece trials. Subsequently, the clinical development of tofacitinib was discontinued in CD. In contrast, efficacy of filgotinib, a selective JAK1 inhibitor, in CD patients was demonstrated in the randomized, double-blinded, placebo-controlled phase II FITZROY study. Upadacitinib also showed promising results in a phase II trial in moderate to severe CD. Subsequently, phase III programmes in CD have been initiated for both substances, which are still ongoing. Several newer molecules of this class of orally administrated immunosuppressants are being tested in clinical programmes. The concern of side effects of systemic JAK inhibition is addressed by either exclusively intestinal action or higher selectivity [Tyk2 inhibitors]. In general, JAK inhibitors constitute a new promising class of drugs for the treatment of CD. BACKGROUND: Atopic dermatitis is a chronic inflammatory skin disease characterized by pruritic skin lesions. OBJECTIVE: We sought to evaluate the safety and efficacy of multiple doses of the selective Janus kinase 1 inhibitor upadacitinib in patients with moderate to severe atopic dermatitis. METHODS: In the 16-week, double-blind, placebo-controlled, parallel-group, dose-ranging portion of this 88-week trial in 8 countries (ClinicalTrials.gov, NCT02925117; ongoing, not recruiting), adults with moderate to severe disease and inadequate control by topical treatment were randomized 1:1:1:1, using an interactive response system and stratified geographically, to once-daily upadacitinib oral monotherapy 7.5, 15, or 30 mg or placebo. The primary end point was percentage improvement in Eczema Area and Severity Index from baseline at week 16. Efficacy was analyzed by intention-to-treat in all randomized patients. Safety was analyzed in all randomized patients who received study medication, based on actual treatment. RESULTS: Patients (N = 167) enrolled from November 21, 2016, to April 20, 2017. All were randomized and analyzed for efficacy (each upadacitinib group, n = 42; placebo, n = 41); 166 were analyzed for safety (each upadacitinib group, n = 42; placebo, n = 40). The mean (SE) primary efficacy end point was 39% (6.2%), 62% (6.1%), and 74% (6.1%) for the upadacitinib 7.5-, 15-, and 30-mg groups, respectively, versus 23% (6.4%) for placebo (P = .03, <.001, and <.001). Serious adverse events occurred in 4.8% (2 of 42), 2.4% (1 of 42), and 0% (0 of 42) of upadacitinib groups (vs 2.5% [1 of 40] for placebo). CONCLUSIONS: A dose-response relationship was observed for upadacitinib efficacy; the 30-mg once-daily dose showed the greatest clinical benefit. Dose-limiting toxicity was not observed. Upadacitinib is a Janus kinase 1 inhibitor developed for treatment of moderate to severe rheumatoid arthritis (RA) and was recently approved by the US Food and Drug Administration for this indication in adults who have had an inadequate response or intolerance to methotrexate. Upadacitinib is currently under regulatory review by other agencies around the world. Ongoing trials are investigating the use of upadacitinib in other inflammatory autoimmune diseases. In this article, we review the clinical pharmacokinetic data available to date for upadacitinib that supported the clinical development program in RA and ultimately regulatory applications for upadacitinib in treatment of patients with moderate to severe RA. BACKGROUND & AIMS: We evaluated the efficacy and safety of upadacitinib, an oral selective Janus kinase 1 inhibitor, in a randomized trial of patients with Crohn's disease (CD). METHODS: We performed a double-blind, phase 2 trial in adults with moderate to severe CD and inadequate response or intolerance to immunosuppressants or tumor necrosis factor antagonists. Patients were randomly assigned (1:1:1:1:1:1) to groups given placebo; or 3 mg, 6 mg, 12 mg, or 24 mg upadacitinib twice daily; or 24 mg upadacitinib once daily and were evaluated by ileocolonoscopy at weeks 12 or 16 of the induction period. Patients who completed week 16 were re-randomized to a 36-week period of maintece therapy with upadacitinib. The primary endpoints were clinical remission at week 16 and endoscopic remission at week 12 or 16 using the multiple comparison procedure and modeling and the Cochran-Mantel-Haenszel test, with a 2-sided level of 10%. RESULTS: Among the 220 patients in the study, clinical remission was achieved by 13% of patients receiving 3 mg upadacitinib, 27% of patients receiving 6 mg upadacitinib (P < .1 vs placebo), 11% of patients receiving 12 mg upadacitinib, and 22% of patients receiving 24 mg upadacitinib twice daily, and by 14% of patients receiving 24 mg upadacitinib once daily, vs 11% of patients receiving placebo. Endoscopic remission was achieved by 10% (P < .1 vs placebo), 8%, 8% (P < .1 vs placebo), 22% (P < .01 vs placebo), and 14% (P < .05 vs placebo) of patients receiving upadacitinib, respectively, vs none of the patients receiving placebo. Endoscopic but not clinical remission increased with dose during the induction period. Efficacy was maintained for most endpoints through week 52. During the induction period, patients in the upadacitinib groups had higher incidences of infections and serious infections vs placebo. Patients in the twice-daily 12 mg and 24 mg upadacitinib groups had significant increases in total, high-density lipoprotein, and low-density lipoprotein cholesterol levels compared with patients in the placebo group. CONCLUSIONS: In a phase 2 trial of patients with CD, upadacitinib induced endoscopic remission in a significant proportion of patients compared with placebo. Upadacitinib's benefit/risk profile supports further development for treatment of CD. (Clinicaltrials.gov, Number: NCT02365649). Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily affecting the joints and is associated with significant levels of disability and reduced quality of life. Janus kinase (JAK) inhibitors are a relatively new class of small molecule oral treatments and offer an alternative for patients with RA who do not respond to conventional or biologic therapy. Upadacitinib is a JAK inhibitor engineered to be selective for JAK1, and has recently been approved for use in patients with moderate-to-severe RA. The purpose of this article is to provide a comprehensive review of upadacitinib, including preclinical development and characterization, phase I and II studies, and the phase III SELECT program. Ongoing trials of upadacitinib in additional indications, including spondyloarthritis, inflammatory bowel disease, and atopic dermatitis, are also discussed. This phase 1 study characterized the effect of multiple doses of upadacitinib, an oral Janus kinase 1 selective inhibitor, on the pharmacokinetics of the cytochrome P450 (CYP) 2B6 substrate bupropion. Healthy subjects (n = 22) received a single oral dose of bupropion 150 mg alone (study period 1) and on day 12 of a 16-day regimen of upadacitinib 30 mg once daily (study period 2). Serial blood samples for measurement of bupropion and hydroxybupropion plasma concentrations were collected in each study period. The central values (90% confidence intervals) for the ratios of change were 0.87 (0.79-0.96) for bupropion maximum plasma concentration (Cmax ), 0.92 (0.87-0.98) for bupropion area under the plasma-concentration time curve from time 0 to infinity (AUCinf ), 0.78 (0.72-0.85) for hydroxybupropion Cmax , and 0.72 (0.67-0.78) for hydroxybupropion AUCinf when administered with, relative to when administered without, upadacitinib. After multiple-dose administration of upadacitinib 30 mg once daily, upadacitinib mean ± SD AUC0-24 was 641 ± 177 ng·h/mL, and Cmax was 83.3 ± 30.7 ng/mL. These results confirm that upadacitinib has no relevant effect on pharmacokinetics of substrates metabolized by CYP2B6. Atopic dermatitis is a common, chronic, immune-mediated disease associated with several comorbidities. Elevated levels of T helper (Th)2, Th22, and also some Th1 and Th17 cytokines are found in atopic dermatitis skin lesions. Similar to psoriasis, there is a tendency towards increased use of more targeted therapies. However, there are still several unmet needs in the treatment of atopic dermatitis concerning long-term efficacy, tolerability, safety, route of administration, and cost. The increased knowledge of atopic dermatitis pathogenesis and the role of Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways has allowed the development of new compounds to inhibit this intracellular signaling pathway implicated in atopic dermatitis-related immune responses. Currently, JAK inhibitors are an important focus of therapeutic research for atopic dermatitis. Upadacitinib and abrocitinib are oral small molecules that inhibit the JAK/STAT pathway by selectively blocking JAK1. Data from phase II and III trials are encouraging, revealing that JAK1 inhibitors are effective and well-tolerated agents for moderate-to-severe atopic dermatitis. Selective JAK1 inhibitors may represent an important therapeutic option to be included in the treatment algorithm of atopic dermatitis, owing to oral administration and a favorable safety and tolerability profile. In this article, we review the current evidence on the efficacy and safety of oral selective JAK1 inhibitors for the treatment of atopic dermatitis.

What is known about natriuretic peptide receptor A?

Atrial natriuretic peptide (ANP) and its natriuretic peptide receptors A (NPR-A) and C (NPR-C) are involved in the regulation of physiological and pathophysiological process of blood pressure. The natriuretic peptide receptor A (NPRA), also known as NPR1 or guanylyl cyclase A, binds ANP and BNP to initiate transmembrane signal transduction by elevating the intracellular levels of cyclic guanosine monophosphate.

BACKGROUND: Atrial natriuretic peptide (ANP) and its natriuretic peptide receptors A (NPR-A) and C (NPR-C) are involved in the regulation of physiological and pathophysiological process of blood pressure. The present study aimed to determine the role of NPR-C in the development of salt-sensitive hypertension. METHODS: The Dahl salt-sensitive (DS) and salt-resistant (DR) rats were used in this study. Animals were matched according to their age and weight, and then placed on either a high-salt (HS, 8%) or a normal-salt (NS, 0.4%) diet for 6 weeks randomly using random number table. The systolic blood pressure (SBP), plasmatic sodium concentration (PLNa), urinary sodium excretion (UVNa), and serum creatinine concentration (Scr) were measured. The concentration of ANP in blood and tissues (heart and kidney) was detected by enzyme-linked immunosorbent assay. The expression of ANP, NPR-A, and NPR-C in kidney was evaluated with western blot analysis. Regarding renal redox state, the concentration changes in malondialdehyde (MDA), lipofuscin, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and nitric oxide synthase (NOS) in kidney were detected by a spectrophotometric method. The kidney damage was evaluated using pathological techniques and the succinodehydrogenase (SDHase) examination. Furthermore, after an intra-peritoneal injection of C-atrial natriuretic peptide (ANP)4-23 (C-ANP4-23), an NPR-C receptor agonist, the SBP, biochemical values in blood and urine, and renal redox state were evaluated. The paired Student's t test and analysis of variance followed by the Bonferroni test were performed for statistical analyses of the comparisons between two groups and multiple groups, respectively. RESULTS: The baseline SBP in all groups was within the normal range. At the end of the 6-week experiment, HS diet significantly increased the SBP in DS rats from 116.63 ± 2.90 mmHg to 162.25 ± 2.15 mmHg (t = -10.213, P < 0.001). The changes of SBP were not significant in DS rats on an NS diet and DR rats on an NS diet or on an HS diet (all P > 0.05). The significant increase of PLNa, UVNa, and Scr related to an HS diet was found in both DS and DR rats (all P < 0.05). However, significant changes in the concentration (t = -21.915, P < 0.001) and expression of renal ANP (t = -3.566, P = 0.016) and the expression of renal NPR-C (t = 5.864, P = 0.002) were only observed in DS hypertensive rats. The significantly higher desmin immunochemical staining score (t = -5.715, P = 0.005) and mitochondrial injury score (t = -6.325, P = 0.003) accompanied by the lower SDHase concentration (t = 3.972, P = 0.017) revealed mitochondrial pathologic abnormalities in podocytes in DS rats with an HS diet. The distinct increases of MDA (t = -4.685, P = 0.009), lipofuscin (t = -8.195, P = 0.001), and Nox (t = -12.733, P < 0.001) but not NOS (t = -0.328, P = 0.764) in kidneys were also found in DS hypertensive rats. C-ANP4-23 treatment significantly decreased the SBP induced by HS in DS rats (P < 0.05), which was still higher than NS groups with the vehicle or C-ANP4-23 treatment (P < 0.05). Moreover, the HS-induced increase of MDA, lipofuscin, Nox concentrations, and Nox4 expression in DS rats was significantly attenuated by C-ANP4-23 treatment as compared with those with HS diet and vehicle injection (all P < 0.05). CONCLUSIONS: The results indicated that the renal NPR-C might be involved in the salt-sensitive hypertension through the damage of mitochondria in podocytes and the reduction of the anti-oxidative function. Hence, C-ANP4-23 might serve as a therapeutic agent in treating salt-sensitive hypertension. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are important biological markers and regulators of cardiac function. The natriuretic peptide receptor A (NPRA), also known as NPR1 or guanylyl cyclase A, binds ANP and BNP to initiate transmembrane signal transduction by elevating the intracellular levels of cyclic guanosine monophosphate. However, the effects and mechanisms downstream of NPRA are largely unknown. The aim of the present study was to evaluate the changes in the global pattern of mRNA and circular RNA (circRNA) expression in NPRA‑/‑ and NPRA+/+ myocardium. Differentially expressed mRNA molecules were characterised using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis and were found to be primarily related to metabolic processes. Moreover, circRNA expression was also examined, and a possible competing endogenous RNA network consisting of circRNA, microRNA (miRNA), and mRNA molecules was constructed. The results of this study indicated that NPRA may play a role in cardiac metabolism, which could be mediated by circRNA through endogenous competition mechanisms. These findings may provide insight into future characterisation of various ceRNA network pathways.

What is the mode of action of dexamethasone?

Glucocorticoids like Dexamethasone have a number of modes of action. While these drugs are used to reduce inflammation, Dexamethasone can also induce apoptosis thru initiation of autophagy, activate glucocorticoid receptors in the treatment of uveitic edema, alter gene expression in allergic asthma prevent tachycardia-induced ionic remodeling by reduction of atrial sodium current I(Na), increase gut permeability and suppress inflammation. in addition, Dexamethasone (Dex) can enhance BMP-2-induced osteoblast differentiation and can differentially modulated dendritic cell maturation and TREM1 signaling pathways in GM-CSF-treated and M-CSF-treated monocytes. Dexamethasone can be used for pain management

Reducing reperfusion injury is effective in reducing flap loss after prolonged ischemia. Anti-inflammatory therapy reduces reperfusion injury in canine cardiac muscle and ex vivo rat cremaster muscle; however, to date, there are no studies involving the use of anti-inflammatory agents in ischemic skin flaps. This study was designed to assess the effects of dexamethasone and indomethacin on the viability of rat island groin flaps subjected to 10 hours of ischemia. The ischemic control and the treatment group flaps were subjected to 10 hours of ischemia by clamping the inferior epigastric vascular pedicle. The treatment groups received either intravenous dexamethasone or intravenous indomethacin after the flap vascular pedicles were clamped. Our results showed significant improvement (p < 0.05, Fisher's exact test) in ischemic flap survival using dexamethasone. The specific mode of action of dexamethasone was not investigated; however, its anti-inflammatory effects were most likely responsible for the improvement of flap survival by suppressing the circulating neutrophil and decreasing reperfusion injury. Dexamethasone is easily available for clinical use, and its use should be considered in cases of prolonged ischemia in skin flaps. Despite the known effectiveness of anti-inflammatory therapy in reducing reperfusion injury, no studies to date involve the use of anti-inflammatory therapy in reducing ischemia-reperfusion injury in fasciocutaneous flaps. Dexamethasone (a phospholipase A2 inhibitor) and specific cyclooxygenase and lipoxygenase inhibitors (indomethacin and BW755C) were administered to rats with ischemic island groin (fasciocutaneous) flaps. Significant improvement in ischemic flap survival was found with dexamethasone and BW755C. The mode of action of dexamethasone was not specifically investigated in our study; however, it may suppress neutrophil function and reduce ischemia-reperfusion injury in its shared ability with BW755C to reduce the formation of leukotrienes. Dexamethasone could be applied in the clinical setting to reduce ischemic flap loss by attenuating the systemic inflammatory response to reperfused ischemic-damaged tissue. In order to evaluate the mode of action of dexamethasone (DEX) on macrophage-mediated cytotoxicity and to understand its association with nitric oxide (NO) production, the effect of DEX on macrophage- and spermine NONOate-mediated cytotoxicity was studied. DEX caused 100% inhibition of cytotoxicity by LPS- and IFN gamma-activated macrophages whereas it caused only partial inhibition of NO production. Inhibition of macrophage-mediated cytotoxicity by DEX was not reversed by supplementation of rTNF alpha. The partial inhibition of NO production by DEX was due to partial inhibition of iNOS mRNA expression. Incubation of macrophages with DEX for up to 24 h prior to activation did not cause further inhibition of NO production. DEX failed to inhibit NO production if added 6 h after addition of LPS and IFN gamma. Addition of P815 cells after the onset of NO production resulted in partial restoration of cytotoxicity in DEX-treated macrophages. Incubation of P815 cells with spermine NONOate, a synthetic NO donor, resulted in P815 cell lysis, which was dose-dependent, had a lag phase of 3 h and was blocked by hemoglobin. DEX also inhibited spermine NONOate-mediated tumor cell lysis, indicating that DEX may have a protective effect on tumor targets. These results indicate that DEX inhibits macrophage-mediated cytotoxicity by decreasing NO production and by inhibiting the cytotoxic effects of NO on the target cells. Glucocorticoids have been shown repeatedly to inhibit the release of prolactin (PRL) in the rat but their site and mode of action is unknown. In the present study, we used an in vitro model to examine the requirement for protein synthesis for dexamethasone to suppress the release of immunoreactive (ir)-PRL release from the rat pituitary gland. In addition we have performed a series of in vitro and in vivo experiments to investigate the potential role in this regard of lipocortin 1 (LC1), a protein shown previously not only to mediate aspects of the anti-inflammatory and anti-proliferative actions of the glucocorticoids but also to contribute to the regulatory actions of the steroids in the brain-neuroendocrine system. In vitro, the release of ir-PRL from rat anterior pituitary tissue initiated by submaximal concentrations of VIP (10 nM). TRH (10 nM) or the adenyl cyclase activator forskolin (100 microM) was reduced significantly (p < 0.01) by preincubation (2 h) of the tissue with dexamethasone (0.1 microM). By contrast, ir-PRL release evoked by a submaximal concentration of the L-Ca2+ channel opener BAY K8644 (10 microM) was unaffected by the steroid although readily antagonised (p < 0.01) by nifedipine (1-100 microM). Exposure of the pituitary tissue to dexamethasone (0.1 microM) also caused a pronounced and highly significant increase in de novo protein synthesis, as assessed by the incorporation of 14C-lysine into the tissue (p < 0.001). This response was reduced markedly by the inclusion of the RNA and protein synthesis inhibitors, actinomycin-D (0.5 micrograms/ml) or cycloheximide (1.0 micrograms/ml), in the incubation medium (p < 0.001), both of which also effectively abrogated (p < 0.01) the dexamethasone-induced inhibition of the release of ir-PRL evoked by TRH. VIP and forskolin. Lipocortin I was readily detectable by Western blotting in protein extracts of freshly excised anterior pituitary tissue: a small proportion of the protein was found to be attached to the outer surface of the cells where it was retained by a Ca(2+)-dependent mechanism. Exposure of the tissue in vitro to dexamethasone (0.1 microM) or corticosterone (0.1 microM) but not 17 beta-oestradiol (0.1 microM) caused a pronounced increase in the amount of LC1 attached to the outer surface of the cells and concomitant decrease in the LC1 content of the intracellular LC1 pool. Addition of an N-terminal LC1 fragment. LC11-188 (10 pg-10 ng/ml), to the incubation medium reduced significantly (p < 0.01) the increases in ir-PRL release induced in vitro by VIP (10 nM) and forskolin (100 microM). By contrast, at all concentrations tested. LC11-188 (10 pg-10 ng/ml) failed to influence (p < 0.05) the highly significant (p < 0.01) ir-PRL response to TRH (10 nM). Similarly, the inhibitory actions of dexamethasone (0.1 microM) on the release of ir-PRL induced by VIP (10 nM) or forskolin (100 microM) but not by TRH (10 nM) were substantially reversed (p < 0.01) by a specific monoclonal anti-LC1 antibody while an isotype-matched control antibody was without effect. In vivo, rats pretreated with either a polyclonal anti LC1 antiserum (anti-LC1 pAb, 1 ml/day s.c. for 2 days) or a corresponding volume of non-immune sheep serum (NSS) responded to stress (laparotomy under ether anaesthesia) with significant (p < 0.05) increases in the serum ir-PRL concentration. In the NSS-treated group, the ir-PRL response to stress was effectively inhibited by dexamethasone (100 micrograms/kg i.p.) which had no effect on the pre-stress serum ir-PRL concentration. By contrast, in rats pretreated with anti-LC1 pAb dexamethasone failed to block the stress-induced release of ir-PRL. The results show clearly that the inhibitory actions of dexamethasone on PRL release are dependent on de novo protein synthesis and provide novel evidence for the involvement of both LC1-dependent and LC1-independent mechanisms. Glucocorticoids (GCs) influence a great variety of cellular functions by at least three important modes of action: the activation (or repression) of genes controlled by binding sites for the glucocorticoid receptor (GR), the induction of apoptosis in lymphocytes and the recently discovered cross-talk to other transcription factors such as NF-kappaB. In this study we systematically compared various natural and synthetic steroid hormones frequently used as therapeutic agents on their ability to mediate these three modes of action. Betamethasone, triamcinolone, dexamethasone and clobetasol turned out to be the best inducers of gene expression and apoptosis. All GCs including the antagonistic compound RU486 efficiently reduced NF-kappaB-mediated transactivation to comparable extents, suggesting that ligand-induced nuclear localization of the GR is sufficient for transrepression. Glucocorticoid treatment of cells did not result in elevated IkappaB-alpha expression, but impaired the tumor necrosis factor (TNF)-alpha-induced degradation of IkappaB-alpha without affecting DNA binding of NF-kappaB. The structural requirements for the various functions of glucocorticoids are discussed. Restenosis after stent implantation is mainly characterized by an inflammatory response to the procedural injury and an intense fibrocellular response including smooth muscle cell (SMC) proliferation. After angioplasty alone, the restenosis process also involves thrombus formation and negative remodeling. Due to the pleiotropic mode of action exerted by glucocorticoids which include profound anti-inflammatory and immunosuppressive effects, direct inhibition on SMC proliferation and apoptosis, their potential in the prevention of restenosis has gained widespread interest. Over the last decade, preclinical and clinical data have not been able to conclusively document a robust therapeutic effect on restenosis after angioplasty or stent implantation. Only recently, preclinical data and limited observations in humans using drug eluting stents for local drug delivery have suggested beneficial effects of dexamethasone on neointimal proliferation. Randomized clinical trials using local drug delivery are expected to start in the near future. In the light of these ongoing developments, this review summarizes the pathophysiological basis of glucocorticoid action in the context of restenosis, provides an overview of the animal data available and discusses the clinical results that have been gathered over the last decade with particular emphasis on dexamethasone. PURPOSE: Atrial fibrillation (AF) results in tachycardia-induced ionic remodeling. Pharmacological prevention of tachycardia-induced ionic remodeling not only with "classical" antiarrhythmics but also with drugs which provide a basis for some of the pillars of the so-called "upstream" therapy of AF like corticosteroids or statins has been proposed as a therapeutic strategy. Amongst other ion currents, atrial sodium current I(Na) and its tachycardia-induced alterations play an important role in AF pathophysiology. Thus, effects of a dexamethasone (DT) and atorvastatin treatment (AT) on atrial sodium current I(Na) and its tachycardia-induced remodeling were studied in a rabbit model. METHODS: 9 groups with 4 animals were examined. Atrial pacemaker leads were implanted in all animals. No rapid atrial pacing (600/min) was performed in the control group but for 24 or 120 hours in the respective pacing groups. Instrumentation and pacing did not differ from the respective drug groups but an additional treatment with dexamethasone or atorvastatin (7 days) was performed. RESULTS: Rapid atrial pacing (RAP, 600/min) reduced I(Na) after 24 hours (≈ -50%) with no further reduction after 120 hours. DT reduced I(Na) (≈ -20%), current densities in consecutively tachypaced animals did not differ from those in untreated animals. AT reduced INa similar as RAP, subsequent RAP did not further diminish I(Na). CONCLUSIONS: Impact of corticosteroids and statins on INa and its tachycardia-induced alterations also contribute to the mode of action of these substances in upstream treatment of atrial fibrillation. Multimodal pain management, combining analgesics with different mode of action in order to minimize occurrence of side-effects still providing safe and efficacious pain management after ambulatory surgery has become standard of care. The combined use of local anaesthesia in order to reduce noxious influx during the procedure and reduce postoperative pain is strongly recommended whenever feasible. Providing oral analgesics paracetamol, and none-steroid anti-inflammatory drugs or selective Cox-II-inhibitors already prior to induction in order to provide effective therapeutic concentrations at end of surgery is a simple and easy way to facilitate the recovery. Single iv. preoperative dose dexamethasone has been shown not only to be effective in reducing postoperative nausea and vomiting but also to improve recovery reduce pain and improve satisfaction. Pregabalin may be used in order to further enhance the recovery and pain management. Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415-23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1β, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects. Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424-102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac treatment on lipids were orthogonal to the effect of dexamethasone underlining differences in the overall mode of action. Heterotopic ossification (HO) consists of ectopic cartilage and bone formation following severe trauma or invasive surgeries, and a genetic form of it characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Recent mouse studies showed that HO was significantly inhibited by systemic treatment with a corticosteroid or the retinoic acid receptor γ agonist Palovarotene. Because these drugs act differently, the data raised intriguing questions including whether the drugs affected HO via similar means, whether a combination therapy would be more effective or whether the drugs may hamper each other's action. To tackle these questions, we used an effective HO mouse model involving subcutaneous implantation of Matrigel plus rhBMP2, and compared the effectiveness of prednisone, dexamathaosone, Palovarotene or combination of. Each corticosteroid and Palovarotene reduced bone formation at max doses, and a combination therapy elicited similar outcomes without obvious interference. While Palovarotene had effectively prevented the initial cartilaginous phase of HO, the steroids appeared to act more on the bony phase. In reporter assays, dexamethasone and Palovarotene induced transcriptional activity of their respective GRE or RARE constructs and did not interfere with each other's pathway. Interestingly, both drugs inhibited the activity of a reporter construct for the inflammatory mediator NF-κB, particularly in combination. In good agreement, immunohistochemical analyses showed that both drugs markedly reduced the number of mast cells and macrophages near and within the ectopic Matrigel mass and reduced also the number of progenitor cells. In sum, corticosteroids and Palovarotene appear to block HO via common and distinct mechanisms. Most importantly, they directly or indirectly inhibit the recruitment of immune and inflammatory cells present at the affected site, thus alleviating the effects of key HO instigators. Although glucocorticoids (GCs) are a mainstay in the clinical management of asthma, the target cells that mediate their therapeutic effects are unknown. Contrary to our expectation, we found that GC receptor (GR) expression in immune cells was dispensable for successful therapy of allergic airway inflammation (AAI) with dexamethasone. Instead, GC treatment was compromised in mice expressing a defective GR in the nonhematopoietic compartment or selectively lacking the GR in airway epithelial cells. Further, we found that an intact GR dimerization interface was a prerequisite for the suppression of AAI and airway hyperresponsiveness by GCs. Our observation that the ability of dexamethasone to modulate gene expression in airway epithelial cells coincided with its potency to resolve AAI supports a crucial role for transcriptional regulation by the GR in this cell type. Taken together, we identified an unknown mode of GC action in the treatment of allergic asthma that might help to develop more specific therapies in the future. Glucocorticoids (GCs) are potent anti-inflammatory drugs whose mode of action is complex and still debatable. One likely cellular target of GCs are monocytes/macrophages. The role of GCs in monocyte survival is also debated. Although both granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) are important regulators of macrophage lineage functions including their survival, the former is often associated with proinflammatory functions while the latter is important in lineage homeostasis. We report here that the GC, dexamethasone, induces apoptosis in GM-CSF-treated human monocytes while having no impact on M-CSF-induced monocyte survival. To understand how GCs, GM-CSF, and M-CSF are regulating monocyte survival and other functions during inflammation, we firstly examined the transcriptomic changes elicited by these three agents in human monocytes, either acting alone or in combination. Transcriptomic and Ingenuity pathway analyses found that dexamethasone differentially modulated dendritic cell maturation and TREM1 signaling pathways in GM-CSF-treated and M-CSF-treated monocytes, two pathways known to be regulated by ERK1/2 activity. These analyses led us to provide evidence that the GC inhibits ERK1/2 activity selectively in GM-CSF-treated monocytes to induce apoptosis. It is proposed that this inhibition of ERK1/2 activity leads to inactivation of p90 ribosomal-S6 kinase and Bad dephosphorylation leading in turn to enhanced caspase-3 activity and subsequent apoptosis. Furthermore, pharmacological inhibition of GC receptor activity restored the ERK1/2 signaling and prevented the GC-induced apoptosis in GM-CSF-treated monocytes. Increased tissue macrophage numbers, possibly from enhanced survival due to mediators such as GM-CSF, can correlate with inflammatory disease severity; also reduction in these numbers can correlate with the therapeutic benefit of a number of agents, including GCs. We propose that the ERK1/2 signaling pathway promotes survival of GM-CSF-treated proinflammatory monocytes, which can be selectively targeted by GCs as a novel mechanism to reduce local monocyte/macrophage numbers and hence inflammation. The development of novel bioactive biomaterials is urgently needed to meet the needs of an aging population. Both sulfated hyaluronic acid and dexamethasone are candidates for the functionalization of bone grafts, as they have been shown to enhance the differentiation of osteoblasts from bone marrow stromal cells in vitro and in vivo. However, the underlying mechanisms are not fully understood. Furthermore, studies combining different approaches to assess synergistic potentials are rare. In this study, we aim to gain insights into the mode of action of both sulfated hyaluronic acid and dexamethasone by a comprehensive analysis of the cellular fraction, released matrix vesicles, and the extracellular matrix, combining classical biochemical assays with mass spectrometry-based proteomics, supported by novel bioinformatical computations. We found elevated differentiation levels for both treatments, which were further enhanced by a combination of sulfated hyaluronic acid and dexamethasone. Single treatments revealed specific effects on osteogenic differentiation. Dexamethasone activates signalling pathways involved in the differentiation of osteoblasts, for example, CXC-motif chemokine receptor type 4 and mitogen-activated protein kinases. The effects of sulfated hyaluronic acid were predomitly linked to an alteration in the composition of the extracellular matrix, affecting the synthesis, secretion, and/or activity of fibrillary (fibronectin and thrombospondin-2) and nonfibrillary (transglutaminase-2, periostin, and lysyloxidase) extracellular matrix components, including proteases and their inhibitors (matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-3). The effects were treatment specific, and less additive or contrary effects were found. Thus, we anticipate that the synergistic action of the treatment-specific effects is the key driver in elevated osteogenesis. Dexamethasone (Dex), a synthetic glucocorticoid (GC), in feed has been shown to increase gut permeability via stress-mediated mechanisms, but the exact mode of action on gut barrier function is not fully understood. Stress has been reported to alter the profile and virulence of intestinal flora predisposing for opportunistic disease. This study aimed to evaluate the relationship between dietary Dex and recoverable intestinal microbial profile in broilers to better understand mode of action and refine future uses of the model. Three experiments were conducted that administered Dex-treated feed for one week in conjunction with the antibiotics BMD (bacitracin methylene disalicylate) or Baytril® (enrofloxacin) to evaluate if enteric microbial mechanisms were important in Dex-induced permeability. Serum fluorescein isothiocyanate-dextran (FITC-d) and bacterial translocation (BT) have been reported to increase after Dex treatment and were used to assess gut epithelial leakage. Shifts in bacterial profiles were also measured on selective agar. Combining Dex with BMD or Baytril resulted in increased (P < 0.05) serum FITC-d versus Dex-only. Additionally, Baytril did not reduce aerobic BT and bacterial profiles remained similar after Dex. These results suggest a minimal role of intestinal microbes in Dex-induced changes to intestinal barrier function. Glucocorticoids (GCs) are widely used to treat acute graft-versus-host disease (aGvHD) due to their immunosuppressive activity, but they also reduce the beneficial graft-versus-leukemia (GvL) effect of the allogeneic T cells contained in the graft. Here, we tested whether aGvHD therapy could be improved by delivering GCs with the help of inorganic-organic hybrid oparticles (IOH-NPs) that preferentially target myeloid cells. IOH-NPs containing the GC betamethasone (BMP-NPs) efficiently reduced morbidity, mortality, and tissue damage in a totally MHC mismatched mouse model of aGvHD. Therapeutic activity was lost in mice lacking the GC receptor (GR) in myeloid cells, confirming the cell type specificity of our approach. BMP-NPs had no relevant systemic activity but suppressed cytokine and chemokine gene expression locally in the small intestine, which presumably explains their mode of action. Most importantly, BMP-NPs delayed the development of an adoptively transferred B cell lymphoma better than the free drug, although the overall incidence was unaffected. Our findings thus suggest that employing IOH-NPs could diminish the risk of relapse associated with GC therapy of aGvHD patients while still allowing to efficiently ameliorate the disease. Bone morphogenetic protein-2 (BMP-2) is considered one of the most effective and extensively used growth factors to induce osteoblast differentiation and accelerate bone regeneration. Dexamethasone (Dex) with suitable dosage can enhance BMP-2-induced osteoblast differentiation. To strengthen this synergistic osteoinductive effect, a pH-responsive chitosan-functionalized mesoporous silica oparticle (chi-MSN) ensemble was fabricated for dual-delivery of BMP-2 and Dex. The MSNs are prepared by a CTAB-templated sol-gel method, and further coated by chitosan via the crosslinking of glycidoxypropyltrimethoxysilane (GPTMS). The small Dex is encapsulated in the mesopores and the large BMP-2 is incorporated into the chitosan coating. These chi-MSNs can quickly release BMP-2 in a bioactive form and can then be efficiently endocytosed and further realize a controlled release of Dex with the decreased pH value into/in cells. With the synergistic action of BMP-2 and Dex outside and inside the cell, this dual hybrid delivery system can significantly stimulate osteoblast differentiation and bone regeneration in vitro and in vivo. Together, this dual-delivery strategy for osteogenic protein delivery may enhance clinical outcomes by retaining the bioactivity and optimizing the release mode of the drug/protein.

What is the phenomenon described as "complex coacervation"?

Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature (TC).

Coacervation was defined as the phenomenon in which a colloidal dispersion separated into colloid-rich (the coacervate), and colloid-poor phases, both with the same solvent. Complex coacervation covered the situation in which a mixture of two polymeric polyions with opposite charge separated into liquid dilute and concentrated phases, in the same solvent, with both phases, at equilibrium, containing both polyions. Voorn and Overbeek provided the first theoretical analysis of complex coacervation by applying Flory-Huggins polymer statistics to model the random mixing of the polyions and their counter ions in solution, assuming completely random mixing of the polyions in each phase, with the electrostatic free energy, ΔG(elect), providing the driving force. However, experimentally complete randomness does not apply: polyion size, heterogeneity, chain stiffness and charge density (σ) all affect the equilibrium phase separation and phase concentrations. Moreover, in pauci-disperse systems multiple phases are often observed. As an alternative, Veis and Aranyi proposed the formation of charge paired Symmetrical Aggregates (SA) as an initial step, followed by phase separation driven by the interaction parameter, χ(23), combining both entropy and enthalpy factors other than the ΔG(elect) electrostatic term. This two stage path to equilibrium phase separation allows for understanding and quantifying and modeling the diverse aggregates produced by interactions between polyampholyte molecules of different charge density, σ, and intrinsic polyion structure. Complex coacervation is an associative liquid/liquid phase separation resulting in the formation of two liquid phases: a polymer-rich coacervate phase and a dilute continuous solvent phase. In the presence of a third liquid phase in the form of disperse oil droplets, the coacervate phase tends to wet the oil/water interface. This affinity has long been known and used for the formation of core/shell capsules. However, while encapsulation by simple or complex coacervation has been used empirically for decades, there is a lack of a thorough understanding of the three-phase wetting phenomena that control the formation of encapsulated, compound droplets and the role of the viscoelasticity of the biopolymers involved. In this contribution, we review and discuss the interplay of wetting phenomena and fluid viscoelasticity in coacervate/oil/water systems from the perspective of colloid chemistry and fluid dynamics, focusing on aspects of rheology, interfacial tension measurements at the coacervate/solvent interface, and on the formation and fragmentation of three-phase compound drops. There has been a resurgence of interest in complex coacervation, a form of liquid-liquid phase separation (LLPS) in systems of oppositely charged macroions, but very few reports describe the somewhat anomalous coacervation between acidic and basic proteins, which occurs under very narrow ranges of conditions. We sought to identify the roles of equilibrium interprotein complexes during the coacervation of β-lactoglobulin dimer (BLG2) with lactoferrin (LF) and found that this LLPS arises specifically from LF(BLG2)2. We followed the progress of complexation and coacervation as a function of r, the LF/BLG molar ratio, using turbidity to monitor the degree of coacervation and proton release and dynamic light scattering (DLS) to assess the stoichiometry and abundance of complexes. Isothermal titration calorimetry (ITC) showed that initial complex formation is endothermic, but a large exotherm related to coacervate formation obscured other regions. On the basis of turbidimetry, proton release, and DLS, we propose a speciation diagram that presents the abundance of various complexes as a function of r. Although multiple species could be simultaneously present, distinct regions could be identified corresponding to equilibria among particular protein pairs. Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles. Oppositely charged polymers can undergo the process of complex coacervation, which refers to a liquid-liquid phase separation driven by electrostatic attraction. These materials have demonstrated considerable promise as the basis for complex, self-assembled materials. In this review, we provide a broad overview of the theoretical tools used to understand the physical properties of polymeric coacervates. In particular, we discuss historic theories (Voorn-Overbeek, Random Phase Approximation), and then describe recent developments in the field (Field Theoretic, Counterion Release, Molecular Simulation, and Polymer Reference Interaction Site Model methods). We provide context for these methods, and map out the patchwork of theoretical models that are used to describe a diverse array of coacervate systems. We use this review of the literature to clarify a number of important theoretical challenges remaining in our physical understanding of complex coacervation. We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid-liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature (TC). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles. Complex coacervation is an emerging liquid/liquid phase separation (LLPS) phenomenon that behaves as a membrane-less organelle in living cells. Yet while one of the critical factors for complex coacervation is temperature, little analysis and research has been devoted to the temperature effect on complex coacervation. Here, we performed a complex coacervation of cationic protamine and multivalent anions (citrate and tripolyphosphate (TPP)). Both mixtures (i.e., protamine/citrate and protamine/TPP) underwent coacervation in an aqueous solution, while a mixture of protamine and sodium chloride did not. Interestingly, the complex coacervation of protamine and multivalent anions showed upper critical solution temperature (UCST) behavior, and the coacervation of protamine and multivalent anions was reversible with solution temperature changes. The large asymmetry in molecular weight between positively charged protamine (~4 kDa) and the multivalent anions (<0.4 kDa) and strong electrostatic interactions between positively charged guanidine residues in protamine and multivalent anions were likely to contribute to UCST behavior in this coacervation system. Underwater adhesion represents a huge technological challenge as the presence of water compromises the performance of most commercially available adhesives. Inspired by natural organisms, we have designed an adhesive based on complex coacervation, a liquid-liquid phase separation phenomenon. A complex coacervate adhesive is formed by mixing oppositely charged polyelectrolytes bearing pendant thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) chains. The material fully sets underwater due to a change in the environmental conditions, namely temperature and ionic strength. In this work, we incorporate silica oparticles forming a hybrid complex coacervate and investigate the resulting mechanical properties. An enhancement of the mechanical properties is observed below the PNIPAM lower critical solution temperature (LCST): this is due to the formation of PNIPAM-silica junctions, which, after setting, contribute to a moderate increase in the moduli and in the adhesive properties only when applying an ionic strength gradient. By contrast, when raising the temperature above the LCST, the mechanical properties are dominated by the association of PNIPAM chains and the ofiller incorporation leads to an increased heterogeneity with the formation of fracture planes at the interface between areas of different concentrations of oparticles, promoting earlier failure of the network-an unexpected and noteworthy consequence of this hybrid system. Complex coacervation is an associative, liquid-liquid phase separation that can occur in solutions of oppositely-charged macromolecular species, such as proteins, polymers, and colloids. This process results in a coacervate phase, which is a dense mix of the oppositely-charged components, and a supernatant phase, which is primarily devoid of these same species. First observed almost a century ago, coacervates have since found relevance in a wide range of applications; they are used in personal care and food products, cutting edge biotechnology, and as a motif for materials design and self-assembly. There has recently been a renaissance in our understanding of this important class of material phenomena, bringing the science of coacervation to the forefront of polymer and colloid science, biophysics, and industrial materials design. In this review, we describe the emergence of a number of these new research directions, specifically in the context of polymer-polymer complex coacervates, which are inspired by a number of key physical and chemical insights and driven by a diverse range of experimental, theoretical, and computational approaches. Multivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluate the impact of lower, more prebiotically-relevant, polyion multivalency on the functional performance of coacervates as compartments. Positively and negatively charged homopeptides with 1-100 residues and adenosine mono-, di-, and triphosphate nucleotides are used as model polyions. Polycation/polyanion pairs are tested for coacervation, and resulting membraneless compartments are analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes than those formed by longer polyions. Hence, coacervates formed by reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, they can outperform higher molecular weight analogues.

Which proteins does RG-7992 target?

BFKB8488A is a bispecific antibody against FGFR1 and KLB.

Is there a role for TFII-I in megakaryopoiesis?

Yes. TFII-I acts as a repressor of β-globin gene transcription and is implicated in the differentiation of erythrocytes into megakaryopoiesis. Mutations in exon 2 interfere with the synthesis of the full-length isoform of TF II-I and lead to the production of a shortened isoform, TFII, in erythroid cells. TF2-I has a role in embryonic development and differentiation of all eukaryotes but its physiological function is still unclear.

TFII-I is a ubiquitously expressed transcription factor that positively or negatively regulates gene expression. TFII-I has been implicated in neuronal and immunologic diseases as well as in thymic epithelial cancer. Williams-Beuren Syndrome (WBS) is caused by a large hemizygous deletion on chromosome 7q11.23 which encompasses 26-28 genes, including GTF2I, the human gene encoding TFII-I. A subset of WBS patients has recently been shown to present with macrocytosis, a mild anemia characterized by enlarged erythrocytes. We conditionally deleted the TFII-I/Gtf2i gene in adult mice by tamoxifen induced Cre-recombination. Bone marrow cells revealed defects in erythro-megakaryopoiesis and an increase in expression of the adult β-globin gene. The data show that TFII-I acts as a repressor of β-globin gene transcription and that it is implicated in the differentiation of erythro-megakaryocytic cells.

Which drugs are included in the VAC regiment for Ewing's sarcoma?

VAC regiment for Ewing's sarcoma includes vincristine, actinomycin, cyclophosphamide.

Vincristine, actinomycin D, and cyclophosphamide (VAC) were administered to 14 patients with Ewing's sarcoma. The primary tumors were treated with radiation therapy and concurrent chemotherapy. Nine patients had no visible metastases at diagnosis: two died following the development of pulmonary metastases and the rest have been free of disease for periods varying from 4 months to 4 1/2 years following completion of treatment. This contrasts with a 27% survival in patients previously treated at this center with single agent chemotherapy. Five other patients had demonstrable metastases at diagnosis: VAC chemotherapy achieved complete regression of pulmonary metastases in three for 9, 9+ and 24+ months, respectively. Following disappearance of tumor in the latter two, pulmonary irradiation was administered in an attempt to consolidate the response, but tumor recurred 6 months later. These patients eventually died of widespread disease although survival appeared prolonged in comparison to that seen in past experience. Chemotherapy was well tolerated, although three patients developed hemorrhagic cystitis, necessitating discontinuation of cyclophosphamide. The data suggest the potential for prolonged control and an increase in the cure rate with this therapeutic approach. Soft tissue sarcomas of the paraspinal region comprised 3.3% (56 of 1,688) of the patients entered and eligible on Intergroup Rhabdomyosarcoma Studies I (IRS-I) and II (IRS-II) (1972 to 1984). These lesions tended to be greater than 5 cm in diameter at diagnosis, invaded the spinal extradural space, and were of the extraosseous Ewing's sarcoma or undifferentiated sarcoma subtype in 55% (30 of 56) of the cases. Patients with tumors in clinical groups II, III, and IV were treated with radiotherapy (XRT) and vincristine-dactinomycin (VA) or VA plus cyclophosphamide (VAC) +/- doxorubicin. Clinical group I patients treated on IRS-II did not receive XRT, while those on IRS-I were randomized to receive VAC +/- XRT. Forty-four of the paraspinal patients (79%) achieved a complete response (CR) compared with 77% (1,260 of 1,632) for patients with disease in other sites. Twenty-seven patients (55%) subsequently relapsed (five local, three regional, four local and distant, and 14 distant). The proportion of patients surviving 5 years by clinical group (stage) from I to IV were 50%, 50%, 62%, and 27%, respectively. Paraspinal patients had somewhat poorer survival than patients with disease in other sites, both in IRS-I and IRS-II; the percentage of paraspinal patients surviving 5 years was 50% and 52% for IRS-I and IRS-II, respectively, whereas these percentages were 55% and 63% for patients with disease in other sites. Histology did not influence the CR rate, but unexpectedly, patients who had embryonal rhabdomyosarcoma (RMS) had the poorest overall survival rate. We concluded that patients with paraspinal lesions may require extended-field radiation therapy to reduce the high local failure rate and more intensive chemotherapy to achieve better local and systemic tumor control. Phase II studies using ifosfamide both alone and combined with vindesine and cisplatin have shown the effectiveness of this drug in patients with Ewing's sarcoma (ES) who had relapsed during VAC (vincristine, actinomycin, cyclosphosphamide)/VAd (vincristine, Adriamycin) therapy. In November 1984, these results led the SFOP to adopt a protocol consisting of (1) initial chemotherapy with three cycles of IVA (ifosfamide, 3 g/m2 on days 1 and 2; actinomycin D, 750 mg/m2 on days 1-3; vincristine, 1.5 mg/m2 on day 1) alternating every 3 weeks with IVAd (vincristine on day 22; ifosfamide on days 21-23; Adriamycin, 60 mg/m2 on day 22); (2) radical surgery if possible; (3) local radiotherapy (RT); and (4) maintece chemotherapy with alternating IVA and VAd (vincristine, Adriamycin) for up to 9 months. In May 1987, 87 patients with previously untreated ES entered the study; 61 had localized ES. To date, 54 patients with localized disease and 22 with metastatic disease have finished initial chemotherapy; 40 patients with localized disease have been evaluated. In all, 28 patients (70%) were in complete remission (17 patients) or had a tumor regression of greater than 50% 11 patients) and were considered to be good responders; 12 patients were considered to be poor responders. After local radiotherapy in all but 7 patients and surgical resection in 29, 52 of 54 were considered to be in clinical remission. A total of 13 patients with metastatic disease were good responders at the completion of the initial chemotherapy. These results confirm the efficacy of primary chemotherapy using ifosfamide for the treatment of ES. A randomized study of 264 children and adults with previously untreated localized Ewing's sarcoma of bone was undertaken between 1973 and 1978 by 83 institutions of three national study groups: Children's Cancer Study Group, Southwest Oncology Group, and Cancer and Leukemia Group B. The Intergroup Study was designed to determine if the addition of adriamycin (ADR) or bilateral pulmonary radiotherapy (RT) to vincristine, dactinomycin, and cyclophosphamide (VAC therapy) would improve survival and reduce local recurrences and metastases. All patients received RT to the primary lesion, and the survival rate after 3 years was 65%. The most effective treatment regimen was VAC plus ADR; 74% of the patients were free of disease at 2 years. The lengths of disease-free status and survival of patients treated with VAC plus ADR or VAC plus RT did not differ. However, both regimens were significantly superior to treatment with VAC alone. The addition of ADR or bilateral pulmonary RT to VAC was highly advantageous to patients with nonpelvic primaries. Bone and lung were the major sites of distant relapse, but the addition of bilateral pulmonary RT showed no advantage over that of ADR in reducing the occurrence of lung metastases. These recent results should eliminate some of the pessimism that has accompanied a diagnosis of Ewing's sarcoma, although distant metastases continued to be a major reason for failure in the control of this tumor. Survival of these patients can be improved through well-controlled clinical trials designed to determine optimal adjuvant chemotherapy and treatment of the primary lesion. PURPOSE: One hundred thirty of 2,792 patients (5%) registered on three Intergroup Rhabdomyosarcoma Study clinical trials (IRS-I, -II, and -III) from 1972 to 1991 had an extraosseous Ewing's sarcoma (EOE). We report here the results of multimodality therapy for this tumor. PATIENTS AND METHODS: The 130 patients were less than 21 years of age; 70 (54%) were males. Primary tumor sites were on the trunk in 41 patients, an extremity in 34, the head/neck in 23, the retroperitoneum/pelvis in 21, and other sites in 11. One hundred fourteen patients had no metastases at diagnosis. In 21 patients, the tumor was completely resected; in 30, the localized or regional tumor was grossly resected, and in 63 patients, grossly visible sarcoma was left behind. Sixteen patients (12%) had distant metastases at diagnosis. All patients were given multiagent chemotherapy and most received irradiation (XRT); none were treated with bone marrow transplantation. RESULTS: One hundred seven patients (82%) achieved a complete response. At 10 years, 62%, 61%, and 77% of the patients were alive after treatment on IRS-I, IRS-II, or IRS-III therapeutic protocols, respectively, similar to figures obtained in all IRS patients. At last follow-up evaluation, 42 patients had died of progressive tumor and one of infection. Survival at 10 years was most likely for patients with tumor that arose in the head and neck, extremities, and trunk, and for those who underwent grossly complete tumor removal before initiation of chemotherapy. For patients with localized, gross residual tumor, adding doxorubicin (DOX) to the combination of vincristine, dactinomycin, cyclophosphamide (VAC), and XRT did not significantly improve survival in 39 patients (62% alive at 10 years) compared with that of 24 patients treated with VAC and XRT without DOX (65% alive at 10 years, P = .93). CONCLUSION: This series indicated that EOE in children is similar to rhabdomyosarcoma (RMS) in its response to multimodal treatment. No benefit was apparent from the addition of DOX to VAC chemotherapy in patients with gross residual EOE. BACKGROUND: Older age and axial location of Ewing's sarcoma have been reported as unfavorable prognostic factors. METHODS: The records of patients older than 15 years with the Ewing's family of tumors were reviewed retrospectively. After the induction chemotherapy consisting of alternating vincristine, adriablastin, cyclophosphamide (VAC) and etoposide, ifosfamide with mesna protection (IE), a local treatment modality was chosen based on tumor and patient characteristics. RESULTS: Twenty-five patients with a median age of 19 years were evaluated. Median follow-up was 26 months (range 4-58). Seventeen patients (68%) had died. In univariate analysis, factors predictive of shorter survival were the patients presenting with metastatic disease, with the primary tumor located at the pelvis, those who never achieved complete response to chemotherapy and those who had chemotherapy for <12 months. Only a negative link with pelvic location was observed in multivariate analysis [risk ratio 7.5; 95% confidence interval (CI) 1.52-37.06; P = 0.0134]. Median progression-free survival (PFS) and overall survival (OS) were 10 months (95% CI 6.2-13.8) and 14 months (95% CI 9.3-18.7), respectively. Cumulative 2-year PFS and OS were 19.0% (95% CI, SD +/-8.4) and 32.7% (95% CI, SD +/-9.8), respectively. CONCLUSIONS: The prognosis of patients with axial Ewing's sarcoma is dismal despite an intensive, multimodality approach including multiagent, alternating chemotherapy, surgery and/or radiotherapy. A more aggressive approach should be considered for this group of Ewing's sarcoma patients. Files of 133 children with Ewing sarcoma (median age 10 years) were reviewed. Frequent primary sites were extremities, trunk, pelvis, and cranium. Half of 43 patients with metastases had disease in the lungs. Ten-year overall and event-free survival rates were 31% and 19%, respectively. Five-year overall survival rates were 42% in localized and 15% in metastatic disease (p < .0001); 66% in cases with primary tumors < 8 cm and 29% in larger tumors (p = .013). VAC (vincristine, actinomycin D, and cyclophosphamide) regimens with anthracyclines resulted in better survival. Presence of distant metastases, large primary tumors, and pelvic localization were related to poor prognosis. Novel therapeutic approaches are needed to produce better results, especially in high-risk patients. BACKGROUND: Filgrastim is an effective granulocyte colony-stimulating factor (G-CSF) used to reduce periods of neutropenia and the risk of infection after chemotherapy courses. Pegfilgrastim is a pegylated filgrastim with a longer plasma half-life that is administered once per cycle. OBJECTIVE: The aim of this study was to compare the efficacy of pegfilgrastim and filgrastim administered after chemotherapy in children with Ewing sarcoma. METHODS: We performed a retrospective chart review of pediatric patients with Ewing sarcoma. Every patient received both types of G-CSF in different treatment courses of chemotherapy, which consisted of vincristine, ifosfamide, doxorubicin, and etoposide (VIDE); vincristine, actinomycin D, and ifosfamide (VAI); or vincristine, actinomycin D, and cyclophosphamide (VAC). A single injection of pegfilgrastim 100 microg/kg SC or a daily injection of filgrastim 5 to 10 microg/kg SC was administered 48 to 72 hours after the completion of chemotherapy. The following data were collected from the medical charts: proportion of chemotherapy courses with grade 4 neutropenia, duration of grade 4 neutropenia, proportion with severe neutropenia, duration of severe neutropenia, proportion with febrile neutropenia, duration of antibiotic treatment, duration of hospitalization, and percentage of patients receiving transfusion. Grade 4 neutropenia was defined as an absolute neutrophil count of <500 x 10(9)/L; severe neutropenia was defined as a count of <200 x 10(9)/L. Adverse events were collected from the medical charts. RESULTS: Twenty children were included (13 girls and 7 boys). The patients' median age was 12.8 years (range, 9-17 years) and median weight was 45.2 kg (range, 28-90 kg). A total of 178 chemotherapy courses (108 VIDE; 70 VAI or VAC) were administered and evaluated, including 134 courses with pegfilgrastim and 44 courses with filgrastim. Considering all types of chemotherapy combined, those courses in which pegfilgrastim was used were associated with a significantly lower incidence of severe neutropenia (0.21 vs 0.85; P = 0.03), a shorter duration of severe neutropenia (0.49 vs 2.36 days; P = 0.01), and a shorter duration of antibiotic treatment (1.07 vs 4.22 days; P = 0.03) compared with courses treated with filgrastim. No statistically significant differences were observed for the proportion and duration of grade 4 neutropenia, proportion of febrile neutropenia, duration of hospitalization, or red blood cell and platelet transfusions. Adverse effects were few and comparable between pegfilgrastim and filgrastim. CONCLUSIONS: In this retrospective chart review of children with Ewing sarcoma, using pegfilgrastim after chemotherapy courses was associated with significantly reduced frequency and shorter duration of severe neutropenia compared with those courses followed by filgrastim. Randomized controlled trials are needed to confirm these preliminary observations. Collaborators: Urban Ch, Meister B, Fink FM, Kerbl R, Stollinger O, Schmitt K, Ebetsberger G, Jones N, Ladenstein R, Gadner H, Maes P, Brichard B, Mazzeo F, Gil T, Dhooge C, Vermorken JB, Klein A, Kruseova J, Krarup-Hansen A, Nielsen O, Capra M, Pautard B, Rialland X, Maillart P, Plouvier E, Vérité C, Bui N'guyen B, Boutard P, Delcambre C, Bay JO, Demeocq F, Couillault G, Isambert N, Plantaz D, Desfachelles AS, Piguet C, Marec-Bérard P, Blay JY, Gentet JC, Duffaud F, Bertucci F, Sirvent N, Schmitt C, Rios M, Corradini N, Rolland F, Deville A, Thyss A, Michon J, Tabone MD, Pierga JY, Millot F, Munzer M, Edan C, Vannier JP, Guillemet C, Brun E, Berger C, Castex MP, Roche H, Lejars O, Linassier C, Oberlin O, Le Cesne A, Bauer S, Ebeling P, Flasshove M, Hartmann JT, Reichardt P, Mertens R, Osieka R, Gnekow A, Schlimok G, Henze G, Thuss-Patience P, Wickmann L, Reichardt P, Potenberg J, Reichardt P, Bode U, Schmidt-Wolf IG, Eberl W, Wolff T, Pekrun A, Hofmann A, Andler W, Schneider D, Lauterbach I, Zickler P, Göbel U, Borkhardt A, Sauerbrey A, Holter W, Bauer S, Ebeling P, Flasshove M, Eggert A, Klingebiel T, Niemeyer C, Heinz J, Reiter A, Rummel M, Runde V, Lakomek M, Trümper L, Beck J, Dölken G, Lindemann HW, Körholz D, Schmoll HJ, Schneppenheim R, Hossfeld DK, Bokemeyer C, Keles H, Welte K, Klein C, Kulozik A, Egerer G, Cyran J, Strumberg D, Freier W, Graf N, Pfreundschuh M, Gruhn B, Leipold A, Bentz M, Wehinger H, Schrappe M, Nolte H, Berthold F, Wolf J, Sternschulte W, Voelpel S, Frieling T, Moessner J, Selle D, Bucsky D, Bartels H, Mohren M, Fischer T, Kluba U, Gutjahr P, Dittrich M, Reiter S, Dürken M, Neubauer A, Beyer J, Christiansen H, Erdlenbruch B, Burdach S, Schmid I, Meyer zum Büschenfelde C, Peschel C, Oduncu F, Jürgens H, Berdel W, Held H, Hofmann-Wackersreuther G, Müller H, Peters O, Andreesen R, Krause S, Heits F, Geib-König R, Kasbohm M, Bürger D, Burghard R, Dickerhoff R, Bielack S, Feddersen I, Clemens MR, Handgretinger R, Hartmann JT, Debatin KM, Mayer-Steinacker R, Schoengen A, Schlegel P, Lee V, Chik KW, van den Berg H, Rodenhuis S, Gelderblom AJ, Hoogerbrugge P, Bökkerink JP, Pieters R, Corbett R, Österlundh G, Behrendtz M, Holmqvist BM, Hjorth L, Petersen C, Jakobson Å, Hjalmars U, Ljungman G, Angst R, Kühne T, Paulussen M, Leyvraz S, Rischewski J, Feldges A, Greiner J, Hess U, Exner GU, Niggli F, Knuth A, King D, McCarthy A, Henry P, Morland B, Rees H, Nicholson J, Traunecker H, Wallace H, Ronghe M, Simpson E, Cowie F, White J, Picton S, Lewis I, Leahy M, Stark D, Selby PJ, Heney D, Pizer B, McDowell H, Michalski A, Whelan J, Chisholm J, Pritchard-Jones K, Judson I, Bren B, Hale J, Verrill M, Walker D, Sokal M, Wheeler K, Lee V, Gerrard M, Woll P, Lorigan P, Robinson M, Kohler J. Owing to its rarity, rhabdomyosarcoma of the head and neck (HNRMS) has seldom been discussed in the literature. As most of the data is based only on the retrospective experiences of tertiary healthcare centers, there are difficulties in formulating a standard treatment protocol. Moreover, the disease is poorly understood at its pathological, genetic, and molecular levels. For instance, 20% of all histological assessment is inaccurate; even an experienced pathologist can confuse rhabdomyosarcoma (RMS) with neuroblastoma, Ewing's sarcoma, and lymphoma. RMS can occur sporadically or in association with genetic syndromes associated with predisposition to other cancers such as Li-Fraumeni syndrome and neurofibromatosis type 1 (von Recklinghausen disease). Such associations have a potential role in future gene therapies but are yet to be fully confirmed. Currently, chemotherapies are ineffective in advanced or metastatic disease and there is lack of targeted chemotherapy or biological therapy against RMS. Also, reported uses of chemotherapy for RMS have not produced reasonable responses in all cases. Despite numerous molecular and biological studies during the past three decades, the chemotherapeutic regimen remains unchanged. This vincristine, actinomycin, cyclophosphamide (VAC) regime, described in Kilman, et al. (1973) and Koop, et al. (1963), has achieved limited success in controlling the progression of RMS. Thus, the pathogenesis of RMS remains poorly understood despite extensive modern trials and more than 30 years of studies exploring the chemotherapeutic options. This suggests a need to explore surgical options for managing the disease. Surgery is the single most critical therapy for pediatric HNRMS. However, very few studies have explored the surgical management of pediatric HNRMS and there is no standard surgical protocol. The aim of this review is to explore and address such issues in the hope of maximizing the number of options available for young patients with HNRMS. BACKGROUND Sarcoma botryoides, known as embryonal rhabdomyosarcoma (ERMS), is a maligt tumor which arises from embryonic muscle cells. The incidence of ERMS in the uterine cervix rarely occurs at a very young age. With sufficient resources, management of this disease is not difficult. However, in limited resources settings, such as in Indonesia, the situation is more challenging. This case report aims to highlight the difficulties encountered in diagnosing and treating patients with sarcoma botryoides. CASE REPORT A 3-year-old female patient came the outpatient clinic of our hospital with a protruding mass from her vagina resembling a bunch of grapes which easily bled. She underwent surgery to remove the mass. After the procedure, she did not return to the hospital for the recommended adjuvant chemotherapy treatment due to limited funds. Three months later, she came to the outpatient clinic with the same complaint, despite smaller size. Due to limited resources, we only evaluated the metastasis using chest x-ray and did not perform intra-operative biopsy. In the second surgery, a wide excision with 1-2 cm margin was performed, followed by adjuvant chemotherapy for 6 series. We achieved a satisfactory outcome in this case, and 18 months after the surgery, the patient was still in remission. CONCLUSIONS Sarcoma botryoides is a rare maligcy. The effective treatment for sarcoma botryoides is wide excision with safe margin of 1-2 cm, followed by 6-12 cycles of vincristine, actinomycin D, and cyclophosphamide (VAC) regiment as an adjuvant chemotherapy. A family's understanding of the treatment plan is important to achieve desired outcomes. Even with limited resources, this maligcy can still be properly treated.

Is YKL-40 used as a biomarker for Alzheimer's disease?

Yes, cerebrospinal fluid (CSF) YKL-40 levels were reported to be a promising candidate biomarker of glial inflammation in Alzheimer's disease (AD).

INTRODUCTION: Synaptic damage, axonal neurodegeneration, and neuroinflammation are common features in Alzheimer's disease (AD), frontotemporal dementia (FTD), and Creutzfeldt-Jakob disease (CJD). METHODS: Unicentric cohort of 353 participants included healthy control (HC) subjects, AD continuum stages, genetic AD and FTD, and FTD and CJD. We measured cerebrospinal fluid neurofilament light (NF-L), neurogranin (Ng), 14-3-3, and YKL-40 proteins. RESULTS: Biomarkers showed differences in HC subjects versus AD, FTD, and CJD. Disease groups differed between them except AD versus FTD for YKL-40. Only NF-L differed between all stages within the AD continuum. AD and FTD symptomatic mutation carriers presented differences with respect to HC subjects. Applying the AT(N) system, 96% subjects were positive for neurodegeneration if 14-3-3 was used, 94% if NF-L was used, 62% if Ng was used, and 53% if YKL-40 was used. DISCUSSION: Biomarkers of synapse and neurodegeneration differentiate HC subjects from neurodegenerative dementias and between AD, FTD, and CJD. NF-L and 14-3-3 performed similar to total tau when AT(N) system was applied. Recently, cerebrospinal fluid (CSF) YKL-40 levels were reported to be a promising candidate biomarker of glial inflammation in Alzheimer's disease (AD). To detect how APOE ε4 affects CSF YKL-40 levels in cognitively normal (CN) states, mild cognitive impairment (MCI) and AD dementia, data from 35 CN subjects, 63 patients with MCI, and 11 patients with AD from a cross-sectional study in the Alzheimer's Disease Neuroimaging Initiative (ADNI) database were investigated. The results showed that CSF YKL-40 concentrations were increased in the AD dementia group than in the CN group. CSF YKL-40 levels were higher in APOE ε4 carriers than in noncarriers with MCI. No statistically significant difference was found in CSF YKL-40 levels between APOE ε4 carrier and noncarriers in AD and CN subjects. CSF YKL-40 concentrations were tightly related to CSF tau and p-tau concentrations in the MCI group. Analysis implied that APOE ε4 might affect CSF YKL-40 levels in MCI subjects, suggesting a crucial role of APOE ε4 in neuroinflammation in detecting individuals who might convert to AD from MCI and, thus, as an effective predictive factor. Neurodegenerative diseases comprise a large number of disorders with high impact on human health. Neurodegenerative processes are caused by various etiological factors and differ in their clinical presentation. Neuroinflammation is widely discussed as both a cause and a consequence in the manifestation of these disorders. The interplay between the two entities is considered as a major contributor to the ongoing disease progression. An attentive search and implementation of new and reliable markers specific for the processes of inflammation and degeneration is still needed. YKL-40 is a secreted glycoprotein produced by activated glial cells during neuroinflammation. Neuron-specific enolase (NSE), expressed mainly by neuronal cells, is a long-standing marker for neuronal damage. The aim of this review is to summarize, clarify, and evaluate the potential significance and relationship between YKL-40 and NSE as biomarkers in the monitoring and prognosis of a set of neurological diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. YKL-40 appears to be a more reliable biomarker in neurological diseases than NSE. The more prominent expression pattern of YKL-40 could be explained with the more obvious involvement of glial cells in pathological processes accompanying each neurodegenerative disease, whereas reduced NSE levels are likely related to low metabolic activity and increased death of neurons.

On what chromosome is the gene for "SILVER" coat color found for the domestic cat?

Linkage mapping defined a genomic region for SILVER as a 3.3-Mb region, (95.87-99.21 Mb) on chromosome D2 in the domestic cat.

Which lncRNAs are induced by heatshock?

Malat1, papas, long noncoding rnas, circrna, neat1, and mirna are induced by heat shock.

Following the initial discovery of the heat shock RNA omega (hsrω) gene of Drosophila melanogaster to be non-coding (nc) and also inducible by cell stress, other stress-inducible long non-coding RNAs (lncRNA) have been described in diverse organisms. In view of the rapid sequence divergence of lncRNAs, present knowledge of stress trasncriptome is limited and fragmented. Several known stress-related lncRNAs, associated with specific nuclear speckled domains or nucleolus, provide structural base for sequestering diverse RNA-processing/regulatory proteins. Others have roles in transcriptional or translational inhibition during stress or in signaling pathways; functions of several other lncRNAs are not yet known. Most stress-related lncRNAs act primarily by modulating activity of the proteins to which they bind or by sequestering specific sets of proteins away from the active pool. A common emerging theme is that a given lncRNA targets one or more protein/s with key role/s in the cascade of events triggered by the stress and therefore has a widespread integrative effect. Since proteins associate with RNA through short sequence motifs, the overall base sequence of functionally similar ncRNAs is often not conserved except for specific motifs. The rapid evolvability of ncRNA sequences provides elegant modules for adaptability to changing environment as binding of one or the other protein to ncRNA can alter its structure and functions in distinct ways. Thus the stress-related lncRNAs act as hubs in the cellular networks to coordinate activities of the members within and between different networks to maintain cellular homeostasis for survival or to trigger cell death. The field of non-coding RNA (ncRNA) has expanded over the last decade following the discoveries of several new classes of regulatory ncRNA. A growing amount of evidence now indicates that ncRNAs are involved even in the most fundamental of cellular processes. The heat shock response is no exception as ncRNAs are being identified as integral components of this process. Although this area of research is only in its infancy, this article focuses on several classes of regulatory ncRNA (i.e., miRNA, lncRNA, and circRNA), while summarizing their activities in mammalian heat shock. We also present an updated model integrating the traditional heat shock response with the activities of regulatory ncRNA. Our model expands on the mechanisms for efficient execution of the stress response, while offering a more comprehensive summary of the major regulators and responders in heat shock signaling. It is our hope that much of what is discussed herein may help researchers in integrating the fields of heat shock and ncRNA in mammals. Genomic studies have revealed that humans possess far fewer protein-encoding genes than originally predicted. These over-estimates were drawn from the inherent developmental and stimuli-responsive complexity found in humans and other mammals, when compared to lower eukaryotic organisms. This left a conceptual void in many cellular networks, as a new class of functional molecules was necessary for "fine-tuning" the basic proteomic machinery. Transcriptomics analyses have determined that the vast majority of the genetic material is transcribed as noncoding RNA, suggesting that these molecules could provide the functional diversity initially sought from proteins. Indeed, as discussed in this review, long noncoding RNAs (lncRNAs), the largest family of noncoding transcripts, have emerged as common regulators of many cellular stressors; including heat shock, metabolic deprivation and DNA damage. These stimuli, while divergent in nature, share some common stress-responsive pathways, notably inhibition of cell proliferation. This role intrinsically makes stress-responsive lncRNA regulators potential tumor suppressor or proto-oncogenic genes. As the list of functional RNA molecules continues to rapidly expand it is becoming increasingly clear that the significance and functionality of this family may someday rival that of proteins. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Flavivirus infection causes host cell death by initiation of an unfolded protein response (UPR). UPR is initiated following activation of three ER-membrane resident sensors, PERK, IRE1α and ATF6, which are otherwise kept inactive through association with the ER-chaperone GRP78. Activation precedes cellular and molecular changes that act to restore homeostasis but might eventually initiate apoptosis. These changes involve influencing function of multiple genes by either transcriptional or post-transcriptional or post-translational mechanisms. Transcriptional control includes expression of transcription factor cascades, which influence cognate gene expression. Malat1 is a long non-coding RNA which is over-expressed in many human oncogenic tissues and regulates cell cycle and survival. In this report, for the first time we show activation of Malat1 following infection by two flaviviruses, both of which activate the UPR in host cells. The temporal kinetics of expression was restricted to later time points. Further, Malat1 was also activated by pharmacological inducer of UPR, to a similar degree. Using drugs that specifically inhibit or activate the PERK or IRE1α sensors, we demonstrate that signalling through the PERK axis activates this expression, through a transcriptional mechanism. To our knowledge, this is the first report of an UPR pathway regulating the expression of an lncRNA. The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders.

What is the prevalence of poor metabolizers of CYP2C19 among Southern Asians compared to East Asians?

Southeast Asians exhibit a higher prevalence of CYP2C19-poor metabolisers compared with Caucasians and East Asians.

Which R/Bioconductor package has been developed for network-based differential expression analysis?

INDEED is an R/Bioconductor package for network based differential expression analysis. INDEED allows users to construct a sparse network based on partial correlation, and to identify biomolecules that have significant changes both at individual expression and pairwise interaction levels.

Can propofol cause green urine?

Yes, propofol can cause green discoloration of urine. It is a rare and benign condition, which occurs when clearance of propofol exceeds the hepatic and extrahepatic elimination.

BACKGROUND: The intragastric balloon is filled with saline and methylene blue dye, to detect balloon deflation early and prevent bowel obstruction, by monitoring the patient's urine for changes in color. METHODS: An intragastric balloon filled with 590 ml of saline plus 10 ml of methylene blue was endoscopically placed under sedation in a 22-year-old man with morbid obesity (BMI 42 kg/m2). 3 days later, the patient's urine changed to dark green, and, suspecting a leaking balloon, endoscopy was repeated under sedation. RESULTS: No signs of balloon deflation were seen, and the urine returned to normal color. The next day, the urine turned green again. 7 days later, the urine discoloration finally disappeared. CONCLUSION: Propofol, a sedative commonly used by anesthesiologists during endoscopic procedures, is known to have several side-effects, and urine discoloration is one of them, albeit rare. This benign side-effect must be known to obesity surgeons to avoid pointless medical expenditure, unnecessary balloon removal and distress for patients and clinicians. We describe a 58-year-old man who developed green urine after operation on a pressure ulcer. The discolouration disappeared gradually after two days. We think that the use of methylene blue dye during the revision of the wounds and the use of the sedative propofol could have caused it. INTRODUCTION: Mild therapeutic hypothermia is an increasingly recognised treatment option to reduce perihemorrhagic edema in severe intracerebral hemorrhage. CASE DESCRIPTION: We report the case of a 77-year old woman with atypical intracerebral hemorrhage that was treated with mild hypothermia in addition to osmotic therapy. The patient's urine subsequently showed a green discoloration. Urine discoloration was completely reversible upon discontinuation of propofol. DISCUSSION AND EVALUATION: Propofol-related urine discoloration may have been provoked by hypothermia. Due to the benign nature of this side effect, propofol should be stopped and gastrointestinal function should be supported. CONCLUSION: More studies are needed to show a causal role of hypothermia and related decreased enzymatic function. The change in the colour of urine is a known occurrence in an intensive care setting and is always a cause of concern to the clinicians who have to differentiate between benign and pathological causes. Herein, we present a case of 62-year-old postoperative lady, noticed to be passing green coloured urine believed to be due to intravenous Propofol administration for induction of general anaesthesia. The green colour of urine due to Propofol occurs when clearance of Propofol exceeds hepatic elimination, and extrahepatic elimination of Propofol occurs. This discolouration of urine is a rare (less than 1% cases) but a benign side effect of Propofol, which is non-nephrotoxic and gets reversed after discontinuation of the drug. BACKGROUND: Propofol is a short-acting, intravenous sedative-hypnotic agent that is widely used for the induction and maintece of general anesthesia and sedation. An uncommon adverse effect of propofol is green discoloration of the urine, which has been reported not only under general anesthesia but also with sedation. Although it is assumed that the phenolic derivatives of propofol can cause green discoloration of the urine, the actual origin remains unknown. The aim of this report was to identify the origin of the green discoloration of the urine using liquid chromatography-mass spectrometry (LC-MS). CLINICAL FEATURES: The patient, a 51-year-old man, was scheduled for his oral surgery under general anesthesia using propofol. Postoperatively, the color of his urine was observed to be green. We compared and analyzed both the green urine and the normal urine using LC-MS. CONCLUSION: We experienced a case of a patient with green discoloration of the urine after general anesthesia using propofol. Although LC-MS analysis showed 2 unique peaks in the green urine at 490 and 590 nm, obvious causes were not revealed. Methylene blue is used to assess the integrity of the bowel and may cause self-limiting bluish or greenish hue to the urine. Green urine is also caused by medications such as propofol and infections such as pseudomonas. Knowledge of the benign nature of this condition prevents unnecessary consultations and anxiety. BACKGROUND: Analgesia and sedation are key items in intensive care. Recently published S3 guidelines specifically address treatment of patients with elevated intracranial pressure. METHODS: The Austrian Society of Anesthesiology, Resuscitation and Intensive Care Medicine carried out an online survey of neurointensive care units in Austria in order to evaluate the current state of practice in the areas of analgosedation and delirium management in this high-risk patient group. RESULTS: The response rate was 88%. Induction of anesthesia in patients with elevated intracranial pressure is carried out with propofol/fentanyl/rocuronium in >80% of the intensive care units (ICU), 60% use midazolam, 33.3% use esketamine, 13.3% use barbiturates and 6.7% use etomidate. For maintece of analgosedation up to 72 h, propofol is used by 80% of the ICUs, followed by remifentanil (46.7%), sufentanil (40%) and fentanyl (6.7%). For long-term sedation, 86.7% of ICUs use midazolam, 73.3% sufentanil and 73.3% esketamine. For sedation periods longer than 7 days, 21.4% of ICUs use propofol. Reasons for discontinuing propofol are signs of rhabdomyolysis (92.9%), green urine, elevated liver enzymes (71.4% each) and elevated triglycerides (57.1%). Muscle relaxants are only used during invasive procedures. Inducing a barbiturate coma is rated as a last resort by 53.3% of respondents. The monitoring methods used are bispectral index (BIS™, 61.5% of ICUs), somatosensory-evoked potentials (SSEP, 53.8%), processed electroencephalography (EEG, 38.5%), intraparenchymal partial pressure of oxygen (pO2, 38.5%) and microdialysis (23.1%). Sedation and analgesia are scored using the Richmond agitation and sedation score (RASS, 86.7%), sedation agitation scale (SAS, 6.7%) or numeric rating scale (NRS, 50%) and behavioral pain scale (BPS, 42.9%), visual analogue scale (VAS), critical care pain observation tool (CCPOT, each 14.3%) and verbal rating scale (VRS, 7.1%). Delirium monitoring is done using the confusion assessment method for intensive care units (CAM-ICU, 46.2%) and intensive care delirium screening checklist (ICDSC, 7.7%). Of the ICUs 46.2% do not carry out delirium monitoring. CONCLUSION: We found good general compliance with the recommendations of the current S3 guidelines. Room for improvement exists in monitoring and the use of scores to detect delirium. The color of urine in patients who receive anesthetic gives much medical information to a medical team. So, we must check the urine color and know the cause of discoloration of the urine from anesthetic patients. Green urine is rare indeed and it is a benign potential side effect of propofol; this phenomenon is related to the metabolism of propofol. We experienced green urine from a long-term anesthetized patient who received a continuous infusion of propofol. We report here on this unusual case and we review the relevant literature. Propofol is commonly used for induction and maintece of anesthesia, and sedation in the intensive care unit. In addition, it is also used as an anesthetic coma treatment for refractory status epilepticus. We present the case of a 52-year-old man, who developed green urine following propofol coma therapy for status epilepticus. The urine color recovered following discontinuation of propofol infusion. The green discoloration of urine is a rare and benign condition, which occurs when clearance of propofol exceeds the hepatic and extrahepatic elimination. Green coloured urine is atypical as it usually signifies the presence of an exogenous substance. Several substances in literature have been associated with green urine including propofol, biliverdin, metoclopramide, methylene blue, indigo blue, amitriptyline, methocarbamol, indomethacin, promethazine, cimetidine and food colourings. We present here a case of middleaged man who presented to our ER with altered mental status and green coloured urine with positive urine toxicology reports for benzodiazepine.

Are Gram positive bacteria able to release extracellular vesicles?

Yes, Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes.

Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri-implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram-negative periodontal pathogens, so-called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP-1 and human oral keratinocyte HOK-16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter-related proteins, metabolism-related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP-1, CCL5, CXCL1, CXCL10, ICAM-1, IL-1β, IL-1ra, IL-6, IL-8, MIF, SerpinE, and TNF-α in THP-1 cells and CXCL1, G-CSF, GM-CSF, IL-6, and IL-8 in HOK-16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram-positive oral bacteria in periodontal diseases. Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have been less thoroughly investigated most probably due to the methodological challenges in separating BEVs from their matrix and host-derived eukaryotic extracellular vesicles (EEVs) such as exosomes and microvesicles. Here, we present a step-by-step procedure to separate and characterize BEVs from human body fluids. BEVs are separated through the orthogonal implementation of ultrafiltration, size-exclusion chromatography (SEC) and density-gradient centrifugation. Size separates BEVs from bacteria, flagella and cell debris in stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, maintains the integrity of BEVs and ensures reproducibility through the use of automated liquid handlers. Post-separation BEVs are characterized using orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in combination with proteomics, electron microscopy and oparticle tracking analysis (NTA) to evaluate BEV quality, abundance, structure and molecular cargo. Separation and characterization of BEVs from body fluids can be done within 72 h, is compatible with EEV analysis and can be readily adopted by researchers experienced in basic molecular biology and extracellular vesicle analysis. We anticipate that this protocol will expand our knowledge on the biological heterogeneity, molecular cargo and function of BEVs in human body fluids and steer the development of laboratory research tools and clinical diagnostic kits. Release of extracellular vesicles (EVs) is a common feature among eukaryotes, archaea, and bacteria. However, the biogenesis and downstream biological effects of EVs released from gram-positive bacteria remain poorly characterized. Here, we report that EVs purified from a community-associated methicillin-resistant Staphylococcus aureus strain were internalized into human macrophages in vitro and that this process was blocked by inhibition of the dynamin-dependent endocytic pathway. Human macrophages responded to S. aureus EVs by TLR2 signaling and activation of NLRP3 inflammasomes through K+ efflux, leading to the recruitment of ASC and activation of caspase-1. Cleavage of pro-interleukin (IL)-1β, pro-IL-18, and gasdermin-D by activated caspase-1 resulted in the cellular release of the mature cytokines IL-1β and IL-18 and induction of pyroptosis. Consistent with this result, a dose-dependent cytokine response was detected in the extracellular fluids of mice challenged intraperitoneally with S. aureus EVs. Pore-forming toxins associated with S. aureus EVs were critical for NLRP3-dependent caspase-1 activation of human macrophages, but not for TLR2 signaling. In contrast, EV-associated lipoproteins not only mediated TLR2 signaling to initiate the priming step of NLRP3 activation but also modulated EV biogenesis and the toxin content of EVs, resulting in alterations in IL-1β, IL-18, and caspase-1 activity. Collectively, our study describes mechanisms by which S. aureus EVs induce inflammasome activation and reveals an unexpected role of staphylococcal lipoproteins in EV biogenesis. EVs may serve as a novel secretory pathway for S. aureus to transport protected cargo in a concentrated form to host cells during infections to modulate cellular functions.

Describe a cytokine release syndrome.

The major factor responsible for acute respiratory distress syndrome is the so-called "cytokine storm," which is an aberrant response from the host immune system that induces an exaggerated release of proinflammatory cytokines/chemokines.

Cytokine-release syndrome is a symptom complex associated with the use of many monoclonal antibodies. Commonly referred to as an infusion reaction, it results from the release of cytokines from cells targeted by the antibody as well as immune effector cells recruited to the area. When cytokines are released into the circulation, systemic symptoms such as fever, nausea, chills, hypotension, tachycardia, asthenia, headache, rash, scratchy throat, and dyspnea can result. In most patients, the symptoms are mild to moderate in severity and are managed easily. However, some patients may experience severe, life-threatening reactions that result from massive release of cytokines. Severe reactions occur more commonly during the first infusion in patients with hematologic maligcies who have not received prior chemotherapy; severe reactions are marked by their rapid onset and the acuity of associated symptoms. Massive cytokine release is an oncologic emergency, and special precautions must be taken to prevent life-threatening complications. This article will present an overview of the etiology and management of cytokine-release syndrome in patients receiving monoclonal antibodies to better prepare oncology nurses to safely care for such patients. Acute cytokine release syndromes are associated with some therapeutic antibodies in man, leading to a spectrum of clinical signs from nausea, chills and fever to more serious dose limiting hypotension and tachycardia. When anticipated this syndrome is typically manageable, however this adverse reaction recently became headline news when a massive and unexpected cytokine release syndrome occurred within a few hours of dosing six healthy volunteers with a therapeutic antibody, putting their lives at risk due to multiple organ failure. Preclinical studies did not predict this adverse event, emphasising the need to compare the relative potency of the product in man and the chosen toxicology species, so that additional margins of safety can be applied when conducting first in man (FIM) studies if there is uncertainty over the predictability of the toxicology species. In vitro human PBMC and whole blood cultures may be useful for predicting cytokine release. However since cytokine release arises through at least two distinct mechanisms, it should be emphasised that the utility of these in vitro methods needs to be established for each antibody product. Immune cells secrete small protein molecules that aim for cell-cell communications. These small molecules are called cytokines. Targeting cancer cells with administration of bispecific antibodies and natural extracts results in elevated circulating levels of inflammatory cytokines, including interferon-γ and interleukin (IL)-6, which lead to cell toxicity. Sustained release of cytokines due to immunotherapy or hormonal issues causes various diseases. Novel T cell-engaging therapies and monoclonal antibodies cause cytokine release syndrome. Efforts are being carried out to maximize the chance for therapeutic benefit from immunotherapy while minimizing the risk for life-threatening complications of sustained cytokine release. Neurodegeneration and cardiac diseases are the prominent diseases caused by inflammatory cytokines. The phenomenon is called cytokine storm. Cytokines can act antagonistically or synergistically. Constitutive expression of proinflammatory cytokines such as IL-3 and IL-6 causes organ damage and unbearable pain. In this review, we will discuss the regulators of cytokine release, its types, its implications on human health, and treatment. OBJECTIVES: To describe a pediatric case of cytokine release syndrome secondary to chimeric antigen receptor-modified T cells associated with acute respiratory distress syndrome. DESIGN: Case report. SETTING: PICU. PATIENTS: A 14-year-old boy with refractory B cell precursor acute lymphoblastic leukemia given chimeric antigen receptor cells developed severe cytokine release syndrome 7 days after the drug product infusion with progressive respiratory failure. He was admitted to PICU with a clinical picture of acute respiratory distress syndrome, requiring mechanical ventilation, and secondary hemophagocytic lymphohistiocytosis. INTERVENTIONS: Hemoadsorption with cartridge column (Cytosorb) in combination with continuous renal replacement therapy was associated to the anti-cytokine therapy (tocilizumab, a monoclonal antibody targeting interleukin-6 receptor). MEASUREMENTS AND MAIN RESULTS: Decrease of the inflammatory biomarkers (ferritin, interleukin-6, interleukin-10) in the first 96 hours associated with a progressive improvement of acute respiratory distress syndrome (Pao2/Fio2 ratio) 7 day after the start of the multimodal treatment. CONCLUSIONS: This case suggests that hemoadsorption with cartridge column in combination with continuous renal replacement therapy and tocilizumab is safe and potentially effective in pediatric patients with severe cytokine release syndrome. In 2019-2020 a new coronavirus named SARS-CoV-2 was identified as the causative agent of a several acute respiratory infection named COVID-19, which is causing a worldwide pandemic. There are still many unresolved questions regarding the pathogenesis of this disease and especially the reasons underlying the extremely different clinical course, ranging from asymptomatic forms to severe manifestations, including the Acute Respiratory Distress Syndrome (ARDS). SARS-CoV-2 showed phylogenetic similarities to both SARS-CoV and MERS-CoV viruses, and some of the clinical features are shared between COVID-19 and previously identified beta-coronavirus infections. Available evidence indicate that the so called "cytokine storm" an uncontrolled over-production of soluble markers of inflammation which, in turn, sustain an aberrant systemic inflammatory response, is a major responsible for the occurrence of ARDS. Chemokines are low molecular weight proteins with powerful chemoattractant activity which play a role in the immune cell recruitment during inflammation. This review will be aimed at providing an overview of the current knowledge on the involvement of the chemokine/chemokine-receptor system in the cytokine storm related to SARS-CoV-2 infection. Basic and clinical evidences obtained from previous SARS and MERS epidemics and available data from COVID-19 will be taken into account. Cytokine release syndrome is a systemic inflammatory condition that may occur after treatment with some types of immunotherapy. Adoptive transfer of T cells modified with chimeric antigen receptors (CAR-T cells) has changed the therapeutic landscape of hematological maligcies, particularly for acute lymphoblastic leukemia and large B cell lymphoma, where two different CAR-T products are now considered standard of care. Furthermore, intense research efforts are under way to expand the clinical application of CAR-T cell therapy for the benefit of patients suffering from other types of cancers. Nevertheless, CAR-T cell treatment is associated with toxicities such as cytokine release syndrome, which can range in severity from mild flu-like symptoms to life-threatening vasodilatory shock, and a neurological syndrome termed ICANS (immune effector cell-associated neurotoxicity syndrome), which can also range in severity from a temporary cognitive deficit lasting only a few hours to lethal cerebral edema. In this review, we provide an in-depth discussion of different types of CAR-T cell-associated toxicities, including an overview of clinical presentation and grading, pathophysiology, and treatment options. We also address future perspectives and opportunities, with a special focus on hematological maligcies. In December 2019, a novel coronavirus, COVID-19, was discovered to be the causal agent of a severe respiratory infection named SARS-CoV-2, and it has since been recognized worldwide as a pandemic. There are still numerous doubts concerning its pathogenesis and particularly the underlying causes of the various clinical courses, ranging from severe manifestations to asymptomatic forms, including acute respiratory distress syndrome. The major factor responsible for acute respiratory distress syndrome is the so-called "cytokine storm," which is an aberrant response from the host immune system that induces an exaggerated release of proinflammatory cytokines/chemokines. In this review, we will discuss the role of cytokine storm in COVID-19 and potential treatments with which counteract this aberrant response, which may be valuable in the clinical translation.

Which genes are the main markers of primitive Endoderm (prEN) formation?

The genes involved in primitive endoderm (prEN) formation are fgf4, lrp2, gata4, pdgfra, p dgfrα, gATA6, nanog, pDgfralpha, egam1 and dab2.

The amount of the heterotrimeric G protein subunit G alpha i2 decreases after the induction of F9 teratocarcinoma cells to become primitive endoderm in the presence of retinoic acid (RA). The reduction of the G alpha i2 protein in F9 cells by antisense RNA expression was associated with (i) loss of receptor-mediated inhibition of adenylyl cyclase; (ii) decreased cell doubling time; (iii) induction of a primitive, endoderm-like phenotype in the absence of RA; and (iv) production of the differentiation marker tissue-type plasminogen activator. Expression of a constitutively active, mutant G alpha i2 blocked RA-induced differentiation. These data suggest the involvement of G alpha i2 in the control of stem cell differentiation and provide insight into the involvement of G proteins in growth regulation. F9 embryonal carcinoma (EC) cells were used as a model system to study endoderm formation during mammalian embryogenesis. F9 cells treated with retinoic acid (RA) or RA plus dibutyryl cyclic AMP (cAMP) were examined for the expression of stage-specific embryonic antigen-3 (SSEA-3), a cell surface marker of primitive and visceral endoderm. SSEA-3 was not detected by indirect immunofluorescence on the surface of undifferentiated stem cells; however, a subset of SSEA-3-positive cells appeared with time in culture, amounting to 20% of cells 10 days after plating. When cultured in the presence of RA, the percentage of SSEA-3-positive cells increased to 70% of cells 10 days after plating. In contrast, treatment of cells with RA plus cAMP yielded differentiated cells that were SSEA-3-negative. These SSEA-3-negative cells exhibited ultrastructural features of parietal yolk sac endoderm. In contrast, SSEA-3-positive cells appearing in cultures treated with RA alone exhibited ultrastructural features of primitive endoderm on day 3, switching to ultrastructural features of parietal endoderm on day 10. Cells with hybrid features, resembling both visceral and parietal yolk sac, were also seen. We suggest that differentiation of F9 EC cells into parietal yolk sac-like cells can occur along two distinct pathways: 1) direct under the combined influence of RA and cAMP; and 2) indirect, under the influence of RA alone, in which cells first differentiate into primitive endoderm. Parietal yolk sac-like cells induced through the latter pathway continue to express SSEA-3, a cell surface marker of primitive endoderm that is not normally found on parietal endodermal cells in vivo. Different types of endoderm, including primitive, definitive and mesendoderm, play a role in the induction and patterning of the vertebrate head. We have studied the formation of the anterior neural plate in chick embryos using the homeobox gene GANF as a marker. GANF is first expressed after mesendoderm ingression from Hensen's node. We found that, after transplantation, neither the avian hypoblast nor the anterior definitive endoderm is capable of GANF induction, whereas the mesendoderm (young head process, prechordal plate) exhibits a strong inductive potential. GANF induction cannot be separated from the formation of a proper neural plate, which requires an intact lower layer and the presence of the prechordal mesendoderm. It is inhibited by BMP4 and promoted by the presence of the BMP antagonist Noggin. In order to investigate the inductive potential of the mammalian visceral endoderm, we used rabbit embryos which, in contrast to mouse embryos, allow the morphological recognition of the prospective anterior pole in the living, pre-primitive-streak embryo. The anterior visceral endoderm from such rabbit embryos induced neuralization and independent, ectopic GANF expression domains in the area pellucida or the area opaca of chick hosts. Thus, the signals for head induction reside in the anterior visceral endoderm of mammals whereas, in birds and amphibia, they reside in the prechordal mesendoderm, indicating a heterochronic shift of the head inductive capacity during the evolution of mammalia. The derivation of the primitive endoderm layer from the pluripotent cells of the inner cell mass is one of the earliest differentiation and morphogenic events in embryonic development. GATA4 and GATA6 are the key transcription factors in the formation of extraembryonic endoderms, but their specific contribution to the derivation of each endoderm lineage needs clarification. We further analyzed the dynamic expression and mutant phenotypes of GATA6 in early mouse embryos. GATA6 and GATA4 are both expressed in primitive endoderm cells initially. At embryonic day (E) 5.0, parietal endoderm cells continue to express both GATA4 and GATA6; however, visceral endoderm cells express GATA4 but exhibit a reduced expression of GATA6. By and after E5.5, visceral endoderm cells no longer express GATA6. We also found that GATA6 null embryos did not form a morphologically recognizable primitive endoderm layer, and subsequently failed to form visceral and parietal endoderms. Thus, the current study establishes that GATA6 is essential for the formation of primitive endoderm, at a much earlier stage then previously recognized, and expression of GATA6 discriminates parietal endoderm from visceral endoderm lineages. Gene knockouts in mice have showed that Grb2 and GATA6 are essential for the formation of primitive endoderm in blastocysts. Here, we confirmed that implanted Grb2-null blastocysts lack primitive or extraembryonic endoderm cells either at E4.5 or E5.5 stages. We analyzed the relationship between Grb2 and GATA6 in the differentiation of embryonic stem (ES) cells to primitive endoderm in embryoid body models. Upon transfection with GATA6 expression vector, Grb2-null ES cells underwent endoderm differentiation as indicated by the expression of the extraembryonic endoderm markers Dab2 and GATA4. Transfection of GATA4 expression vector also had the same differentiation potency. When GATA6- or GATA4-transfected Grb2-null ES cells were allowed to aggregate, fragments of an endoderm layer formed on the surface of the spheroids. The results suggest that GATA6 is downstream of Grb2 in the inductive signaling pathway and the expression of GATA6 is sufficient to compensate for the defects caused by Grb2 deficiency in the development of the primitive and extraembryonic endoderm. During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages--the epiblast and the primitive endoderm (PrE)--whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4--later markers of the PrE--depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program. One of the earliest epithelial-to-mesenchymal transitions in mouse embryogenesis involves the differentiation of inner cell mass cells into primitive and then into parietal endoderm. These processes can be recapitulated in vitro using F9 teratocarcinoma cells, which differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm with subsequent treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP). Our previous work on how primitive endoderm develops revealed that the Wnt6 gene is upregulated by RA, leading to the activation of the canonical WNT-β-catenin pathway. The mechanism by which Wnt6 is regulated was not determined, but in silico analysis of the human WNT6 promoter region had suggested that the GATA6 and FOXA2 transcription factors might be involved [1]. Subsequent analysis determined that both Gata6 and Foxa2 mRNA are upregulated in F9 cells treated with RA or RA and db-cAMP. More specifically, overexpression of Gata6 or Foxa2 alone induced molecular and morphological markers of primitive endoderm, which occurred concomitantly with the upregulation of the Wnt6 gene. Gata6- or Foxa2-overexpressing cells were also found to have increased levels in T-cell factor (TCF)-dependent transcription, and when these cells were treated with db-cAMP, they developed into parietal endoderm. Chromatin immunoprecipitation analysis revealed that GATA6 and FOXA2 were bound to the Wnt6 promoter, and overexpression studies showed that these transcription factors were sufficient to switch on the gene expression of a Wnt6 reporter construct. Together, these results provide evidence for the direct regulation of Wnt6 that leads to the activation of the canonical WNT-β-catenin pathway and subsequent induction of primitive extraembryonic endoderm. Embryonic stem (ES) cells have been considered as a valuable renewable source of materials in regenerative medicine. Recently, we identified the homeoprotein EGAM1 both in preimplantation mouse embryos and mouse ES cells. Expression of the Egam1 transcript and its encoded protein was detectable in differentiating mouse ES cells, while it was almost undetectable in undifferentiated cells. In the present study, in order to clarify the effect of forced expression of EGAM1 on the differentiation of mouse ES cells in vitro, transfectants expressing exogenous EGAM1 were generated. Egam1 transfectants promoted differentiation into cell types expressing Gata6, Gata4, Afp, or Plat, genes associated with emergence of the extra-embryonic endoderm lineages. On the other hand, Egam1 transfectants inhibited the expression of specific genes for the embryonic lineages, including Fgf5 (epiblast) and T (mesoderm), in addition to Cdx2, a specific gene for the extra-embryonic trophectoderm lineages. Changes in the percentage of cells recognizing by antibodies against specific marker proteins closely correlated with the expression patterns of their transcripts. Taken together, the results obtained in this study suggested that mouse ES cells expressing exogenous EGAM1 preferentially differentiate into extra-embryonic primitive endoderm lineages, rather than embryonic lineages or extra-embryonic trophectoderm lineages. Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification. A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P < 0.05)-which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan-FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2-KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle. Mol. Reprod. Dev. 83: 516-525, 2016. © 2016 Wiley Periodicals, Inc.

What are the EMA and FDA recommendations regarding pharmacogenetic testing for abacavir?

Abacavir HSRs are highly associated with the major histocompatibility complex class I. Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results the abacavir label has been modified: the European Medicines Agency (EMA) and the FDA recommend/suggested that the administration of abacavir must be preceded by a specific genotyping test. The HLA locus is extremely polymorphic, exhibiting many closely related alleles, making it difficult to discriminate HLA-B*57:01 from other related alleles, and a number of different molecular techniques have been developed recently to detect the presence of HLA-B*57:01.

Many pharmacogenomic biomarkers (PGBM) were identified and translated into clinical practice, affecting the usage of drugs via label updates. In this context, abacavir is one of the most brilliant examples of pharmacogenetic studies translated into clinical practice. Pharmacogenetic studies have revealed that abacavir HSRs are highly associated with the major histocompatibility complex class I. Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results the abacavir label has been modified: the European Medicines Agency (EMA) and the FDA recommend/suggested that the administration of abacavir must be preceded by a specific genotyping test. The HLA locus is extremely polymorphic, exhibiting many closely related alleles, making it difficult to discriminate HLA-B*57:01 from other related alleles, and a number of different molecular techniques have been developed recently to detect the presence of HLA-B*57:01. In this review, we provide a summary of the available techniques used by laboratories to genotype HLA-B*57:01, outlining the scientific and pharmacoeconomics pros and cons.

Describe PWMScan

PWMScan is used to scan a position weight matrix (PWM) against a genome or, in general, a large set of DNA sequences. The PWM is the most commonly used mathematical model to describe the DNA binding specificity of a transcription factor (TF).

SUMMARY: Transcription factors regulate gene expression by binding to specific short DNA sequences of 5-20 bp to regulate the rate of transcription of genetic information from DNA to messenger RNA. We present PWMScan, a fast web-based tool to scan server-resident genomes for matches to a user-supplied PWM or transcription factor binding site model from a public database. AVAILABILITY AND IMPLEMENTATION: The web server and source code are available at http://ccg.vital-it.ch/pwmscan and https://sourceforge.net/projects/pwmscan, respectively. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Is vocimagene amiretrorepvec effective for glioblastoma?

No. Treatment with vocimagene amiretrorepvec did not improve survival of glioblastoma patients.

PURPOSE: High-grade gliomas (HGGs) are central nervous system tumors with poor prognoses and limited treatment options. Vocimagene amiretrorepvec (Toca 511) is a retroviral replicating vector encoding cytosine deaminase, which converts extended release 5-fluorocytosine (Toca FC) into the anticancer agent, 5-fluorouracil. According to preclinical studies, this therapy kills cancer cells and immunosuppressive myeloid cells in the tumor microenvironment, leading to T-cell-mediated antitumor immune activity. Therefore, we sought to elucidate this immune-related mechanism of action in humans, and to investigate potential molecular and immunologic indicators of clinical benefit from therapy. PATIENTS AND METHODS: In a phase I clinical trial (NCT01470794), patients with recurrent HGG treated with Toca 511 and Toca FC showed improved survival relative to historical controls, and some had durable complete responses to therapy. As a part of this trial, we performed whole-exome DNA sequencing, RNA-sequencing, and multiplex digital ELISA measurements on tumor and blood samples. RESULTS: Genetic analyses suggest mutations, copy-number variations, and neoantigens are linked to survival. Quantities of tumor immune infiltrates estimated by transcript abundance may potentially predict clinical outcomes. Peak values of cytokines in peripheral blood samples collected during and after therapy could indicate response. CONCLUSIONS: These results support an immune-related mechanism of action for Toca 511 and Toca FC, and suggest that molecular and immunologic signatures are related to clinical benefit from treatment. Conflict of interest statement: Conflict of Interest Disclosures: Dr Gruber is a member of the board for Tocagen Inc. Drs Rao, Hogan, Accomando, Ostertag, Montellano, Kheoh, and Kabbinavar were Tocagen employees. Dr Cloughesy reported personal fees from Roche, personal fees from Trizel, personal fees from Medscape, personal fees from Bayer, personal fees from Amgen, personal fees from Odonate Therapeutics, personal fees from Pascal Biosciences, personal fees from Del Mar Pharmaceuticals, personal fees from Tocagen, personal fees from Kayopharm, personal fees from GW Pharma, personal fees from Kiyatec, personal fees from AbbVie, personal fees from Boehringer Ingelheim, personal fees from VBL, personal fees from VBI, personal fees from Deciphera, personal fees from Agios, personal fees from QED, personal fees from Merck, personal fees from Genocea, personal fees from Celgene, personal fees from Puma, personal fees from Lilly, and personal fees from BMS outside the submitted work; in addition, Dr Cloughesy had a patent to 62/819322 issued and licensed; and Member of the board for the 501c3 Global Coalition for Adaptive Research and CMO for the entity; Co-founder and board member of Katmai Pharmaceuticals. Dr Petrecca reported other from Tocagen during the conduct of the study. Dr Walbert reported personal fees from Tocagen outside the submitted work. Dr Damek reported grants from Tocagen during the conduct of the study; grants from NovoCure, grants from Kazia Therapeutics, grants from Genentech, grants from Orbus, grants from Roche, and grants from Forma outside the submitted work. Dr Bota reported personal fees from NovoCure and personal fees from Zai Lab outside the submitted work. Dr Bettegowda reported he is a consultant for Depuy-Synthes and Bionaut Pharmaceuticals. The activities associated with those entities are not related to the work presented in this manuscript. Dr Zhu reported grants from Tocagen, Inc and personal fees from Tocagen, Inc during the conduct of the study. Dr Iwamoto reported personal fees from Tocagen during the conduct of the study; personal fees from Merck, personal fees from Guidepoint, grants from BMS, personal fees from NovoCure, personal fees from Alexion, personal fees from AbbVie, and personal fees from Regeneron outside the submitted work. Dr Placantonakis reported personal fees from Tocagen during the conduct of the study; personal fees from Monteris, personal fees from Synaptive, and personal fees from Robeaute outside the submitted work; in addition, Dr Placantonakis had a patent to “Method to treat high grade glioma” pending. Dr Brem reported personal fees from Tocagen during the conduct of the study. Dr Piccioni reported personal fees from Tocagen during the conduct of the study. Dr Chen reported other from Tocagen during the conduct of the study; personal fees from Tocagen outside the submitted work. Dr Gruber reported grants from the US Food and Drug Administration orphan drug grant, other from Apollo Bio, other from Abentis, and other from Denovo Pharma during the conduct of the study; other from Apollo Bio, other from Abentis, and other from Denovo pharma outside the submitted work; in addition, Dr Gruber had a patent to many pending, issued, licensed, and with royalties paid, a patent to many pending, issued, and licensed, and a patent to many pending, issued, and licensed; and Stock and option ownership in Tocagen. Dr Hogan reported other from Tocagen Inc during the conduct of the study. Dr Accomando reported personal fees from Tocagen Inc. during the conduct of the study; personal fees from Tocagen Inc. outside the submitted work. Dr Ostertag reported a patent to US20130130986A1 issued, a patent to US20130323301A1, a patent to US20180021365A1, and a patent to US20140178340A1 . Dr Montellano reported grants from the US Food and Drug Administration Office of Orphan Products Development during the conduct of the study. Dr Kheoh reported other from Tocagen Inc during the conduct of the study. Dr Kabbinavar reported other from Tocagen Inc during the conduct of the study; other from Tocagen outside the submitted work; and Employee of Tocagen Inc. Dr Vogelbaum reported personal fees and other from Tocagen during the conduct of the study; other from Infuseon Theraepeutics and personal fees from Celgene outside the submitted work. No other disclosures were reported.

List the major royal jelly proteins in Apis mellifera.

The genome of the western honeybee (Apis mellifera) harbors nine transcribed major royal jelly protein genes (mrjp1-9) which originate from a single-copy precursor via gene duplication.

The honey from chestnut, acacia, sunflower, eucalyptus and orange was analysed for its proteome content, in order to see if any plant proteins present would allow the proteo-typing of these different varieties. Since the total protein content turned out to be minute, 200g of each honey type were diluted to 1L and then added with ProteoMiner to enhance the visibility of the proteinaceous material. All bands visible in the SDS-PAGE profile of each type of honey were eluted, digested and identified by mass spectrometry in a LTQ-XL instrument. It turned out that all proteins identified (except one, the enzyme glyceraldehyde-3-phosphate dehydrogenase from Mesembryanthemum crystallinum) were not of plant origin but belonged to the Apis mellifera proteome. Among the total proteins identified (eight, but only seven as basic constituents of all types of honey) five belonged to the family of major royal jelly proteins 1-5, and were also the most abundant ones in any type of honey, together with α-glucosidase and defensin-1. It thus appears that honey has a proteome resembling the royal jelly proteome (but with considerably fewer species), except that its protein concentration is lower by three to four orders of magnitude as compared to royal jelly. Attempts at identifying additional plant (pollen, nectar) proteins via peptidome analysis were unsuccessful. BACKGROUND: In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee's genome. RESULTS: We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals. CONCLUSIONS: The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee. The genome of the western honeybee (Apis mellifera) harbors nine transcribed major royal jelly protein genes (mrjp1-9) which originate from a single-copy precursor via gene duplication. The first MRJP was identified in royal jelly, a secretion of the bees' hypopharyngeal glands that is used by young worker bees, called nurses, to feed developing larvae. Thus, MRJPs are frequently assumed to mainly have functions for developing bee larvae and to be expressed in the food glands of nurse bees. In-depth knowledge on caste- and age-specific role and abundance of MRJPs is missing. We here show, using combined quantitative real-time PCR with quantitative mass spectrometry, that expression and protein amount of mrjp1-5 and mrjp7 show an age-dependent pattern in worker's hypopharyngeal glands as well as in brains, albeit lower relative abundance in brains than in glands. Expression increases after hatching until the nurse bee period and is followed by a decrease in older workers that forage for plant products. Mrjp6 expression deviates considerably from the expression profiles of the other mrjps, does not significantly vary in the brain, and shows its highest expression in the hypopharyngeal glands during the forager period. Furthermore, it is the only mrjp of which transcript abundance does not correlate with protein amount. Mrjp8 and mrjp9 show, compared to the other mrjps, a very low expression in both tissues. Albeit mrjp8 mRNA was detected via qPCR, the protein was not quantified in any of the tissues. Due to the occurrence of MRJP8 and MRJP9 in other body parts of the bees, for example, the venom gland, they might not have a hypopharyngeal gland- or brain-specific function but rather functions in other tissues. Thus, mrjp1-7 but not mrjp8 and mrjp9 might be involved in the regulation of phenotypic plasticity and age polyethism in worker honeybees. Understanding the effect of pesticides on the survival of honeybee colonies is important because these pollinators are reportedly declining globally. In the present study, we examined the changes in the head proteome of nurse honeybees exposed to individual and combined pesticides (the fungicide pyraclostrobin and the insecticide fipronil) at field-relevant doses (850 and 2.5 ppb, respectively). The head proteomes of bees exposed to pesticides were compared with those of bees that were not exposed, and proteins with differences in expression were identified by mass spectrometry. The exposure of nurse bees to pesticides reduced the expression of four of the major royal jelly proteins (MRJP1, MRJP2, MRJP4, and MRJP5) and also several proteins associated with carbohydrate metabolism and energy synthesis, the antioxidant system, detoxification, biosynthesis, amino acid metabolism, transcription and translation, protein folding and binding, olfaction, and learning and memory. Overall, when pyraclostrobin and fipronil were combined, the changes in protein expression were exacerbated. Our results demonstrate that vital proteins and metabolic processes are impaired in nurse honeybees exposed to pesticides in doses close to those experienced by these insects in the field, increasing their susceptibility to stressors and affecting the nutrition and maintece of both managed and natural colonies.

What is BEL(Biological Expression Language) used for?

Biological Expression Language (BEL) is a novel method for the statistical extraction of causal relation relationships from large biomedical literature datasets.

Towards the development of a systems biology-based risk assessment approach for environmental toxicants, including tobacco products in a systems toxicology setting such as the "21st Century Toxicology", we are building a series of computable biological network models specific to non-diseased pulmonary and cardiovascular cells/tissues which capture the molecular events that can be activated following exposure to environmental toxicants. Here we extend on previous work and report on the construction and evaluation of a mechanistic network model focused on DNA damage response and the four main cellular fates induced by stress: autophagy, apoptosis, necroptosis, and senescence. In total, the network consists of 34 sub-models containing 1052 unique nodes and 1538 unique edges which are supported by 1231 PubMed-referenced literature citations. Causal node-edge relationships are described using the Biological Expression Language (BEL), which allows for the semantic representation of life science relationships in a computable format. The Network is provided in .XGMML format and can be viewed using freely available network visualization software, such as Cytoscape. Neurodegenerative as well as autoimmune diseases have unclear aetiologies, but an increasing number of evidences report for a combination of genetic and epigenetic alterations that predispose for the development of disease. This review examines the major milestones in epigenetics research in the context of diseases and various computational approaches developed in the last decades to unravel new epigenetic modifications. However, there are limited studies that systematically link genetic and epigenetic alterations of DNA to the aetiology of diseases. In this work, we demonstrate how disease-related epigenetic knowledge can be systematically captured and integrated with heterogeneous information into a functional context using Biological Expression Language (BEL). This novel methodology, based on BEL, enables us to integrate epigenetic modifications such as DNA methylation or acetylation of histones into a specific disease network. As an example, we depict the integration of epigenetic and genetic factors in a functional context specific to Parkinson's disease (PD) and Multiple Sclerosis (MS). Biological expression language (BEL) is one of the most popular languages to represent the causal and correlative relationships among biological events. Automatically extracting and representing biomedical events using BEL can help biologists quickly survey and understand relevant literature. Recently, many researchers have shown interest in biomedical event extraction. However, the task is still a challenge for current systems because of the complexity of integrating different information extraction tasks such as named entity recognition (NER), named entity normalization (NEN) and relation extraction into a single system. In this study, we introduce our BelSmile system, which uses a semantic-role-labeling (SRL)-based approach to extract the NEs and events for BEL statements. BelSmile combines our previous NER, NEN and SRL systems. We evaluate BelSmile using the BioCreative V BEL task dataset. Our system achieved an F-score of 27.8%, ∼7% higher than the top BioCreative V system. The three main contributions of this study are (i) an effective pipeline approach to extract BEL statements, and (ii) a syntactic-based labeler to extract subject-verb-object tuples. We also implement a web-based version of BelSmile (iii) that is publicly available at iisrserv.csie.ncu.edu.tw/belsmile. Biological expression language (BEL) is one of the main formal representation models of biological networks. The primary source of information for curating biological networks in BEL representation has been literature. It remains a challenge to identify relevant articles and the corresponding evidence statements for curating and validating BEL statements. In this paper, we describe BELTracker, a tool used to retrieve and rank evidence sentences from PubMed abstracts and full-text articles for a given BEL statement (per the 2015 task requirements of BioCreative V BEL Task). The system is comprised of three main components, (i) translation of a given BEL statement to an information retrieval (IR) query, (ii) retrieval of relevant PubMed citations and (iii) finding and ranking the evidence sentences in those citations. BELTracker uses a combination of multiple approaches based on traditional IR, machine learning, and heuristics to accomplish the task. The system identified and ranked at least one fully relevant evidence sentence in the top 10 retrieved sentences for 72 out of 97 BEL statements in the test set. BELTracker achieved a precision of 0.392, 0.532 and 0.615 when evaluated with three criteria, namely full, relaxed and context criteria, respectively, by the task organizers. Our team at Mayo Clinic was the only participant in this task. BELTracker is available as a RESTful API and is available for public use.Database URL: http://www.openbionlp.org:8080/BelTracker/finder/Given_BEL_Statement. Automatic extraction of biological network information is one of the most desired and most complex tasks in biological and medical text mining. Track 4 at BioCreative V attempts to approach this complexity using fragments of large-scale manually curated biological networks, represented in Biological Expression Language (BEL), as training and test data. BEL is an advanced knowledge representation format which has been designed to be both human readable and machine processable. The specific goal of track 4 was to evaluate text mining systems capable of automatically constructing BEL statements from given evidence text, and of retrieving evidence text for given BEL statements. Given the complexity of the task, we designed an evaluation methodology which gives credit to partially correct statements. We identified various levels of information expressed by BEL statements, such as entities, functions, relations, and introduced an evaluation framework which rewards systems capable of delivering useful BEL fragments at each of these levels. The aim of this evaluation method is to help identify the characteristics of the systems which, if combined, would be most useful for achieving the overall goal of automatically constructing causal biological networks from text. Success in extracting biological relationships is mainly dependent on the complexity of the task as well as the availability of high-quality training data. Here, we describe the new corpora in the systems biology modeling language BEL for training and testing biological relationship extraction systems that we prepared for the BioCreative V BEL track. BEL was designed to capture relationships not only between proteins or chemicals, but also complex events such as biological processes or disease states. A BEL opub is the smallest unit of information and represents a biological relationship with its provece. In BEL relationships (called BEL statements), the entities are normalized to defined namespaces mainly derived from public repositories, such as sequence databases, MeSH or publicly available ontologies. In the BEL opubs, the BEL statements are associated with citation information and supportive evidence such as a text excerpt. To enable the training of extraction tools, we prepared BEL resources and made them available to the community. We selected a subset of these resources focusing on a reduced set of namespaces, namely, human and mouse genes, ChEBI chemicals, MeSH diseases and GO biological processes, as well as relationship types 'increases' and 'decreases'. The published training corpus contains 11 000 BEL statements from over 6000 supportive text excerpts. For method evaluation, we selected and re-annotated two smaller subcorpora containing 100 text excerpts. For this re-annotation, the inter-annotator agreement was measured by the BEL track evaluation environment and resulted in a maximal F-score of 91.18% for full statement agreement. In addition, for a set of 100 BEL statements, we do not only provide the gold standard expert annotations, but also text excerpts pre-selected by two automated systems. Those text excerpts were evaluated and manually annotated as true or false supportive in the course of the BioCreative V BEL track task.Database URL: http://wiki.openbel.org/display/BIOC/Datasets. Network-based approaches have become extremely important in systems biology to achieve a better understanding of biological mechanisms. For network representation, the Biological Expression Language (BEL) is well designed to collate findings from the scientific literature into biological network models. To facilitate encoding and biocuration of such findings in BEL, a BEL Information Extraction Workflow (BELIEF) was developed. BELIEF provides a web-based curation interface, the BELIEF Dashboard, that incorporates text mining techniques to support the biocurator in the generation of BEL networks. The underlying UIMA-based text mining pipeline (BELIEF Pipeline) uses several named entity recognition processes and relationship extraction methods to detect concepts and BEL relationships in literature. The BELIEF Dashboard allows easy curation of the automatically generated BEL statements and their context annotations. Resulting BEL statements and their context annotations can be syntactically and semantically verified to ensure consistency in the BEL network. In summary, the workflow supports experts in different stages of systems biology network building. Based on the BioCreative V BEL track evaluation, we show that the BELIEF Pipeline automatically extracts relationships with an F-score of 36.4% and fully correct statements can be obtained with an F-score of 30.8%. Participation in the BioCreative V Interactive task (IAT) track with BELIEF revealed a systems usability scale (SUS) of 67. Considering the complexity of the task for new users-learning BEL, working with a completely new interface, and performing complex curation-a score so close to the overall SUS average highlights the usability of BELIEF.Database URL: BELIEF is available at http://www.scaiview.com/belief/. SUMMARY: Biological Expression Language (BEL) assembles knowledge networks from biological relations across multiple modes and scales. Here, we present PyBEL; a software package for parsing, validating, converting, storing, querying, and visualizing networks encoded in BEL. AVAILABILITY AND IMPLEMENTATION: PyBEL is implemented in platform-independent, universal Python code. Its source is distributed under the Apache 2.0 License at https://github.com/pybel. CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. The BioCreative-V community proposed a challenging task of automatic extraction of causal relation network in Biological Expression Language (BEL) from the biomedical literature. Previous studies on this task largely used models induced from other related tasks and then transformed intermediate structures to BEL statements, which left the given training corpus unexplored. To make full use of the BEL training corpus, in this work, we propose a deep learning-based approach to extract BEL statements. Specifically, we decompose the problem into two subtasks: entity relation extraction and entity function detection. First, two attention-based bidirectional long short-term memory networks models are used to extract entity relation and entity function, respectively. Then entity relation and their functions are combined into a BEL statement. In order to boost the overall performance, a strategy of threshold filtering is applied to improve the precision of identified entity functions. We evaluate our approach on the BioCreative-V Track 4 corpus with or without gold entities. The experimental results show that our method achieves the state-of-the-art performance with an overall F1-measure of 46.9% in stage 2 and 21.3% in stage 1, respectively. BACKGROUND: Extracting relations between bio-entities from biomedical literature is often a challenging task and also an essential step towards biomedical knowledge expansion. The BioCreative community has organized a shared task to evaluate the robustness of the causal relationship extraction algorithms in Biological Expression Language (BEL) from biomedical literature. METHOD: We first map the sentence-level BEL statements in the BC-V training corpus to the corresponding text segments, thus generating hierarchically tagged training instances. A hierarchical sequence labeling model was afterwards induced from these training instances and applied to the test sentences in order to construct the BEL statements. RESULTS: The experimental results on extracting BEL statements from BioCreative V Track 4 test corpus show that our method achieves promising performance with an overall F-measure of 31.6%. Furthermore, it has the potential to be enhanced by adopting more advanced machine learning approaches. CONCLUSION: We propose a framework for hierarchical relation extraction using hierarchical sequence labeling on the instance-level training corpus derived from the original sentence-level corpus via word alignment. Its main advantage is that we can make full use of the original training corpus to induce the sequence labelers and then apply them to the test corpus. Knowledge of the molecular interactions of biological and chemical entities and their involvement in biological processes or clinical phenotypes is important for data interpretation. Unfortunately, this knowledge is mostly embedded in the literature in such a way that it is unavailable for automated data analysis procedures. Biological expression language (BEL) is a syntax representation allowing for the structured representation of a broad range of biological relationships. It is used in various situations to extract such knowledge and transform it into BEL networks. To support the tedious and time-intensive extraction work of curators with automated methods, we developed the BEL track within the framework of BioCreative Challenges. Within the BEL track, we provide training data and an evaluation environment to encourage the text mining community to tackle the automatic extraction of complex BEL relationships. In 2017 BioCreative VI, the 2015 BEL track was repeated with new test data. Although only minor improvements in text snippet retrieval for given statements were achieved during this second BEL task iteration, a significant increase of BEL statement extraction performance from provided sentences could be seen. The best performing system reached a 32% F-score for the extraction of complete BEL statements and with the given named entities this increased to 49%. This time, besides rule-based systems, new methods involving hierarchical sequence labeling and neural networks were applied for BEL statement extraction.

Which database contains gene expression data for yeast?

We developed the ExpressDB database for yeast RNA expression data and loaded it with approximately 17.5 million pieces of data reported by 11 studies with three different kinds of high-throughput RNA assays.

We report steps toward the systematic management, standardization, and analysis of functional genomics data. We developed the ExpressDB database for yeast RNA expression data and loaded it with approximately 17.5 million pieces of data reported by 11 studies with three different kinds of high-throughput RNA assays. A web-based tool supports queries across the data from these studies. We examined comparability of data by converting data from 9 studies (217 conditions) into mRNA relative abundance estimates (ERAs) and by clustering of conditions by ERAs. We report on generation of ERAs and condition clustering for non-microarray data (5 studies, 63 conditions) and describe initial attempts to generate microarray-based ERAs (4 studies, 154 conditions), which exhibit increased error, on our web site http://arep.med.harvard. edu/ExpressDB. We recommend standards for data reporting, suggest research into improving comparability of microarray data through quantifying and standardizing control condition RNA populations, and also suggest research into the calibration of different RNA assays. We introduce a model for a database that integrates different kinds of functional genomics data, Biomolecule Interaction, Growth and Expression Database (BIGED).

Which CYP gene polymorphism is a well-known predictor of efavirenz disposition?

Cytochrome P450 (CYP) CYP2B6 G516T (rs3745274) is a well-known predictor of efavirenz disposition.

Background and Objectives: Severe, recalcitrant cases of pediatric psoriasis or atopic dermatitis may necessitate treatment with biological agents; however, this may be difficult due to lack of treatment options and standardized treatment guidelines. This review evaluates the biological treatment options available, including off-label uses, and provides a basic therapeutic guideline for pediatric psoriasis and atopic dermatitis. Materials and Methods: A PubMed review of biological treatments for pediatric psoriasis and atopic dermatitis with information regarding age, efficacy, dosing, contra-indications, adverse events, and off-label treatments. Results: Currently there are three European Medicines Agency (EMA)-approved biological treatment options for pediatric psoriasis: etanercept, ustekinumab, and adalimumab. While dupilumab was recently Food and Drug Administration (FDA)- and EMA-approved for adult atopic dermatitis, it is still not yet approved for pediatric atopic dermatitis. Conclusions: Given the high morbidity associated with pediatric atopic dermatitis and psoriasis, there is a need for more treatment options. Further research and post-marketing registries are needed to extend the use of biologics into pediatric patients. Author information: (1)Clinical Pharmacology Department, Instituto Teófilo Herdo, Universidad Autónoma de Madrid (UAM) Instituto de Investigación Sanitaria La Princesa (IP), Hospital Universitario de La Princesa, Madrid, Spain. (2)UICEC Hospital Universitario de La Princesa, Plataforma SCReN (Spanish Clinical Research Network), Instituto de Investigación Sanitaria La Princesa (IP), Madrid, Spain. (3)Clinical Pharmacology Department, Instituto Teófilo Herdo, Universidad Autónoma de Madrid (UAM) Instituto de Investigación Sanitaria La Princesa (IP), Hospital Universitario de La Princesa, Madrid, Spain. [email protected]. (4)UICEC Hospital Universitario de La Princesa, Plataforma SCReN (Spanish Clinical Research Network), Instituto de Investigación Sanitaria La Princesa (IP), Madrid, Spain. [email protected]. (5)Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain. [email protected]. (6)Pharmacology Department, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain. [email protected].

What is the role of the IRE1a-XBP1 pathway?

The IRE1a-XBP1 pathway is a conserved adaptive mediator of the unfolded protein response, playing an important role in the regulation of cell differentiation.

BACKGROUND: The IRE1a-XBP1 pathway is a conserved adaptive mediator of the unfolded protein response. The pathway is indispensable for the development of secretory cells by facilitating protein folding and enhancing secretory capacity. In the immune system, it is known to function in dendritic cells, plasma cells, and eosinophil development and differentiation, while its role in T helper cell is unexplored. Here, we investigated the role of the IRE1a-XBP1 pathway in regulating activation and differentiation of type-2 T helper cell (Th2), a major T helper cell type involved in allergy, asthma, helminth infection, pregcy, and tumor immunosuppression. METHODS: We perturbed the IRE1a-XBP1 pathway and interrogated its role in Th2 cell differentiation. We performed genome-wide transcriptomic analysis of differential gene expression to reveal IRE1a-XBP1 pathway-regulated genes and predict their biological role. To identify direct target genes of XBP1 and define XBP1's regulatory network, we performed XBP1 ChIPmentation (ChIP-seq). We validated our predictions by flow cytometry, ELISA, and qPCR. We also used a fluorescent ubiquitin cell cycle indicator mouse to demonstrate the role of XBP1 in the cell cycle. RESULTS: We show that Th2 lymphocytes induce the IRE1a-XBP1 pathway during in vitro and in vivo activation. Genome-wide transcriptomic analysis of differential gene expression by perturbing the IRE1a-XBP1 pathway reveals XBP1-controlled genes and biological pathways. Performing XBP1 ChIPmentation (ChIP-seq) and integrating with transcriptomic data, we identify XBP1-controlled direct target genes and its transcriptional regulatory network. We observed that the IRE1a-XBP1 pathway controls cytokine secretion and the expression of two Th2 signature cytokines, IL13 and IL5. We also discovered that the IRE1a-XBP1 pathway facilitates activation-dependent Th2 cell proliferation by facilitating cell cycle progression through S and G2/M phase. CONCLUSIONS: We confirm and detail the critical role of the IRE1a-XBP1 pathway during Th2 lymphocyte activation in regulating cytokine expression, secretion, and cell proliferation. Our high-quality genome-wide XBP1 ChIP and gene expression data provide a rich resource for investigating XBP1-regulated genes. We provide a browsable online database available at http://data.teichlab.org .

Can Panitumumab cause trichomegaly?

Yes. Panitumumab is EGFR inhibitor that is associated with eyelash trichomegaly.

Eyelash trichomegaly is an uncommon drug-associated sequelae experienced during treatment with epidermal growth factor receptor (EGFR) inhibitors. Elongation of the eyelashes induced by these agents has predomitly been observed in oncology patients with either colorectal or lung cancer. It is most frequently associated with cetuximab and erlotinib; however, it has also been described in individuals treated with gefitinib or panitumumab. We describe cetuximab-associated eyelash trichomegaly in a woman with metastatic rectal carcinoma. We review the clinical presentation, adverse effects, and management of EGFR inhibitor-related eyelash trichomegaly. The long eyelashes are not a drug-limiting adverse effect and some patients consider the change to be cosmetically enhancing. Trimming the lashes with scissors can usually ameliorate local symptoms. The eyelashes often return to their original length at variable time periods after EGFR inhibitor therapy is discontinued. A wide spectrum of skin toxicities has been described in patients receiving epidermal growth factor receptor (EGFR), inhibitors, including papulopustular rash, xerosis and fissures, pruritus, mucositis, paronychia, and hair changes.Trichomegaly of the eyelashes is a rare adverse effect of EGFR inhibitor therapy and is characterized by a paradoxical overgrowth of eyelashes. We present 3 cases of trichomegaly occurred during EGFR inhibitor therapy.

List proteins that are contained in atherosclerotic plaques?

extracellular matrix proteins biglycan Lumican Apolipoprotein A-I

Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms. To gain deeper insights into the composition of atherosclerotic plaques, we created quantitative proteome profiles of advanced plaque tissues of six male patients undergoing carotid endarterectomy for stroke prevention. Using a quantitative, data-independent proteome approach, we identified 4181 proteins with an average protein coverage of 45%. An analysis of the quantitative composition of the tissue revealed key players of vascular remodeling processes. Moreover, compared with proximal arterial tissue, 20 proteins in mature plaques were enriched, whereas 52 proteins were found in lower quantities. Among the proteins with increased abundance were prominent extracellular matrix proteins such as biglycan and lumican, whereas cytoskeletal markers for contractile smooth muscle cells (SMCs) were decreased. Taken together, this study provides the most comprehensive quantitative assessment of mature human plaque tissue to date, which indicates a central role of SMCs in the structure of advanced atherosclerotic plaques. To evaluate the presence of serum protein biomarkers associated with the early phases of formation of carotid atherosclerotic plaques, label-free quantitative proteomics analyses were made for serum samples collected as part of The Cardiovascular Risk in Young Finns Study. Samples from subjects who had an asymptomatic carotid artery plaque detected by ultrasound examination (N = 43, Age = 30-45 years) were compared with plaque free controls (N = 43) (matched for age, sex, body weight and systolic blood pressure). Seven proteins (p < 0.05) that have been previously linked with atherosclerotic phenotypes were differentially abundant. Fibulin 1 proteoform C (FBLN1C), Beta-ala-his-dipeptidase (CNDP1), Cadherin-13 (CDH13), Gelsolin (GSN) and 72 kDa type IV collagenase (MMP2) were less abundant in cases, whereas Apolipoproteins C-III (APOC3) and apolipoprotein E (APOE) were more abundant. Using machine learning analysis, a biomarker panel of FBLN1C, APOE and CDH13 was identified, which classified cases from controls with an area under receiver-operating characteristic curve (AUROC) value of 0.79. Furthermore, using selected reaction monitoring mass spectrometry (SRM-MS) the decreased abundance of FBLN1C was verified. In relation to previous associations of FBLN1C with atherosclerotic lesions, the observation could reflect its involvement in the initiation of the plaque formation, or represent a particular risk phenotype. Arterial foam cells are central players of atherogenesis. Cholesterol acceptors, apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL), take up cholesterol and phospholipids effluxed from foam cells into the circulation. Due to the high abundance of cholesterol in foam cells, most previous studies focused on apoA-I/HDL-mediated free cholesterol (FC) transport. However, recent lipidomics of human atherosclerotic plaques also identified that oxidized sterols (oxysterols) and non-sterol lipid species accumulate as atherogenesis progresses. While it is known that these lipids regulate expression of pro-inflammatory genes linked to plaque instability, how cholesterol acceptors impact the foam cell lipidome, particularly oxysterols and non-sterol lipids, remains unexplored. Using lipidomics analyses, we found cholesterol acceptors remodel foam cell lipidomes. Lipid subclass analyses revealed various oxysterols, sphingomyelins, and ceramides, species uniquely enriched in human plaques were significantly reduced by cholesterol acceptors, especially by apoA-I. These results indicate that the function of lipid-poor apoA-I is not limited to the efflux of cholesterol and phospholipids but suggest that apoA-I serves as a major regulator of the foam cell lipidome and might play an important role in reducing multiple lipid species involved in the pathogenesis of atherosclerosis. Cardiovascular disease (CVD) is the most common cause of death in industrialized countries. One underlying cause is atherosclerosis, which is a systemic disease characterized by plaques of retained lipids, inflammatory cells, apoptotic cells, calcium and extracellular matrix (ECM) proteins in the arterial wall. The biologic composition of an atherosclerotic plaque determines whether the plaque is more or less vulnerable, that is prone to rupture or erosion. Here, the ECM and tissue repair play an important role in plaque stability, vulnerability and progression. This review will focus on ECM remodelling in atherosclerotic plaques, with focus on how ECM biomarkers might predict plaque vulnerability and outcome.

List characteristics of Developmental and Epileptic Encephalopathies (DEEs).

yes, developmental and epileptic encephalopathies (dees) are a group of severe, early onset epilepsies characterized by refractory seizures, developmental delay or regression associated with ongoing epileptic activity, and generally poor prognosis.

Author information: (1)Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC H3T1C5, Canada. (2)Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, WA 98195, USA. (3)Centre Hospitalier de l'Université de Montréal Research Center, Montreal, QC H2X 0A9, Canada; Department of Neurosciences, Université de Montréal, Montreal, QC H3T1J4, Canada. (4)Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A2B4, Canada. (5)McGill University and Genome Quebec Innovation Center, Montreal, QC H3A 1A4, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 1B1, Canada. (6)Centre Hospitalier de l'Université de Montréal Research Center, Montreal, QC H2X 0A9, Canada; Center for Pediatric Genomic Medicine, Children's Mercy Kansas City, Kansas City, MO 64108, USA; Department of Pathology and Laboratory Medicine, Children's Mercy Kansas City, Kansas City, MO 64108, USA. (7)Centre Hospitalier de l'Université de Montréal Research Center, Montreal, QC H2X 0A9, Canada. (8)GeneDx, Gaithersburg, MD 20877, USA. (9)Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. (10)Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Baylor Miraca Genetics Laboratories, Baylor College of Medicine, Houston, TX 77021, USA. (11)Program in Genetics and Genome Biology, Division of Neurology, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON M5G 0A4, Canada. (12)Division of Neurology, Epilepsy Genetics Program, Krembil Neuroscience Centre, Toronto Western Hospital, University of Toronto, Toronto, ON M5G 2C4, Canada. (13)Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia. (14)Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. (15)MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. (16)Clinical Genetics Service, University Hospitals Bristol NHS Foundation Trust, St. Michael's Hospital, St. Michael's Hill, Bristol BS2 8DT, UK. (17)North West Thames Regional Genetics Service, London North West Healthcare NHS Trust, Northwick Park Hospital, Watford Road, Harrow HA1 3UJ, UK. (18)Oxford Centre for Genomic Medicine, ACE building Nuffield Orthopaedic Centre, Oxford University Hospitals NHS Foundation Trust, Oxford OX3 7HE, UK. (19)Manchester Centre for Genomic Medicine, St. Mary's Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester M13 9WL, UK. (20)Institute of Medical Genetics, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK. (21)MRC Centre for Neuropsychiatric Genetics & Genomics, Hadyn Ellis Building, Cathays, Cardiff University, Cardiff CF24 4HQ, UK. (22)West of Scotland Regional Genetics Service, Queen Elizabeth University Hospital, Glasgow G51 4TF, UK. (23)North East Thames Regional Genetics Service, Great Ormond Street Hospital for Children, London WC1N 3JH, UK. (24)Yorkshire Regional Genetics Service, Leeds Teaching Hospitals NHS Trust, Department of Clinical Genetics, Chapel Allerton Hospital, Chapeltown Road, Leeds LS7 4SA, UK. (25)Department of Pediatrics, Section of Medical Genetics, SUNY Upstate Medical University, Syracuse, NY 13210, USA. (26)University of Groningen, University Medical Center Groningen, Department of Genetics, 9700 RB Groningen, the Netherlands. (27)University of South Dakota Sanford School of Medicine, Sioux Falls, SD 57117, USA. (28)Augustana-Sanford Genetic Counseling Graduate Program, Sioux Falls, SD 57197, USA. (29)Departments of Medicine and Pediatrics, Columbia University Medical Center, New York, NY 10032, USA. (30)Baptist Hospital, Miami, FL 33176 USA. (31)Joe DiMaggio Children's Hospital, Hollywood, FL 33021, USA. (32)Division of Genetics and Genomic Medicine, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA. (33)Department of Human Genetics, Donders Centre for Brain, Cognition and Behavior, Radboud University Medical Center, 6500 HB Nijmegen, the Netherlands. (34)Centre de Génétique des Anomalies du Développement, Centre Hospitalier Universitaire de Dijon, 21000 Dijon, France; Équipe INSERM 1231, Génétique des Anomalies du Développement, Université de Bourgogne, 21000 Dijon, France. (35)Genetics Department, Assistance Publique - Hôpitaux de Paris, Robert-Debré University Hospital, 75000 Paris, France. (36)Department of Clinical Genetics, United Laboratories, Tartu University Hospital and Institute of Clinical Medicine, University of Tartu, Tartu 51014, Estonia. (37)Division of Genetics and Genomics and Division of Newborn Medicine, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. (38)Nationwide Children's Hospital and Ohio State University, Department of Pediatrics, Division of Neurology, Columbus, OH 43205, USA. (39)Division of Neurology, Children's Hospital of Eastern Ontario, Ottawa, ON K1H 8L1, Canada. (40)University of Tasmania, Royal Hobart Hospital, Department of Paediatrics, Hobart, TAS 7000, Australia. (41)School of Medicine, University of Tasmania, Hobart, TAS 7000, Australia. (42)Population Health and Immunity Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia. (43)Children's Hospital at Westmead Clinical School, University of Sydney, Westmead, NSW 2145, Australia. (44)Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Texas Children's Hospital, Houston, TX 77030, USA. (45)Dipartimento di Pediatria e di Neuropsichiatria Infantile, Università La Sapienza, 00185 Rome, Italy. (46)Dipartimento di Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, 00161 Rome, Italy. (47)Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, Istituto di Ricovero e Cura a Carattere Scientifico, 00165 Rome, Italy. (48)Metabolic Neurogenetic Clinic and Pediatric Movement Disorders Clinic, Wolfson Medical Center, Holon 5822012, Israel. (49)University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. (50)University of British Columbia, BC Children's Hospital, Vancouver, BC V6H 3N1, Canada. (51)Department of Medical Genetics, University of British Columbia, Vancouver, BC V6H 3N1, Canada. (52)Département de Génétique, Centre de Référence des Déficiences Intellectuelles de Causes Rares, Groupe de Recherche Clinique "Déficiences Intellectuelles et Autisme," Université Pierre et Marie Curie, Hôpital de la Pitié-Salpêtrière, Paris 75013, France; Sorbonne Universités, Université Pierre et Marie Curie (Université Paris 06), UMRS 1127, INSERM U 1127, CNRS UMR 7225, Institut du Cerveau et de la Moelle Épinière, Paris 75013, France. (53)Assistance Publique - Hôpitaux de Paris, Hôpital d'Enfants Armand Trousseau, Service de Neuropédiatrie, Paris 75012, France. (54)Université Paris Diderot, Sorbonne Paris Cité, INSERM UMR 1141, Paris 75019, France; Assistance Publique - Hôpitaux de Paris, Hôpital Robert Debré, Service de Neurologie Pédiatrique, Paris 75019, France. (55)HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA. (56)Department of Neurology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. (57)Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; Arkansas Children's Research Institute, Little Rock, AR 72205, USA. (58)Texas Children's Hospital and Baylor College of Medicine, Houston, TX 77030, USA. (59)Centre Hospitalier Rouyn-Noranda, Rouyn-Noranda, QC J9X 2B2, Canada. (60)Division of Neurology, Centre Hospitalier Universitaire de Québec, Quebec, QC G1V 4G2, Canada. (61)Department of Pediatrics, Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. (62)Department of Pediatrics, McGill University, Montreal, QC H3A 1A4, Canada; Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A4, Canada. (63)Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC H3T1C5, Canada; Department of Neurosciences, Université de Montréal, Montreal, QC H3T1J4, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC H3T1C5, Canada. (64)Department of Neurosciences, Université de Montréal, Montreal, QC H3T1J4, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC H3T1C5, Canada. (65)Department of Pediatrics and Child Health, University of Otago, Wellington 9016, New Zealand. (66)Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC H3T1C5, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC H3T1C5, Canada. (67)Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC H3T1C5, Canada; Department of Neurosciences, Université de Montréal, Montreal, QC H3T1J4, Canada. (68)Centre Hospitalier de l'Université de Montréal Research Center, Montreal, QC H2X 0A9, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 1B1, Canada; Département des Sciences Fondamentales, Université du Québec à Chicoutimi, Chicoutimi, QC G7H 2B1, Canada. (69)Division of Clinical and Metabolic Genetics, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada. (70)Division of Neurology, BC Children's Hospital, Vancouver, BC V6H 3N1, Canada. (71)Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia; Department of Pediatrics, University of Melbourne Royal Children's Hospital, Parkville, VIC 3052, Australia; Florey Institute of Neuroscience and Mental Health, Melbourne, VIC 3084, Australia. (72)Program in Genetics and Genome Biology, Division of Neurology, Department of Pediatrics, Hospital for Sick Children and University of Toronto, Toronto, ON M5G 0A4, Canada; Division of Child Neurology, Department of Pediatrics, University of Texas Southwestern, Dallas, TX 75390, USA. Electronic address: [email protected]. (73)Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC H3T1C5, Canada; Department of Neurosciences, Université de Montréal, Montreal, QC H3T1J4, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC H3T1C5, Canada. Electronic address: [email protected]. Author information: (1)UF Innovation en diagnostic genomique des maladies rares, CHU Dijon Bourgogne, Dijon, France. [email protected]. (2)INSERM UMR1231 GAD, F-21000, Dijon, France. [email protected]. (3)Universite Claude Bernard Lyon I, CHU de Lyon, Lyon, France. (4)Service de Radiologie, Hopital-Femme-Mère-Enfant, Hospices Civils de Lyon, Lyon, France. (5)INSERM UMR1231 GAD, F-21000, Dijon, France. (6)Departement de Genetique, Hopital Pitie-Salpetriere, Paris, France. (7)Division of Genetics and Metabolic Phoenix Children's Hospital, Phoenix, Arizona, USA. (8)Inserm U 1127, CNRS UMR 7225, Sorbonne Universites, UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle epinière, ICM, Paris, France. (9)Reference Center for Adult Neurometabolic Diseases, Pitie-Salpêtrière University Hospital, Paris, France. (10)Department of Genetics, University Medical Center, Utrecht, The Netherlands. (11)Centre de Reference maladies rares « Anomalies du Developpement et syndrome malformatifs » de l'Est, Centre de Genetique, Hopital d'Enfants, FHU TRANSLAD, CHU Dijon Bourgogne, Dijon, France. (12)UF Innovation en diagnostic genomique des maladies rares, CHU Dijon Bourgogne, Dijon, France. (13)Department of Child Neurology, Brain Center Rudolf Magnus, University Medical Center, Utrecht, The Netherlands. (14)Department of Pediatrics, Division of Medical Genetics, Cedars-Sinai Medical Center and Harbor-UCLA Medical Center, Los Angeles, California, USA. (15)Division of Pediatric Neurology, Department of Pediatrics, Harbor-UCLA Medical Center, Los Angeles, California, USA. (16)GeneDx, Gaithersburg, Maryland, USA. (17)Genomed Ltd., Moscow, Russia. (18)Veltischev Research and Clinical Institute for Pediatrics of the Pirogov Russian National Research Medical University, Moscow, Russia. (19)Division of Medical Genetics, Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California, USA. (20)Departments of Human Genetics and Psychiatry, David Geffen School of Medicine at UCLA, Los Angeles, California, USA. (21)Institute of Medical Genetics, University of Zurich, Schlieren, Zurich, Switzerland. (22)Division of Pediatric Neurology, Children's Hospital, Lucerne, Switzerland. (23)Department of Neurosciences and Pediatrics UCSD/Rady Children's Hospital San Diego, Rady Children's Institute for Genomic Medicine, San Diego, California, USA. (24)UF Innovation en diagnostic genomique des maladies rares, CHU Dijon Bourgogne, Dijon, France. [email protected]. (25)INSERM UMR1231 GAD, F-21000, Dijon, France. [email protected]. Author information: (1)Division of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. (2)Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tübingen, 72076 Tübingen, Germany. (3)Department of Physiology & Pharmacology, Hotchkiss Brain Institute and Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB T2N 1N4, Canada. (4)Division of Genetic Medicine, University of Washington, Seattle, WA 98195, USA. (5)APHP, Département de Génétique, Centre de Référence Déficiences Intellectuelles de Causes Rares, Groupe Hospitalier Pitié Salpêtrière et GHUEP Hôpital Trousseau; Sorbonne Université, GRC "Déficience Intellectuelle et Autisme," 75013 Paris, France. (6)Sorbonne Université, GRC n°19, Pathologies Congénitales du Cervelet-LeucoDystrophies, Département de Génétique et Embryologie Médicale, AP-HP, Hôpital d'Enfants Armand Trousseau, Centre de Référence des Déficits Intellectuels de Causes Rares, 75012 Paris, France. (7)Sorbonne Université, GRC n°19, Pathologies Congénitales du Cervelet-LeucoDystrophies, Service de Neuropédiatrie, AP-HP, Hôpital d'Enfants Armand Trousseau; Centre de Référence des Déficits Intellectuels de Causes Rares; Inserm U 1141, 75012 Paris, France. (8)Department of Clinical Genetics, Odense University Hospital, 5000 Odense, Denmark; H.C. Andersen Children's Hospital, Odense University Hospital, 5000 Odense, Denmark. (9)Department of Clinical Genetics, Odense University Hospital, 5000 Odense, Denmark. (10)Department of Human Genetics, Radboud University Medical Center, 6525 Nijmegen, the Netherlands. (11)Stichting Epilepsie Instellingen Nederland, 8025 Zwolle, the Netherlands. (12)Department of Neurology, Academic Center for Epileptology, Kempenhaeghe and Maastricht UMC, 5591 Heeze, the Netherlands. (13)Barrow Neurological Institute, Phoenix Children's Hospital, Departments of Child Health, Genetics, Neurology, and Cellular & Molecular Medicine, University of Arizona College of Medicine, Phoenix, AZ 85013, USA. (14)Division of Genetics and Metabolism, Phoenix Children's Hospital, Phoenix, AZ 85016, USA. (15)Yale School of Medicine, New Haven, CT 06510, USA. (16)Department of Pediatric Neurology, Penteli Children's Hospital, 152 36 Athens, Greece. (17)Institute of Biomedical Science and Children's Hospital Fudan University, 201102 Shanghai, China. (18)Perinatal Center, Sahlgrenska Academy, Gothenburg University, 413 46 Gothenburg, Sweden; Hospital of Zhengzhou University, 450001 Zhengzhou, China. (19)Epilepsy Research Centre, Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia. (20)Department of Health Science Research, Mayo Clinic, Rochester, MN 55905, USA. (21)Department of Neurology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. (22)Department of Health Science Research, Mayo Clinic, Rochester, MN 55905, USA; Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA. (23)Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA; Department of Clinical Genomics, Mayo Clinic, Rochester, MN 55905, USA. (24)University of Illinois Chicago College of Medicine, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA. (25)Adult Genetics Unit, Royal Adelaide Hospital, and School of Medicine, University of Adelaide, Adelaide, SA 5000, Australia. (26)Department of Neurology, Women's and Children's Hospital, University of Adelaide, North Adelaide, SA 5006, Australia. (27)Center for Human Disease Modeling, Duke University Medical Center, Durham, NC 27701, USA. (28)Adelaide Medical School, Robinson Research Institute, University of Adelaide, North Adelaide, SA 5006, Australia. (29)CeGaT, 72076 Tübingen, Germany. (30)Department of Human Genetics, University of Tübingen, 72076 Tübingen, Germany. (31)Division of Genetics and Genomics and Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02215, USA. (32)Northeast Regional Epilepsy Group, Hackensack University Medical Center, Hackensack, NJ 07601, USA. (33)University of Louisville, Louisville, KY 40292, USA. (34)Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA. (35)Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, UT 84113, USA. (36)Division of Pediatric Neurology, Departments of Pediatrics and Neurology, University of Utah, Salt Lake City, UT 84113, USA. (37)ARUP Laboratories, Salt Lake City, UT 84108, USA. (38)Division of Metabolic Disorders CHOC Children's Hospital, Orange, CA 92868, USA. (39)Division of Metabolic Disorders CHOC Children's Hospital, Orange, CA 92868, USA; Department of Pediatrics, University of California-Irvine School of Medicine, Irvine, CA 92617, USA. (40)Centre for Brain Research and School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand. (41)Department of Neurology, Starship Children's Health, Auckland 1023, New Zealand. (42)Department of Paediatrics and Child Health, University of Otago Wellington, Wellington South 6242, New Zealand. (43)Department of Clinical Genetics, Karolinska University Hospital, 171 76 Stockholm, Sweden; Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 77 Stockholm, Sweden. (44)Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 77 Stockholm, Sweden. (45)Department of Neuroscience, Azienda Ospedaliero-Universitaria Meyer, University of Florence, 50139 Florence, Italy. (46)Greenwood Genetic Center, Greenwood, SC 29646, USA. (47)Medical Genetics, Sanford Health, Bemidji, MN 56601, USA. (48)Medical Genetics, Sanford Health, Fargo, ND 58102, USA. (49)Epilepsy Genetics Program, Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Harvard Medical School, Boston, MA 02215, USA. (50)Epilepsy Genetics Program, Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA. (51)Department of Neurology, Harvard Medical School, Boston, MA 02215, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA. (52)Department of Haematology, University of Cambridge, NHS Blood and Transplant Centre, Cambridge CB2 0QQ, UK; NIHR BioResource - Rare Diseases, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK. (53)Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter EX2 5DW, UK. (54)Department of Neurology, Royal Devon and Exeter NHS Foundation Trust, Exeter EX2 5DW, UK. (55)NIHR BioResource - Rare Diseases, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK; Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK. (56)Yorkshire Regional Genetics Service, Chapel Allerton Hospital Leeds Teaching Hospitals NHS Trust, Leeds LS7 4SA, UK. (57)Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK; Manchester Centre for Genomic Medicine, St. Mary's Hospital, Manchester University Foundation NHS Trust, Health Innovation, Manchester M13 9WL, UK. (58)Department of Paediatric Neurology, Royal Manchester Children's Hospital, Manchester University Foundation NHS Trust, Health Innovation, Manchester M13 9WL, UK. (59)Manchester Centre for Genomic Medicine, St. Mary's Hospital, Manchester University Foundation NHS Trust, Health Innovation, Manchester M13 9WL, UK. (60)Wellcome Sanger Institute, Cambridge CB10 1SA, UK. (61)Epilepsy Research Centre, Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, VIC 3084, Australia; The Florey Institute and Murdoch Children's Research Institute, Parkville, VIC 3052, Australia; Department of Paediatrics, The University of Melbourne, Royal Children's Hospital, Parkville, VIC 3052, Australia; Department of Neurology, Royal Children's Hospital, Parkville, VIC 3052, Australia. (62)Division of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Neuropediatrics, Christian-Albrechts-University of Kiel, 24105 Kiel, Germany. (63)Division of Genetic Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address: [email protected]. De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy With Contractures, Macrocephaly, and Dyskinesias Helbig KL, Lauerer RJ, Bahr JC, et al. Am J Hum Genet. 2019;104(3):562. Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on electroencephalogram (EEG), and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high-voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all 4 S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the antiepileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders. OBJECTIVE: Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene. METHODS: We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on CaV 2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling. RESULTS: We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M. SIGNIFICANCE: Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies. Developmental and Epileptic encephalopathies (DEE) describe heterogeneous epilepsy syndromes, characterized by early-onset, refractory seizures and developmental delay (DD). Several DEE associated genes have been reported. With increased access to whole exome sequencing (WES), new candidate genes are being identified although there are fewer large cohort papers describing the clinical phenotype in such patients. We describe 6 unreported individuals and provide updated information on an additional previously reported individual with heterozygous de novo missense variants in YWHAG. We describe a syndromal phenotype, report 5 novel, and a recurrent p.Arg132Cys YWHAG variant and compare developmental trajectory and treatment strategies in this cohort. We provide further evidence of causality in YWHAG variants. WES was performed in five patients via Deciphering Developmental Disorders Study and the remaining two were identified via Genematcher and AnnEX databases. De novo variants identified from exome data were validated using Sanger sequencing. Seven out of seven patients in the cohort have de novo, heterozygous missense variants in YWHAG including 2/7 patients with a recurrent c.394C > T, p.Arg132Cys variant; 1/7 has a second, pathogenic variant in STAG1. Characteristic features included: early-onset seizures, predomitly generalized tonic-clonic and absence type (7/7) with good response to standard anti-epileptic medications; moderate DD; Intellectual Disability (ID) (5/7) and Autism Spectrum Disorder (3/7). De novo YWHAG missense variants cause EE, characterized by early-onset epilepsy, ID and DD, supporting the hypothesis that YWHAG loss-of-function causes a neurological phenotype. Although the exact mechanism of disease resulting from alterations in YWHAG is not fully known, it is possible that haploinsufficiency of YWHAG in developing cerebral cortex may lead to abnormal neuronal migration resulting in DEE. BACKGROUND: Early-onset developmental and epileptic encephalopathy (DEE) is characterized by repeated seizures beginning within 3 months of birth and severe interictal epileptiform discharge, including burst suppression. This study assessed the utility of targeted gene panel sequencing in the genetic diagnosis of this disease. MATERIALS AND METHODS: Targeted gene panel sequencing was performed in 150 early infantile-onset DEE patients (≤3 months of age), and we extensively reviewed their clinical characteristics, including therapeutic efficacy, according to genotype. RESULTS: Of the early infantile-onset DEE patients, 70 were neonatal-onset DEE and the other 80 patients began experiencing seizures from 1 to 3 months after birth. There were 11 different pathogenic or likely pathogenic variants among 34.7% (52/150) of patients with early infantile-onset DEE, in whom KCNQ2, STXBP1, CDKL5, and SCN1A were the major pathogenic variants. Among the neonatal-onset DEE patients, pathological genes were identified in 42.9% (30/70), indicating a significantly higher diagnostic yield than in 27.5% (22/80) of patients who experienced seizure onset 1 to 3 months after birth (p = 0.048). Among the neonatal-onset DEE group, variants in KCNQ2, STXBP1, and CDKL5 were detected at high frequencies, accounting for 66.7% (20/30) of the pathogenic or likely pathogenic variants found in this study. CONCLUSION: Targeted gene panel sequencing demonstrated a high yield of pathogenic variants in the diagnosis of early-onset epileptic encephalopathy, especially in those with neonatal-onset DEE. Early diagnosis of early-onset epileptic encephalopathy may improve the prognosis of patients by earlier selection of appropriate treatment based on pathogenic variant. Developmental and epileptic encephalopathies (DEEs) are a group of severe, early onset epilepsies characterized by refractory seizures, developmental delay or regression associated with ongoing epileptic activity, and generally poor prognosis. DEE is genetically and phenotypically heterogeneous, and there is a plethora of genetic testing options to investigate the rapidly growing list of epilepsy genes. However, more than 50% of patients with DEE remain without a genetic diagnosis despite state-of-the-art genetic testing. In this review, we discuss the major advances in epilepsy genomics that have surfaced in recent years. The goal of this review is to reach a larger audience and build a better understanding of pathogenesis and genetic testing options in DEE. Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of disorders characterized by epilepsy with comorbid intellectual disability. Recently, two de novo heterozygous mutations in the gene encoding TRPM3, a calcium permeable ion channel, were identified as the cause of DEE in eight probands, but the functional consequences of the mutations remained elusive. Here we demonstrate that both mutations (V990M and P1090Q) have distinct effects on TRPM3 gating, including increased basal activity, higher sensitivity to stimulation by the endogenous neurosteroid pregnenolone sulfate (PS) and heat, and altered response to ligand modulation. Most strikingly, the V990M mutation affected the gating of the non-canonical pore of TRPM3, resulting in large inward cation currents via the voltage sensor domain in response to PS stimulation. Taken together, these data indicate that the two DEE mutations in TRPM3 result in a profound gain of channel function, which may lie at the basis of epileptic activity and neurodevelopmental symptoms in the patients. AIM: Developmental and epileptic encephalopathies (DEEs) are a group of devastating disorders caused by epileptic activity, resulting in deterioration in developmental, cognitive, and motor functions. The number of genes identified as being responsible for DEEs has been increasing rapidly. However, despite a comprehensive molecular analysis, a molecular diagnosis can only be established in 50% of cases. The aim of this project is to use whole exome sequencing (WES) to determine the molecular etiology of DEEs in undiagnosed patients with a pedigree suggestive of an autosomal recessive single gene disease. METHODS: Three DEE families, having either consanguineous parents of an affected individual and/or having more than one affected offspring, were enrolled in the project. Prior to this project, the families had been evaluated using a next-generation sequencing panel including 16 DEE genes in a previous study; however, no molecular diagnosis could be established. In five cases from the three selected DEEs families in our study, the genetic etiology was investigated using WES. RESULTS: All patients in the study group had infantile onset epileptic seizures; however, semiologies varied. All patients presented with severe developmental delay. WES revealed biallelic disease causing mutations in DENDD5A, GRN, and TBCD genes in family 1, family 2, and family 3, respectively. In each family, the identified variants associated with the disease were segregated. Reverse phenotyping supported the molecular analysis. CONCLUSION: This study provided a valuable contribution to the genotype-phenotype relationship by determining rare epilepsy syndromes in undiagnosed patients previously. WES is a useful diagnostic alternative, particularly in consanguineous families. Developmental and epileptic encephalopathies (DEEs) are the spectrum of severe epilepsies characterized by early-onset, refractory seizures occurring in the context of developmental regression or plateauing. Early infantile epileptic encephalopathy (EIEE) is one of the earliest forms of DEE, manifesting as frequent epileptic spasms and characteristic electroencephalogram findings in early infancy. In recent years, next-generation sequencing approaches have identified a number of monogenic determits underlying DEE. In the case of EIEE, 85 genes have been registered in Online Mendelian Inheritance in Man as causative genes. Model organisms are indispensable tools for understanding the in vivo roles of the newly identified causative genes. In this review, we first present an overview of epilepsy and its genetic etiology, especially focusing on EIEE and then briefly summarize epilepsy research using animal and patient-derived induced pluripotent stem cell (iPSC) models. The Drosophila model, which is characterized by easy gene manipulation, a short generation time, low cost and fewer ethical restrictions when designing experiments, is optimal for understanding the genetics of DEE. We therefore highlight studies with Drosophila models for EIEE and discuss the future development of their practical use. OBJECTIVE: SCN2A-associated developmental and epileptic encephalopathies (DEEs) present with seizures, developmental impairments, and often both. We sought to characterize the level and pattern of development in children with SCN2A variants, and to address the sensitivity of the Vineland Adaptive Behavior Scales (VABS) in measuring changes over time in children with SCN2A-DEEs. METHODS: Clinical histories for participants with pathogenic SCN2A variants in the Simons SearchLight project were analyzed for descriptive purposes. VABS scores obtained at study entry and yearly thereafter were analyzed for floor and ceiling effects, change with age, and association with epilepsy through use of regression and longitudinal regression methods. RESULTS: Sixty-four participants (50 with epilepsy, 30 [47%] female, median age 49 months, interquartile range [IQR] 28 to 101) were included. Histories of birth complications (N = 34, 54%), neonatal neurological signs (N = 45, 74%), and other neurological symptoms (N = 31, 48%) were common and similar in epilepsy and nonepilepsy subgroups. Mean standardized VABS scores (Composite 53.5; Motor, 55.8, Communication, 54.1, Socialization, 59.4, and Daily living skills, 55.1) reflected performance ~3 standard deviations below the normative test average. In longitudinal regression analyses, standardized scores decreased between 1.3 and 2.8 points per year, suggesting regression of abilities. Raw score analyses, however, revealed several subdomains with substantial floor effects (eg, community use); other raw scores increased with increasing age. Participants with epilepsy scored 0.6 to 1 SD lower than those without epilepsy (all P's < .05). SIGNIFICANCE: The VABS, as standardly administered, has shortcomings for addressing growth or regression in individuals with SCN2A-DEEs. Some subdomain raw scores reflected substantial floor effects. Raw scores increased so slowly over time that standardized scores declined. Alternative measures sensitive to incremental meaningful change are required if outcomes such as adaptive behavior are to be primary outcomes in short-term clinical trials.

Which methods exist for efficient calculation of Elementary flux modes (EFMs) in genome-scale metabolic networks (GSMNs)?

EFM-Ta is a novel algorithm that uses a linear programming-based tree search and efficiently enumerates a subset of EFMs in genome-scale metabolic networks (GSMNs). The stand-alone software TreeEFM is implemented in C++ and interacts with the open-source linear solver COIN-OR Linear program Solver (CLP).

MOTIVATION: Elementary flux modes (EFMs) analysis constitutes a fundamental tool in systems biology. However, the efficient calculation of EFMs in genome-scale metabolic networks (GSMNs) is still a challenge. We present a novel algorithm that uses a linear programming-based tree search and efficiently enumerates a subset of EFMs in GSMNs. RESULTS: Our approach is compared with the EFMEvolver approach, demonstrating a significant improvement in computation time. We also validate the usefulness of our new approach by studying the acetate overflow metabolism in the Escherichia coli bacteria. To do so, we computed 1 million EFMs for each energetic amino acid and then analysed the relevance of each energetic amino acid based on gene/protein expression data and the obtained EFMs. We found good agreement between previous experiments and the conclusions reached using EFMs. Finally, we also analysed the performance of our approach when applied to large GSMNs. AVAILABILITY AND IMPLEMENTATION: The stand-alone software TreeEFM is implemented in C++ and interacts with the open-source linear solver COIN-OR Linear program Solver (CLP).

What is the mechanism of action of vosoritide?

Vosoritide is a biologic analogue of C-type natriuretic peptide, a potent stimulator of endochondral ossification.

TransCon CNP is a C-type natriuretic peptide (CNP-38) conjugated via a cleavable linker to a polyethylene glycol carrier molecule, designed to provide sustained systemic CNP levels upon weekly subcutaneous administration. TransCon CNP is in clinical development for the treatment of comorbidities associated with achondroplasia. In both mice and cynomolgus monkeys, sustained exposure to CNP via TransCon CNP was more efficacious in stimulating bone growth than intermittent CNP exposure. TransCon CNP was well tolerated with no adverse cardiovascular effects observed at exposure levels exceeding the expected clinical therapeutic exposure. At equivalent dose levels, reductions in blood pressure and/or an increase in heart rate were seen following single subcutaneous injections of the unconjugated CNP-38 molecule or a daily CNP-39 molecule (same amino acid sequence as Vosoritide, USAN:INN). The half-life of the daily CNP-39 molecule in cynomolgus monkey was estimated to be 20 minutes, compared with 90 hours for CNP-38, released from TransCon CNP. C max for the CNP-39 molecule (20 µg/kg) was approximately 100-fold higher, compared with the peak CNP level associated with administration of 100 µg/kg CNP as TransCon CNP. Furthermore, CNP exposure for the daily CNP-39 molecule was only evident for up to 2 hours postdose (lower limit of quantification 37 pmol/l), whereas TransCon CNP gave rise to systemic exposure to CNP-38 for at least 7 days postdose. The prolonged CNP exposure and associated hemodynamically safe peak serum concentrations associated with TransCon CNP administration are suggested to improve efficacy, compared with short-lived CNP molecules, due to better therapeutic drug coverage and decreased risk of hypotension. SIGNIFICANCE STATEMENT: The hormone C-type natriuretic peptide (CNP) is in clinical development for the treatment of comorbidities associated with achondroplasia, the most common form of human dwarfism. The TransCon Technology was used to design TransCon CNP, a prodrug that slowly releases active CNP in the body over several days. Preclinical data show great promise for TransCon CNP to be an effective and well-tolerated drug that provides sustained levels of CNP in a convenient once-weekly dose, while avoiding high systemic CNP bolus concentrations that can induce cardiovascular side effects. BACKGROUND: Achondroplasia is a genetic disorder that inhibits endochondral ossification, resulting in disproportionate short stature and clinically significant medical complications. Vosoritide is a biologic analogue of C-type natriuretic peptide, a potent stimulator of endochondral ossification. METHODS: In a multinational, phase 2, dose-finding study and extension study, we evaluated the safety and side-effect profile of vosoritide in children (5 to 14 years of age) with achondroplasia. A total of 35 children were enrolled in four sequential cohorts to receive vosoritide at a once-daily subcutaneous dose of 2.5 μg per kilogram of body weight (8 patients in cohort 1), 7.5 μg per kilogram (8 patients in cohort 2), 15.0 μg per kilogram (10 patients in cohort 3), or 30.0 μg per kilogram (9 patients in cohort 4). After 6 months, the dose in cohort 1 was increased to 7.5 μg per kilogram and then to 15.0 μg per kilogram, and in cohort 2, the dose was increased to 15.0 μg per kilogram; the patients in cohorts 3 and 4 continued to receive their initial doses. At the time of data cutoff, the 24-month dose-finding study had been completed, and 30 patients had been enrolled in an ongoing long-term extension study; the median duration of follow-up across both studies was 42 months. RESULTS: During the treatment periods in the dose-finding and extension studies, adverse events occurred in 35 of 35 patients (100%), and serious adverse events occurred in 4 of 35 patients (11%). Therapy was discontinued in 6 patients (in 1 because of an adverse event). During the first 6 months of treatment, a dose-dependent increase in the annualized growth velocity was observed with vosoritide up to a dose of 15.0 μg per kilogram, and a sustained increase in the annualized growth velocity was observed at doses of 15.0 and 30.0 μg per kilogram for up to 42 months. CONCLUSIONS: In children with achondroplasia, once-daily subcutaneous administration of vosoritide was associated with a side-effect profile that appeared generally mild. Treatment resulted in a sustained increase in the annualized growth velocity for up to 42 months. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov numbers, NCT01603095, NCT02055157, and NCT02724228.).

What is the "flight-or-fight response"?

This is also known as " flight-or- fight response" and is defined as an individual's response to a stimulus such as stress, that includes loss of sleep, anxiety, shortness of breath, muscle and joint pain.

The brain is the key organ of the response to stress because it determines what is threatening and, therefore, potentially stressful, as well as the physiological and behavioral responses which can be either adaptive or damaging. Stress involves two-way communication between the brain and the cardiovascular, immune, and other systems via neural and endocrine mechanisms. Beyond the "flight-or-fight" response to acute stress, there are events in daily life that produce a type of chronic stress and lead over time to wear and tear on the body ("allostatic load"). Yet, hormones associated with stress protect the body in the short-run and promote adaptation ("allostasis"). The brain is a target of stress, and the hippocampus was the first brain region, besides the hypothalamus, to be recognized as a target of glucocorticoids. Stress and stress hormones produce both adaptive and maladaptive effects on this brain region throughout the life course. Early life events influence life-long patterns of emotionality and stress responsiveness and alter the rate of brain and body aging. The hippocampus, amygdala, and prefrontal cortex undergo stress-induced structural remodeling, which alters behavioral and physiological responses. As an adjunct to pharmaceutical therapy, social and behavioral interventions such as regular physical activity and social support reduce the chronic stress burden and benefit brain and body health and resilience. The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack. Although it is not known whether invertebrates possess an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a sympathetic-like response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli. Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state. Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems. More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors. In the complex microcosm of a cell, information security and its faithful transmission are critical for maintaining internal stability. To achieve a coordinated response of all its parts to any stimulus the cell must protect the information received from potentially confounding signals. Physical segregation of the information transmission chain ensures that only the entities able to perform the encoded task have access to the relevant information. The cAMP intracellular signaling pathway is an important system for signal transmission responsible for the ancestral 'flight or fight' response and involved in the control of critical functions including frequency and strength of heart contraction, energy metabolism and gene transcription. It is becoming increasingly apparent that the cAMP signaling pathway uses compartmentalization as a strategy for coordinating the large number of key cellular functions under its control. Spatial confinement allows the formation of cAMP signaling "hot spots" at discrete subcellular domains in response to specific stimuli, bringing the information in proximity to the relevant effectors and their recipients, thus achieving specificity of action. In this report we discuss how the different constituents of the cAMP pathway are targeted and participate in the formation of cAMP compartmentalized signaling events. We illustrate a few examples of localized cAMP signaling, with a particular focus on the nucleus, the sarcoplasmic reticulum and the mitochondria. Finally, we discuss the therapeutic potential of interventions designed to perturb specific cAMP cascades locally. Although the function of the autonomic nervous system (ANS) in mediating the flight-or-fight response was recognized decades ago, the crucial role of peripheral innervation in regulating cell behavior and response to the microenvironment has only recently emerged. In the hematopoietic system, the ANS regulates stem cell niche homeostasis and regeneration and fine-tunes the inflammatory response. Additionally, emerging data suggest that cancer cells take advantage of innervating neural circuitry to promote their progression. These new discoveries outline the need to redesign therapeutic strategies to target this underappreciated stromal constituent. Here, we review the importance of neural signaling in hematopoietic homeostasis, inflammation, and cancer.

How is Burke-Fahn-Marsden Dystonia scale used?

Burck-Fahn-Marsden Dystonia scale (BBS) is a simple, reliable, and valid measure of disease severity in patients with dystonia.

Object. Deep brain stimulation (DBS) of the globus pallidus internus (GPi) is a promising new procedure for the treatment of dystonia. The authors present their technical approach for placement of electrodes into the GPi in awake patients with dystonia, including the methodology used for electrophysiological mapping of the GPi in the dystonic state, clinical outcomes and complications, and the location of electrodes associated with optimal benefit. Methods. Twenty-three adult and pediatric patients who had various forms of dystonia were included in this study. Baseline neurological status and improvement in motor function resulting from DBS were measured using the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). Implantation of the DBS lead was performed using magnetic resoce (MR) imaging-based stereotaxy, single-cell microelectrode recording, and intraoperative test stimulation to determine thresholds for stimulation-induced adverse effects. Electrode locations were measured on computationally reformatted postoperative MR images according to a prospective protocol. Conclusions. Physiologically guided implantation of DBS electrodes in patients with dystonia is technically feasible in the awake state in most cases, with low morbidity rates. Spontaneous discharge rates of GPi neurons in dystonia are similar to those of globus pallidus externus neurons, such that the two nuclei must be distinguished by neuronal discharge patterns rather than by rates. Active electrode locations associated with robust improvement (> 50% decrease in BFMDRS score) were located near the intercommissural plane, at a mean distance of 3.7 mm from the pallidocapsular border. Patients with juvenile-onset primary dystonia and those with the tardive form benefited greatly from this procedure, whereas benefits for most secondary dystonias and the adult-onset craniocervical form of this disorder were more modest. Seven children between 2 and 15 years of age with cerebral palsy and upper extremity dystonia were enrolled in an open-label, dose-escalation pilot clinical trial of botulinum toxin type B (Myobloc), injected into the biceps and brachioradialis muscles of I or both arms. The primary outcome measure was the change in maximum speed of hand movement during attempted forward reaching. Escalating doses of 12.5, 25, and 50 U/kg per muscle were injected at each of 3 visits. Reaching speed improved in response to injection, and dystonia scores on the Burke-Fahn-Marsden dystonia scale, the Unified Dystonia Rating Scale, and the Unified Parkinson's Disease Rating Scale improved. There was not a dose-related effect on efficacy. There were no serious adverse events. Two children reported transient weakness. These results support the use of botulinum toxin type B as a safe and effective treatment for upper extremity dystonia in children with cerebral palsy. Larger controlled trials are needed to confirm these results. Neurodegeneration with brain iron accumulation encompasses a heterogeneous group of rare neurodegenerative disorders that are characterized by iron accumulation in the brain. Severe generalized dystonia is frequently a prominent symptom and can be very disabling, causing gait impairment, difficulty with speech and swallowing, pain and respiratory distress. Several case reports and one case series have been published concerning therapeutic outcome of pallidal deep brain stimulation in dystonia caused by neurodegeneration with brain iron degeneration, reporting mostly favourable outcomes. However, with case studies, there may be a reporting bias towards favourable outcome. Thus, we undertook this multi-centre retrospective study to gather worldwide experiences with bilateral pallidal deep brain stimulation in patients with neurodegeneration with brain iron accumulation. A total of 16 centres contributed 23 patients with confirmed neurodegeneration with brain iron accumulation and bilateral pallidal deep brain stimulation. Patient details including gender, age at onset, age at operation, genetic status, magnetic resoce imaging status, history and clinical findings were requested. Data on severity of dystonia (Burke Fahn Marsden Dystonia Rating Scale-Motor Scale, Barry Albright Dystonia Scale), disability (Burke Fahn Marsden Dystonia Rating Scale-Disability Scale), quality of life (subjective global rating from 1 to 10 obtained retrospectively from patient and caregiver) as well as data on supportive therapy, concurrent pharmacotherapy, stimulation settings, adverse events and side effects were collected. Data were collected once preoperatively and at 2-6 and 9-15 months postoperatively. The primary outcome measure was change in severity of dystonia. The mean improvement in severity of dystonia was 28.5% at 2-6 months and 25.7% at 9-15 months. At 9-15 months postoperatively, 66.7% of patients showed an improvement of 20% or more in severity of dystonia, and 31.3% showed an improvement of 20% or more in disability. Global quality of life ratings showed a median improvement of 83.3% at 9-15 months. Severity of dystonia preoperatively and disease duration predicted improvement in severity of dystonia at 2-6 months; this failed to reach significance at 9-15 months. The study confirms that dystonia in neurodegeneration with brain iron accumulation improves with bilateral pallidal deep brain stimulation, although this improvement is not as great as the benefit reported in patients with primary generalized dystonias or some other secondary dystonias. The patients with more severe dystonia seem to benefit more. A well-controlled, multi-centre prospective study is necessary to enable evidence-based therapeutic decisions and better predict therapeutic outcomes. Primary Meige syndrome is an idiopathic movement disorder that manifests as craniofacial and often cervical dystonias. Deep brain stimulation (DBS) of the globus pallidus internus (GPi) has emerged as a powerful surgical option in the treatment of primary generalized or segmental dystonia. However, the experience with GPi-DBS in Meige syndrome is limited. We followed 5 patients with disabling Meige syndrome treated by bilateral GPi-DBS for 49 ± 43.7 (mean ± SD) months. All patients were assessed before surgery and at the last follow-up after surgery using the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) which includes both the movement and disability scales. Bilateral GPi-DBS produced a sustained and long-lasting improvement in dystonia symptoms associated with Meige syndrome. At the last follow-up, the mean scores of BFMDRS movement and disability scales improved significantly by 84 ± 6.8% (range, 75-94%) and 89 ± 8.1% (range, 80-100%), respectively. Bilateral pallidal stimulation is a beneficial therapeutic option for long-term relief of the disabling dystonia symptoms in Meige syndrome. BACKGROUND: The preoperative evaluation in dystonia aims at characterizing the severity and topography of motor symptoms in patients, who have previously been selected for deep brain stimulation (DBS). METHODS: The literature search was performed using PubMed, CINAHL, and the Cochrane Collaborative databases. RESULTS: Commonly used scales for clinical assessment are the Burke-Fahn-Marsden dystonia rating scale for generalized dystonia and the Toronto Western Spasmodic Torticollis Scale for cervical dystonia. Motor assessment is completed by quality of life and functional scales, such as the Short-Form Health Survey (SF-36) or the Parkinson's Disease Questionnaire 39. Validated rating scales for cranial or upper limb dystonia are lacking. DISCUSSION: In common clinical practice, these outcome measures can be administered in an open-label fashion because double blind assessment is only required for ascertaining new treatment indications or research purposes. The same measures are to be used postoperatively to revaluate outcome after DBS. Brain MRI is required to confirm diagnosis and assess structural abnormalities. Other imaging techniques, particularly functional imaging, are used for research purposes. Secondary dystonia encompasses a heterogeneous group with different etiologies. Cerebral palsy is the most common cause. Pharmacological treatment is often unsatisfactory. There are only limited data on the therapeutic outcomes of deep brain stimulation in dyskinetic cerebral palsy. The published literature regarding deep brain stimulation and secondary dystonia was reviewed in a meta-analysis to reevaluate the effect on cerebral palsy. The Burke-Fahn-Marsden Dystonia Rating Scale movement score was chosen as the primary outcome measure. Outcome over time was evaluated and summarized by mixed-model repeated-measures analysis, paired Student t test, and Pearson's correlation coefficient. Twenty articles comprising 68 patients with cerebral palsy undergoing deep brain stimulation assessed by the Burke-Fahn-Marsden Dystonia Rating Scale were identified. Most articles were case reports reflecting great variability in the score and duration of follow-up. The mean Burke-Fahn-Marsden Dystonia Rating Scale movement score was 64.94 ± 25.40 preoperatively and dropped to 50.5 ± 26.77 postoperatively, with a mean improvement of 23.6% (P < .001) at a median follow-up of 12 months. The mean Burke-Fahn-Marsden Dystonia Rating Scale disability score was 18.54 ± 6.15 preoperatively and 16.83 ± 6.42 postoperatively, with a mean improvement of 9.2% (P < .001). There was a significant negative correlation between severity of dystonia and clinical outcome (P < .05). Deep brain stimulation can be an effective treatment option for dyskinetic cerebral palsy. In view of the heterogeneous data, a prospective study with a large cohort of patients in a standardized setting with a multidisciplinary approach would be helpful in further evaluating the role of deep brain stimulation in cerebral palsy. © 2013 Movement Disorder Society. BACKGROUND/AIMS: Reports of outcomes in treating dystonia secondary to stroke with deep brain stimulation (DBS) are limited. We report our experience with 3 patients, all with infarcts involving the striatum, who developed hemidystonia and were treated with unilateral globus pallidus interna DBS. METHODS: Case series describing characteristics and outcomes based on the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) scores before and after DBS at 3, 6 and at least 12 months. RESULTS: All patients reported subjective improvements after surgery. At 1 year or more after surgery, none of the 3 patients displayed a measureable improvement in the BFMDRS movement score. CONCLUSION: Our findings are consistent with previous reports of limited benefits from pallidal DBS in secondary dystonia. Future work should focus on predictive factors for DBS outcomes and the development of more sensitive assessment tools specifically for secondary dystonias as well as the exploration of alternative brain targets for stimulation. PURPOSE: To evaluate the functional goal-directed outcomes of Deep Brain Stimulation (DBS) in childhood dystonia according to aetiology and to explore relationship with a traditional impairment-based measure. METHOD: This is a prospective case series study involving thirty children with dystonia with a 1-year follow-up post-DBS. The Canadian Occupational Performance Measure (COPM) and Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) were used as primary outcome measures. Results were analysed based on aetiology in 3 groups: 1. primary/primary plus dystonia; 2. secondary dystonia-cerebral palsy (CP); 3. secondary dystonia-non-CP group. Correlation between functional outcome using COPM and dystonia improvement as captured by BFMDRS was measured. RESULTS: All groups demonstrated significant improvement in individualised goal attainment, measured with the COPM, at 1-year post-DBS. The secondary dystonia-CP group also achieved significant improvement at 6 months for performance and satisfaction scores. In the majority of secondary dystonias, the BFMDRS failed to demonstrate significant improvement. A linear correlation between change in BFMDRS and COPM scores was observed when the entire cohort was analysed. INTERPRETATION/CONCLUSIONS: DBS improved functional performance, independently of the dystonic phenotype. Improvements in individualized COPM functional goal areas were seen in the absence of significant changes in BFMDRS scores, highlighting the relative insensitivity of impairment scales in this patient group. AIM: Hyperkinetic movement disorders (HMDs) can be assessed using impairment-based scales or functional classifications. The Burke-Fahn-Marsden Dystonia Rating Scale-movement (BFM-M) evaluates dystonia impairment, but may not reflect functional ability. The Gross Motor Function Classification System (GMFCS), Manual Ability Classification System (MACS), and Communication Function Classification System (CFCS) are widely used in the literature on cerebral palsy to classify functional ability, but not in childhood movement disorders. We explore the concordance of these three functional scales in a large sample of paediatric HMDs and the impact of dystonia severity on these scales. METHOD: Children with HMDs (n=161; median age 10y 3mo, range 2y 6mo-21y) were assessed using the BFM-M, GMFCS, MACS, and CFCS from 2007 to 2013. This cross-sectional study contrasts the information provided by these scales. RESULTS: All four scales were strongly associated (all Spearman's rank correlation coefficient rs >0.72, p<0.001), with worse dystonia severity implying worse function. Secondary dystonias had worse dystonia and less function than primary dystonias (p<0.001). A longer proportion of life lived with dystonia is associated with more severe dystonia (rs =0.42, p<0.001). INTERPRETATION: The BFM-M is strongly linked with the GMFCS, MACS, and CFCS, irrespective of aetiology. Each scale offers interrelated but complementary information and is applicable to all aetiologies. Movement disorders including cerebral palsy can be effectively evaluated using these scales. Pallidal deep brain stimulation (DBS) is an established treatment for patients with severe isolated dystonia. However, clinical evidence for the long-term use of DBS in children is limited and controlled trials have not yet been conducted. Here, we provide the long-term results of up to 13 years of pallidal DBS in eight pediatric patients with generalized idiopathic or hereditary isolated dystonia (five males, mean age at surgery 12.5 ± 3.5 years), as assessed by retrospective video rating. Video rating was performed at three time points: pre-operative, 1-year short-term follow-up (1y-FU) and long-term last FU (LT-FU, up to 13 years). Symptom severity and disability were assessed using the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). Disability scores were obtained from clinical charts and during the last FU. The mean improvement in BFMDRS motor score was 54.4 ± 8.9 % at 1y-FU and 42.9 ± 11.6 % at LT-FU; the disability scores improved by 59.8 ± 10.3 and 63.3 ± 7.8 %, respectively. Electrode dislocation was noted in one patient and implantable pulse generator dislocation in another, both requiring surgical intervention; no further serious adverse events occurred. Our study presents the first blinded video rating assessment of the short- and long-term effects of pallidal DBS in children with idiopathic or hereditary isolated dystonia. Results confirm that pallidal DBS is a safe and efficacious long-term treatment in children, with overall motor improvement similar to that described in controlled trials in adults. BACKGROUND AND PURPOSE: Huntington's disease (HD) is an autosomal domit, neurodegenerative movement disorder, typically characterized by chorea. Dystonia is also recognized as part of the HD motor phenotype, although little work detailing its prevalence, distribution, severity and impact on functional capacity has been published to date. METHODS: Patients (>18 years of age) were recruited from the Cardiff (UK) HD clinic, each undergoing a standardized videotaped clinical examination and series of functional assessment questionnaires (Unified Huntington's Disease Rating Scale, Burke-Fahn-Marsden Dystonia Rating Scale and modified version of the Toronto Western Spasmodic Torticollis Rating Scale). The presence and severity of dystonia were scored by four independent neurologists using the Burke-Fahn-Marsden Dystonia Rating Scale and Unified Huntington's Disease Rating Scale. Statistical analysis included Fisher's exact test, Wilcoxon test, anova and calculation of correlation coefficients where appropriate. RESULTS: Forty-eight patients [91% (48/53)] demonstrated evidence of dystonia, with the highest prevalence in the left upper limb (n = 44, 83%), right upper limb most severely affected and eyes least affected. Statistically significant positive correlations (P < 0.05) were observed between dystonia severity and increasing HD disease stage and motor disease duration. Deterioration in functional capacity also correlated with increasing dystonia severity. No significant relationship was observed with age at motor symptom onset or CAG repeat length. CONCLUSIONS: We report a high prevalence of dystonia in adult patients with HD, with worsening dystonia severity with increasing HD disease stage and motor disease duration. The recognition and management of dystonic symptoms in routine clinical practice will aid overall symptomatic treatment and functional improvement. BACKGROUND: The Burke-Fahn-Marsden Dystonia Rating Scale is a universally applied instrument for the quantitative assessment of dystonia in both children and adults. However, immature movements by healthy young children may also show "dystonic characteristics" as a consequence of physiologically incomplete brain maturation. This could implicate that Burke-Fahn-Marsden scale scores are confounded by pediatric age. OBJECTIVE: In healthy young children, we aimed to determine whether physiologically immature movements and postures can induce an age-related effect on Burke-Fahn-Marsden movement and disability scale scores. METHODS: Nine assessors specializied in movement disorders (3 adult neurologists, 3 pediatric neurologists, and 3 MD/PhD students) independently scored the Burke-Fahn-Marsden movement scale in 52 healthy children (4-16 years of age; 2 boys and 2 girls per year of age). Independent of that, parents scored their children's functional motor development according to the Burke-Fahn-Marsden disability scale in another 52 healthy children (4-16 years of age; 2 boys and 2 girls per year of age). By regression analysis, we determined the association between Burke-Fahn-Marsden movement and disability scales outcomes and pediatric age. RESULTS: In healthy children, assessment of physiologically immature motor performances by the Burke-Fahn-Marsden movement and disability scales showed an association between the outcomes of both scales and age (until 16 years and 12 years of age, β = -0.72 and β = -0.60, for Burke-Fahn-Marsden movement and disability scale, respectively [both P < 0.001]). CONCLUSIONS: The Burke-Fahn-Marsden movement and disability scales are influenced by the age of the child. For accurate interpretation of longitudinal Burke-Fahn-Marsden Dystonia Rating Scale scores in young dystonic children, consideration of pediatric age-relatedness appears advisory. OBJECTIVES: The pallidothalamic tract connects the globus pallidus internus with the ventroanterior and ventrolateral parts of the thalamus. Lesioning or stimulation of the pallidothalamic tract has ameliorating effects on dyskinesia and dystonia in patients with Parkinson disease. However, the effect of the procedure on dystonia due to other etiologies has not been reported. METHODS: We retrospectively analyzed patients with dystonia who underwent unilateral pallidothalamic tractotomy between July 2017 and October 2018 at Tokyo Women's Medical University Hospital. The Burke-Fahn-Marsden Dystonia Rating Scale-Movement Scale was used to evaluate the severity of dystonia at three time points (before surgery, 3 months postoperatively, and the last available follow-up). Adverse events were also evaluated. RESULTS: Eleven patients underwent unilateral pallidothalamic tractotomy, including 5 with generalized dystonia, 4 with segmental dystonia, and 2 with focal (cervical) dystonia. All patients had undergone unilateral pallidotomy before contralateral pallidothalamic tractotomy. The mean interval between the previous surgery (pallidotomy) and pallidothalamic tractotomy was 9.5 ± 3.1 months. The mean follow-up period was 11.5 ± 4.2 months. The Burke-Fahn-Marsden Dystonia Rating Scale-Movement Scale scores at 3 months after pallidothalamic tractotomy (5.8 ± 8.4) and at the last available follow-up (5.6 ± 8.3, P < 0.001) were significantly improved compared with that before pallidothalamic tractotomy (21.8 ± 16.3). The most common adverse event was reduced voice volume (6 patients), which was mild and did not interfere with the patient's daily activities. CONCLUSIONS: This study suggests that pallidothalamic tractotomy can be an alternative treatment target for dystonia. A larger and longer prospective study is needed to elucidate the safety and efficacy of pallidothalamic tractotomy for dystonia. OBJECTIVE: To evaluate the short-term and long-term clinical effectiveness and safety of subthalamic nucleus deep brain stimulation (STN-DBS) for medically intractable pediatric isolated dystonia. METHODS: Using a longitudinal retrospective design, we assessed the clinical outcomes of nine patients who underwent STN-DBS for treatment-refractory pediatric isolated dystonia one decade ago (mean age at surgery: 15.9 ± 4.5 years). The primary clinical outcome used was assessed by retrospective video analyses of patients' dystonia symptoms using the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). Clinical assessments were performed at baseline, 1-year follow-up (1-yr FU), and 10-year follow-up (10-yr FU). Adverse side effects, including surgery-related, device-related, and stimulation-related effects, were also documented. RESULTS: After STN-DBS surgery, the mean improvement in the BFMDRS motor score was 77.1 ± 26.6% at 1-yr FU and 90.4 ± 10.4% at 10-yr FU. Similarly, the mean BFMDRS disability score was improved by 69.5 ± 13.6% at 1-yr FU and by 86.5 ± 13.9% at 10-yr FU. The clinical improvements gained at 10-yr FU were significantly larger than those observed at 1-yr FU. Negative correlations were found between the duration of disease to age at surgery ratio (DD/AS) and the improvements in the BFMDRS motor score and total score at 1-yr FU and 10-yr FU. CONCLUSION: To our knowledge, this study provides the first clinical evidence for the short- and long-term effectiveness and safety of STN-DBS for pediatric isolated dystonia. Additionally, putative evidence is provided that earlier STN-DBS intervention in patients with refractory pediatric isolated dystonia may improve short- and long-term clinical outcomes. AIM: To establish the prevalence of dystonic pain in children and their response to deep brain stimulation (DBS). METHOD: Dystonic pain was assessed in a cohort of 140 children, 71 males and 69 females, median age 11 years 11 months (range 3y-19y 1mo), undergoing DBS in our centre over a period of 10 years. The cohort was divided into aetiological dystonia groups: 1a, inherited; 1b, heredodegenerative; 2, acquired; and 3, idiopathic. Motor responses were measured with the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). RESULTS: Dystonic pain was identified in 63 (45%) patients, 38% of whom had a diagnosis of cerebral palsy (CP). Dystonic pain improved in 90% of children and in all aetiological subgroups 1 year after DBS, while the BFMDRS motor score improved in 70%. Statistically significant improvement (p<0.01) was noted for the whole cohort on the Numerical Pain Rating Scale (n=27), Paediatric Pain Profile (n=17), and Caregivers Priorities and Child Health Index of Life with Disabilities questionnaire (n=48). There was reduction of pain severity, frequency, and analgesia requirement. Findings were similar for the whole cohort and aetiological subgroups other than the inherited heredodegenerative group where the improvement did not reach statistical significance. INTERPRETATION: Dystonic pain is frequent in children with dystonia, including those with CP, who undergo DBS; this can be an important, realizable goal of surgery irrespective of aetiology. We encourage the use of multimodal approach in pain research to reduce the risk of bias. BACKGROUND: Although an increasing number of trials are reported on the treatment of generalized or segmental isolated dystonia, the minimal clinically important difference thresholds for the most frequently reported outcome measures are still undetermined. OBJECTIVES: To estimate the minimal clinically important difference for the Burke-Fahn-Marsden Dystonia Rating Scale and the 36-Item Short-Form Health Survey in generalized or segmental dystonia. METHODS: A total of 898 paired examinations of 198 consecutive patients, aged >18 years, with idiopathic and inherited (torsin family 1 member A positive) segmental and generalized isolated dystonia were analyzed. To calculate the minimal clinically important difference thresholds, both anchor- and distribution-based methods were used simultaneously. RESULTS: Any improvement >16.6% or worsening larger than 21.5% on the Burke-Fahn-Marsden Dystonia Rating Scale indicates a minimal, yet clinically relevant, change. Threshold values for the Burke-Fahn-Marsden Dystonia Disability Scale were 0.5 points for both decline and improvement. Cut-off scores for the Physical Component Summary, the Mental Component Summary, and the Global (Total or Overall) Score of the 36-Item Short-Form Health Survey were 5.5 and 5.5, 6.5 and 7.5, and 7.5 and 8.5 points for clinically meaningful improvement and deterioration, respectively. CONCLUSIONS: The minimal clinically important difference represents the smallest change in an outcome measure that is meaningful to patients. Our estimates for the Burke-Fahn-Marsden Dystonia Rating Scale and the 36-Item Short-Form Health Survey may allow more reliable judgment of the clinical relevance of different treatments for segmental and generalized isolated dystonia. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. BACKGROUND: The Dyskinesia Impairment Scale (DIS) is a new assessment scale for dystonia and choreoathetosis in children and youth with dyskinetic cerebral palsy. Today, the Burke-Fahn-Marsden Dystonia Rating Scale (BFM) is mostly used to assess dystonia in children with inherited dystonia. The aim of this study was to assess reliability and validity of the DIS in children and youth with inherited or idiopathic dystonia. METHODS: Reliability was measured by (1) the intraclass correlation coefficients (ICCs) for inter-rater and test-retest reliability, as well as (2) standard error of measurement (SEM) and minimal detectable difference (MDD). For concurrent validity of the DIS-dystonia subscale, the BFM was administered. RESULTS: In total, 11 males and 9 females (median age 16 years and 7 months, range 6 to 24 years) were included. For inter-rater reliability, the ICCs for the DIS total score and the dystonia and choreoathetosis subscale scores were 0.83, 0.87, and 0.71, respectively. For test-retest reliability, the ICCs for the DIS total score and the dystonia and choreoathetosis subscale scores were 0.95, 0.88, and 0.93, respectively. The SEM and MDD for the total DIS were 3.98% and 11.04%, respectively. The Spearman correlation coefficient between the dystonia subscale and the BFM was 0.88 (p < 0.01). CONCLUSIONS: Good to excellent inter-rater, test-retest reliability, and validity were found for the total DIS and the dystonia subscale. The choreoathetosis subscale showed moderate inter-rater reliability and excellent test-retest reliability. The DIS may be a promising tool to assess dystonia and choreoathetosis in children and young adults with inherited or idiopathic dystonia. BACKGROUND: Mutations in TUBB4A are associated with a wide phenotypic spectrum including generalized dystonia with whispering dysphonia (DYT-TUBB4A). METHODS: We report the case of a 44-year-old patient with DYT-TUBB4A with a clinical presentation of disabling progressive dystonia, with a prominent laryngeal, cervical and facial involvement. RESULTS: Bipallidal deep brain stimulation (DBS) resulted in a 55% reduction of dystonia severity assessed by the Burke-Fahn-Marsden scale score 6 months after surgery. The effect was obvious on the cervical and facial components of dystonia. CONCLUSION: We suggest that bipallidal DBS should be considered in patients with disabling dystonia related to TUBB4A variants.

Is methotrexate used for the treatment of Rheumatoid Arthritis (RA)?

Yes, methotrexate is effective for the treatment of Rheumatoid Arthritis. MTX can be administered in combination with other chemotherapeutic agents or as mono-therapy. Response rate to MTX is more than 90%. MTX therapy is associated with improved survival of RA patients.

Methotrexate (MTX) is currently under study for use in juvenile rheumatoid arthritis. One complication of MTX is hepatotoxicity. Although liver function tests may be abnormal with its use, in this setting they do not correlate well with the development of hepatic fibrosis. Periodic liver biopsy is required to monitor for the hepatotoxic changes secondary to MTX. We describe and discuss the case of a 17-year-old woman who developed evidence of hepatic fibrosis after 3 years of MTX therapy. Twenty-nine patients participated in a prospective study of the safety and efficacy of oral methotrexate in the treatment of refractory rheumatoid arthritis. Patients received a mean dosage of 12.4 mg weekly over a mean duration of 29.1 months. All patients had liver biopsies at baseline, 2 years, and annually thereafter. Patients improved significantly by all clinical measures of efficacy after 1 month; maximum improvement tended to occur after approximately 6 months of therapy. Radiographs showed improvement of erosive disease in 7 of 11 patients measured. There was a significant reduction in mean prednisone dosage. Four patients required an increase in the dosage of methotrexate after prolonged therapy, because of declining clinical response. Toxicity was noted at some time in 26 of 29 patients (90%), but reactions universally became mild and tolerable after adjustment of the dosage. No significant hepatotoxicity was found in 60 sequential liver biopsies, although elevated transaminase levels were noted at some time in 20 of 29 patients (70%). The use of methotrexate in rheumatoid arthritis is reviewed. Methotrexate, a folic acid antagonist, is sometimes employed in an attempt to symptomatically control patients whose disease does not respond adequately to conventional therapies. Systemic administration of 7.5-15 mg/wk in a "pulse" fashion appears to be effective without precipitating severe adverse effects. However, concern over potentially serious side effects and a lack of well-controlled clinical trials have limited its use to severe, refractory disease. Further studies are needed before its role in rheumatoid arthritis can justifiably be expanded. Low dose pulse methotrexate (MTX) has become a widely used therapy for rheumatoid arthritis (RA) because of its good response rate profile. With the increased use of MTX, reports of opportunistic infections associated with MTX therapy have appeared. Fourteen cases of pneumocystis carinii (PC) pneumonia in patients receiving low dose MTX have been previously reported. Yet, no case of PC pneumonia associated with low dose MTX has so far been reported in Japan. We report the first case in Japan of PC pneumonia occurring in a patient with rheumatoid vasculitis who was receiving low dose MTX. A 70-year old woman with 13 year history of RA presented with 3-day history of rapidly aggravating dyspnea, dry cough and fever. She had been receiving MTX 7.5 mg/week for 2.5 months because of her vasculitis symptoms. She had also been receiving prednisolone 7.5 mg/day which had been successfully tapered from an initial dose of 15 mg/day. At the time of her presentation with respiratory symptoms, all of her vasculitis symptoms had been alleviated. A chest radiograph revealed diffuse interstitial shadowing bilaterally and bilateral hilar and right lower lung field infiltrates. Her arterial blood gas showed severe hypoxemia (PaO2 27.7 torr). Polymerase chain reaction assay of bronchoalveolar lavage fluid showed PC. Although the patient required ventilatory support for 9 days, she was successfully treated with trimethoprime-sulphamethoxazole and methylprednisolone pulse therapy. Eight months later, the patient was well with no evidence of vasculitis or respiratory symptoms. A number of studies show the efficacy of methotrexate (MTX) for rheumatoid arthritis (RA) in general. However, is there any reason to single this drug out for early RA? Mechanistically, it probably works differently in RA than in cancer, at least in part. Thus, in addition to dihydrofolate reductase-related effects, MTX inhibits aminoimidazocarboxamide transformylase, decreases leukotriene B4 production, and increases adenosine release at concentrations achieved with low-dose MTX regimens. Clinically, it is well tolerated over relatively long periods. Further, a recent meta-analysis of radiology studies shows that MTX compares favorably with intramuscular gold and is better than azathioprine. Toxicity remains a concern in treating early RA, particularly as pulmonary "hypersensitivity reactions" continue (1% to 7.6%), infections (both fungal and perioperative) are documented, and more cirrhosis is found. With all of the above in mind, the use of MTX seems reasonable but not necessarily uniformly appropriate and not yet proved for early RA. Studies of MTX in early RA, particularly in combination with other drugs, are only beginning. Increasingly, methotrexate (MTX) and sulphasalazine (SASP) are used initially for second-line therapy of rheumatoid arthritis (RA). Although SASP and MTX are commonly used, the mechanism(s) by which these drugs control the inflammation that characterizes RA have remained obscure. Results from my laboratory indicate that these agents share a mode of action; the anti-inflammatory effects of both SASP and MTX are due, in both in vitro and in vivo studies, to their capacity to enhance adenosine release at inflamed sites. This mode of action suggests that the development of agents that directly alter adenosine metabolism may lead to new, more effective and safer antirheumatic drugs than those currently available. OBJECTIVES: The folate antagonist methotrexate (MTX) has become established as the most commonly used disease-modifying anti-rheumatic drug (DMARD) in the treatment of rheumatoid arthritis (RA) but is commonly discontinued due to adverse effects. Adverse effects are thought to be mediated via folate antagonism. In this paper we summarize the current data on the use of folates as a supplement to MTX use in RA for the prevention of adverse effects and as a potential modulator of cardiovascular risk, and propose guidelines for standard practice. METHODS: A Medline search was performed using the search terms "methotrexate", "folic acid", "folinic acid", "folate" and "homocysteine". Literature relevant to the use of folates as a supplement to MTX in the treatment of RA was reviewed and other papers referred to as references were explored. RESULTS: The use of supplemental folates, including folic and folinic acid, in RA patients treated with MTX has been shown to improve continuation rates by reducing the incidence of liver function test abnormalities and gastrointestinal intolerance. Folate supplements do not appear to significantly reduce the effectiveness of MTX in the treatment of RA. Furthermore, supplemental folic acid offsets the elevation in plasma homocysteine associated with the use of MTX. This may in turn reduce the risk of cardiovascular disease, which is over-represented amongst patients with RA, and for which hyperhomocysteinaemia is now recognized as an independent risk factor. CONCLUSIONS: We propose that folic acid supplements be prescribed routinely to all patients receiving MTX for the treatment of RA. We recommend a pragmatic dosing schedule of 5 mg of oral folic acid given on the morning following the day of MTX administration. Methotrexate (MTX) has been the anchor treatment in rheumatoid arthritis (RA) over the last 15 years, and is used in combination with biologic agents to enhance efficacy over the last decade or so. The safety profile of MTX has been studied over 25 years with very few clinically important adverse events in the weekly low-doses used for RA treatment. The importance of MTX in earlier and more aggressive management of RA patients cannot be overstated. MTX courses show some of the longest continuation rates reported in clinical medicine, due to both effectiveness and safety. The safety profile of MTX indicates that it is among the safest of any mediation used for the treatment of any arthritis. Better information on the effectiveness and safety of weekly-low dose MTX should be communicated to all health professionals involved in the management of RA patients. Methotrexate (MTX) is currently the most frequently used drugs in the treatment of rheumatoid arthritis (RA). The drug had been synthesized in 1948 and first tests to treat patients with psoriasis and RA were published in 1951. However, until the 1980s there was only limited use of MTX in the treatment of RA. Since the 1990s MTX is the disease-modifying antirheumatic drug (DMARD) of first choice for the treatment of RA in most countries worldwide. By definition, DMARDs in RA are those compounds for which an inhibiting effect on radiographic progression has been demonstrated. Several combinations of DMARDs have been tested, most commonly with MTX as the anchor drug. Regarding the route of administration of MTX there is some evidence that the parenteral route, most often performed subcutaneously, has some additional benefits over the oral route. In MTX monotherapy, dosages up to 30 mg/week are now used. There are now three main combinations that are playing an important role: MTX + sulfasalazine (SSZ) + hydroxychloroquine, MTX + leflunomide (LEF), and MTX + biologics such as antitumour necrosis factor (anti-TNF) and other new compounds which block the interleukin 6 (IL6) receptor or T-cell activation and delete B cells. Regarding clinical efficacy, MTX monotherapy has performed almost similarly well in comparison with biologic mono-therapy, both usually combined with glucocorticoids. However, structural damage is usually inhibited to a significantly greater degree with the biologics. The combination of MTX with biologics has proven superior to either agent alone in all aspects. Current strategic regimens which concentrate on systematic ways to bring patients into remission all include MTX as first choice. The objective of this review is to update the recommendations of the 2010 Italian Consensus on the use of methotrexate (MTX) in rheumatoid arthritis (RA) and other rheumatic diseases. The literature published between 2008 and 2012 was systematically reviewed and updated recommendations on MTX use in rheumatic diseases, particularly RA, were formulated. These recommendations were approved by a panel of expert Italian Rheumatologists. A total of 10,238 references were identified, among which 70 studies were selected for critical evaluation. Sufficient evidence had accumulated to warrant changes to several of the recommendations in the new version. A new recommendation for patients with RA who are in MTX-induced clinical remission was also proposed and approved by the panel. Updated recommendations for the use of MTX in patients with RA or other rheumatologic disease are proposed. The association of rheumatoid arthritis (RA) and immune thrombocytopenic purpura (ITP) has been reported rarely. Methotrexate, which is used for RA treatment, causes thrombocytopenia. Therefore, in medical practice, physicians avoid using methotrexate for RA in patients who have both RA and ITP. Here, we report an RA case that also had ITP, which did not decrease in platelet count after methotrexate therapy. A 50-year-old woman was diagnosed with diabetes mellitus in 1990, RA in 1995, and ITP in 2000. She had received hydroxychloroquine for more than 5 years. She was treated with prednisolone 16 mg/daily between 2006 and 2007, but she discontinued this therapy because of weight gain. Laboratory findings were not remarkable, except for thrombocytopenia. We started methotrexate therapy 10 mg per week for treatment of RA, and hydroxychloroquine therapy was stopped due to nonresponse. The methotrexate dose was increased up to 15 mg/week. Her complete blood cell count was monitored frequently. We did not observe any decrease in platelet count, while active arthritis symptoms of the patient were relieved. This case shows that methotrexate may be used in patients diagnosed with RA that is associated with ITP under strict monitoring. BACKGROUND: Treatment with methotrexate (MTX) in patients with rheumatoid arthritis (RA) leads to decreased total immunoglobulin (Ig) levels and impairs vaccine-specific IgG antibody levels following pneumococcal vaccination. The mechanisms by which MTX exerts these effects in RA are unknown. We aimed to evaluate whether MTX reduces vaccine-specific serum Ig levels and their functionality in RA patients following vaccination with pneumococcal conjugate vaccine, and if numbers of antigen-specific circulating plasmablasts are affected. METHODS: Ten patients with RA on MTX and 10 RA patients without disease modifying anti-rheumatic drug (DMARD) were immunized with a dose 13-valent pneumococcal conjugate vaccine (Prevenar13). Circulating plasmablasts producing total IgG and IgA as well as specific IgG and IgA against two pneumococcal capsular serotypes (6B and 23F) were enumerated using ELISPOT 6days after vaccination. IgG levels against both these serotypes were determined with ELISA before and 4-6weeks after vaccination. Positive antibody response was defined as ⩾2-fold increase of pre-vaccination antibody levels. The functionality of vaccine specific antibodies to serotype 23F was evaluated by measuring their ability to opsonize bacteria using opsonophagocytic assay (OPA) in 4 randomly chosen RA patients on MTX and 4 RA patients without DMARD. RESULTS: After vaccination, RA patients on MTX showed significant increase in pre- to postvaccination antibody levels for 6B (p<0.05), while patients without DMARD had significant increases for both 6B and 23F (p<0.05 and p<0.01, respectively). Only 10% of RA on MTX and 40% of RA patients without DMARD showed positive post-vaccination antibody responses for both serotypes. Increased opsonizing ability after vaccination was detected in 1 of 4 RA patients on MTX and 3 of 4 patients on RA without DMARD. However, numbers of circulating total and vaccine-specific IgG- or IgA-producing plasmablasts did not differ between RA patients with or without MTX. CONCLUSIONS: MTX treatment in RA leads to reduced vaccine-specific antibody responses and their functionality compared to untreated RA following pneumococcal vaccination using polysaccharide-protein conjugate vaccine. However, since there was no reduction in numbers of circulating total or vaccine-specific antibody-producing plasmablasts after vaccination this effect is probably not due to reduced activation of B cells in lymphoid tissue. CLINICAL TRIAL REGISTRATION: NCT02240888. In rheumatoid arthritis (RA) treatment, the concomitant use of methotrexate has been shown to reduce the incidence of antibodies to infliximab (ATI), on the other hand, it is unclear whether azathioprine can reduce ATI production. We enrolled a total of 10 Japanese adult patients with RA who were treated with infliximab concomitantly with methotrexate or azathioprine. Serum concentrations of infliximab and ATI of these patients were measured. The mean serum infliximab concentrations was 1.6±1.3 μg/ml in patients with methotrexate and 1.0±0.5 μg/ml in patients with azathioprine. Serum ATI concentrations were below the limit of quantitation in 4 of 5 patients in each group. The results from the present study suggest that azathioprine suppresses ATI production. Methotrexate has been used in treatment of rheumatoid arthritis (RA) since the 1980s and to this day is often the first line medication for RA treatment. In this review, we examine multiple hypotheses to explain the mechanism of methotrexate efficacy in RA. These include folate antagonism, adenosine signaling, generation of reactive oxygen species (ROS), decrease in adhesion molecules, alteration of cytokine profiles, and polyamine inhibition amongst some others. Currently, adenosine signaling is probably the most widely accepted explanation for the methotrexate mechanism in RA given that methotrexate increases adenosine levels and on engagement of adenosine with its extracellular receptors an intracellular cascade is activated promoting an overall anti-inflammatory state. In addition to these hypotheses, we examine the mechanism of methotrexate in RA from the perspective of its adverse effects and consider some of the newer genetic markers of methotrexate efficacy and toxicity in RA. Lastly, we briefly discuss the mechanism of additive methotrexate in the setting of TNF-α inhibitor treatment of RA. Ultimately, finding a clear explanation for the pathway and mechanism leading to methotrexate efficacy in RA, there may be a way to formulate more potent therapies with fewer side effects. Conflict of interest statement: CONFLICT OF INTEREST: SS has received speaking fees from Chugai Pharmaceutical, Eisai, Bristol–Myers K.K, Asahikasei Pharma Corp, Pfizer Japan, and consultant fees from Asahikasei Pharma Corp. TT has received research grants from Astellas Pharma Inc, Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., and Taisho Toyama Pharmaceutical Co., Ltd., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, speaking fees from AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co,. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., and Astellas Pharma Inc, and Daichi Sankyo Co., Ltd, and consultant fees from Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbvie GK, Nipponkayaku Co., Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc. Despite the introduction of numerous biologic agents for the treatment of rheumatoid arthritis (RA) and other forms of inflammatory arthritis, low-dose methotrexate therapy remains the gold standard in RA therapy. Methotrexate is generally the first-line drug for the treatment of RA, psoriatic arthritis and other forms of inflammatory arthritis, and it enhances the effect of most biologic agents in RA. Understanding the mechanism of action of methotrexate could be instructive in the appropriate use of the drug and in the design of new regimens for the treatment of RA. Although methotrexate is one of the first examples of intelligent drug design, multiple mechanisms potentially contribute to the anti-inflammatory actions of methotrexate, including the inhibition of purine and pyrimidine synthesis, transmethylation reactions, translocation of nuclear factor-κB (NF-κB) to the nucleus, signalling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and nitric oxide production, as well as the promotion of adenosine release and expression of certain long non-coding RNAs.

What methodology does the FoundationOne CDx test use?

FoundationOne CDx is a next generation sequencing (NGS) based test.

BACKGROUND: Immunotherapy has demonstrated encouraging clinical benefits in patients with advanced breast carcinomas and Programmed death ligand 1 (PD-L1) expression has been proposed as an immunotherapy biomarker. Challenges with current PD-L1 testing exist and tumor mutation burden (TMB) is emerging as a biomarker to predict clinical response to immunotherapy in melanoma and non-small cell lung cancer patients. However, TMB has not been well characterized in breast carcinomas. METHODS: The study cohort included 62 advanced breast cancer patients (13 primary and 49 metastatic). Genetic alterations and TMB were determined by FoundationOne CDx next generation sequencing (NGS) and the association with clinicopathologic features was analyzed. RESULTS: High TMB was observed in a relatively low frequency (3/62, 4.8%). TMB levels were positively associated tumor infiltrating lymphocytes and significantly higher TMB was observed in breast carcinomas with DNA damage repair gene mutation(s). There was no significant association between TMB levels and other analyzed clinicopathologic characteristics. CONCLUSIONS: Our data indicate the importance of DNA damage repair proteins in maintaining DNA integrity and immune reaction and breast carcinoma patients with DDR mutation may benefit from immunotherapy.

Which R/bioconductor package exists for discovery of intergenic transcripts?

To increase the power of transcript discovery from large collection of RNA-seq data sets, a novel '1-Step' approach named pooling RNA-Seq and Assembling Models (PRAM) has been developed that build transcript models from pooled RNA- sequencing data sets. PRAM is implemented as an R/Bioconductor package.

Publicly available RNA-seq data is routinely used for retrospective analysis to elucidate new biology. Novel transcript discovery enabled by joint analysis of large collections of RNA-seq data sets has emerged as one such analysis. Current methods for transcript discovery rely on a '2-Step' approach where the first step encompasses building transcripts from individual data sets, followed by the second step that merges predicted transcripts across data sets. To increase the power of transcript discovery from large collections of RNA-seq data sets, we developed a novel '1-Step' approach named Pooling RNA-seq and Assembling Models (PRAM) that builds transcript models from pooled RNA-seq data sets. We demonstrate in a computational benchmark that 1-Step outperforms 2-Step approaches in predicting overall transcript structures and individual splice junctions, while performing competitively in detecting exonic nucleotides. Applying PRAM to 30 human ENCODE RNA-seq data sets identified unotated transcripts with epigenetic and RAMPAGE signatures similar to those of recently annotated transcripts. In a case study, we discovered and experimentally validated new transcripts through the application of PRAM to mouse hematopoietic RNA-seq data sets. We uncovered new transcripts that share a differential expression pattern with a neighboring gene Pik3cg implicated in human hematopoietic phenotypes, and we provided evidence for the conservation of this relationship in human. PRAM is implemented as an R/Bioconductor package.

What is the mechanism of action of idasanutlin?

Idasanutlin is a small-molecule inhibitor of MDM2, a negative regulator of tumor suppressor p53.

BACKGROUND: Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. METHODS: The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. RESULTS: Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies. CONCLUSIONS: Our findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies. PURPOSE: Idasanutlin, a selective small-molecule MDM2 antagonist in phase 3 testing for refractory/relapsed AML, is a non-genotoxic oral p53 activator. To optimize its dosing conditions, a number of clinical pharmacology characteristics were examined in this multi-center trial in patients with advanced solid tumors. METHOD: This was an open-label, single-dose, crossover clinical pharmacology study investigating the effects of strong CYP3A4 inhibition with posaconazole (Part 1), two new oral formulations (Part 2), as well as high-energy/high-fat and low-energy/low-fat meals (Part 3) on the relative bioavailability of idasanutlin. After completing Part 1, 2, or 3, patients could have participated in an optional treatment with idasanutlin. Clinical endpoints were pharmacokinetics (PK), pharmacodynamics (PD) of MIC-1 elevation (Part 1 only), and safety/tolerability. RESULTS: The administration of posaconazole 400 mg BID × 7 days with idasanutlin 800 mg resulted in a slight decrease (7%) in Cmax and a modest increase (31%) in AUC for idasanutlin, a marked reduction in Cmax (~ 60%) and AUC0 (~ 50%) for M4 metabolite, and a minimal increase (~ 24%) in serum MIC-1 levels. Cmax and AUC were both 45% higher for the SDP formulation. While the low-fat meal caused a less than 20% increase in all PK exposure parameters with the 90% CI values just outside the upper end of the equivalence criteria (80-125%), the high-fat meal reached bioequivalence with dosing under fasting. CONCLUSION: In patients with solid tumors, multiple doses of posaconazole, a strong CYP3A4 inhibitor, minimally affected idasanutlin PK and PD without clinical significance. The SDP formulation improved rBA/exposures by ~ 50% without major food effect. PURPOSE: Idasanutlin, a selective small-molecule MDM2 antagonist in phase 3 testing for refractory/relapsed AML, is a non-genotoxic oral p53 activator. The aim of this analysis is to examine the potential of idasanutlin to prolong the corrected QT (QTc) interval by evaluating the relationship between plasma idasanutlin concentration and QTc interval. METHOD: Intensive plasma concentration QTc interval data were collected at the same timepoints, from three idasanutlin (RO5503781) phase 1 studies in patients with solid tumors and AML. QTc data in absolute values and changes from baseline (Δ) were analyzed for a potential association with plasma idasanutlin concentrations with a linear mixed effect model. Categorical analysis was also performed. RESULTS: A total of 282 patients were exposed to idasanutlin and had at least one observation of QTc and idasanutlin plasma concentration. There was no apparent increase of QTcF or ΔQTcF in a wide idasanutlin plasma concentration range, even at concentrations exceeding the exposure matching the dose adopted in the ongoing phase 3 study (300-mg BID). Categorical analysis did not detect a potential signal of QT prolongation. CONCLUSION: The concentration-QTc analysis indicates that idasanutlin does not prolong the QT interval within the targeted concentration range currently in consideration for clinical development. Venetoclax (ABT-199) and idasanutlin (RG7388) are efficient anticancer drugs targeting two essential apoptosis markers, Bcl-2 and MDM2, respectively. Recent studies have shown that the combination of these two drugs leads to remarkable enhancement of anticancer efficacy, both in vitro and in vivo. In an attempt to disclose the relationships of their protein targets, competitive affinity-based proteome profiling coupled with bioimaging was employed to characterize their protein targets in the same cancer cell line and tumor tissue. A series of protein hits, including ITPR1, GSR, RER1, PDIA3, Apoa1, and Tnfrsf17 were simultaneously identified by pull-down/LC-MS/MS with the two sets of affinity-based probes. Dual imaging was successfully carried out, with the simultaneous detection of Bcl-2 and MDM2 expression in various cancer cells. This could facilitate the novel diagnostic and therapeutic strategies of dual targeting of Bcl-2/MDM2. A concise asymmetric synthesis has been developed to prepare idasanutlin, a small molecule MDM2 antagonist. Idasanutlin is currently being investigated as a potential treatment for various solid tumors and hematologic maligcies. The highly congested pyrrolidine core, containing four contiguous stereocenters, was constructed via a Cu(I)/(R)-BINAP catalyzed [3+2]-cycloaddition reaction. This optimized copper(<small>I</small>)-catalyzed process has been used to produce more than 1500 kg of idasanutlin. The manufacturing process will be described, highlighting the exceptionally selective and consistent cycloaddition/isomerization/hydrolysis sequence. The excellent yields, short cycle times and reduction in waste streams result in a sustainable production process with low environmental impact. The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations. BACKGROUND AND OBJECTIVE: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells. MATERIALS AND METHODS: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods. RESULTS: The RG7388 treatment was able to induce cell death by elevating p21WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21WAF1/CIP1 in MCF-7 cells. CONCLUSION: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi. Maligt rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential. Purpose MDM2 is a negative regulator of the tumor suppressor p53. RO6839921 is an inactive pegylated prodrug of idasanutlin, an MDM2 antagonist, developed for intravenous administration. On cleavage by plasma esterases, the active principle (AP = idasanutlin) is released. This phase 1 study investigated the safety, pharmacokinetics, and pharmacodynamics of RO6839921 in patients with advanced solid tumors (NCT02098967). Methods Patients were evaluated on a 5-day dosing schedule every 28 days. Dose escalation used the Bayesian new continual reassessment model. Accelerated dose titration was permitted until grade ≥2 drug-related AEs were observed. The target DLT rate to define the MTD was 16-25%. p53 activation was assessed by measuring macrophage inhibitory cytokine-1 (MIC-1). Results Forty-one patients received 14-120 mg AP; 39 were DLT evaluable. The MTD was 110-mg AP (8% DLT rate), whereas 120-mg AP had a 44% DLT rate. DLTs were neutropenia, thrombocytopenia, and stridor. The most common treatment-related AEs (≥30%) were nausea, fatigue, vomiting, and thrombocytopenia. Pharmacokinetic analyses indicated rapid conversion of prodrug to AP and an approximately linear and dose-proportional dose-exposure relationship, with a 2-fold increase in exposure between Days 1 and 5 of AP. MIC-1 increases were exposure dependent. Stable disease was observed in 14 patients (34%). Conclusions RO6839921 showed reduced pharmacokinetic exposure variability and a safety profile comparable with that of oral idasanutlin. Although this study indicated that RO6839921 could be administered to patients, the results did not provide sufficient differentiation or improvement in the biologic or safety profile compared with oral idasanutlin to support continued development. In acute myeloid leukemia (AML), TP53 mutations and dysregulation of wild-type p53 is common and supports an MDM2 antagonist as a therapy. RO6839921 is an inactive pegylated prodrug of the oral MDM2 antagonist idasanutlin (active principle [AP]) that allows for IV administration. This phase 1 monotherapy study evaluated the safety, pharmacokinetics, and pharmacodynamics of RO6839921 in patients with AML. Primary objectives identified dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD). Secondary objectives assessed pharmacokinetic, pharmacodynamic, and antileukemic activity. A total of 26 patients received 120-300 mg AP of idasanutlin. The MTD was 200 mg, with DLTs at 250 (2/8 patients) and 300 mg (2/5). Treatment-related adverse events in >20% of patients were diarrhea, nausea, vomiting, decreased appetite, and fatigue. Six deaths (23.1%) occurred, all unrelated to treatment. Pharmacokinetics showed rapid and near-complete conversion of the prodrug to AP and dose-proportional exposure across doses. Variability ranged from 30%-47% (22%-54% for idasanutlin). TP53 was 21 (87.5%) wild-type and 3 mutant (12.5%). The composite response rate (complete remission [CR], CR with incomplete hematologic recovery/morphological leukemia-free state [CRi/MLFS], or CR without platelet recovery [CRp]) was 7.7%. Antileukemic activity (CR, CRi/MLFS, partial response, hematologic improvement/stable disease) was observed in 11 patients (disease control rate, 42%): 10/11 were TP53 wild-type; 1 had no sample. p53 activation was demonstrated by MIC-1 induction and was associated with AP exposure. There was not sufficient differentiation or improvement in the biologic or safety profile compared with oral idasanutlin to support continued development of RO6839921. NCT02098967. Author information: (1)Departamento de Hematologia, Hospital Universitari i Politècnic La Fe, València, Spain. (2)CIBERONC, Instituto Carlos III, Madrid, Spain. (3)F. Hoffmann-La Roche Ltd, Basel, Switzerland. (4)Hospital Clinic de Barcelona, IDIBAPS, Barcelona, Spain. (5)Department of Leukemia, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (6)Department of Hematology and Sciences Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. (7)Department of Experimental, Diagnostic and Specialty Medicine Institute of Hematology & Medical Oncology L & A Seràgnoli, Bologna, Italy. (8)Hoffmann-La Roche Ltd, Mississauga, ON, Canada. (9)Serviced'Hématologie, Institut Universitaire du Cancer Toulouse - Oncopole,Toulouse, France. (10)Department of Internal Medicine, Universitätsklinikum Carl Gustav Carus, Dresden, Germany. (11)Hematology Department, Aix-Marseille University, Institut Paoli-Calmettes, Marseille, France. (12)Department of Haematology, The Alfred Hospital & Monash University, Melbourne, VIC, Australia. (13)Division of Hematology/Medical Oncology, Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea. (14)Service d'Hématologie Séniors Hôpital Saint-Louis, Assistance Publique - Hôpitaux de Paris, Université de Paris, Paris, France. BACKGROUND: Despite intensive treatment protocols and recent advances, neuroblastomas still account for approximately 15% of all childhood cancer deaths. In contrast with adult cancers, p53 pathway inactivation in neuroblastomas is rarely caused by p53 mutation but rather by altered MDM2 or p14ARF expression. Moreover, neuroblastomas are characterised by high proliferation rates, frequently triggered by pRb pathway dysfunction due to aberrant expression of cyclin D1, CDK4 or p16INK4a. Simultaneous disturbance of these pathways can occur via co-amplification of MDM2 and CDK4 or homozygous deletion of CDKN2A, which encodes both p14ARF and p16INK4a. METHODS AND RESULTS: We examined whether both single and combined inhibition of MDM2 and CDK4/6 is effective in reducing neuroblastoma cell viability. In our panel of ten cell lines with a spectrum of aberrations in the p53 and pRb pathway, idasanutlin and abemaciclib were the most potent MDM2 and CDK4/6 inhibitors, respectively. No correlation was observed between the genetic background and response to the single inhibitors. We confirmed this lack of correlation in isogenic systems overexpressing MDM2 and/or CDK4. In addition, combined inhibition did not result in synergistic effects. Instead, abemaciclib diminished the pro-apoptotic effect of idasanutlin, leading to slightly antagonistic effects. In vivo treatment with idasanutlin and abemaciclib led to reduced tumour growth compared with single drug treatment, but no synergistic response was observed. CONCLUSION: We conclude that p53 and pRb pathway aberrations cannot be used as predictive biomarkers for neuroblastoma sensitivity to MDM2 and/or CDK4/6 inhibitors. Moreover, we advise to be cautious with combining these inhibitors in neuroblastomas. Author information: (1)Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, ON, Canada. Electronic address: [email protected]. (2)Institute of Hematology "L. and A. Seràgnoli", University Hospital S. Orsola-Malpighi, Bologna, Italy. (3)Department of Hematology, Aix-Marseille University, Institut Paoli-Calmettes, Marseille, France. (4)Clinical Haematology, Peter MacCallum Cancer Centre and The Royal Melbourne Hospital, and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia. (5)Division of Hematology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, United States. (6)Division of Hematologic Oncology, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada. (7)Department of Medical Oncology, Thomas Jefferson University, Philadelphia, PA, United States. (8)Department of Medicine, Division of Oncology, New York Medical College, Valhalla, NY, United States. (9)Department of Haemato-Oncology, Beatson West of Scotland Cancer Centre, Glasgow, UK. (10)Division of Hematology/Medical Oncology, Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea. (11)Department of Hematology, Asan Medical Center, Seoul, Republic of Korea. (12)Translational Medicine-Oncology, Roche Innovation Center, New York, NY, United States. (13)Clinical Pharmacology, Roche Innovation Center, New York, NY, United States. (14)Clinical Pharmacology, F. Hoffmann-La Roche, Basel, Switzerland. (15)Product Development Oncology, Genentech, Inc, South San Francisco, CA, United States. (16)Department of Biostatistics Oncology, F. Hoffmann-La Roche, Basel, Switzerland. (17)Clinical Development Oncology, F. Hoffmann-La Roche, Basel, Switzerland. (18)Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), IRCCS, Meldola, Italy.

Where is corticosterone synthesized?

Following a stressful event, the hypothalamus-pituitary-adrenal axis mediates the release of the stress hormone cortisol (corticosterone in rodents; CORT).

Arecoline has biomedical importance, but it has untoward side effects on endocrine functions. The aim is to investigate its role on adrenal activity under thermal stress by ultrastructural and hormonal parameters in mice. Cold (4 °C) or heat (37 °C) stress, or arecoline (10 mg/kg body wt), each for 7 days in cold or heat stress stimulated adrenocortical activity ultrastructurally with an elevation of corticosterone level. Adrenomedullary activity was suppressed in cold stress with depletion of catecholamine levels. In heat stress, adrenomedullary activity was stimulated ultrastructurally with an elevation of catecholamine levels. Arecoline treatment alone, or in cold or heat stress suppressed adrenomedullary activity, judged by ultrastructural and hormonal parameters. Arecoline treatment caused hypoglycemia with an elevation of glycogen level, but cold or heat stress, or arecoline treatment in thermal stress caused hyperglycemia, with a fall in glycogen profile. Thus, arecoline in thermal stress plays a dual role on adrenal function and glucose-glycogen homeostasis in mice. In full-term elective caesarian sections, fetal flow of adrenal substrate steroids to products differs by sex, with males (M) in molar equilibrium whereas females (F) add net molarity and synthesize more cortisol. Using the same sampling design, paired, full-term, arterial, and venous umbilical cord samples and intrapartum chart records were obtained at the time of vaginal delivery (N = 167, 85 male) or emergency C-section (N = 38, 22 male). Eight steroids were quantified by liquid chromatography coupled to tandem mass spectrometry (adrenal glucocorticoids [cortisol, corticosterone], sequential cortisol precursor steroids [17-hydroxyprogesterone, 11-deoxycortisol], cortisol and corticosterone metabolites [cortisone and 11-dehydrocorticosterone], and gonadal steroids [androstenedione, testosterone]). Fetal sex was not significant in any analytic models. Going through both phase 1 and phase 2 labor increased fetal adrenal steroidogenesis and decreased male testosterone relative to emergency C-sections that do not reach stage 2 of labor (ie, head compressions) and elective C-sections with no labor. Sum adrenal steroid molarity arriving in venous serum was almost double the equivalent metric for deliveries without labor. No effects of operative vaginal delivery were noted. Maternal regional anesthetic suppressed venous concentrations, and fetal synthesis replaced that steroid. Approximate molar equivalence between substrate pool depletion and net glucocorticoid synthesis was seen. Paired venous and arterial umbilical cord serum has the potential to identify sex differences that underlie antenatal programming of hypothalamic-pituitary-adrenal axis function in later life. However, stage 2 labor before the collection of serum, and regional anesthetic for the mother, mask those sex differences.

Where is the klotho protein primarily expressed in the body

The klotho protein is primarily expressed in the lungs, kidney, lens, cerebellum, trpc6, renal cells and the brain.

The Klotho mouse is a recently developed model that exhibits phenotypes resembling human aging. We used this model to investigate sensorineural hearing loss from the point of view that it may be considered an issue of aging. Using reverse transcription-polymerase chain reaction, Western blotting and immunohistochemical staining, we were able to confirm klotho gene transcription and protein synthesis in the kidney and inner ear. Klotho protein was mainly expressed in the stria vascularis and spiral ligament of the inner ear and in the distal convoluted tubule of the kidney, likely serving a common function in the two organs, i.e., modulating ion transport. The threshold for the auditory brainstem response was significantly higher in Klotho mice than in wild-type mice, and wave I latencies were prolonged. On the other hand, Klotho mice exhibited a normal distribution of I-IV interpeak intervals. No obvious morphological abnormalities were detected in Klotho mice, although no expression of Klotho protein was detected, and there was an apparent hearing disorder. Taken together, these findings suggest that by contributing to the maintece ion homeostasis in the endolymph, Klotho protein serves as a key mediator of auditory function. To escape aging and aging-related disorders has been one of mankind's biggest dreams from the beginning of history. However, our knowledge regarding the molecular mechanisms of aging has been limited. We recently developed a unique short lifespan mouse strain in which a single gene mutation caused multiple aging-related disorders and identified the responsible gene as klotho. The most characteristic phenotypes seem to be caused by abnormalities in calcium metabolism. Furthermore, the klotho gene is expressed principally in the important tissues for calcium homeostasis such as distal tubule cells of the kidney, choroid plexus in the brain, and the main cells of the parathyroid gland. Klotho plays a critical role for the regulation of calcium and phosphorus homeostasis by negatively regulating the synthesis of active Vitamin D. The deficiency of the klotho gene results in degradation of cells by the activation of calcium-dependent proteolysis in kidney, lung and heart. Importantly, the increased activation of calcium-dependent proteolysis occurs in the tissues of old mice together with the down regulation of klotho gene expression. What is Klotho protein required for and how does it act? In this review, I will discuss our working hypotheses on the biological roles and molecular functions of Klotho protein. Klotho mutant mouse (kl-/-), a mouse model for human aging, exhibits various phenotypes in a wide range of organs including arteriosclerosis, neural degeneration, skin and gonadal atrophy, pulmonary emphysema, calcification of soft tissues, and cognition impairment. Klotho mRNA, however, is expressed only in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland. Therefore it remains to be elucidated how lack of Klotho protein in these limited organs leads to the variety of phenotypes. To shed light on mechanisms by which Klotho protein acts on distant targets, we examined localization of Klotho protein in brain, kidney, and reproductive organs, and analyzed brain and kidney in kl-/- mice searching for changes in target regions in these organs. In brain, Klotho proteins were localized at choroid plexus, where the proteins were domitly localized at the apical plasma membrane of ependymal cells. In kl-/- brain, reduction of synapses was evident in hippocampus, suggesting a role of Klotho as a humoral factor in cerebrospinal fluid. Klotho proteins in kidney localized at distal renal tubules. Interestingly, in kl-/-mice, type IIa Na/phosphate (Pi) cotransporters, which function at the proximal renal tubules in reabsorption of phosphate ions, were translocated. This suggests that Klotho protein in kidney is implicated in calcium homeostasis which regulates localization of type IIa Na/Pi cotransporters via parathyroid hormone (PTH). Klotho proteins in reproductive organs were expressed only in mature germ cells, although in kl-/- mice germ cell maturation was arrested at earlier stages. Thus, Klotho proteins not only function as a humoral factor, but also are implicated in hormonal regulation, which may explain why mutation of klotho gene results in a variety of phenotypes. Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho(-/-)) than in their wild-type littermates (klotho(+/+)). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D(3)(1,25(OH)(2)D(3)) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho(-/-) mice. In a variety of cells, 1,25(OH)(2)D(3) stimulates Ca(2+) entry. In erythrocytes, increased Ca(2+) entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca(2+) concentration was significantly larger in klotho(-/-) erythrocytes as compared to klotho(+/+) erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho(-/-) erythrocytes as compared to klotho(+/+) erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho(-/-) erythrocytes as compared to klotho(+/+) erythrocytes. Reticulocyte number was significantly increased in klotho (-/-) mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca(2+) entry and annexin V-binding of klotho(-/-) erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca(2+) activity in and suicidal death of erythrocytes. Serum phosphate concentration value is controlled by the kidney, which adapts phosphate reabsorption in the proximal tubule to the needs of the body and regulates intestine absorption of phosphate and calcium through calcitriol synthesis. Fibroblast Growth Factor 23 (FGF23) is a hormone that controls sodium-phosphate transporter and 1-alpha 25(OH) vitamin D hydroxylase expression in the renal proximal tubule. FGF23 is synthesized by bone cells in response to an increase in serum phosphate or calcitriol concentrations. The binding of FGF23 to a FGF receptor requires a protein named Klotho that is expressed in the kidney in the distal but not in the proximal tubule. The mechanism by which FGF23 controls proximal tubule function remains to be established. The alteration of the FGF23-Klotho axis in animal models and various human disorders is associated with abnormal control of body phosphate content confirming the major role played by these protein in phosphate homeostasis. This review details FGF23 and Klotho functions in normal conditions and in genetics or acquired disorders. The klotho gene (KL) was identified first as a putative aging-suppressor gene that extended life span when overexpressed and accelerated aging-like phenotypes when disrupted in mice. It encodes a single-pass transmembrane protein and is expressed predomitly in kidney, where it functions as an obligate coreceptor for fibroblast growth factor 23 (FGF-23). FGF-23 is a bone-derived hormone that suppresses phosphate reabsorption and 1,25 dihydroxyvitamin D(3) (vitamin D) synthesis in the kidney. Klotho also is expressed in the parathyroid gland, where FGF-23 decreases parathyroid hormone expression and secretion, further suppressing vitamin D synthesis in kidney. Thus, FGF-23 functions as a phosphaturic hormone and a counter-regulatory hormone for vitamin D, thereby inducing negative phosphate balance. Mice lacking either FGF-23 or Klotho show hyperphosphatemia in addition to developing multiple aging-like phenotypes, which can be rescued by resolving phosphate retention. These findings have unveiled an unexpected link between aging and phosphate. In patients with chronic kidney disease (CKD), phosphate retention is seen universally and has been associated with increased mortality risk. Patients with CKD have high serum FGF-23 levels with decreased klotho expression in the kidney and parathyroid, rendering FGF-23 and Klotho as potential biomarkers and therapeutic targets for CKD. The Klotho protein not only serves as a coreceptor for FGF-23, but also functions as a humoral factor. Klotho's extracellular domain is released into blood and urine by ectodomain shedding and exerts various functions independently of FGF-23, including regulation of multiple ion channels and transporters. Decreased urinary Klotho protein level has been identified as one of the earliest biomarkers of CKD progression. This review focuses on the current understanding of Klotho protein function, with emphasis on its potential involvement in the pathophysiologic process of CKD. The Klotho gene has been identified as an aging suppressor gene that encodes a transmembrane protein, which is expressed primarily in renal tubules. There are 2 forms of Klotho, membrane and secreted. However, there is a paucity of data on levels of soluble Klotho in diseases like diabetes and kidney disease. We validated an enzyme-linked immunosorbent assay for Klotho and quantitated Klotho levels separately in patients with diabetes and also in patients with chronic kidney disease (CKD). The Klotho assay showed good precision and was linear down to 19 ng/mL. There were no significant effects on Klotho levels with the addition of common interferents such as ascorbate, triglycerides, or hemolysis; only bilirubin (250 mg/L) significantly reduced Klotho levels (P < .05). There was a significant reduction in Klotho levels in samples with glycated hemoglobin (HbA(1c)) levels of 6.5% or more compared with control samples (HbA(1c) < 6.5%; P < .001). We also documented significantly higher levels of Klotho with CKD. Thus, we validated an assay for Klotho and made the novel observation that levels are decreased in diabetes and increased in CKD. BACKGROUND: Klotho, a single-pass transmembrane protein primarily expressed in the kidneys, parathyroid glands, and choroid plexus of the brain, has a short cytoplasmic tail and a long extracellular domain, which can be cleaved and released as a soluble form. However, information regarding the origins and kinetics of soluble serum Klotho remains poorly understood. We evaluated serial changes in serum Klotho levels among living donors before and after retroperitoneoscopic nephrectomy as well as in their renal transplant recipients. METHODS: The levels of soluble Klotho in serum obtained from 10 living donors and their renal transplant recipients were determined using a sandwich enzyme-linked immunosorbent assay system. RESULTS: Serum soluble Klotho was detectable in all subjects. The baseline serum Klotho concentrations in the living donors ranged from 726.4 to 1417.1 pg/mL (median, 909.8 pg/mL; interquartile ranges [IR], 754.8-1132.4), whereas that in the concomitant renal transplant recipients ranged from 397.5 to 1047.2 pg/mL (median, 613.0 pg/mL; IR, 445.9-750.8; P = .003). The levels of soluble serum Klotho measured 5 days after retroperitoneoscopic nephrectomy (median, 619.0 pg/mL; IR, 544.6-688.5; P = .001) were significantly lower than the baseline values. Among the renal transplant recipients, no significant changes in serum Klotho levels were observed during the observation period. CONCLUSION: Our data regarding soluble serum Klotho levels obtained from living donors support the idea that the kidneys are a major source of soluble serum Klotho in human subjects without a deterioration of renal function. In recipients, concomitant acute kidney injuries and immunosuppressive protocols might modulate the release of soluble Klotho from the grafts into the circulation. AIM: Active vitamin D (1,25-dihydroxyvitamin D₃), PTH, fibroblast growth factor-23 (FGF-23) and Klotho protein are key regulators of phosphate metabolism. Hyperphosphatemia and increased FGF-23 level in patients with end-stage renal disease are associated with increased morbidity and mortality. The relationships among key regulators of phosphate metabolism are still being investigated. FGF-23, the humoral factor involved in phosphate metabolism, is strongly associated with serum phosphorus level. Klotho, a transmembrane protein expressed primarily in renal tubules, functions as an obligatory co-receptor for FGF-23. The soluble form of Klotho, produced by the shedding of the transmembrane protein, is detectable in body fluids. The purpose of the study was to assess if serum soluble alpha-Klotho level was related to phosphate metabolism parameters and residual renal function (RRF) in incident peritoneal dialysis (PD) patients. METHODS: Thirty-five clinically stable patients 4 to 6 weeks after the onset of PD were included in the study. For each patient, clinical and laboratory data were reviewed. Serum phosphorus concentration, urinary and peritoneal phosphate clearance, serum FGF-23 and soluble Klotho protein concentrations were determined. RESULTS: Serum soluble alpha-Klotho was strongly negatively correlated with 24-hour diuresis (Rs = -0.55, p = 0.004) and renal phosphate clearance (Rs = -0.40, p = 0.049), but not with RRF. CONCLUSIONS: Serum soluble Klotho protein concentration is inversely related to residual diuresis and renal phosphate clearance in incident PD patients. Klotho is a recently discovered anti-aging gene and is primarily expressed in kidneys. In humans, the klotho level decreases with age whereas the prevalence of chronic kidney disease (CKD) increases with age. Diabetic nephropathy is the most common form of CKD, which leads to end-stage renal disease. A decrease in klotho has been found in kidneys of patients with diabetic nephropathy. The purpose of this study is to assess whether klotho gene deficiency affects early diabetic nephropathy in a mouse of model of type 1 diabetes induced by streptozotocin (STZ). Male KL(+/-) mutant and wild-type mice (6-8 weeks) were injected with multiple low doses of STZ. Renal functions and renal blood flow were assessed. Kidneys were collected for histological examination and molecular assays of TGFβ1 and mammalian targets of rapamycin (mTOR) signaling. Klotho deficiency in KL(+/-) mutant mice exacerbated STZ-induced increases in urine albumin, blood urea nitrogen, expansion of mesangial matrix in renal glomeruli, and kidney hypertrophy, suggesting a protective role of klotho in kidney function and structure. Klotho deficiency did not affect renal blood flow. Notably, klotho deficiency significantly increased phosphorylation of Smad2, indicating enhanced TGFβ1 signaling in kidneys. Klotho deficiency also increased phosphorylation of mTOR and S6 (a downstream effector of mTOR), indicating enhanced mTOR signaling in kidneys of early diabetic mice. Thus, klotho gene deficiency may make kidneys more susceptible to diabetic injury. Klotho gene deficiency exacerbated early diabetic nephropathy via enhancing both TGFβ1 and mTOR signaling in kidneys. Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determit of aging and life span.Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K(+) channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombit Klotho protein (30 ng/mL) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 μM DSA L (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I(Ks)) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombit Klotho protein (30 ng/mL) and inhibited by DSA L (10 μM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by “mainly” enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein. The antiaging protein of Klotho is a transmembrane protein mainly expressed in the kidney, parathyroid glands and choroid plexus of the brain. The Klotho protein exists in two forms, a full-length membrane form and a soluble secreted form. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it acts as β-glucuronidase and a hormone. Soluble Klotho can be found in the blood, cerebrospinal fluid, and the urine of mammals. Klotho deficiency results in early appearance of multiple age-related disorders and premature death, whereas overexpression of Klotho exerts the opposite effect. Klotho may influence cellular transport processes across the cell membrane by inhibiting calcitriol (1,25(OH) (2)D(3)), formation or by directly affecting transporter proteins, including ion channels, carriers and pumps. Accordingly, Klotho protein is a powerful regulator of transport mechanisms across the cell membrane. Klotho regulates diverse calcium and potassium ion channels, as well as several carriers including the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, the Na(+)-coupled phosphate cotransporters, NaPi-IIa and NaPi-IIb, and a Na(+)/K(+)-ATPase. All those cellular transport regulations contribute in the aging suppressor role of Klotho. Future studies will help to determine if the Klotho protein regulates cell-surface expression of other transport proteins and is affecting underlying mechanisms. Klotho is a membrane-bound protein predomitly expressed in the kidney, where it acts as a permissive co-receptor for Fibroblast Growth Factor 23. In its shed form, Klotho exerts anti-fibrotic effects in several tissues. Klotho-deficient mice spontaneously develop fibrosis and Klotho deficiency exacerbates the disease progression in fibrotic animal models. Furthermore, Klotho overexpression or supplementation protects against fibrosis in various models of renal and cardiac fibrotic disease. These effects are mediated at least partially by the direct inhibitory effects of soluble Klotho on TGFβ1 signaling, Wnt signaling, and FGF2 signaling. Soluble Klotho, as present in the circulation, appears to be the primary mediator of anti-fibrotic effects. Similarly, through inhibition of the TGFβ1, Wnt, FGF2, and IGF1 signaling pathways, Klotho also inhibits tumorigenesis. The Klotho promoter gene is generally hypermethylated in cancer, and overexpression or supplementation of Klotho has been found to inhibit tumor growth in various animal models. This review focuses on the protective effects of soluble Klotho in inhibiting renal fibrosis and fibrosis in distant organs secondary to renal Klotho deficiency. We also discuss the structure-function relationships of Klotho domains and biological effects in the context of potential targeted treatment strategies. BACKGROUND: The hormone klotho, encoded by the gene klotho, is primarily expressed in the kidney and choroid plexus of the brain. Higher klotho concentrations have been linked to better physical performance; however, it is unknown whether klotho relates to frailty status in older adults. METHODS: Plasma klotho was measured in 774 participants aged ≥65 years enrolled in InCHIANTI, a prospective cohort study comprising Italian adults. Frailty status was assessed at 3 and 6 years after enrollment. Frailty was defined as presence of at least three out of five criteria of unintentional weight loss, exhaustion, sedentariness, muscle weakness, and slow walking speed; prefrailty was defined as presence of one or two criteria; and robustness was defined as zero criteria. We assessed whether plasma klotho concentrations measured at the 3-year visit related to frailty. RESULTS: Each additional natural logarithm of klotho (pg/mL) was associated with lower odds of frailty versus robustness after adjustment for covariates (odds ratio [OR] 0.46; 95% confidence interval 0.21, 0.98; p-value = .045). Higher klotho was particularly associated with lower odds of exhaustion (OR 0.57; 95% CI 0.36, 0.89; p-value = .014). Participants with higher klotho also had lower estimated odds of weight loss and weakness, but these findings were not statistically significant. CONCLUSIONS: Higher plasma klotho concentrations were associated with lower likelihoods of frailty and particularly exhaustion. Future studies should investigate modifiable mechanisms through which klotho may affect the frailty syndrome. Aerobic exercise induces many adaptive changes in the whole body and improves metabolic characteristics. Klotho, an anti-aging gene, is mainly expressed in the brain and kidney. The roles of Klotho in the brain and kidney during aerobic exercise remain largely unknown. The present study aimed to determine whether aerobic exercise could influence the expression of Klotho, decrease reactive oxygen species (ROS) and prolong life span. Sprague Dawley rats were exercised on a motor treadmill. Klotho mRNA and protein expression levels in rat brain and kidney tissues were examined using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. ROS production was detected following intermittent aerobic exercise (IAE) or continuous aerobic exercise (CAE). Kaplan-Meier curve analysis demonstrated that aerobic exercise significantly improved rat survival (P<0.001). The ROS levels in rat brain and kidney tissues were decreased in the aerobic exercise groups compared with the control group (P<0.05). In addition, Klotho mRNA and protein expression levels were increased significantly following aerobic exercise compared with controls (P<0.05). There was no significant difference between the IAE and CAE groups in any experiments (P>0.05). These results suggest that aerobic exercise-stimulated Klotho upregulation extends the life span by attenuating the excess production of ROS in the brain and kidney. As Klotho exhibits a potential anti-aging effect, promoting Klotho expression through aerobic exercise may be a novel approach for the prevention and treatment of aging and aging-related diseases. Klotho is a protein primarily expressed in renal tubular epithelial cells. Studies have suggested that Klotho is an antiaging protein that reduces renal fibrosis after acute kidney injury (AKI) and inhibits stem cell senescence. Bone marrow mesenchymal stem cells (BMSCs) have consistent proliferation ability and multidirectional differentiation ability and have been used to treat tissue injury. Thus, we hypothesized that Klotho expressed in BMSCs could increase the renal protective effects of BMSCs. To verify the hypothesis, we isolated BMSCs from C57BL/6 mice, transfected them with Klotho-GFP-adenovirus and investigated the change in BMSC proliferation. We then transplanted Klotho-GFP-BMSCs into mice with AKI and investigated the therapeutic effect compared with that of sham-treated mice and GFP-BMSC-transplanted mice. Kidney fibrosis after ischemia/reperfusion injury (IRI) was relieved by BMSC transplantation, and the antifibrotic effect of BMSCs was significantly enhanced by overexpressing the Klotho gene. Mechanistic studies showed that Klotho increased pluripotency gene expression in BMSCs. Klotho produced by Klotho-GFP-BMSCs inhibited the Wnt/β-catenin pathway in renal tubular epithelial cells (TECs). Klotho-GFP-BMSCs showed increased proliferative ability and more potent immuno-regulation ability than did GFP-BMSCs. Our findings suggested that Klotho gene-modified BMSCs may be a better choice for cell therapy after AKI. α-Klotho, a multifunctional protein, has been demonstrated to protect tissues from injury via anti-oxidation and anti-inflammatory effects. The expression of α-klotho is regulated by several physiological and pathological factors, including acute inflammatory stress, oxidative stress, hypertension, and chronic renal failure. Exhaustive exercise has been reported to result in tissue damage, which is induced by inflammation, oxidative stress, and energy metabolism disturbance. However, little is known about the effects of exhaustive exercise on the expression of α-klotho in various tissues. To determine the effects, the treadmill exhaustion test in mice was performed and the mice were sacrificed at different time points following exhaustive exercise. Our results confirmed that the full-length (130 kDa) and shorter-form (65 kDa) α-klotho were primarily expressed in the kidneys. Moreover, we found that, except for the kidneys and brain, other tissues primarily expressed the shorter-form α-klotho, including liver, which was in contrast to previous reports. Furthermore, the shorter-form α-klotho was decreased immediately following the acute exhaustive exercise and was then restored to the pre-exercise level or even higher levels in the next few days. Our results indicate that α-klotho may play a key role in the body exhaustion and recovery following exhaustive exercise. Klotho interacts with various membrane proteins such as receptors for transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF). Renal expression of klotho is diminished in polycystic kidney disease (PKD). In the present study, the effects of klotho supplementation on PKD were assessed. Recombit human klotho protein (10 μg·kg-1·day-1) or a vehicle was administered daily by subcutaneous injection to 6-wk-old mice with PKD (DBA/2-pcy). Blood pressure was measured using tail-cuff methods. After 2 mo, mice were killed, and the kidneys were harvested for analysis. Exogenous klotho protein supplementation reduced kidney weight, cystic area, systolic blood pressure, renal angiotensin II levels, and 8-epi-PGF2α excretion (P < 0.05). Klotho protein supplementation enhanced glomerular filtration rate, renal expression of superoxide dismutase, and klotho itself (P < 0.05). Klotho supplementation attenuated renal expressions of TGF-β and collagen type I and diminished renal abundance of Twist, phosphorylated Akt, and mammalian target of rapamycin (P < 0.05). Pathological examination revealed that klotho decreased the fibrosis index and nuclear staining of Smad in PKD kidneys (P < 0.05). Our data indicate that klotho protein supplementation ameliorates the renin-angiotensin system, reducing blood pressure in PKD mice. Furthermore, the present results implicate klotho supplementation in the suppression of Akt/mammalian target of rapamycin signaling, slowing cystic expansion. Finally, our findings suggest that klotho protein supplementation attenuated fibrosis at least partly by inhibiting epithelial mesenchymal transition in PKD. The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease. NEW FINDINGS: What is the central question of this study? Can short-term high-intensity interval training (HIIT) contribute to the reduction of ischaemia-reperfusion (IR) injury by enhancing the levels of Klotho and its related axes, including myocardial TRPC6 expression, and antioxidant defence as novel possible mechanisms of exercise-induced cadioprotection (EICP) against IR injury? What is the main finding and its importance? The increase of plasma and myocardial levels of Klotho as a result of preconditioning with HIIT and prevention of a significant reduction of Klotho during IR injury can promote cardioprotection and reduce damage by attenuating myocardial TRPC6 expression and increasing antioxidant defence. The present findings may provide a new mechanism in EICP and IR injury, and provide the knowledge to develop preventive and therapeutic approaches. ABSTRACT: Cardiovascular disease, especially coronary artery disease, remains a major cause of morbidity and mortality in the world, and ischaemia-reperfusion (IR) insult is the main pathological cause leading to death. Exercise training is associated with a reduced risk of cardiovascular disease and the development of cardioprotection against IR injury. Therefore, the purpose of this study was to investigate the effect of preconditioning with high-intensity interval training (HIIT) on myocardial and plasma levels of Klotho and its related axes as novel mechanisms of exercise-induced cardioprotection against IR injury. Seventy male Wistar rats were randomly divided into five groups of control, HIIT, sham, IR and HIIT group that underwent IR injury (H-IR). The training group performed five sessions of HIIT on the treadmill. The cardiac IR injury was induced by ligation of the left anterior descending coronary artery for 30 min followed by 24 h reperfusion. Infarct size and histopathological assessment of cardiac tissues were determined through Evans Blue-triphenyltetrazolium chloride and haematoxylin-eosin staining, respectively. We investigated lipid peroxidation and markers of cardiac injury, antioxidant enzymes and the plasma levels of Klotho using enzyme-linked immunosorbent assays. Also, myocardial levels of Klotho and TRPC6 expression were determined by western blot assays. The results demonstrated a significant increase in myocardial and plasma levels of Klotho following HIIT and a significant decrease during IR injury. Myocardial TRPC6 channel expression increased following IR. HIIT also prevented a significant reduction of Klotho during IR and consequently reduced the expression of the TRPC6 channel in the H-IR group compared with the IR group. Furthermore, HIIT decreased the infarct size, cardiac injury, lipid peroxidation, lactate dehydrogenase, creatine kinase myocardial band and cardiac troponin-I, and improved total antioxidant capacity and catalase, superoxide dismutase and glutathione peroxidase activities following IR injury. The findings of the present study suggest that HIIT improves cardioprotection against IR injury and reduces cardiac damages through an increase in myocardial and plasma levels of Klotho and its related axes (TRPC6 and antioxidant defence). These findings can help to develop preventive and therapeutic approaches. Increased oxidative stress and inflammation play an important role in the pathogenesis of diabetic cataract. Klotho, known as an anti-ageing protein, has antioxidative and anti-inflammatory properties. Klotho is expressed in limited tissues including the lens. Here we examined whether klotho expression is decreased in diabetic lens and, if so, whether klotho treatment can prevent diabetic cataract formation. Streptozotocin (STZ)-induced diabetic rats and age-matched control rats were treated with vehicle or klotho protein, starting at 1 week after STZ injection. Twelve weeks after treatment, cataract formation was observed in diabetic rats but not control rats. Cataract formation and scores were significantly less in klotho-treated diabetic rats than vehicle-treated diabetic rats. Levels of klotho in plasma, aqueous humor and lens were significantly decreased in vehicle-treated diabetic rats, compared with control rats, but were restored in klotho-treated diabetic rats. Additionally, vehicle-treated diabetic rats had increased oxidative stress and inflammation in the lens, which were associated with decreased antioxidant transcriptional master regulator Nrf2 activity and increased transcription factor NF-κB activity. All of these findings were ameliorated in klotho-treated diabetic rats. Notably, klotho treatment did not alter blood glucose in diabetic rats. These results indicate that klotho reduction may increase susceptibility of the lens to oxidative and inflammatory insults, promoting cataract formation under diabetic conditions. Klotho treatment can ameliorate the onset and progression of diabetic cataract via enhancing Nrf2-mediated antioxidant defense and suppressing NF-κB-mediated inflammatory responses. Klotho in the lens may be a novel therapeutic target for prevention of cataract formation in diabetes. While radiation nephropathy is a major problem associated with radiotherapy, the exact mechanisms underlying its pathogenesis and the mediators involved in kidney deterioration remain to be elucidated. In view of the finding that senescence is typically increased post‑irradiation, the present study examined whether ionizing radiation may cause kidney injury by enhancing premature senescence. The present study explored the relevance of the aging suppressor, Klotho, which has anti‑aging activity and is highly expressed in murine renal cells/kidney tissues, under irradiation conditions. Firstly, the effects of radiation on mouse inner medullary collecting duct‑3 (mIMCD‑3) cells and kidney tissues of mice were assessed. Subsequently, the mRNA expression levels of Klotho, TNF‑α and ADAM metallopeptidase domain (ADAM)9/10/17 were analyzed by reverse transcription‑quantitative PCR following exposure to radiation. In addition, the levels of these proteins were measured by western blotting or ELISA. The results revealed that irradiation of mIMCD‑3 cells clearly triggered cellular senescence. Notably, Klotho gene expression was considerably decreased in radiation‑exposed mIMCD‑3 cells and in the kidney tissues of irradiated BALB/c mice, and the corresponding translated protein was consistently expressed following radiation exposure. Moreover, expression of TNF‑α, a negative regulator of Klotho, was significantly increased, whereas ADAM9/10/17, an ectodomain shedding enzyme of Klotho, was decreased in irradiated mIMCD‑3 cells and in the kidney tissues of BALB/c mice. Collectively, these data suggested that TNF‑α‑mediated inhibition of Klotho expression and blockage of soluble Klotho formation via decreased ADAM expression following irradiation may contribute to the development of renal dysfunction through acceleration of radiation‑induced cellular senescence.

Which particular intersex phenotype is related to steroid reductase?

Steroid reductase mutations are associated with both sex-determining and non-syndromic hypospadias

In the 20 yr since it was established that impairment of dihydrotestosterone formation is the cause of a rare form of human intersex, a wealth of information has accumulated about the genetics, endocrinology, and variable phenotypic manifestations, culminating in the cloning of cDNAs encoding two 5 alpha-reductase genes and documentation that mutations in the steroid 5 alpha-reductase 2 gene are the cause of 5 alpha-reductase deficiency. Perplexing and difficult problems remain unresolved, e.g. whether the variability in manifestations is due to variable expressions of steroid 5 alpha-reductase 1 or to effects of testosterone itself. It is also imperative to establish whether defects in steroid 5 alpha-reductase 2, perhaps in the heterozygous state, are responsible for a portion of cases of sporadic hypospadias, to determine whether 5 alpha-reductase plays a role in progesterone action in women, and to elucidate the relation between androgen action and gender role behavior. Conversion of testosterone (T) to dihydrotestosterone (DHT) in genital tissue is catalysed by the enzyme 5 alpha-reductase 2, which is encoded by the SRD5A2 gene. The potent androgen DHT is required for full masculinization of the external genitalia. Mutations of the SRD5A2 gene inhibit enzyme activity, diminish DHT formation, and hence cause masculinization defects of varying degree. The classical syndrome, formerly described as pseudovaginal perineoscrotal hypospadias, is characterized by a predomitly female phenotype at birth and significant virilization without gynecomastia at puberty. We investigated nine patients with steroid 5 alpha-reductase 2 deficiency (SRD). Phenotypes, which were classified according to the severity of the masculinization defect, varied between completely female (SRD type 5), predomitly female (SRD type 4), ambiguous (SRD type 3), predomitly male with micropenis and hypospadias (SRD type 2), and completely male without overt signs of undermasculinization (SRD type 1). T/DHT-ratios were highly increased ( > 50) in the classical syndrome (SRD type 5), but variable in the less severe affected patients (SRD types 1-4) (14-35). Mutations in the SRD5A2 gene had been characterized using PCR-SSCP analysis and direct DNA sequencing. A small deletion was encountered in two patients, while all other patients had single base mutations which result in amino acid substitutions. We conclude that phenotypes may vary widely in patients with SRD5A2 gene mutations spanning the whole range from completely female to normal male without distinctive clinical signs of the disease. Hence, steroid 5 alpha-reductase deficiency should be considered not only in sex reversed patients with female or ambiguous phenotypes, but also in those with mild symptoms of undermasculinization as encountered in patients with hypospadias and/or micropenis. A classification based on the severity of the masculinization defect may be used for correlation of phenotypes with enzyme activities and genotypes, and for comparisons of phenotypes between different patients as the basis for clinical decisions to be made in patients with pseudohermaphroditism due to steroid 5 alpha-reductase 2 deficiency. Between 1986 and 1995, a pedigree of six Arabs with male pseudohermaphroditism due to 5 alpha reductase-2 deficiency have been identified. All, were raised as girls since birth. At the time of diagnosis, three were post-pubertal, one pubertal and two pre-pubertal. The external genitalia of 'pseudo-vaginal perineoscrotal hypospadias' was identical in these subjects. Although these individuals were a homogeneous group in terms of their sex of upbringing, phenotypic appearance, endocrinological profile and socio-cultural background, the development of the gender identity and role was not uniform in these six cases. Their psycho-sexual make-up was closely related to the transaction of their life experiences. These cases provide further insight into the interaction between various factors involved in the development of gender identity and role in male pseudohermaphrodites in an Eastern culture. Steroid 5-alpha-reductase 2 deficiency is an autosomal recessive disorder with clinical spectrum ranges from a male phenotype with hypospadia to a female phenotype with normal wolffian structures. Over 50 different mutations of SRD5A2 gene has been described in affected patients and several mutations were detected in specific populations. DNAs of two 46,XY DSD Indonesian siblings, aged 13 and 18 years old, with clinically suspected of 5-alpha-reductase deficiency and their mother were analysed for molecular defects of SRD5A2 gene. Different from other reports, in our series three mutations were found in each patient. Two novel mutations were detected in these patients and their mother, which are p.Gly34fs and c.699-1G>T. The other mutation detected was c.680G>A or p.Arg227Gln, which commonly described in Far East Asian population. Whether the p.Arg227Gln mutation is considered a polymorphism or a mutation in Indonesian population warrants further study. Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5'- and 3'-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes. Steroid 5-alpha-reductase 2 deficiency is a rare disorder leading to male pseudohermaphroditism, a condition characterized by incomplete differentiation of male genitalia in 46,XY patients. Here, we report a case of a 21-year-old woman from Ardabil who presented with primary amenorrhea, ambiguous genitalia, and lack of breast development. All of the serum hormone profiles were normal except for raised serum total testosterone. Testosterone to DHT ratio (T/DHT) was elevated before (15.72) and further increased after hCG stimulation (32.46). A chromosomal study revealed a 46,XY karyotype. A bilateral gonadectomy, recessive cliteroplasty, urethroplasty, and vaginoplasty were performed and hormonal replacement therapy using estrogen was started. In conclusion, the diagnosis of 5-alpha-reductase 2 deficiency may be suspected in infants with ambiguous genitalia or in adolescents or young adults with the characteristic phenotype and serum hormone profiles. Inactivating mutations of the 5α-steroid reductase type-2 (SRD5A2) gene result in a broad spectrum of masculinization defects, ranging from a male phenotype with hypospadias to a female phenotype with Wolffian structures. Molecular studies of the SRD5A2 revealed a new heterozygous gene variant within the coding region that results in phenotypic expression. A c.92C>T transition changing serine to phenylalanine at codon 31 of exon 1 (p.Ser31Phe) was identified in a patient with 46,XY disorder of sexual development who displayed glandular hypospadias with micropenis and bilateral cryptorchidism. The restoration of the p.Ser31Phe mutation by site-directed mutagenesis and transient expression assays using cultured HEK-293 cells showed that this novel substitution does not abolish but does deregulate the catalytic efficiency of the enzyme. Thus, the maximum velocity (V max) value was higher for the mutant enzyme (22.5 ± 6.9 nmol DHT mg protein(-1) h(-1)) than for the wild-type enzyme (9.8 ± 2.0 nmol DHT mg protein(-1) h(-1)). Increased in vitro activity of the p.Ser31Phe mutant suggested an activating effect. This case provides evidence that heterozygous missense mutations in SRD5A2 may induce the abnormal development of male external genitalia. In 1974, a lack of 5α-dihydrotestosterone (5α-DHT), the most potent androgen across species except for fish, was shown to be the origin of a type of pseudohermaphrodism in which boys have female-like external genitalia. This human intersex condition is linked to a mutation in the steroid-5α-reductase type 2 (SRD5α2) gene, which usually produces an important enzyme capable of reducing the Δ4-ene of steroid C-19 and C-21 into a 5α-stereoisomer. Seeing the potential of SRD5α2 as a target for androgen synthesis, pharmaceutical companies developed 5α-reductase inhibitors (5ARIs), such as finasteride (FIN) and dutasteride (DUT) to target SRD5α2 in benign prostatic hyperplasia and androgenic alopecia. In addition to human treatment, the development of 5ARIs also enabled further research of SRD5α functions. Therefore, this review details the morphological, physiological, and molecular effects of the lack of SRD5α activity induced by both SRD5α mutations and inhibitor exposures across species. More specifically, data highlights 1) the role of 5α-DHT in the development of male secondary sexual organs in vertebrates and sex determination in non-mammalian vertebrates, 2) the role of SRD5α1 in the synthesis of the neurosteroid allopregolone (ALLO) and 5α-androstane-3α,17β-diol (3α-diol), which are involved in anxiety and sexual behavior, respectively, and 3) the role of SRD5α3 in N-glycosylation. This review also features the lesser known functions of SRD5αs in steroid degradation in the uterus during pregcy and glucocorticoid clearance in the liver. Additionally, the review describes the regulation of SRD5αs by the receptors of androgens, progesterone, estrogen, and thyroid hormones, as well as their differential DNA methylation. Factors known to be involved in their differential methylation are age, inflammation, and mental stimulation. Overall, this review helps shed light on the various essential functions of SRD5αs across species.

Is atenolol metabolized by CYP2D6?

No, atenolol is metabolized in a CYP2D6-independent manner.

AIMS: The study aimed to investigate the clinical adherence to drug label recommendations on important drug-drug interactions (DDIs). Dispensing data on drug combinations involving selective serotonin reuptake inhibitor (SSRI) antidepressants could help to identify areas for intensified medical education. METHODS: This was a retrospective, cross-sectional analysis of individual dispensing data regarding all individuals > or =15 years old in Sweden. The study analysed the prescribing and dispensing of CYP2D6 drugs (metoprolol, donepezil, galantamine, codeine, tamoxifen) together with CYP2D6-blocking SSRIs (paroxetine/fluoxetine) or SSRIs without significant CYP2D6 inhibition (citalopram/escitalopram/sertraline), and the related prescribing of CYP2D6-independent comparator drugs (atenolol, rivastigmine, propoxyphene, anastrozole). Odds were calculated between each CYP2D6 drug and the corresponding comparator drug in patients on fluoxetine/paroxetine and citalopram/escitalopram/sertraline, respectively. The odds ratio (OR) was calculated by dividing the obtained odds in patients on fluoxetine/paroxetine by the corresponding odds in patients on citalopram/escitalopram/sertraline. RESULTS: Compared with patients that were dispensed citalopram/escitalopram/sertraline, patients dispensed fluoxetine/paroxetine had lower prescribing rates of metoprolol (adjusted OR 0.80; 95% confidence interval 0.76, 0.85), donepezil (0.65; 0.49, 0.86) and galantamine (0.58; 0.41, 0.81). In contrast, the use of prodrugs codeine (compared woth propoxyphene) or tamoxifen (compared with anastrozole) was similar among patients on fluoxetine/paroxetine and citalopram/escitalopram/sertraline (adjusted OR 1.03; 0.94, 1.12 and 1.29; 0.96, 1.73, respectively). CONCLUSIONS: Clinically important DDIs that are associated with impaired bioactivation of prodrugs might be more easily neglected in clinical practice compared with DDIs that cause drug accumulation and symptomatic adverse drug reactions.

Describe DeepTRIAGE

DeepTRIAGE (Deep learning for the TRactable Individualised Analysis of Gene Expression) is a novel deep learning architecture which uses an attention mechanism to obtain personalised biomarker scores that describe how important each gene is in predicting the cancer sub-type for each sample. DeepTRIAge simultaneously reveals heterogeneity within the luminal A biomarker score that significantly associate with tumour stage, placing all luminal samples along a continuum of severity.

What is Pseudomelanosis duodeni?

Pseudomelanosis duodeni is a rare incidental finding seen on endoscopy and has the characteristic appearance of flat, black-speckled pigmented mucosa that can be associated with gastrointestinal bleeding, hypertension, chronic heart failure, chronic renal failure and consumption of different drugs.

Despite the common practice of upper gastrointestinal endoscopy, the unique phenomenon of punctate black pigmentation of the duodenal mucosa, now known as pseudomelanosis duodeni, still remains a rare entity. Four patients with normal renal function at presentation were found to have this pigment on endoscopy. One developed renal failure subsequently but the pigmentation persisted and this observation has not been reported before. Histochemical and ultrastructural studies were made. It is postulated that the pigment is heterogenous and represents a form of stored iron. Anti-hypertensive drugs may have a role in the causation of the pigmentation formation. Pseudomelanosis duodeni, speckled black pigmentation of the duodenal mucosa, presents a striking appearance at endoscopy. Among the 14 reported cases there is a predomice of black women greater than 40 years old, but it can occur in any race and age group. There is no known association with pigmentation elsewhere in the gastrointestinal tract or with the use of laxatives. However, most reported patients were hypertensive (many treated with hydralazine and propranolol) and significant numbers suffered from upper gastrointestinal bleeding, chronic renal failure, or diabetes mellitus. The pigment is usually located in mucosal macrophages, in lysosomes. Histochemical studies and electron probe microanalysis suggest that several pigments may result in this endoscopic appearance, including lipomelanin, ceroid, iron sulfide, and hemosiderin. Additional studies, possibly using tissue from surgical resections or autopsies, are needed to determine the etiology and clinical significance of this heterogeneous entity. Pseudomelanosis duodeni is an uncommon endoscopic sign characterized by diffuse small black spots on the first and second portions of the duodenum. It occurs predomitly in female and elderly patients and is linked to chronic illnesses and related medications. Between 1988 and 1994, the authors saw eight patients with pseudomelanosis duodeni. To evaluate the nature of the pigments, special staining was performed in seven cases. Iron stain was strongly positive in three cases. Electron microscopy was performed in two cases. This revealed amorphous bodies within macrophage lysosomes in one case and angular crystals in another case. These tests suggest that in pseudomelanosis duodeni iron metabolism may be impaired and iron is pooled within macrophages. Pseudomelanosis duodeni is rarely seen in children. It manifests endoscopically as peppery speckles in the duodenal mucosa. This pigment corresponds principally to accumulation of ferrous sulfide in macrophages within the lamina propria. We report the case of a 16-year-old boy with ectodermal dysplasia who underwent renal transplantation for vesicoureteral reflux and later developed epigastric pain. Endoscopic and pathologic findings in the duodenal mucosa were typical of pseudomelanosis duodeni. A review of the literature reveals shared clinical features among reported adult and pediatric cases, including chronic renal failure, use of antihypertensive medication and oral iron supplementation, and/or presence of gastric hemorrhage. Pseudomelanosis duodeni is a rare entity characterized by dark pigmentation of duodenal mucosa of uncertain etiology and clinical significance. We report a case with endoscopic and pathologic correlation. Some aspects about etiology, clinical and histopathologic characteristics are discussed. Pseudomelanosis duodeni is a rare benign condition. It manifests endoscopically as discrete, flat, small brown-black spots in the duodenal mucosa. It produces no symptoms and may be reversible. The cause and natural history of the pigmentation have not been clarified, although it is associated with a variety of systemic illnesses and medications. With electron microscopy and electron-probe energy dispersive X-ray analysis, the pigment corresponds principally to an accumulation of ferrous sulfide (FeS) in macrophages within the lamina propria. We report the case of a 56-year-old female patient with a past history of diabetes mellitus and hypertension. She was admitted because of nausea, vomiting, and diarrhea and underwent esophagogastroduodenoscopy because of stool occult blood test results of 3+. Endoscopy revealed diffusely scattered black spots in the bulb and second portion of the duodenum. Histological examination showed numerous pigment-laden macrophages in the lamina propria of mucosal villi. The diagnosis requires further confirmation by electron microscopy and electron-probe energy dispersive X-ray microanalysis. No special therapy is indicated for this rare lesion. Pseudomelanosis duodeni is seen endoscopically as dark spots in the duodenal mucosa and is generally considered to be local deposition of iron from oral iron intake. However, pseudomelanosis duodeni may be identified histologically even before it becomes endoscopically evident; iron stainability within the mucosa is uneven and unpredictable, and multiple clinical conditions other than oral iron intake may be associated. We reviewed 17 adult patients with histologically detected pseudomelanosis duodeni, their endoscopic appearances, iron stainability, and clinical findings including oral iron and drug intake. Only 6/17 (35 %) had endoscopically apparent dark spots. Perl's iron stain was entirely positive in 18 %, partially positive in 64 %, and negative in 18 % of cases. History of oral iron was present in 76 % of patients, but other clinical conditions consistently associated were hypertension in 88 %, end stage renal disease in 59 %, and diabetes mellitus in 35 % of patients. Pseudomelanosis duodeni (PD) is a rare dark speckled appearance of the duodenum associated with gastrointestinal bleeding, hypertension, chronic heart failure, chronic renal failure and consumption of different drugs. We report four cases of PD associated with chronic renal failure admitted to the gastroenterology outpatient unit due to epigastric pain, nausea, melena and progressive reduction of hemoglobin index. Gastroduodenal endoscopy revealed erosions in the esophagus and stomach, with no active bleeding at the moment. In addition, the duodenal mucosa presented marked signs of melanosis; later confirmed by histopathological study. Even though PD is usually regarded as a benign condition, its pathogenesis and clinical significance is yet to be defined. Pseudomelanosis duodeni is a rare entity characterised by dark pigmented intracellular granules seen within macrophages that lie within the lamina propria of the duodenal villi. There is no known treatment, and the clinical significance and long-term sequelae of this entity are unclear. We present a case of pseudomelanosis duodeni in a 54-year-old woman who presented with a 1-month history of nausea, vomiting and non-bloody diarrhoea. The medical history was significant for diabetes mellitus type 2, end-stage renal disease status postkidney transplant, hypertension, anaemia of chronic disease and hypothyroidism. A gastroduodenal endoscopy revealed pigmented dark lesions in the duodenal mucosa. Biopsies from the second part of the duodenum and duodenal bulb showed pigmented macrophages in the lamina propria. The findings were consistent with duodenal melanosis. In spite of renal transplant with normalisation of renal function, the duodenal melanosis persists, which raises questions on the role of renal impairment in this entity. A 76-year-old female patient with a past medical history of diabetes mellitus, stage 3 chronic renal failure and iron deficiency anemia was referred for esophagogastroduodenoscopy (EGD) for evaluation of solid food dysphagia. She had been on oral therapy with ferrous sulfate for several years. Besides a Schatzki's ring the EGD revealed a duodenal mucosa with black-speckled pigmentation. Biopsies were performed and disclosed the deposition of brown (hemosiderin) pigment within macrophages in the lamina propria of normal villi. This endoscopic appearance is called pseudomelanosis duodeni (PD). Melanosis of the stomach and duodenum is a rare entity and a striking finding diagnosed by upper gastrointestinal endoscopy. Here, we describe the case of an 83-year-old female, with a complicated medical history, who was referred to gastroenterologist to assess bleeding risk. From the endoscopy, it was determined that she had both melanosis gastri and duodeni. Although both are rare, gastric melanosis appears to be even more unusual than duodenal melanosis, with only a few reported cases documented in the literature thus far. Duodenal pseudomelanosis (or pseudomelanosis duodeni) is a rare benign condition characterized by black-brown speckled pigmentation of the duodenal mucosa. Collections of pigment-laden macrophages are found in the tips of duodenal villi. The pigment is thought to be mostly composed of ferrous sulfide. Histochemichal stains for iron (Perl's prussian blue) or melanin (Masson-Fontana) may be positive, but are usually negative or unpredictable. Duodenal pseudomelanosis occurs predomitly in middle-aged to old adults and more commonly in females. It is associated with chronic renal failure, arterial hypertension, diabetes mellitus and gastrointestinal bleeding. Medications such as ferrous sulfate, hydralazine, propranolol, hydrochlorothiazide and furosemide are thought to play a role as well. We report a case of a 86-year-old female who presented with a history of watery diarrhea and melena. The patient had a history of high blood pressure and ischemic stroke episodes. She was on multiple medication including hidralazine, captopril, hydrochlorthiazide and aspirin. She was dehydrated, her blood pressure was 96 × 60 mmHg and neurologic examination showed complete left hemiplegia with central VII nerve palsy. Laboratory tests showed normal serum electrolytes and renal function. Hemoglobin level was 10.7 g%. An upper endoscopy showed multiple diminutive black spots throughout the distal duodenal bulb and second portion. Histology showed multiple foci of a brown-black granular pigment inside macrophages within the tips of the villi (pseudomelanosis). Stains for iron and melanin were negative. She was treated with omeprazol, parenteral fluid replacement with saline and partial fasting. After complete recovery she was discharged for ambulatory follow up. Pseudomelanosis duodeni is a rare incidental finding seen on endoscopy and has the characteristic appearance of flat, black-speckled pigmented mucosa. We present the case of an 83-year-old woman who presented with gastrointestinal bleeding and was found to have pseudomelanosis duodeni. The finding has no diagnostic or prognostic significance. Therapeutic chelation or endoscopic follow-up is not recommended.

Is the glucocorticoid receptor a transcription factor?

Yes, The glucocorticoid receptor (GR) is a ligand-activated transcription factor that translocates to the nucleus upon hormone stimulation and distributes between the nucleoplasm and membraneless compartments named nuclear foci.

An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Krüppel-like transcription factors (KLF), i.e., KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives bovine infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR and KLF family members transactivate the bICP0 E promoter. GR and KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity in a ligand-independent manner in mouse neuroblastoma cells (Neuro-2A). Furthermore, GR and KLF4 stimulated productive infection. Mutating both half GR binding sites did not significantly reduce GR- and KLF4-mediated transactivation of the bICP0 E promoter, suggesting that a novel mechanism exists for transactivation. GR and KLF15 cooperatively stimulated bICP0 activity less efficiently than GR and KL4: however, KLF6, PLZF, and GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest that cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli.IMPORTANCE Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone. We predict that increased corticosteroid levels activate the glucocorticoid receptor (GR). Consequently, viral gene expression is stimulated by the activated GR. The immediate early transcription unit 1 promoter (IEtu1) drives expression of two viral transcriptional regulatory proteins, bovine infected cell protein 0 (bICP0) and bICP4. Interestingly, a separate early promoter also drives bICP0 expression. Two pioneer transcription factors, GR and Krüppel-like transcription factor 4 (KLF4), cooperatively transactivate the bICP0 early (E) promoter. GR and KLF15 cooperate to stimulate bICP0 E promoter activity but significantly less than GR and KLF4. The bICP0 E promoter contains enhancer-like domains necessary for GR- and KLF4-mediated transactivation that are distinct from those for GR and KLF15. Stress-induced pioneer transcription factors are proposed to activate key viral promoters, including the bICP0 E promoter, during early stages of reactivation from latency.

What are the five traits associated with metabolic syndrome?

Metabolic syndrome is the concurrent presentation of multiple cardiovascular risk factors, including obesity, insulin resistance, hyperglycemia, dyslipidemia and hypertension.

BACKGROUND: The close association of type 2 diabetes and atherosclerotic cardiovascular disease (CVD) suggests that they share a common physiologic antecedent, postulated to be tissue resistance to insulin. Insulin resistance is associated with a cluster of risk factors recognized as the metabolic syndrome. OBJECTIVE: To describe the epidemiology of the insulin resistance syndrome, also known as the metabolic syndrome. METHODS: Overall obesity, central obesity, dyslipidemia characterized by elevated levels of triglycerides and low levels of high-density lipoprotein cholesterol, hyperglycemia, and hypertension are common traits that, when they occur together, constitute the metabolic syndrome. The World Health Organization and the National Cholesterol Education Program Adult Treatment Panel III have proposed working definitions for the syndrome based on these traits. Cross-sectional and longitudinal epidemiologic studies provide an emerging picture of the prevalence and outcomes of the syndrome. RESULTS: National survey data suggest the metabolic syndrome is very common, affecting about 24% of US adults who are 20 to 70 years of age and older. The syndrome is more common in older people and in Mexican Americans. People with the syndrome are about twice as likely to develop CVD and over 4 times as likely to develop type 2 diabetes compared with subjects who do not have metabolic syndrome. While this syndrome may have a genetic basis, environmental factors are important modifiable risk factors for the condition. CONCLUSIONS: The metabolic syndrome is very common and will become even more common as populations age and become more obese. Treatment for component traits is known to reduce the risk for type 2 diabetes and CVD; whether risk is reduced by treatment of the syndrome, specifically, remains uncertain. Primary care physicians must recognize that the co-occurrence of risk factors for type 2 diabetes and CVD represents an extremely adverse metabolic state warranting aggressive risk factor intervention. BACKGROUND: Insulin resistance, obesity, dyslipidemia, and high blood pressure characterize the metabolic syndrome. In an effort to explore the utility of different multivariate methods of data reduction to better understand the genetic influences on the aggregation of metabolic syndrome phenotypes, we calculated phenotypic, genetic, and genome-wide LOD score correlation matrices using five traits (total cholesterol, high density lipoprotein cholesterol, triglycerides, systolic blood pressure, and body mass index) from the Framingham Heart Study data set prepared for the Genetic Analysis Workshop 13, clinic visits 10 and 1 for the original and offspring cohorts, respectively. We next applied factor analysis to summarize the relationship between these phenotypes. RESULTS: Factors generated from the genetic correlation matrix explained the most variation. Factors extracted using the other matrices followed a different pattern and suggest distinct effects. CONCLUSIONS: Given these results, different methods of multivariate data reduction may provide unique clues on the clustering of this complex syndrome. Prevention of the metabolic syndrome and treatment of its main characteristics are now considered of utmost importance in order to combat the epidemic of type 2 diabetes mellitus and to reduce the increased risk of cardiovascular disease and all-cause mortality. Insulin resistance/hyperinsulinaemia are consistently linked with a clustering of multiple clinical and subclinical metabolic risk factors. It is now widely recognised that obesity (especially abdominal fat accumulation), hyperglycaemia, dyslipidaemia and hypertension are common metabolic traits that, concurrently, constitute the distinctive insulin resistance or metabolic syndrome. Cross-sectional and prospective data provide an emerging picture of associations of both physical activity habits and cardiorespiratory fitness with the metabolic syndrome. The metabolic syndrome, is a disorder that requires aggressive multi-factorial intervention. Recent treatment guidelines have emphasised the clinical utility of diagnosis and an important treatment role for 'therapeutic lifestyle change', incorporating moderate physical activity. Several previous narrative reviews have considered exercise training as an effective treatment for insulin resistance and other components of the syndrome. However, the evidence cited has been less consistent for exercise training effects on several metabolic syndrome variables, unless combined with appropriate dietary modifications to achieve weight loss. Recently published randomised controlled trial data concerning the effects of exercise training on separate metabolic syndrome traits are evaluated within this review. Novel systematic review and meta-analysis evidence is presented indicating that supervised, long-term, moderate to moderately vigorous intensity exercise training, in the absence of therapeutic weight loss, improves the dyslipidaemic profile by raising high density lipoprotein-cholesterol and lowering triglycerides in overweight and obese adults with characteristics of the metabolic syndrome. Lifestyle interventions, including exercise and dietary-induced weight loss may improve insulin resistance and glucose tolerance in obesity states and are highly effective in preventing or delaying the onset of type 2 diabetes in individuals with impaired glucose regulation. Randomised controlled trial evidence also indicates that exercise training decreases blood pressure in overweight/obese individuals with high normal blood pressure and hypertension. These evidence-based findings continue to support recommendations that supervised or partially supervised exercise training is an important initial adjunctive step in the treatment of individuals with the metabolic syndrome. Exercise training should be considered an essential part of 'therapeutic lifestyle change' and may concurrently improve insulin resistance and the entire cluster of metabolic risk factors. The diagnostic categories of impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) were stablished in an effort to identify populations at risk for developing type 2 diabetes mellitus (T2DM). Both IGT and IFG are associated with increased risk of developing T2DM, but recent analyses found that the thresholds of risk vary among different populations and an even lower diagnostic threshold of IFG may be appropriate. IGT has been linked with an increased risk of cardiovascular events and some analyses have demonstrated an increased mortality risk compared with patients with normal glucose tolerance. In contrast, a continuum of increased risk of microvascular manifestations of T2DM has been demonstrated with IFG but an association of IFG with cardiovascular events has not been well established. Although both IGT and IFG are associated with resistance to insulin and increased insulin secretion, they do not identify the identical patient populations and are not equivalent in predicting development of T2DM or cardiovascular events. IFG and IGT have been associated with other features of insulin resistance, including dyslipidaemia, hypertension, abdominal obesity, microalbuminuria, endothelial dysfunction, and markers of inflammation and hypercoagulability, traits collectively referred to as the metabolic syndrome. Analyses of combinations of these components have also been associated with progression to T2DM, cardiovascular disease and increased mortality. The foundation of treatment for IGT, IFG, and the metabolic syndrome is lifestyle modification, including both dietary change and routine exercise. To date, several clinical trials have found that lifestyle modification is the most efficacious strategy to prevent progression to T2DM. Alternative treatments include pharmacotherapy with metformin or acarbose, both of which have been demonstrated to decrease the development of T2DM. Ongoing clinical trials are evaluating newer pharmacotherapies, including angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, metglitinides and thiazolidinediones, to prevent both T2DM and cardiovascular events. In combination with lifestyle modification, these therapies offer hope for effective prevention of T2DM and its consequences in high-risk patients. Metabolic syndrome (MetS) is a common complex trait consisting of the clustering of abdominal obesity, hypertension, dyslipidemia, and dysglycemia. MetS is found in about 25% of the population in the United States and is associated with increased risk for type 2 diabetes and cardiovascular disease. Despite research into possible genetic influences for MetS, no consistently reproducible genetic markers have been obtained, partially due to lack of agreement on the definition of the phenotype. Because phenotypic precision is essential for genomic interrogation, the evolving discipline of clinical phenomics, which uses objective and systematic acquisition of phenotypic data (ie, "deep phenotyping"), may help evaluate the genetic influences of MetS. This article reviews evidence that MetS has a genetic component and the potential applicability of clinical phenomics for the genetic evaluation of MetS using the example of hierarchical cluster analysis of phenotypic components of lipodystrophy syndromes, which serve as monogenic models of MetS. Obesity is associated with increased susceptibility to dyslipidemia, insulin resistance, and hypertension, a combination of traits that comprise the traditional definition of the metabolic syndrome. Recent evidence suggests that obesity is also associated with the development of nonalcoholic fatty liver disease (NAFLD). Despite the high prevalence of obesity and its related conditions, their etiologies and pathophysiology remains unknown. Both genetic and environmental factors contribute to the development of obesity and NAFLD. Previous genetic analysis of high-fat, diet-induced obesity in C57BL/6J (B6) and A/J male mice using a panel of B6-Chr(A/J)/NaJ chromosome substitution strains (CSSs) demonstrated that 17 CSSs conferred resistance to high-fat, diet-induced obesity. One of these CSS strains, CSS-17, which is homosomic for A/J-derived chromosome 17, was analyzed further and found to be resistant to diet-induced steatosis. In the current study we generated seven congenic strains derived from CCS-17, fed them either a high-fat, simple-carbohydrate (HFSC) or low-fat, simple-carbohydrate (LFSC) diet for 16 weeks and then analyzed body weight and related traits. From this study we identified several quantitative trait loci (QTLs). On a HFSC diet, Obrq13 protects against diet-induced obesity, steatosis, and elevated fasting insulin and glucose levels. On the LFSC diet, Obrq13 confers lower hepatic triglycerides, suggesting that this QTL regulates liver triglycerides regardless of diet. Obrq15 protects against diet-induced obesity and steatosis on the HFSC diet, and Obrq14 confers increased final body weight and results in steatosis and insulin resistance on the HFSC diet. In addition, on the LFSC diet, Obrq 16 confers decreased hepatic triglycerides and Obrq17 confers lower plasma triglycerides on the LFSC diet. These congenic strains provide mouse models to identify genes and metabolic pathways that are involved in the development of NAFLD and aspects of diet-induced metabolic syndrome. Moderate-to-high levels of physical activity are established as preventive factors in metabolic syndrome development. However, there is variability in the phenotypic expression of metabolic syndrome under distinct physical activity conditions. In the present study we applied a Genotype X Environment interaction method to examine the presence of GxEE interaction in the phenotypic expression of metabolic syndrome. A total of 958 subjects, from 294 families of The Portuguese Healthy Family study, were included in the analysis. Total daily energy expenditure was assessed using a 3 day physical activity diary. Six metabolic syndrome related traits, including waist circumference, systolic blood pressure, glucose, HDL cholesterol, total cholesterol and triglycerides, were measured and adjusted for age and sex. GxEE examination was performed on SOLAR 4.3.1. All metabolic syndrome indicators were significantly heritable. The GxEE interaction model fitted the data better than the polygenic model (p<0.001) for waist circumference, systolic blood pressure, glucose, total cholesterol and triglycerides. For waist circumference, glucose, total cholesterol and triglycerides, the significant GxEE interaction was due to rejection of the variance homogeneity hypothesis. For waist circumference and glucose, GxEE was also significant by the rejection of the genetic correlation hypothesis. The results showed that metabolic syndrome traits expression is significantly influenced by the interaction established between total daily energy expenditure and genotypes. Physical activity may be considered an environmental variable that promotes metabolic differences between individuals that are distinctively active. Metabolic syndrome (MS), conventionally defined by the presence of at least three out of five dysmetabolic traits (abdominal obesity, hypertension, low plasma HDL-cholesterol, high plasma glucose and high triglycerides), has been associated with an increased risk of several age-related chronic diseases, including breast cancer (BC). This may have prognostic implications for BC survivors. 2,092 early stage BC survivors aged 35-70, recruited in eleven Italian centres 0-5 years after surgical treatment (1.74 years on average), were followed-up over 2.8 years on average for additional BC-related events, including BC-specific mortality, distant metastasis, local recurrences and contralateral BC. At recruitment, 20 % of the patients had MS. Logistic regression models were carried out to generate OR and 95 % confidence intervals (CI) for new BC events associated with MS, adjusting for baseline pathological prognostic factors. New BC events occurred in 164 patients, including 89 distant metastases. The adjusted ORs for women with MS versus women without any MS traits were 2.17 (CI 1.31-3.60) overall, and 2.45 (CI 1.24-4.82) for distant metastasis. The OR of new BC events for women with only one or two MS traits was 1.40 (CI 0.91-2.16). All MS traits were positively associated with new BC events, and significantly so for low HDL and high triglycerides. MS is an important prognostic factor in BC. As MS is reversible through lifestyle changes, interventions to decrease MS traits in BC patients should be implemented in BC clinics. The metabolic syndrome represents a cluster of closely connected premorbid risk factors or diseases with visceral obesity type 2 diabetes, hypertension and low HLD/hypertriglyceridemia as established traits affecting about 20% in the adult European populations. This syndrome develops on a complex soil with overnutrition, low physical activity and psychosocial stress. Common comorbidities are fatty liver, sleep apnoe and endothelial dysfunction with cardiovascular complications, nephropathy and type 2 diabetes as "end-stage" diseases. Thus, a rational diagnostic is needed to elucidate the complex cluster of diseases as basis for an integrated therapy. There is a clear priority for life style intervention, however, most diseases of the metabolic syndrome need medical treatment. Medical treatment of single traits has to take into account possible pleiotropic or adverse effects on the other traits. This paper presents the pros and cons of major drug intervention for type 2 diabetes, hypertension, dyslipidemia and hypercoagulation in the context with the metabolic syndrome. In ancient Greek medicine the concept of a distinct syndrome (going together) was used to label 'a group of signs and symptoms' that occur together and 'characterize a particular abnormality and condition'. The (dys)metabolic syndrome is a common cluster of five pre-morbid metabolic-vascular risk factors or diseases associated with increased cardiovascular morbidity, fatty liver disease and risk of cancer. The risk for major complications such as cardiovascular diseases, NASH and some cancers develops along a continuum of risk factors into clinical diseases. Therefore we still include hyperglycemia, visceral obesity, dyslipidemia and hypertension as diagnostic traits in the definition according to the term 'deadly quartet'. From the beginning elevated blood pressure and hyperglycemia were core traits of the metabolic syndrome associated with endothelial dysfunction and increased risk of cardiovascular disease. Thus metabolic and vascular abnormalities are in extricable linked. Therefore it seems reasonable to extend the term to metabolic-vascular syndrome (MVS) to signal the clinical relevance and related risk of multimorbidity. This has important implications for integrated diagnostics and therapeutic approach. According to the definition of a syndrome the rapid global rise in the prevalence of all traits and comorbidities of the MVS is mainly caused by rapid changes in life-style and sociocultural transition resp. with over- and malnutrition, low physical activity and social stress as a common soil. OBJECTIVE: Metabolic syndrome and the presence of metabolic syndrome components are risk factors for cardiovascular disease (CVD). However, the association between personality traits and metabolic syndrome remains controversial, and few studies have been conducted in East Asian populations. METHODS: We measured personality traits using the Japanese version of the Eysenck Personality Questionnaire (Revised Short Form) and five metabolic syndrome components-elevated waist circumference, elevated triglycerides, reduced high-density lipoprotein cholesterol, elevated blood pressure, and elevated fasting glucose-in 1322 participants aged 51.1±12.7years old from Kakegawa city, Japan. Metabolic syndrome score (MS score) was defined as the number of metabolic syndrome components present, and metabolic syndrome as having the MS score of 3 or higher. We performed multiple logistic regression analyses to examine the relationship between personality traits and metabolic syndrome components and multiple regression analyses to examine the relationship between personality traits and MS scores adjusted for age, sex, education, income, smoking status, alcohol use, and family history of CVD and diabetes mellitus. We also examine the relationship between personality traits and metabolic syndrome presence by multiple logistic regression analyses. RESULTS: "Extraversion" scores were higher in those with metabolic syndrome components (elevated waist circumference: P=0.001; elevated triglycerides: P=0.01; elevated blood pressure: P=0.004; elevated fasting glucose: P=0.002). "Extraversion" was associated with the MS score (coefficient=0.12, P=0.0003). No personality trait was significantly associated with the presence of metabolic syndrome. CONCLUSIONS: Higher "extraversion" scores were related to higher MS scores, but no personality trait was significantly associated with the presence of metabolic syndrome. Metabolic syndrome is a cluster of the most dangerous heart attack risk factors (diabetes and raised fasting plasma glucose, abdominal obesity, high cholesterol and high blood pressure), and has become a major global threat to human health. A number of studies have demonstrated that hundreds of non-coding RNAs, including miRNAs and lncRNAs, are involved in metabolic syndrome-related diseases such as obesity, type 2 diabetes mellitus, hypertension, etc. However, these research results are distributed in a large number of literature, which is not conducive to analysis and use. There is an urgent need to integrate these relationship data between metabolic syndrome and non-coding RNA into a specialized database. To address this need, we developed a metabolic syndrome-associated non-coding RNA database (ncRNA2MetS) to curate the associations between metabolic syndrome and non-coding RNA. Currently, ncRNA2MetS contains 1,068 associations between five metabolic syndrome traits and 627 non-coding RNAs (543 miRNAs and 84 lncRNAs) in four species. Each record in ncRNA2MetS database represents a pair of disease-miRNA (lncRNA) association consisting of non-coding RNA category, miRNA (lncRNA) name, name of metabolic syndrome trait, expressive patterns of non-coding RNA, method for validation, specie involved, a brief introduction to the association, the article referenced, etc. We also developed a user-friendly website so that users can easily access and download all data. In short, ncRNA2MetS is a complete and high-quality data resource for exploring the role of non-coding RNA in the pathogenesis of metabolic syndrome and seeking new treatment options. The website is freely available at http://www.biomed-bigdata.com:50020/index.html. BACKGROUND AND AIMS: Metabolic syndrome is the concurrent presentation of multiple cardiovascular risk factors, including obesity, insulin resistance, hyperglycemia, dyslipidemia and hypertension. It has been suggested that some of these risk factors can have detrimental effects on the skeletal muscle while others can be a direct result of skeletal muscle abnormalities, showing a two-way directionality in the pathogenesis of the condition. This review aims to explore this bidirectional correlation by discussing the impact of metabolic syndrome on skeletal muscle tissue in general and will also discuss ways in which skeletal muscle alterations may contribute to the pathogenesis of metabolic syndrome. METHODS: Literature searches were conducted with key words (e.g. metabolic syndrome, skeletal muscle, hyperglycemia) using PubMed, EBSCOhost, Science Direct and Google Scholar. All article types were included in the search. RESULTS: The pathological mechanisms associated with metabolic syndrome, such as hyperglycemia and inflammation, have been associated with changes in skeletal muscle fiber composition, metabolism, insulin sensitivity, mitochondrial function, and strength. Additionally, some skeletal muscle alterations, particularly mitochondrial dysfunction and insulin resistance, are suggested to contribute to the development of metabolic syndrome. For example, the suggested underlying mechanisms of sarcopenia development are also contributors to metabolic syndrome pathogenesis. CONCLUSION: Whilst numerous studies have identified a relationship between metabolic syndrome and skeletal muscle abnormalities, further investigation into the underlying mechanisms is needed to elucidate the best prevention and management strategies for these conditions.

Which gene is responsible for the Liebenberg syndrome?

Liebenberg syndrome is a genetic disease caused by heterozygous mutations or deletions of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. Patients present with prominent neurological, medical, and behavioral symptoms.

The study of homeotic-transformation mutants in model organisms such as Drosophila revolutionized the field of developmental biology, but how these mutants relate to human developmental defects remains to be elucidated. Here, we show that Liebenberg syndrome, an autosomal-domit upper-limb malformation, shows features of a homeotic limb transformation in which the arms have acquired morphological characteristics of a leg. Using high-resolution array comparative genomic hybridization and paired-end whole-genome sequencing, we identified two deletions and a translocation 5' of PITX1. The structural changes are likely to remove active PITX1 forelimb suppressor and/or insulator elements and thereby move active enhancer elements in the vicinity of the PITX1 regulatory landscape. We generated transgenic mice in which PITX1 was misexpressed under the control of a nearby enhancer and were able to recapitulate the Liebenberg phenotype. We report a case of Liebenberg syndrome in a 6-year-old girl, including the clinical, radiological, angiographic, and operative findings. We note that the forearm and hand malformations have similarities to leg and foot anatomy. Our observations may help provide insight into the etiology of this unusual condition. The Liebenberg syndrome was first described in 1973 in a five- generation family. A sixth generation was added in 2001, and in 2009 a hitherto unknown branch of the same family with similar anomalies extended the family tree significantly. This article describes the clinical findings and illustrates the abnormalities with radiographs and three-dimensional computed tomography scans. We discuss the genetic abnormality that causes Liebenberg syndrome, the genomic rearrangement at the PITX1 locus on chromosome 5.The structural variations seem to result in an ectopic expression of paired-like homeodomain transcription factor 1 (PITX1) in the forelimb causing a partial arm-to-leg transformation in these patients. BACKGROUND: Structural variants (SVs) affecting non-coding cis-regulatory elements are a common cause of congenital limb malformation. Yet, the functional interpretation of these non-coding variants remains challenging. The human Liebenberg syndrome is characterised by a partial transformation of the arms into legs and has been shown to be caused by SVs at the PITX1 locus leading to its misregulation in the forelimb by its native enhancer element Pen. This study aims to elucidate the genetic cause of an unsolved family with a mild form of Liebenberg syndrome and investigate the role of promoters in long-range gene regulation. METHODS: Here, we identify SVs by whole genome sequencing (WGS) and use CRISPR-Cas9 genome editing in transgenic mice to assign pathogenicity to the SVs. RESULTS: In this study, we used WGS in a family with three mildly affected individuals with Liebenberg syndrome and identified the smallest deletion described so far including the first non-coding exon of H2AFY. To functionally characterise the variant, we re-engineered the 8.5 kb deletion using CRISPR-Cas9 technology in the mouse and showed that the promoter of the housekeeping gene H2afy insulates the Pen enhancer from Pitx1 in forelimbs; its loss leads to misexpression of Pitx1 by the pan-limb activity of the Pen enhancer causing Liebenberg syndrome. CONCLUSION: Our data indicate that housekeeping promoters may titrate promiscuous enhancer activity to ensure normal morphogenesis. The deletion of the H2AFY promoter as a cause of Liebenberg syndrome highlights this new mutational mechanism and its role in congenital disease.

Which pharmacogenetic test is recommended prior to administering carbamazepine and why?

HLA-B∗15:02 is known as a biomarker for carbamazepine (CBZ) induced Steven-Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) in some Asian populations. Hence United States Federal Drug Administration (USFDA) recommends HLA-B∗15:02 screening for Asian and other populations with a high prevalence of HLA-B∗15:02, prior to the administration of carbamazepine.

HLA-B∗15:02 is known as a biomarker for carbamazepine (CBZ) induced Steven-Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) in some Asian populations. Hence United States Federal Drug Administration (USFDA) recommends HLA-B∗15:02 screening for Asian and other populations with a high prevalence of HLA-B∗15:02, prior to the administration of carbamazepine. This study was conducted to estimate the prevalence of HLA-B∗15:02 in a cohort of Sri Lankans. We observed an overall prevalence of 4.3% (4/93) among 93 Sri Lankans comprising 32 Sinhalese, 30 Sri Lankan Tamils and 31 Moors. The allele was detected in 3 [9.3%; 3/32] Sinhalese, 0 [0%; 0/30] Sri Lankan Tamils and in 1 [3%; 1/31] Moor. The overall prevalence of HLA-B∗15:02 in this population was close to that of other populations where the USFDA has recommended HLA-B∗15:02 screening. A larger study is required to confirm these findings, especially among the Sinhalese where the frequency appears to be high.

Are there sex differences in oncogenic mutational processes?

Yes. Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. There are sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes.

Which drugs are included in the MAID chemotherapy regimen for sarcoma?

MAID chemotherapy regimen for sarcomas include mesna, adriamycin, ifosfamide and dacarbazine.

Although carcinosarcoma occurs in various locations throughout the body, it rarely originates in the ovary. Chemotherapy has been minimally beneficial. This case describes a patient with carcinosarcoma of the ovary who responded minimally to chemotherapy used for epithelial carcinomas but had a complete response after receiving chemotherapy used for sarcomas. The patient relapsed within 1 year after receiving cisplatin therapy. She was treated with mesna, ifosfamide, Adriamycin, and dacarbazine (MAID) chemotherapy and after one cycle of chemotherapy she had no evidence of tumor. She has received six cycles of chemotherapy without evidence of progression 13+ months since beginning MAID therapy. MAID chemotherapy may be useful in the treatment of carcinosarcoma of the ovary. The mesna, doxorubicin, ifosfamide, dacarbazine regimen produced a 47% response rate (including 10% complete responses) in 105 eligible adults with advanced sarcoma. The major dose-limiting toxicity was granulocytopenia. There was one toxic death from sepsis. Central nervous system and renal toxicity occurred infrequently, perhaps as a result of the continuous-infusion schedule. This regimen is being evaluated further in advanced disease, the adjuvant setting, and in combination with bone marrow colony-stimulating factors. In this phase II trial, 105 eligible patients with no prior chemotherapy and advanced sarcoma received doxorubicin, ifosfamide, and dacarbazine (DTIC) with mesna uroprotection (MAID). Starting doses of these drugs were 60, 7,500, and 900 mg/m2 divided over 72 hours by continuous infusion, respectively. Mesna was given for 84 to 96 hours at 2,500 mg/m2/d. Myelosuppression was dose limiting, causing the only toxic death (sepsis). Nonhematologic toxicity consisted predomitly of anorexia and vomiting. Severe mucositis, macroscopic hematuria, renal tubular acidosis, renal failure, and CNS toxicity occurred in less than 5% of cycles. No cardiotoxicity was detected. The overall response rate (10% complete response [CR]) was 47% (95% confidence intervals, 5% to 18% and 37% to 57%, respectively). Most responses (approximately 70%) were observed within two cycles. Median times to progression were 10 and 9 months, respectively. Histologic high tumor grade, lesions less than 5 cm, and less than 1 year from diagnosis to study entry correlated with the probability of response. The median survival was 16 months. Time from diagnosis to study entry, performance status, and extent of disease, but not histologic grade, correlated with survival. Following CR, two patients remain disease-free at 32 and 16 months. Of the 15 additional patients rendered disease-free with surgery, two remain disease-free at 30 and 18 months with no further therapy. While most relapses occurred in sites of prior involvement, death from CNS metastases occurred in 11 of the 80 patients with high-grade sarcomas, of whom seven were still responding systematically (three complete responders). Because of its substantial response in this phase II trial, the MAID regimen is being compared with doxorubicin and DTIC alone in advanced sarcomas and to observation in the adjuvant treatment of high-grade sarcomas in randomized trials. Four patients with metastatic ovarian mixed Müllerian sarcoma (2 homologous, 2 heterologous) were treated with mesna, doxorubicin, ifosfamide, and dacarbazine (MAID) chemotherapy. Two of four patients had optimal debulking. Three of four patients responded to chemotherapy, with two complete responses of 34- and 46-month duration. The MAID regimen appears to be active in patients with ovarian sarcoma. Since dose intensity of doxorubicin is correlated with the clinical response of patients with soft tissue sarcomas and since doxorubicin dose intensity may be compromised in combination chemotherapy, we evaluated the use of recombit granulocytemacrophage colony-stimulating factor (rGM-CSF) to ameliorate myelosuppression and allow doxorubicin dose escalation in a phase I trial utilizing the MAID combination [Mesna 2.5 g/m2/day x 4 days, Adriamycin (doxorubicin) 15 mg/m2/day x 4 days, ifosfamide 2.0 g/m2/day x 3 days, dacarbazine 250 mg/m2/day x 4 days; to be repeated every 21 days]. Thirteen patients were treated. The doxorubicin dose for the first 6 patients was at the standard dose of 15 mg/m2/day x 4 days (level 1), while the doxorubicin dose for the next 7 patients was escalated by 25% to 18.75 mg/m2/day x 4 days (level 2). rGM-CSF was given at 5 micrograms/kg/day, days 5-14. All patients experienced moderate to severe myelosuppression, with all patients at dose level 2 requiring doxorubicin dose reduction to dose level 1 or lower by their third course of treatment. rGM-CSF failed to allow sustained escalation of the doxorubicin dose in the MAID regimen. This study was conducted to determine the maximum tolerated dose of an intensified MAID (mesna, adriamycin, ifosfamide, dacarbazine) regimen with the support of lenograstim in patients with advanced soft tissue sarcomas. Following 1 cycle of MAID at the standard dose, four patients were to be treated at each of five dosage levels: +25%, +45%, +65%, +85%, +100%. Sixteen patients were treated. Because there were no significant differences in hematologic toxicity between patients receiving lenograstim 5 or 10 microg/kg/day (levels 1-5 and 1-10), the data were pooled for comparison with level 2. The median duration of absolute neutrophil count < 0.5 x 10(9)/l was 3 days at level 1 and 7 days at level 2 (p < 0.01). The median platelet nadir was 25 x 10(9)/l at level 1 and 10 x 10(9)/l at level 2 (p < 0.01). The median duration of toxicity-related hospitalization was 3.5 days and 11 days at levels 1 and 2, respectively, (p < 0.001). Mucositis > or = grade III occurred after 3/29 cycles at level 1 and 10/15 cycles at level 2 (p < 0.001). After 4 cycles at level 1, 8/8 patients still had performance status scores < or = 2, and only 4/8 had performance status scores < or = 2 after the second cycle at level 2. Lenograstim enabled an increase of 25% of the MAID regimen. At higher dose levels, severe mucositis and deterioration in performance status were dose limiting. The sarcomatoid variant of renal-cell carcinoma (SRCC), a clinically aggressive subtype of renal parenchymal tumors, is typically resistant to systemic treatments and carries a poor prognosis. The authors report a case of a 57-year-old male with advanced SRCC who had a durable complete response after MAID (mesna, adriamycin, ifosfamide and dacarbazine) chemotherapy, and remains free of disease four years after completing treatment. To the authors' knowledge, this is the first report of a remission from MAID chemotherapy in SRCC. A review of published literature revealed occasional responses after systemic chemotherapy. Notably, all responses were seen with doxorubicin containing regimens, suggesting that doxorubicin is a critical component in chemotherapy regimens for SRCC. BACKGROUND & OBJECTIVES: Chemotherapy combined with other therapeutic modalities is the main option for advanced and metastatic soft tissue sarcoma(STS). So far there is no standard regimen for STS yet. Adrimycin, ifosfamide, and dacarbazine are the most effective agents at present. The purpose of this clinical trial was to evaluate the efficacy and toxicity of MAID regimen (mesna/ifosfamide + Adriamycin + dacarbazine) in the treatment of advanced soft tissue sarcoma. METHODS: Twenty-two patients with advanced STS were treated by MAID(Adriamycin 60 mg/m2, ifosfamide 6,000 mg/m2, and dacarbazine 1,000 mg/m2). These drugs were administered as continuous intravenous infusion for 72 hours while mesna was infused continuously for 96 hours. RESULTS: Partial response rate was 36.4% without complete remission. The duration of response ranged from 2-10 months with median of 4.6 months. Main toxicities were myelosuppression, gastrointestinal toxicity and alopecia. Percentage of leucopenia, nausea/vomiting, and alopecia in WHO grade III and IV were 63.6%, 27.3%, and 50%, respectively. CONCLUSIONS: The response rate of MAID for advanced STS was not satisfactory with evident myelosuppression. Further study on new anti-cancer agents and regimen are needed. PURPOSE: On the basis of a positive reported single-institution pilot study, the Radiation Therapy Oncology Group initiated phase II trial 9514 to evaluate its neoadjuvant regimen in a multi-institutional Intergroup setting. PATIENTS AND METHODS: Eligibility included a high-grade soft tissue sarcoma > or = 8 cm in diameter of the extremities and body wall. Patients received three cycles of neoadjuvant chemotherapy (CT; modified mesna, doxorubicin, ifosfamide, and dacarbazine [MAID]), interdigitated preoperative radiation therapy (RT; 44 Gy administered in split courses), and three cycles of postoperative CT (modified MAID). RESULTS: Sixty-six patients were enrolled, of whom 64 were analyzed. Seventy-nine percent of patients completed their preoperative CT and 59% completed all planned CT. Three patients (5%) experienced fatal grade 5 toxicities (myelodysplasias, two patients; infection, one patient). Another 53 patients (83%) experienced grade 4 toxicities; 78% experienced grade 4 hematologic toxicity and 19% experienced grade 4 nonhematologic toxicity. Sixty-one patients underwent surgery. Fifty-eight of these were R0 resections, of which five were amputations. There were three R1 resections. The estimated 3-year rate for local-regional failure is 17.6% if amputation is considered a failure and 10.1% if not. Estimated 3-year rates for disease-free, distant-disease-free, and overall survival are 56.6%, 64.5%, and 75.1%, respectively. CONCLUSION: This combined-modality treatment can be delivered successfully in a multi-institutional setting. Efficacy results are consistent with previous single-institution results. BACKGROUND: Metastatic soft tissue sarcoma (STS) prognosis remains poor and few cytotoxic agents offer proven efficacy. This randomized open phase III study examines whether high-dose (HD) chemotherapy with peripheral blood stem cells (PBSCs) could improve overall survival (OS) of chemosensitive patients. PATIENTS AND METHODS: Advanced STS patients aged 18-65 years received four courses of standard mesna, adryamycin, ifosfamide and dacarbazine (MAID) treatment. Chemotherapy-responding patients and patients with at least stable disease amenable to complete surgical resection were randomized to receive standard dose (SD) with two successive MAID cycles or HD treatments of one MAID then MICE intensification: mesna (3.6 g/m(2), day 1-5), ifosfamide (2.5 g/m(2), day 1-4), carboplatin [area under the curve (AUC) 5/day 2-4] and etoposide (300 mg/m(2), day 1-4) with PBSC reinjection at day 7. RESULTS: From 2000 to 2008, 207 patients received four cycles of MAID and 87 assessable patients were randomly assigned to receive the following: 46 SD, 41 HD, with 45 and 38 maintained for analyses after secondary centralized histological review. Futility analyses led to study closure in November 2008. Three-year OS was 49.4% for the SD group versus 32.7% for HD arm, hazard ratio= 1.26, 95% confidence interval 0.70-2.29; progression-free survival was 32.4% and 14.0%, respectively. HD treatment led to higher grades 3-4 toxicity. CONCLUSION: This study failed to show an OS advantage for advanced STS patients treated with dose-intensified chemotherapy with PBSC. BACKGROUND: Good local control of high-grade non-small round cell soft tissue sarcomas (NSRCSTSs) has been achieved with significant advances in surgical techniques and radiotherapy. However, the role of chemotherapy remains controversial. Our aim was to investigate the efficacy, feasibility and adverse effects of neoadjuvant and adjuvant chemotherapy with modified mesna, adriamycin, ifosfamide and dacarbazine (MAID) regimen for NSRCSTSs. METHODS: We conducted a retrospective review of 40 consecutive patients (29 men, 11 women; median age 47 years) with high-grade NSRCSTSs treated in two referral centers between 2004 and 2009 (median follow-up 38.5 months). Patients with distant or nodal metastases at diagnosis were excluded. The regimen consisted of ifosfamide 2,500 mg/m(2)/6 h (days 1-3), mesna 2,500 mg/m(2)/6 h (days 1-3), tetrahydropyranyl adriamycin 20 mg/m(2)/0.5 h (days 1-3), and dacarbazine 300 mg/m(2)/1 h (days 1-3). RESULTS: Among the 26 evaluable patients, there were 8 with a partial response, 15 with stable disease, and 3 with progressive disease. Two- and 5-year overall survival rates were 92 and 86%, respectively, and corresponding disease-free survival rates were 80 and 77%. All relapses were metastases without local recurrence. Grade 3-4 neutropenia, anemia and thrombocytopenia were observed in 38, 18, and 21 patients, respectively. No serious infectious complications occurred due to the administration of granulocyte colony-stimulating factor and prophylactic antibiotics. No other life-threatening serious adverse events were observed. CONCLUSION: The modified MAID regimen achieved a better outcome with less serious adverse events than previously reported and is a potential option in the management of NSRCSTSs. Further evaluation with long-term follow-up is required. BACKGROUND: Patients with large, high-grade extremity and truncal soft tissue sarcomas (STS) are at considerable risk for recurrence. A regimen of pre-operative chemotherapy consisting of mesna, adriamycin, ifosfamide and dacarbazine (MAID), interdigitated with radiotherapy (RT), followed by resection and post-operative chemotherapy with or without RT, has demonstrated high rates of local and distant control. The goal of this study is to assess outcomes in a recent cohort of patients treated on this regimen. METHODS: We retrospectively reviewed records of 66 consecutive patients with STS of the extremity or trunk who were treated with the aforementioned regimen from May 2000 to April 2011. Clinicopathologic characteristics and patient outcomes were analysed. RESULTS: Sixty-six patients were analysed and were equally divided between grade 2 and 3 tumours. Margins were negative in 57 (89%) patients and positive in seven (11%) patients. At a median follow-up of 46 months, there were six (9%) locoregional and 20 (30%) distant recurrences. The locoregional and distant 5-year recurrence-free survival (RFS) rates were 91% and 64%, respectively. The 5-year overall (OS) and disease-specific survival rates were 86% and 89%, respectively. There were no treatment-related deaths or secondary myelodysplasias. Thirty-four (52%) patients had grade 3 or 4 acute haematologic chemotherapy-related toxicity. There were no statistically significant predictors of OS or RFS. CONCLUSIONS: For a contemporary cohort of patients with high-risk extremity and truncal STS, a regimen of neoadjuvant chemoradiotherapy and surgery continues to result in high rates of survival with tolerable short- and long-term toxicity. Publisher: HINTERGRUND: Für Patienten mit anthrazyklinresistentem metastasierten Angiosarkom existiert momentan keine Standard-Zweitlinientherapie, und es besteht Bedarf an neuen effektiven Regimen zur Verbesserung der Ansprechraten. FALLBERICHT: Wir berichten von einem Fall von primärem, bilateralen Angiosarkom der Brust bei einer 34-jährigen Patientin mit Lungenmetastasen. Nach 3 Zyklen des MAID-Regimes (Mesna, Adriamycin, Ifosfamid, Dacarbazin) zeigte die computertomographische Untersuchung ein Fortschreiten der Erkrankung. Daraufhin wurde eine Zweitlinienchemotherapie mit dem GVP-Regime (Gemcitabin, Vincristin, Cisplatin) begonnen. Nach 6 Behandlungszyklen konnte ein komplettes Ansprechen der Lungenmetastasen verzeichnet werden. SCHLUSSFOLGERUNG: In Abwesenheit einer effektiven Therapie für Patienten mit anthrazyklinresistentem metastasierten Angiosarkom der Brust stellt das GVP-Chemothe-rapieregime eine Behandlungsmöglichkeit dar. OBJECTIVES: Pulmonary pleomorphic carcinoma (PC) is a rare type of lung tumor with a dismal prognosis. There is no consensus on a chemotherapy regimen for PC, and conventional platinum-based chemotherapy has been associated with disappointing response rates and PFS. In searches for a new regimen, the sarcomatoid (spindle or giant cell) component has been assumed to be susceptible to chemotherapy used for soft tissue sarcoma. MATERIALS AND METHODS: The medical records of 17 patients who received mesna, doxorubicin, ifosfamide, and dacarbazine (MAID) for advanced PC between January 2010 and February 2017 were retrospectively analyzed for clinicopathological features and outcomes. RESULTS AND CONCLUSION: The median age was 59 years. Sixteen patients were male, and only one patient had never smoked. Six patients achieved partial response to MAID, leading to an objective response rate of 35%. The median PFS was 2.8 months, and the median OS was 8.7 months. Hematologic toxicity-related adverse events were the most frequent, which comprised grade 3-4 anemia in 35% of patients, neutropenia in 47%, thrombocytopenia in 24%, and febrile neutropenia in 29%. No febrile neutropenia was reported in patients who received 5-day granulocyte-colony stimulating factor (G-CSF) prophylaxis. Most adverse events resolved without complications, except for one death due to sepsis. MAID is an effective, and possibly important, regimen for PC. MAID could be more safely used in clinical practice with appropriate dose modifications and G-CSF primary prophylaxis according to patients' status. RATIONALE: Ameloblastoma is generally characterized as a benign tumor originating in odontogenic epithelium. However, few cases of metastatic maligt ameloblastoma have also been reported. Due to the low incidence of maligt ameloblastoma, there is no established treatment regimen. To explore effective treatment for maligt ameloblastoma, we reported this case study. PATIENTS CONCERNS: This report described a case of a 28-year-old maligt ameloblastoma female patient with multiple metastasis (brain and lung). DIAGNOSES: The patient presented ameloblastoma of the left mandible in 2012. Three years later, local recurrence and brain metastasis was observed during a follow-up examination. Five years later, maligt ameloblastoma was detected by imaging and immunohistochemistry in the bilateral multiple pulmonary nodules and mediastinal lymph nodes. INTERVENTIONS: The patient was initially treated with tumor resection. Three years later after local recurrence and brain metastasis, she was accepted the extensive mandibulectomy supplemented with brain stereotactic body radiotherapy (SBRT). When diagnosed with pulmonary metastasis, the patient received combined chemotherapy regimen of MAID (mesna, adriamycin, ifosfamide and dacarbazine) for 6 cycles. OUTCOMES: The efficacy evaluation was partial remission (PR) after the 6 cycles of MAID. The last patient follow-up was July 24th 2018, and no evidence of progression was observed. The progression-free survival (PFS) of the patient was more than 9 months. LESSONS: Surgical resection is the optimal treatment for locally recurrent ameloblastoma. SBRT may be an effective treatment for unresectable oligometastasis of maligt ameloblastoma. Finally, combined chemotherapy of MAID showed encouraging effects in the management of metastatic maligt ameloblastoma. The role of chemotherapy in the treatment of myxofibrosarcoma is unclear. There are no randomized clinical trials evaluating the therapeutic effect of chemotherapy on myxofibrosarcoma. We report, to the best of our knowledge, the first case of myxofibrosarcoma successfully treated with mesna, pirarubicin, ifosfamide and dacarbazine (modified MAID) regimen. The patient achieved complete remission evaluated according to Response Evaluation Criteria in Solid Tumours (RECIST).

Which is the main ligand for the glucocorticoid receptor?

Glucocorticoids (GC) such as cortisol regulate multiple physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene in humans).

Glucocorticoids (GC) such as cortisol regulate multiple physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene in humans). GC signaling follows several consecutive steps leading to target gene transactivation, including ligand binding, nuclear translocation of ligand-activated GR complexes, DNA binding, and recruitment of functional transcriptional machinery. Generalized glucocorticoid resistance syndrome, due to GR loss-of-function mutations, may be related to the impairment of one of the GC signaling steps. To date, 31 NR3C1 loss-of-function mutations have been reported in patients presenting with various clinical signs such as hypertension, adrenal hyperplasia, hirsutism or metabolic disorders associated with biological hypercortisolism but without Cushing syndrome signs and no negative regulatory feedback loop on the hypothalamic-pituitary-adrenal axis. Functional characterization of GR loss-of-function mutations often demonstrates GR haploinsufficiency and a decrease of GR target gene induction in relevant cell types. The main signs at presentation are very variable from resistant hypertension, bilateral adrenal hyperplasia likely related to increased ACTH levels but not exclusively, hirsutism to isolated renin-angiotensin-aldosterone system abnormalities in a context of 11βHSD2 deficiency. Some mutated GR patients are obese or overweight together with a healthier metabolic profile that remains to be further explored in future studies. Deciphering the molecular mechanisms altered by GR mutations should enhance our knowledge on GR signaling and ultimately facilitate management of GC-resistant patients. This review also focuses on the criteria facilitating identification of novel NR3C1 mutations in selected patients. Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.This article has an associated First Person interview with the first author of the paper. A decline in normal physiological functions characterizes the aging process. While some of these changes are benign, the decrease in the function of the cardiovascular system that occurs during aging leads to the activation of pathological processes associated with an increased risk for heart disease and its complications. Imbalances in endocrine function are also common occurrences during the aging process. Glucocorticoids are primary stress hormones and are critical regulators of energy metabolism, inflammation, and cardiac function. Glucocorticoids exert their actions by binding the glucocorticoid receptor (GR) and, in some instances, to the mineralocorticoid receptor (MR). GR and MR are members of the nuclear receptor family of ligand-activated transcription factors. There is strong evidence that imbalances in GR and MR signaling in the heart have a causal role in cardiac disease. The extent to which glucocorticoids play a role in the aging heart, however, remains unclear. This review will summarize the positive and negative direct and indirect effects of glucocorticoids on the heart and the latest molecular and physiological evidence on how alterations in glucocorticoid signaling lead to changes in cardiac structure and function. We also briefly discuss the effects of other hormones systems such as estrogens and GH/IGF-1 on different cardiovascular cells during aging. We will also review the link between imbalances in glucocorticoid levels and the molecular processes responsible for promoting cardiomyocyte dysfunction in aging. Finally, we will discuss the potential for selectively manipulating glucocorticoid signaling in cardiomyocytes, which may represent an improved therapeutic approach for preventing and treating age-related heart disease.

Who received the Nobel prize for development of CRISPR?

The 2020 Nobel Prize in Chemistry was awarded to CRISPR-Cas pioneers Emmanuelle Charpentier and Jennifer Doudna. Charpentier and Doudna pioneered the site-specific CRISPR gene-editing technology that has revolutionized cancer research and treatment.

Emmanuelle Charpentier, PhD, and Jennifer Doudna, PhD, who pioneered the site-specific CRISPR gene-editing technology that has revolutionized cancer research and treatment, were awarded the 2020 Nobel Prize in Chemistry. Many CRISPR-based therapies are already in human testing, with gene-edited T cells for blood cancers and solid tumors leading the way. Conflict of interest statement: Conflict of interest: JB has served on advisory boards for GenSight Biologics; SparingVision; Akouos, Inc; Life Biosciences; and Odylia Therapeutics and has consulted for Spark Therapeutics. She served as the scientific director for clinical trials run by Spark Therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) is a prokaryotic immune surveillance system that is used by bacteria to recognize genetic material of infectious organisms, such as phage viruses. Using CRISPR-associated (Cas) proteins, this system cleaves foreign nucleic acid into fragments, thus defending the bacterium against the attacker. The 2020 Nobel Prize in Chemistry was awarded to CRISPR-Cas pioneers Emmanuelle Charpentier and Jennifer Doudna, who developed the CRISPR-Cas system to precisely edit genomic DNA. This technology has exploded at a breathtaking pace and is now used by almost every molecular biology laboratory around the world in a myriad of organisms. In this Virtual Issue, the FEBS Journal features articles reviewing the development of CRISPR/Cas9 technology and its applications to understand the functions of proteins in vivo.

How are super enhancers defined?

Super-enhancers are defined as genomic regions spanned by highly conserved non-coding elements (HCNEs), most of which serve as regulatory inputs of one target gene in the region.

Super-enhancers are large clusters of transcriptional enhancers that drive expression of genes that define cell identity. Improved understanding of the roles that super-enhancers play in biology would be afforded by knowing the constellation of factors that constitute these domains and by identifying super-enhancers across the spectrum of human cell types. We describe here the population of transcription factors, cofactors, chromatin regulators, and transcription apparatus occupying super-enhancers in embryonic stem cells and evidence that super-enhancers are highly transcribed. We produce a catalog of super-enhancers in a broad range of human cell types and find that super-enhancers associate with genes that control and define the biology of these cells. Interestingly, disease-associated variation is especially enriched in the super-enhancers of disease-relevant cell types. Furthermore, we find that cancer cells generate super-enhancers at oncogenes and other genes important in tumor pathogenesis. Thus, super-enhancers play key roles in human cell identity in health and in disease. Enhancers are critical genomic elements that define cellular and functional identity through the spatial and temporal regulation of gene expression. Recent studies suggest that key genes regulating cell type-specific functions reside in enhancer-dense genomic regions (i.e., super enhancers, stretch enhancers). Here we report that enhancer RNAs (eRNAs) identified by global nuclear run-on sequencing are extensively transcribed within super enhancers and are dynamically regulated in response to cellular signaling. Using Toll-like receptor 4 (TLR4) signaling in macrophages as a model system, we find that transcription of super enhancer-associated eRNAs is dynamically induced at most of the key genes driving innate immunity and inflammation. Unexpectedly, genes repressed by TLR4 signaling are also associated with super enhancer domains and accompanied by massive repression of eRNA transcription. Furthermore, we find each super enhancer acts as a single regulatory unit within which eRNA and genic transcripts are coordinately regulated. The key regulatory activity of these domains is further supported by the finding that super enhancer-associated transcription factor binding is twice as likely to be conserved between human and mouse than typical enhancer sites. Our study suggests that transcriptional activities at super enhancers are critical components to understand the dynamic gene regulatory network. Super-enhancers (SEs) are regions of the genome consisting of clusters of regulatory elements bound with very high amounts of transcription factors, and this architecture appears to be the hallmark of genes and noncoding RNAs linked with cell identity. Recent studies have identified SEs in CD4(+) T cells and have further linked these regions to single nucleotide polymorphisms (SNPs) associated with immune-mediated disorders, pointing to an important role for these structures in the T cell differentiation and function. Here we review the features that define SEs, and discuss their function within the broader understanding of the mechanisms that define immune cell identity and function. We propose that SEs present crucial regulatory hubs, coordinating intrinsic and extrinsic differentiation signals, and argue that delineating these regions will provide important insight into the factors and mechanisms that define immune cell identity. Author information: (1)Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Genome #02-01, Singapore 138672, Singapore. (2)Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore. (3)NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074, Singapore. (4)Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, #12-01, Singapore 117599, Singapore. (5)Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2 Medical Drive #04-01, Singapore 117597, Singapore. (6)Department of Human Genetics, Genome Institute of Singapore, 60 Biopolis Street, Genome #02-01, Singapore 138672, Singapore. (7)Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, UK. (8)Department of Upper Gastrointestinal &Bariatric Surgery, Singapore General Hospital, Singapore 169608, Singapore. (9)Division of Surgical Oncology, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore. (10)Department of General Surgery, Singapore General Hospital, Singapore 169608, Singapore. (11)Department of Medical Oncology, Yonsei University College of Medicine, Seoul 120-752, South Korea. (12)SingHealth/Duke-NUS Institute of Precision Medicine, National Heart Centre Singapore, Singapore 168752, Singapore. (13)Laboratory of Cancer Epigenome, Department of Medical Sciences, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore. (14)School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. (15)Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore. The "hallmarks" of pancreatic ductal adenocarcinoma (PDAC) include proliferative, invasive, and metastatic tumor cells and an associated dense desmoplasia comprised of fibroblasts, pancreatic stellate cells, extracellular matrix, and immune cells. The oncogenically activated pancreatic epithelium and its associated stroma are obligatorily interdependent, with the resulting inflammatory and immunosuppressive microenvironment contributing greatly to the evolution and maintece of PDAC. The peculiar pancreas-specific tumor phenotype is a consequence of oncogenes hacking the resident pancreas regenerative program, a tissue-specific repair mechanism regulated by discrete super enhancer networks. Defined as genomic regions containing clusters of multiple enhancers, super enhancers play pivotal roles in cell/tissue specification, identity, and maintece. Hence, interfering with such super enhancer-driven repair networks should exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. Novel drugs that directly or indirectly inhibit processes regulating epigenetic status and integrity, including those driven by histone deacetylases, histone methyltransferase and hydroxylases, DNA methyltransferases, various metabolic enzymes, and bromodomain and extraterminal motif proteins, have shown the feasibility of disrupting super enhancer-dependent transcription in treating multiple tumor types, including PDAC. The idea that pancreatic adenocarcinomas rely on embedded super enhancer transcriptional mechanisms suggests a vulnerability that can be potentially targeted as novel therapies for this intractable disease. Clin Cancer Res; 23(7); 1647-55. ©2017 AACRSee all articles in this CCR Focus section, "Pancreatic Cancer: Challenge and Inspiration." Metabolic changes are linked to epigenetic reprogramming and play important roles in several tumor types. PGC-1α is a transcriptional coactivator controlling mitochondrial biogenesis and is linked to oxidative phosphorylation. We provide evidence that melanoma models with elevated PGC-1α levels are characteristic of the proliferative phenotype and are sensitive to bromodomain and extra-terminal domain (BET) inhibitor treatment. A super-enhancer region highly occupied by the BET family member BRD4 was identified for the PGC-1α gene. BET inhibitor treatment prevented this interaction, leading to a dramatic reduction of PGC-1α expression. Accordingly, BET inhibition diminished respiration and mitochondrial function in cells. In vivo, melanoma models with high PGC-1α expression strongly responded to BET inhibition by reduction of PGC-1α and impaired tumor growth. Altogether, our findings identify epigenetic regulatory elements that define a subset of melanomas with high sensitivity to BET inhibition, which opens up the opportunity to define melanoma patients most likely to respond to this treatment, depending on their tumor characteristics. Super-enhancers comprise of clusters of enhancers that are typically defined by the ChIP-seq analysis for active histone marks. Although the biological significance of super-enhancers is still controversial, this concept is gaining prominence as useful characteristics of genes that play crucial roles in normal development and pathogenesis of cancer. In various cancer cells, super-enhancers are often associated with genes involved in carcinogenesis. For example, in T-cell acute lymphoblastic leukemia, the oncogenic transcription factor TAL1 and its regulatory partners (GATA3, RUNX1 and MYB) are regulated by super-enhancers; these genes are sensitive to transcriptional inhibition, for example, via the pharmacological approach using a small-molecule CDK7 inhibitor. This preferential inhibition of cancer genes can also be observed for other types of cancer. Based on these findings, we recently performed super-enhancer profiling combined with gene expression analysis in adult T-cell leukemia/lymphoma, which is a genetically complicated hematological maligcy, to identify critical genes responsible for the pathogenesis. This review article aims to discuss the concept of super-enhancers, their significance in biomedical research, and their potential utility in elucidating the molecular pathogenesis of cancer. Super-enhancers and stretch enhancers represent classes of transcriptional enhancers that have been shown to control the expression of cell identity genes and carry disease- and trait-associated variants. Specifically, super-enhancers are clusters of enhancers defined based on the binding occupancy of master transcription factors, chromatin regulators, or chromatin marks, while stretch enhancers are large chromatin-defined regulatory regions of at least 3,000 base pairs. Several studies have characterized these regulatory regions in numerous cell types and tissues to decipher their functional importance. However, the differences and similarities between these regulatory regions have not been fully assessed. We integrated genomic, epigenomic, and transcriptomic data from ten human cell types to perform a comparative analysis of super and stretch enhancers with respect to their chromatin profiles, cell type-specificity, and ability to control gene expression. We found that stretch enhancers are more abundant, more distal to transcription start sites, cover twice as much the genome, and are significantly less conserved than super-enhancers. In contrast, super-enhancers are significantly more enriched for active chromatin marks and cohesin complex, and more transcriptionally active than stretch enhancers. Importantly, a vast majority of super-enhancers (85%) overlap with only a small subset of stretch enhancers (13%), which are enriched for cell type-specific biological functions, and control cell identity genes. These results suggest that super-enhancers are transcriptionally more active and cell type-specific than stretch enhancers, and importantly, most of the stretch enhancers that are distinct from super-enhancers do not show an association with cell identity genes, are less active, and more likely to be poised enhancers. Molecular subtyping of cancer offers tremendous promise for the optimization of a precision oncology approach to anticancer therapy. Recent advances in pancreatic cancer research uncovered various molecular subtypes with tumors expressing a squamous/basal-like gene expression signature displaying a worse prognosis. Through unbiased epigenome mapping, we identified deltaNp63 as a major driver of a gene signature in pancreatic cancer cell lines, which we report to faithfully represent the highly aggressive pancreatic squamous subtype observed in vivo, and display the specific epigenetic marking of genes associated with decreased survival. Importantly, depletion of deltaNp63 in these systems significantly decreased cell proliferation and gene expression patterns associated with a squamous subtype and transcriptionally mimicked a subtype switch. Using genomic localization data of deltaNp63 in pancreatic cancer cell lines coupled with epigenome mapping data from patient-derived xenografts, we uncovered that deltaNp63 mainly exerts its effects by activating subtype-specific super enhancers. Furthermore, we identified a group of 45 subtype-specific super enhancers that are associated with poorer prognosis and are highly dependent on deltaNp63. Genes associated with these enhancers included a network of transcription factors, including HIF1A, BHLHE40, and RXRA, which form a highly intertwined transcriptional regulatory network with deltaNp63 to further activate downstream genes associated with poor survival. Super-enhancers (SEs) are clusters of transcriptional enhancers which control the expression of cell identity and disease-associated genes. Current studies demonstrated the role of multiple factors in SE formation; however, a systematic analysis to assess the relative predictive importance of chromatin and sequence features of SEs and their constituents is lacking. In addition, a predictive model that integrates various types of data to predict SEs has not been established. Here, we integrated diverse types of genomic and epigenomic datasets to identify key signatures of SEs and investigated their predictive importance. Through integrative modeling, we found Cdk8, Cdk9, and Smad3 as new features of SEs, which can define known and new SEs in mouse embryonic stem cells and pro-B cells. We compared six state-of-the-art machine learning models to predict SEs and showed that non-parametric ensemble models performed better as compared to parametric. We validated these models using cross-validation and also independent datasets in four human cell-types. Taken together, our systematic analysis and ranking of features can be used as a platform to define and understand the biology of SEs in other cell-types. Cellular identity relies on cell-type-specific gene expression controlled at the transcriptional level by cis-regulatory elements (CREs). CREs are unevenly distributed across the genome, giving rise to individual CREs and clusters of CREs (COREs). Technical and biological features hinder CORE identification. We addressed these issues by developing an unsupervised machine learning approach termed clustering of genomic regions analysis method (CREAM). CREAM automates CORE detection from chromatin accessibility profiles that are enriched in CREs strongly bound by master transcription regulators, proximal to highly expressed and essential genes, and discriminating cell identity. Although COREs share similarities with super-enhancers, we highlight differences in terms of the genomic distribution and structure of these cis-regulatory units. We further show the enhanced value of COREs over super-enhancers to identify master transcription regulators, highly expressed and essential genes defining cell identity. COREs enrich at topologically associated domain (TAD) boundaries. They are also preferentially bound by the chromatin looping factors CTCF and cohesin, in contrast to super-enhancers, forming clusters of CTCF and cohesin binding regions and defining homotypic clusters of transcription regulator binding regions (HCTs). Finally, we show the clinical utility of CREAM to identify COREs across chromatin accessibility profiles to stratify more than 400 tumor samples according to their cancer type and to delineate cancer type-specific active biological pathways. Collectively, our results support the utility of CREAM to delineate COREs underlying, with greater accuracy than individual CREs or super-enhancers, the cell-type-specific biological underpinning across a wide range of normal and cancer cell types. Super-enhancers (SE) have become a popular concept and are widely used as a feature defining key identity genes. Here, we provide perspectives on the use of SE to define and identify cell/tissue-identity genes. By mining SE and their associated genes using murine functional genomics data, we highlight and discuss current limitations and open questions regarding both the sensitivity and specificity of identity genes/transcription factors predicted by SE. In this context, we point to cell/tissue-specific promoters as an important additional level of information, which we propose to combine with SE when aiming to define potential identity genes.

Is metoprolol metabolized by CYP2D6?

Yes, metoprolol is metabolized by CYP2D6.

Patients with cardiovascular diseases are often treated by concurrent multiple drug therapy. It is therefore plausible that with an increasing number of drugs the risk of drug interactions increases. Such interactions can be either pharmacodynamic (and are due to the mechanism of the administered drugs) or they can be pharmacokinetic (resulting in a reduction or enhancement of drug elimination). Pharmacokinetic interactions can be either due to interactions at the level of drug metabolizing enzymes (most important cytochrome P450 (CYP) enzymes) or interactions at the level of drug transporter proteins (for example P-glycoprotein (MDR1)). It is important to distinguish between both mechanisms because interactions at transporter proteins can be attributed to those drugs that are not enzymatically metabolized. The scope of this article is to give an overview on clinically relevant interactions of the four beta-blockers widely used in the therapy of cardiovascular diseases namely atenolol (CAS 29122-68-7), bisoprolol (CAS 66722-44-9), metoprolol (CAS 37350-58-6) (each beta-1 selective), and carvedilol (CAS 72956-09-3) (beta-1 and beta-2 nonselective). Among these beta-blockers atenolol is mainly eliminated by renal excretion, bisoprolol is in part excreted as parent compound via the renal route (50%), the other 50% are hepatically metabolised, whereas metoprolol and carvedilol are metabolised by CYP2D6. In addition, evidence is accumulating that carvedilol is a substrate for P-glycoprotein. For these four beta-blockers various pharmacodynamic and pharmacokinetic interactions have been demonstrated. Such interactions that result in an altered pharmacokinetics are mainly observed with those beta-blockers that are excreted via metabolism (metoprolol and carvedilol). Accordingly these drugs have a higher potential for drug interactions. However, it should be emphasized that, in general, beta-blockers are well tolerated safe drugs with a large therapeutic index.

List versions of ExpansionHunter

ExpansionHunter and ExpansionHunter Denovo

SUMMARY: We describe a novel computational method for genotyping repeats using sequence graphs. This method addresses the long-standing need to accurately genotype medically important loci containing repeats adjacent to other variants or imperfect DNA repeats such as polyalanine repeats. Here we introduce a new version of our repeat genotyping software, ExpansionHunter, that uses this method to perform targeted genotyping of a broad class of such loci. AVAILABILITY AND IMPLEMENTATION: ExpansionHunter is implemented in C++ and is available under the Apache License Version 2.0. The source code, documentation, and Linux/macOS binaries are available at https://github.com/Illumina/ExpansionHunter/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

What are the uses of Nirsevimab?

A single injection of nirsevimab resulted in fewer medically attended RSV-associated lower respiratory tract infections and hospitalizations than placebo throughout the RSV season in healthy preterm infants.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants, and a need exists for prevention of RSV in healthy infants. Nirsevimab is a monoclonal antibody with an extended half-life that is being developed to protect infants for an entire RSV season with a single intramuscular dose. METHODS: In this trial conducted in both northern and southern hemispheres, we evaluated nirsevimab for the prevention of RSV-associated lower respiratory tract infection in healthy infants who had been born preterm (29 weeks 0 days to 34 weeks 6 days of gestation). We randomly assigned the infants in a 2:1 ratio to receive nirsevimab, at a dose of 50 mg in a single intramuscular injection, or placebo at the start of an RSV season. The primary end point was medically attended RSV-associated lower respiratory tract infection through 150 days after administration of the dose. The secondary efficacy end point was hospitalization for RSV-associated lower respiratory tract infection through 150 days after administration of the dose. RESULTS: From November 2016 through November 2017, a total of 1453 infants were randomly assigned to receive nirsevimab (969 infants) or placebo (484 infants) at the start of the RSV season. The incidence of medically attended RSV-associated lower respiratory tract infection was 70.1% lower (95% confidence interval [CI], 52.3 to 81.2) with nirsevimab prophylaxis than with placebo (2.6% [25 infants] vs. 9.5% [46 infants]; P<0.001) and the incidence of hospitalization for RSV-associated lower respiratory tract infection was 78.4% lower (95% CI, 51.9 to 90.3) with nirsevimab than with placebo (0.8% [8 infants] vs. 4.1% [20 infants]; P<0.001). These differences were consistent throughout the 150-day period after the dose was administered and across geographic locations and RSV subtypes. Adverse events were similar in the two trial groups, with no notable hypersensitivity reactions. CONCLUSIONS: A single injection of nirsevimab resulted in fewer medically attended RSV-associated lower respiratory tract infections and hospitalizations than placebo throughout the RSV season in healthy preterm infants. (Funded by AstraZeneca and Sanofi Pasteur; ClinicalTrials.gov number, NCT02878330.).

What is the proteoglycan Tsukushi?

Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan (SLRP) family, plays multifunctional roles by interacting with signaling molecules during development. In lung cancer cells, TSK is expressed more highly than the other SLRPs family members, and regulates the EMT and proliferation. Thus, TSK may be a key coordinator of multiple pathways and an important structural element in the lung cancer microenvironment. Gain- and loss-of-function analyses showed that the small leucine-rich proteoglycan, tsukushi, contributes to vitamin K2-mediated enhancement of collagen accumulation.

Vitamin K2 is a critical nutrient required for blood coagulation. It also plays a key role in bone homeostasis and is a clinically effective therapeutic agent for osteoporosis. We previously demonstrated that vitamin K2 is a transcriptional regulator of bone marker genes in osteoblastic cells and that it may potentiate bone formation by activating the steroid and xenobiotic receptor, SXR. To explore the SXR-mediated vitamin K2 signaling network in bone homeostasis, we identified genes up-regulated by both vitamin K2 and the prototypical SXR ligand, rifampicin, in osteoblastic cells using oligonucleotide microarray analysis and quantitative reverse transcription-PCR. Fourteen genes were up-regulated by both ligands. Among these, tsukushi, matrilin-2, and CD14 antigen were shown to be primary SXR target genes. Moreover, collagen accumulation in osteoblastic MG63 cells was enhanced by vitamin K2 treatment. Gain- and loss-of-function analyses showed that the small leucine-rich proteoglycan, tsukushi, contributes to vitamin K2-mediated enhancement of collagen accumulation. Our results suggest a new function for vitamin K2 in bone formation as a transcriptional regulator of extracellular matrix-related genes, that are involved in the collagen assembly. The nuclear receptor steroid and xenobiotic receptor (SXR) is a transcriptional regulator activated by various biological and xenobiotic substances. We have recently shown that SXR is expressed in bone and that this receptor is critical for bone metabolism, particularly in osteoblastic cells. Vitamin K2, one of the critical nutrients in bone metabolism, has been demonstrated that it is a potent SXR agonist and modulates the expression of various bone-related genes in osteoblastic cells. Using microarray analysis, we identified novel SXR target genes that were activated by vitamin K2 in osteoblastic cells. Among them, a small leucine-rich repeat proteoglycan, tsukushi, has been shown to contribute to collagen accumulation, and the protein may interact with another vitamin K2-inducible SXR target, matrilin-2, a member of the matrilin family that functions as collagen adaptors. Besides functioning as a xenobiotic biosensor, our findings show that SXR is also a vitamin K2 target and an important transcriptional factor that regulates bone homeostasis in bone cells.

Does Curare function by stimulating the acetylcholine receptor?

No. Curare function does not stimulate the acetylcholine receptor.

We have studied the effects of curare on responses resulting from iontophoretic application of several putative neurotransmitters onto Aplysia neurons. These neurons have specific receptors for acetylcholine (ACh), dopamine, octopamine, phenylethanolamine, histamine, gamma-aminobutyric acid (GABA), aspartic acid, and glutamic acid. Each of these substances may on different specific neurons elicit at least three types of response, caused by a fast depolarizing Na+, a fast hyperpolarizing Cl-, or a slow hyperpolarizing K+ conductance increase. All responses resulting from either Na+ or Cl- conductance increases, irrespective of which putative transmitter activated the response, were sensitive to curare. Most were totally blocked by less than or equal to 10-4 M curare. GABA responses were less sensitive and were often only depressed by 10-3 M curare. K+ conductance responses, irrespective of the transmitter, were not curare sensitive. These results are consistent with a model of receptor organization in which one neurotransmitter receptor may be associated with any of at least three ionophores, mediating conductance increase responses to Na+, Cl-, and K+, respectively. In Aplysia nervous tissue, curare appears not to be a specific antagonist for the nicotinic ACh receptor, but rather to be a specific blocking agent for a class of receptor-activated Na+ and Cl- responses. The perhydro derivative of histrionicotoxin reversibly blocks the excitatory ionic transduction system in the synaptic and sarcolemmal membranes of mammalian skeletal muscle cells. The efficacy of perhydrohistrionicotoxin as an antagonist at the post-synaptic membrane is increased by the transient presence of acetylcholine in the endplate of innervated muscles and at extrajunctional receptors in denervated muscles. alpha-Bungarotoxin and [(3)H]monoacetyl-alpha-bungarotoxin block the endplate acetylcholine receptors, each binding to the same extent. The effect of bungarotoxin is partially reversible. These electrophysiological results, together with the effects of perhydrohistrionicotoxin and/or d-tubocurarine on the binding of [(3)H]monoacetyl-alpha-bungarotoxin at endplates of murine diaphragm muscle and on the bungarotoxin-elicited irreversible blockade of neuromuscular transmission, suggest that at least two types of sites participate in the synaptic excitation by acetylcholine. One site, competitively blocked by bungarotoxin and by curare, is presumably the acetylcholine receptor. Binding of bungarotoxin at this site is responsible for an irreversible blockade of neuromuscular transmission. The second site, competitively blocked by bungarotoxin and perhydrohistrionicotoxin, is proposed to be part of the cholinergic ion conductance modulator. Binding of bungarotoxin to this site does not result in an irreversible blockade. The mode of action of curare, a well-known competitive antagonist of acetylcholine at the nicotinic receptor, was examined with the single channel recording technique. Curare can open cholinergic channels in rat myotubes, as suggested by Ziskind and Dennis (1978). Moreover another curare molecule can then block the curare-activated open channel, in line with previous results concerning such a mode of action. In adult rat muscle, the partial agonist activity of curare can also be demonstrated, though it is much weaker than in embryonic muscle. It is also shown that in adult muscle cell, the conductance of the channel (activated either by ACh or by curare) is 50-60 pS, i.e., higher than in the myotubes (35 pS). d-Tubocurarine (curare) is a well-characterized competitive antagonist of nicotinic acetylcholine receptors (AChRs), and it is usually assumed that curare and agonists share a common binding site. We have examined the role of several highly conserved residues of the alpha-, gamma-, and delta-subunits in the interaction of curare with the Torpedo acetylcholine receptor (AChR). Curare inhibition of wild-type receptors is consistent with curare binding to a single high-affinity binding site [inhibitor constant (Ki) = 20 nM]. Phenylalanine substitutions for two tyrosine residues implicated as being in the ligand binding site (alpha Y93F, alpha Y190F) reduce curare affinity, indicating that these residues are also important for high-affinity curare binding. Phenylalanine substitution for alpha Y198 [alpha Y198F (notation used here: subunit/amino acid in wild-type/residue number/substitution)] causes a 10-fold increase in curare affinity (Ki = 3.1 nM), and measurement of the recovery from curare inhibition indicates that this increase in affinity is due to a reduction in the rate of curare dissociation from the receptor. In addition to the alpha-subunits, portions of the ligand binding sites also reside on the gamma- and delta-subunits, and photoaffinity studies have implicated two residues (gamma W55 and delta W57) as forming part of the curare sites. The gamma W55L mutation results in an eightfold decrease in curare affinity (Ki = 170 nM), whereas the delta W57L mutation has no effect. These data support the notion that the high-affinity curare binding site is formed by segments of the alpha- and gamma-subunits.(ABSTRACT TRUNCATED AT 250 WORDS) Spinal motoneurons (MNs) in the chick embryo undergo programmed cell death coincident with the establishment of nerve-muscle connections and the onset of synaptic transmission at the neuromuscular junction. Chronic treatment of embryos during this period with nicotinic acetylcholine receptor (nAChR)-blocking agents [e.g., curare or alpha-bungarotoxin (alpha-BTX)] prevents the death of MNs. Although this rescue effect has been attributed previously to a peripheral site of action of the nAChR-blocking agents at the neuromuscular junction (NMJ), because nAChRs are expressed in both muscle and spinal cord, it has been suggested that the rescue effect may, in fact, be mediated by a direct central action of nAChR antagonists. By using a variety of different nAChR-blocking agents that target specific muscle or neuronal nAChR subunits, we find that only those agents that act on muscle-type receptors block neuromuscular activity and rescue MNs. However, paralytic, muscular dysgenic mutant chick embryos also exhibit significant increases in MN survival that can be further enhanced by treatment with curare or alpha-BTX, suggesting that muscle paralysis may not be the sole factor involved in MN survival. Taken together, the data presented here support the argument that, in vivo, nAChR antagonists promote the survival of spinal MNs primarily by acting peripherally at the NMJ to inhibit synaptic transmission and reduce or block muscle activity. Although a central action of these agents involving direct perturbations of MN activity may also play a contributory role, further studies are needed to determine more precisely the relative roles of central versus peripheral sites of action in MN rescue. Nicotinic acetylcholine receptors are members of the ligand-gated ion channel superfamily, that includes also gamma-amino-butiric-acid(A), glycine, and 5-hydroxytryptamine(3) receptors. Functional nicotinic acetylcholine receptors result from the association of five subunits each contributing to the pore lining. The major neuronal nicotinic acetylcholine receptors are heterologous pentamers of alpha4beta2 subunits (brain), or alpha3beta4 subunits (autonomic ganglia). Another class of neuronal receptors that are found both in the central and peripheral nervous system is the homomeric alpha7 receptor. The muscle receptor subtypes comprise of alphabetadeltagamma (embryonal) or alphabetadeltaepsilon (adult) subunits. Although nicotinic acetylcholine receptors are not directly involved in the hypnotic component of anesthesia, it is possible that modulation of central nicotinic transmission by volatile agents contributes to analgesia. The main effect of anesthetic agents on nicotinic acetylcholine receptors is inhibitory. Volatile anesthetics and ketamine are the most potent inhibitors both at alpha4beta2 and alpha3beta4 receptors with clinically relevant IC(50) values. Neuronal nicotinic acetylcholine receptors are more sensitive to anesthetics than their muscle counterparts, with the exception of the alpha7 receptor. Several intravenous anesthetics such as barbiturates, etomidate, and propofol exert also an inhibitory effect on the nicotinic acetylcholine receptors, but only at concentrations higher than those necessary for anesthesia. Usual clinical concentrations of curare cause competitive inhibition of muscle nicotinic acetylcholine receptors while higher concentrations may induce open channel blockade. Neuronal nAChRs like alpha4beta2 and alpha3beta4 are inhibited by atracurium, a curare derivative, but at low concentrations the alpha4beta2 receptor is activated. Inhibition of sympathetic transmission by clinically relevant concentrations of some anesthetic agents is probably one of the factors involved in arterial hypotension during anesthesia. To elucidate innervation in the upper esophageal sphincter (UES) muscle of the eel, a key muscle in swallowing, repetitive electrical field stimulation (EFS; 30 mA, 40 V, 300 micros, 10 Hz, 10 trains) was employed. Anatomically, the eel UES muscle consists of striated fibers. The EFS-induced contraction of the UES was completely blocked by tetrodotoxin and curare, and abolished in Ca2+ -free Ringer solution. These results suggest that the EFS stimulates nerve fibers specifically and releases acetylcholine as a neurotransmitter. In fact, acetylcholine and carbachol constricted the UES in a concentration-dependent manner. Even after blocking neuronal firing with tetrodotoxin, acetylcholine constricted the UES muscle, suggesting the existence of acetylcholine receptors on the UES muscle cells. Both EFS- and carbachol-evoked contractions of the UES were blocked by curare at a lower concentration than by atropine or hexamethonium, suggesting that the acetylcholine receptor is nicotinic. Even in Ca2+ -free Ringer solution, a direct current stimulus (2 s duration) constricted the UES muscle to an extent similar to that in the presence of Ca2+, indicating that the muscle contraction itself does not need extracellular Ca2+, i.e., the muscle can be constricted by a release of Ca2+ from the sarcoplasmic reticulum. 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." The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K(+)) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion. Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138-1149, 2016.

Are somatic mutations positioned towards the nuclear periphery?

lamina-associated regions, which are typically localized at the nuclear periphery, displayed higher somatic mutation frequencies than did the interlamina regions at the nuclear core. Smoking and UV-related signatures, as well as substitutions at certain motifs, were more enriched in the nuclear periphery.

The nuclear matrix (NM) is the structural framework of the nucleus that consists of the peripheral lamins and pore complexes, an internal ribonucleic protein network, and residual nucleoli. Differences between the nuclear matrix protein (NMP) composition of transformed cells and their normal homologues were detected in numerous cases. Actually several tumor-specific nuclear matrix proteins (NMPs) are proposed for diagnostic of bladder, breast, colon and some other cancers. According to the role of NMPs in development and phenotype of a given neoplasms the tumors can be classified as follows: I. Tumors bearing mutations in the genes encoding NMPs. The group consists of following subgroups: 1) hereditary cancer syndromes with mutations in the NM-attached oncoproteins or tumor suppressor genes; 2) sporadic tumors with somatic mutations in the NM-attached oncoproteins, tumor suppressor genes or replication enzymes; 3) leukemias with fused NMPs. II. Tumors with phenotypic quantitative or qualitative changes of the NMP spectrum. Nuclear organization of genomic DNA affects processes of DNA damage and repair, yet its effects on mutational landscapes in cancer genomes remain unclear. Here we analyzed genome-wide somatic mutations from 366 samples of six cancer types. We found that lamina-associated regions, which are typically localized at the nuclear periphery, displayed higher somatic mutation frequencies than did the interlamina regions at the nuclear core. This effect was observed even after adjustment for features such as GC percentage, chromatin, and replication timing. Furthermore, mutational signatures differed between the nuclear core and periphery, thus indicating differences in the patterns of DNA-damage or DNA-repair processes. For instance, smoking and UV-related signatures, as well as substitutions at certain motifs, were more enriched in the nuclear periphery. Thus, the nuclear architecture may influence mutational landscapes in cancer genomes beyond the previously described effects of chromatin structure and replication timing.

Which biological drugs are EMA approved for pediatric psoriasis?

Currently there are three European Medicines Agency (EMA)-approved biological treatment options for pediatric psoriasis: etanercept, ustekinumab, and adalimumab.

Background and Objectives: Severe, recalcitrant cases of pediatric psoriasis or atopic dermatitis may necessitate treatment with biological agents; however, this may be difficult due to lack of treatment options and standardized treatment guidelines. This review evaluates the biological treatment options available, including off-label uses, and provides a basic therapeutic guideline for pediatric psoriasis and atopic dermatitis. Materials and Methods: A PubMed review of biological treatments for pediatric psoriasis and atopic dermatitis with information regarding age, efficacy, dosing, contra-indications, adverse events, and off-label treatments. Results: Currently there are three European Medicines Agency (EMA)-approved biological treatment options for pediatric psoriasis: etanercept, ustekinumab, and adalimumab. While dupilumab was recently Food and Drug Administration (FDA)- and EMA-approved for adult atopic dermatitis, it is still not yet approved for pediatric atopic dermatitis. Conclusions: Given the high morbidity associated with pediatric atopic dermatitis and psoriasis, there is a need for more treatment options. Further research and post-marketing registries are needed to extend the use of biologics into pediatric patients.

What is the role of Adamts18 in hormone receptor signaling?

Adamts18 links luminal hormone receptor signaling to basement membrane remodeling and stem cell activation.

Is belimumab effective for the lupus nephritis?

Yes, belimumab appears to effective for the lupus nephritis.

We report the case of a 19-year-old woman with progressive proliferative lupus nephritis (LN) class III after induction and maintece therapy with mycophenolate mofetil (MMF). Despite a satisfying clinical improvement proteinuria progressed under this medication. We treated the patient with additional belimumab after discussing other options. Following treatment with belimumab, proteinuria rapidly improved to almost normal levels and clinical remission lasted. Belimumab might hold promise for this indication. Recently introduced into the market, belimumab (Benlysta) is a monoclonal antibody that has potential clinically efficacious applications for the treatment of lupus nephritis. Lupus nephritis is a major complication of systemic lupus erythematosus (SLE) that can lead to significant illness or even death without proper intervention and treatment. With vast implications through a novel mechanism, belimumab offers a new standard of treatment for physicians in the complications associated with SLE, specifically lupus nephritis. By targeting B cell signaling and maturation, belimumab is able to mitigate the underlying pathological complications surrounding SLE. Phase 3 clinical trials with belimumab have depicted clinically efficacious applications, suggesting belimumab as a revolutionary breakthrough in the treatment armamentarium for practicing clinicians. This article explains the precise mechanism of action of belimumab on the soluble protein BlyS that plays a major role in the pathogenesis of lupus nephritis. In addition, the extensive pharmacokinetics and clinical implications are exemplified in this review with belimumab's comparison with standard therapeutic guidelines for the treatment of lupus nephritis. BACKGROUND: The treatment of Lupus Nephritis (LN) is an unmet need in the management of patients with Systemic Lupus Erythematosus (SLE). CASE PRESENTATION : We report two cases of women affected by Lupus Nephritis (LN) ISN/RNP Class IV with serological active disease, high disease activity and marked fatigue. In both cases, Mycophenolate mofetil (MMF), as induction therapy, was poorly tolerated because of gastrointestinal toxicity. Belimumab, together with low-doses of MMF, was effective as induction treatment leading to early achievement of complete renal response in these two selected cases of LN. CONCLUSIONS: We also report a literature review concerning the efficacy and safety of Belimumab in Lupus Nephritis. Further studies are needed to evaluate the use of Belimumab to manage the renal involvement in patients with Systemic Lupus Erythematosus, waiting for the results of ongoing randomized clinical trials. Background and Objectives: Belimumab (BEL) is a monoclonal antibody approved for the treatment of active systemic lupus erythematosus (SLE) but not for lupus nephritis (LN) and neuropsychiatric systemic lupus erythematosus (NPSLE). We aimed to assess BEL's effects on these severe, potentially life-threatening manifestations. Methods: Retrospective observational cohort study using routine clinical data in a case series of patients with SLE receiving BEL. Results: Sixteen patients received BEL therapy for active SLE. Nine were excluded because they had no LN or NPSLE. Six suffered from LN, and one patient had NPSLE. All LN patients received BEL in addition to standard therapy including glucocorticoids, hydroxychloroquine, and mycophenolate mofetil in five cases, and tacrolimus in one case. Three patients with proteinuria >1,000 mg/g creatinine responded well (one complete, two partial renal responses); all other patients had decreasing proteinuria and a reduction in anti-dsDNA levels. The patient with NPSLE who had failed previous therapies had persistent clinical improvement of cutaneous and neuropsychiatric manifestations. There was one mild allergic reaction and one lower respiratory tract infection, but no other adverse events. One patient discontinued therapy due to a lack of improvement in clinical symptoms, another because of clinical remission. Conclusions: In our series, BEL led to a decrease of proteinuria in patients with proteinuria of more than 1,000 mg/g creatinine despite standard of care treatment, and led to a marked clinical improvement in one patient with NPSLE. No adverse events were observed. Routinely administered BEL shows clinical efficacy on non-approved manifestations, but careful patient selection is warranted. BACKGROUND: Anti-CD20 B-cell depletion has not shown superior efficacy to standard immunosuppression in patients with systemic lupus erythematosus (SLE). Besides trial design, potential explanations are incomplete B-cell depletion in relation to substantial surges in B-cell-activating factor (BAFF). To improve B-cell targeting strategies, we conducted the first study in SLE patients aimed at investigating immunological effects and feasibility of combining rituximab (RTX; anti-CD20) and belimumab (BLM; anti-BAFF). METHODS: Reported is the long-term follow-up of a Phase 2 proof-of-concept study in 15 patients with SLE including 12 (80%) with lupus nephritis (LN). RESULTS: In 10/15 (67%) patients, a clinical response was observed by achievement of lupus low disease activity state, of which 8 (53%) continued treatment (BLM + ≤7.5 mg prednisolone) for the complete 2 years of follow-up. Five patients (33%) were referred to as 'non-responders' due to persistent LN, major flare or repetitive minor flares. Out of 12 LN patients, 9 (75%) showed a renal response including 8 (67%) complete renal responders. All anti-dsDNA+ patients converted to negative, and both anti-C1q and extractable nuclear antigen autoantibodies showed significant reductions. CD19+ B cells showed a median decrease from baseline of 97% at 24 weeks, with a persistent reduction of 84% up to 104 weeks. When comparing responders with non-responders, CD20+ B cells were depleted significantly less in non-responders and double-negative (DN) B cells repopulated significantly earlier. CONCLUSIONS: Combined B-cell targeted therapy with RTX and BLM prevented full B-cell repopulation including DN B cells, with concomitant specific reduction of SLE-relevant autoantibodies. The observed immunological and clinical benefits in a therapy-refractory SLE population prompt further studies on RTX + BLM. BACKGROUND: In adults with active lupus nephritis, the efficacy and safety of intravenous belimumab as compared with placebo, when added to standard therapy (mycophenolate mofetil or cyclophosphamide-azathioprine), are unknown. METHODS: In a phase 3, multinational, multicenter, randomized, double-blind, placebo-controlled, 104-week trial conducted at 107 sites in 21 countries, we assigned adults with biopsy-proven, active lupus nephritis in a 1:1 ratio to receive intravenous belimumab (at a dose of 10 mg per kilogram of body weight) or matching placebo, in addition to standard therapy. The primary end point at week 104 was a primary efficacy renal response (a ratio of urinary protein to creatinine of ≤0.7, an estimated glomerular filtration rate [eGFR] that was no worse than 20% below the value before the renal flare (pre-flare value) or ≥60 ml per minute per 1.73 m2 of body-surface area, and no use of rescue therapy), and the major secondary end point was a complete renal response (a ratio of urinary protein to creatinine of <0.5, an eGFR that was no worse than 10% below the pre-flare value or ≥90 ml per minute per 1.73 m2, and no use of rescue therapy). The time to a renal-related event or death was assessed. RESULTS: A total of 448 patients underwent randomization (224 to the belimumab group and 224 to the placebo group). At week 104, significantly more patients in the belimumab group than in the placebo group had a primary efficacy renal response (43% vs. 32%; odds ratio, 1.6; 95% confidence interval [CI], 1.0 to 2.3; P = 0.03) and a complete renal response (30% vs. 20%; odds ratio, 1.7; 95% CI, 1.1 to 2.7; P = 0.02). The risk of a renal-related event or death was lower among patients who received belimumab than among those who received placebo (hazard ratio, 0.51; 95% CI, 0.34 to 0.77; P = 0.001). The safety profile of belimumab was consistent with that in previous trials. CONCLUSIONS: In this trial involving patients with active lupus nephritis, more patients who received belimumab plus standard therapy had a primary efficacy renal response than those who received standard therapy alone. (Funded by GlaxoSmithKline; BLISS-LN ClinicalTrials.gov number, NCT01639339.). PURPOSE OF REVIEW: Despite ground-breaking innovations for most autoimmune diseases, the treatment of lupus nephritis has remained largely the same for decades because none of the tested drugs demonstrated superiority over standard-of-care in randomized controlled clinical trials. RECENT FINDINGS: Recently, the Belimumab in Subjects with Systemic Lupus Erythematosus - Lupus Nephritis trial tested belimumab, an inhibitor of B-cell activating factor, as an add-on therapy to steroids and either mycophenolate mofetil (MMF) or cyclophosphamide when given IV monthly over a period of 104 weeks at an effect size of 11% for a Primary Efficacy Renal Response. The NOBILITY trial reported positive results for the B-cell-depleting agent obinutuzumab as an add-on therapy to steroids and MMF when given IV every 6 months over a period of 76 weeks at an effect size of 22% for a complete renal response (CRR). The AURORA trial reported positive results for the calcineurin inhibitor voclosporin as an oral add-on therapy to low dose steroids and MMF when given twice daily over a period of 52 weeks at an effect size of 18.5% for a CRR. SUMMARY: These studies will change the treatment landscape of lupus nephritis. In which way is discussed in this article.

Which are the lactate isomers?

Lactate contains a chiral carbon and thus has two optical isomers-d-lactate and l-lactate.

Lactate contains a chiral carbon and thus has two optical isomers-d-lactate and l-lactate. l-Lactate is the predomit form that is produced by the body and can be delivered to the organs. On the other hand, gut microbiota produce both isomers, which can then flow into the body. Although both d-lactate and l-lactate can contribute to energy metabolism, their potential roles in adipocyte differentiation remain to be elucidated. Here, we investigated the effects of l-lactate and d-lactate on the differentiation of 3T3-L1 preadipocytes. Both lactate etiomers were demonstrated to enhance triglyceride accumulation by stimulating the early phase of adipocyte differentiation. Notably, d-lactate was more potent than l-lactate in inducing triglyceride accumulation. The degree of triglyceride accumulation induced by l-lactate was similar to that induced by pyruvate. d-Lactate was more potent than l-lactate in increasing the activity of glycerol-3-phosphate dehydrogenase. Both lactate etiomers did not affect cell viability. Moreover, both etiomers upregulated the expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, sterol regulatory element-binding protein-1c, and fatty acid synthase, with d-lactate exerting stronger effects than l-lactate. By contrast, lactate did not influence the expression of C/EBPβ and C/EBPδ. d-Lactate significantly increased and l-lactate tended to increase p38 MAPK phosphorylation, and the p38 MAPK inhibitor SB203580 inhibited the stimulation of adipocyte differentiation by d-lactate and l-lactate. These findings showed that both lactate etiomers stimulate preadipocyte differentiation, with d-lactate showing more potent effects than l-lactate. In addition, our study demonstrated that d-lactate and l-lactate exert different effects on physiological events.

What is the function of the Eyeless associated gene in Drosophila?

Eyeless (ey) also known as Pax6, is one of the most critical transcription factors for initiating the entire eye development in Drosophila.

The Drosophila gene eyeless (ey) encodes a transcription factor with both a paired domain and a homeodomain. It is homologous to the mouse Small eye (Pax-6) gene and to the Aniridia gene in humans. These genes share extensive sequence identity, the position of three intron splice sites is conserved, and these genes are expressed similarly in the developing nervous system and in the eye during morphogenesis. Loss-of-function mutations in both the insect and in the mammalian genes have been shown to lead to a reduction or absence of eye structures, which suggests that ey functions in eye morphogenesis. By targeted expression of the ey complementary DNA in various imaginal disc primordia of Drosophila, ectopic eye structures were induced on the wings, the legs, and on the antennae. The ectopic eyes appeared morphologically normal and consisted of groups of fully differentiated ommatidia with a complete set of photoreceptor cells. These results support the proposition that ey is the master control gene for eye morphogenesis. Because homologous genes are present in vertebrates, ascidians, insects, cephalopods, and nemerteans, ey may function as a master control gene throughout the metazoa. Development of the vertebrate eye requires a series of steps including specification of the anterior neural plate, evagination of the optic vesicles from the ventral forebrain, and the cellular differentiation of the lens and retina. Homeobox-containing genes, especially the transcription regulator Pax6, play a critical role in vertebrate and invertebrate eye formation. Mutations in Pax6 function result in eye malformations known as Aniridia in humans and Small eye syndrome in mice. The Drosophila homologue of Pax6, eyeless, is also necessary for correct invertebrate eye development, and its misexpression leads to formation of ectopic eyes in Drosophila. Here we show that a conserved vertebrate homeobox gene, Rx, is essential for normal eye development, and that its misexpression has profound effects on eye morphology. Xenopus embryos injected with synthetic Rx RNA develop ectopic retinal tissue and display hyperproliferation in the neuroretina. Mouse embryos carrying a null allele of this gene do not form optic cups and so do not develop eyes. The Rx gene family plays an important role in the establishment and/or proliferation of retinal progenitor cells. The development of the Drosophila compound eye requires the function of a set of evolutionarily conserved genes. Among these, the Drosophila Pax-6 gene eyeless (ey) plays a major role. ey has been considered a master control gene of eye development in the animal kingdom because targeted expression of ey and vertebrate as well as invertebrate homologs lead to the formation of ectopic eyes in Drosophila. We demonstrate that an intron of the ey gene contains an enhancer that regulates the eye specific expression of the gene in the eye disc primordia of embryos and in the eye imaginal discs of third instar larvae. Moreover, a 212-bp enhancer element is necessary and sufficient for the enhancer function. It is partially conserved in Drosophila hydei and contains putative Pax-6 Paired domain binding sites. We show that several binding sites are required for the eye specific expression, and, therefore, we propose a Pax-6-like molecule to be a positive transactivator for the eye specific ey expression. This transactivator recently has been identified as twin of eyeless, the second Pax-6 gene in Drosophila. We describe here the role of the transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and EGFR signaling in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobe primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwig's organ cells that we call Bolwig's organ founders. Phenotypic analysis, using specific markers for Bolwig's organ and the optic lobe, of tll loss- and gain-of-function mutant embryos reveals that tll functions to drive cells to optic lobe as opposed to Bolwig's organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwig's organ fate. Since we can show that tll and ato do not regulate each other, we propose a model wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwig's organ pioneers. Our data further suggest that the Bolwig's organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwig's organ cells where it activates the EGFR signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive, as we were unable to find evidence for regulation by the 'early eye genes' so, eya and ey, or by EGFR signaling. A role for the Pax-6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax-6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed. We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain. Pax-6 genes, known to be essential for eye development, encode an evolutionarily conserved transcription factor with two DNA-binding domains. To corroborate the contribution of each DNA-binding domain to eye formation, we generated truncated forms of the Drosophila Pax-6 gene eyeless and tested their capacity to rescue the ey(2) mutant. Surprisingly, EY deleted of the homeodomain rescued the ey(2) mutant and triggered ectopic eyes morphogenesis. In contrast, EY lacking the paired domain failed to rescue the ey(2) mutant, led to truncation of appendages, and repressed Distal-less when misexpressed. This result suggests distinct functions mediated differentially by the two DNA-binding domains of eyeless. In 1995, the eyeless (ey) gene was dubbed the "master-regulator" of eye development in Drosophila. Not only is ey required for eye development, but its misexpression can convert many other tissues into eye, including legs, wings and antennae.(1) ey is remarkable for its ability to drive coordinate differentiation of the multiple cell types that have to differentiate in a very precise pattern to construct the fly eye, and for its power to override the previous differentiation programs of many other diverse tissues. Even more remarkable, the ey homolog Pax6 and homologs of other eye determination genes from Drosophila are also required for eye development in vertebrates,(2,3) prompting reassessment of the evolution of vision throughout the animal kingdom.(4,5) Now Kumar and Moses have published a study that throws a new light on ey function in Drosophila.(6) According to their work, ey becomes a master regulator of eye development much later than previously thought, and is regulated by signalling through the Notch and EGFR signaling pathways. Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. We examined the expression patterns of four eye specification genes, eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac) throughout all time points of embryogenesis and show that only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. We also compare the expression of Ey with that of a putative antennal specifying gene Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field. The Drosophila compound eye is specified by the simultaneous and interdependent activity of transcriptional regulatory genes from four families: PAX6 (eyeless, twin of eyeless, eyegone), EYA (eyes absent), SIX (sine oculis, Optix) and DACH (dachshund). Mammals have homologues of all these genes, and many of them are expressed in the embryonic or adult eye, but the functional relationships between them are currently much less clear than in Drosophila. Nevertheless, mutations in the mammalian genes highlight their requirement both within and outside the eye in embryos and adults, and emphasize that they can be deployed in many different contexts. We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy. Loss of Pax 6 function leads to an eyeless phenotype in both mammals and insects, and ectopic expression of both the Drosophila and the mouse gene leads to the induction of ectopic eyes in Drosophila, which suggested to us that Pax 6 might be a universal master control gene for eye morphogenesis. Here, we report the reciprocal experiment in which the RNAs of the Drosophila Pax 6 homologs, eyeless and twin of eyeless, are transferred into a vertebrate embryo; i.e., early Xenopus embryos at the 2- and 16-cell stages. In both cases, ectopic eye structures are formed. To understand the genetic program specifying eye morphogenesis, we have analyzed the regulatory mechanisms of Pax 6 expression that initiates eye development. Previously, we have demonstrated that Notch signaling regulates the expression of eyeless and twin of eyeless in Drosophila. Here, we show that in Xenopus, activation of Notch signaling also induces eye-related gene expression, including Pax 6, in isolated animal caps. In Xenopus embryos, the activation of Notch signaling causes eye duplications and proximal eye defects, which are also induced by overexpression of eyeless and twin of eyeless. These findings indicate that the gene regulatory cascade is similar in vertebrates and invertebrates. The two Pax6 gene homologs eyeless and twin of eyeless play decisive early roles in Drosophila eye development. Strong mutants of twin of eyeless or of eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs. The activity of these genes is crucial at the very beginning of eye-antennal development in the primordia of eye-antennal discs when eyeless is first activated by the twin of eyeless gene product. This activation does not strictly depend on the Twin of eyeless protein, but is temperature-dependent in its absence. Twin of eyeless acts also in parallel to the eyeless gene and exerts functions that are partially redundant with those of Eyeless, while Eyeless is mainly required to prevent early cell death and promote eye development in eye-antennal discs. eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene. Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila, two Pax6 genes function in a pathway in which twin of eyeless (toy) directly regulates eyeless (ey), which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, we used different truncated forms of toy and small eyes (sey), and tested their capacity to induce ectopic eye development in an ey-independent manner. Even though the Paired domains of TOY and SEY have DNA-binding properties that differ from those of the Paired domain of EY, they all are capable of inducing ectopic eye development in an ey mutant background. We also show that one of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, we show that only the homeodomain (HD) of EY is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of TOY and EY, and significant evolutionary divergence between the two Drosophila Pax6 genes. Pax genes encode DNA binding proteins that play pivotal roles in the determination of complex tissues. Members of one subclass, Pax6, function as selector genes and play key roles in the retinal development of all seeing animals. Mutations within the Pax6 homologs including fly eyeless, mouse Small eye and human Pax6 lead to severe retinal defects in their respective systems. In Drosophila eyeless and twin of eyeless, play non-redundant roles in the developing retina. One particularly interesting characteristic of these genes is that, although expression of either gene can induce ectopic eye formation in non-retinal tissues, there are differences in the location and frequencies at which the eyes develop. eyeless induces much larger ectopic eyes, at higher frequencies, and in a broader range of tissues than twin of eyeless. In this report we describe a series of experiments conducted in both yeast and flies that has identified protein modules that are responsible for the differences in tissue transformation. These domains appear to contain transcriptional activator and repressor activity of distinct strengths. We propose a model in which the selective presence of these activities and their relative strengths accounts, in part, for the disparity to which ectopic eyes are induced in response to the forced expression of eyeless and twin of eyeless. The identification of both transcriptional activator and repressor activity within the Pax6 protein furthers our understanding of how this gene family regulates tissue determination. Pax6 genes encode evolutionarily highly conserved transcription factors that are required for eye and brain development. Despite the characterization of mutations in Pax6 homologs in a range of organisms, and despite functional studies, it remains unclear what the relative importance is of the various parts of the Pax6 protein. To address this, we have studied the Drosophila Pax6 homolog eyeless. Specifically, we have generated new eyeless alleles, each with single missense mutations in one of the four domains of the protein. We show that these alleles result in abnormal eye and brain development while maintaining the OK107 eyeless GAL4 activity from which they were derived. We performed in vivo functional rescue experiments by expressing in an eyeless-specific pattern Eyeless proteins in which either the paired domain, the homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain phenotypes was only observed when full-length Eyeless was expressed, while all deletion constructs failed to rescue. These data, along with the phenotypes observed in the four newly characterized eyeless alleles, demonstrate the requirement for an intact Eyeless protein for normal Drosophila eye and brain development. They also suggest that some endogenous functions may be obscured in ectopic expression experiments. Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the "retinal determination gene network," composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development. Pax6 transcription factors are essential upstream regulators in the developing anterior brain and peripheral visual system of most bilaterian animals. While a single homolog is in charge of these functions in vertebrates, two Pax6 genes are in Drosophila: eyeless (ey) and twin of eyeless (toy). At first glance, their co-existence seems sufficiently explained by their differential involvement in the specification of two types of insect visual organs: the lateral compound eyes (ey) and the dorsal ocelli (toy). Less straightforward to understand, however, is their genetic redundancy in promoting defined early and late growth phases of the precursor tissue to these organs: the eye-antennal imaginal disc. Drawing on comparative sequence, expression, and gene function evidence, I here conclude that this gene regulatory network module dates back to the dawn of arthropod evolution, securing the embryonic development of the ocular head segment. Thus, ey and toy constitute a paradigm to explore the organization and functional significance of longterm conserved genetic redundancy of duplicated genes. Indeed, as first steps in this direction, recent studies uncovered the shared use of binding sites in shared enhancers of target genes that are under redundant (string) and, strikingly, even subfunctionalized control by ey and toy (atonal). Equally significant, the evolutionarily recent and paralog-specific function of ey to repress the transcription of the antenna fate regulator Distal-less offers a functionally and phylogenetically well-defined opportunity to study the reconciliation of shared, partitioned, and newly acquired functions in a duplicated developmental gene pair. Eyeless (ey) is one of the most critical transcription factors for initiating the entire eye development in Drosophila. However, the molecular mechanisms through which Ey regulates target genes and pathways have not been characterized at the genomic level. Using ChIP-Seq, we generated an endogenous Ey-binding profile in Drosophila developing eyes. We found that Ey binding occurred more frequently at promoter compared to non-promoter regions. Ey promoter binding was correlated with the active transcription of genes involved in development and transcription regulation. An integrative analysis revealed that Ey directly regulated a broad and highly connected genetic network, including many essential patterning pathways, and known and novel eye genes. Interestingly, we observed that Ey could target multiple components of the same pathway, which might enhance its control of these pathways during eye development. In addition to protein-coding genes, we discovered Ey also targeted non-coding RNAs, which represents a new regulatory mechanism employed by Ey. These findings suggest that Ey could use multiple molecular mechanisms to regulate target gene expression and pathway function, which might enable Ey to exhibit a greater flexibility in controlling different processes during eye development. The insect central complex (CX) is a conserved brain region containing 60 + neuronal subtypes, several of which contribute to navigation. It is not known how CX neuronal diversity is generated or how developmental origin of subtypes relates to function. We mapped the developmental origin of four key CX subtypes and found that neurons with similar origin have similar axon/dendrite targeting. Moreover, we found that the temporal transcription factor (TTF) Eyeless/Pax6 regulates the development of two recurrently-connected CX subtypes: Eyeless loss simultaneously produces ectopic P-EN neurons with normal axon/dendrite projections, and reduces the number of E-PG neurons. Furthermore, transient loss of Eyeless during development impairs adult flies' capacity to perform celestial navigation. We conclude that neurons with similar developmental origin have similar connectivity, that Eyeless maintains equal E-PG and P-EN neuron number, and that Eyeless is required for the development of circuits that control adult navigation.

Are enhancers directional in their targeting of gene promoters?

Promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. Most enhancers are able to regulate promoters on either side.

In cell lines established from Marek's disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek's Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.

Has dupilumab been FDA approved for atopic dermatitis?

Yes, dupilumab has been approved by FDA for atopic dermatitis.

Atopic dermatitis (AD) is a chronic, inflammatory skin disease characterized by pruritus, inflammatory erythematous skin lesions, and skin-barrier defect. Current mainstay treatments of emollients, steroids, calcineurin inhibitors, and immunosuppressants have limited efficacy and potentially serious side effects. Recent advances and understanding of the pathogenesis of AD have resulted in new therapies that target specific pathways with increased efficacy and the potential for less systemic side effects. New FDA-approved therapies for AD are crisaborole and dupilumab. The JAK-STAT inhibitors (baricitinib, upadacitinib, PF-04965842, ASN002, tofacitinib, ruxolitinib, and delgocitinib) have the most promising results of the emerging therapies. Other drugs with potential include the aryl hydrocarbon receptor modulating agent tapinarof, the IL-4/IL-13 antagonists lebrikizumab and tralokinumab, and the IL-31Rα antagonist nemolizumab. In this review, new and emerging AD therapies will be discussed along with their mechanisms of action and their potential based on clinical study data. Dupilumab inhibits the interleukin-4 receptor subunit α and is FDA approved for treatment of moderate-to-severe atopic dermatitis. It is a relatively new drug, and whether it is efficacious for other diseases in dermatology is an area of increasing interest. We searched the literature and ClinicalTrials.gov database for uses of dupilumab beyond atopic dermatitis in dermatology and for ongoing studies on new uses for dupilumab. Off-label reports identified described use of dupilumab for several different dermatologic conditions, including allergic contact dermatitis, hand dermatitis, chronic spontaneous urticaria, prurigo nodularis, and alopecia areata. Overall, there is limited but promising data for dupilumab use beyond atopic dermatitis in dermatology. The relatively safe adverse effect profile of dupilumab may make it an option for certain recalcitrant diseases in dermatology, but further studies will be needed to assess its efficacy and determine its best possible use. J Drugs Dermatol. 2019;18(10):1053-1055. Dupilumab is the first US FDA approved biologic for treatment of atopic dermatitis. It is a human monoclonal antibody which blocks the shared receptor component, the interleukin (IL)-4α subunit, of IL-4 and IL-13 signaling pathways. Occurrence of "conjunctivitis", mostly in atopic dermatitis trials, has been the main side effect reported thus far. The etiology of "conjunctivitis" associated with dupilumab treatment is unclear and might be similar to atopic keratoconjunctivitis. There is evidence in the published literature that unlike the Th2-like profile in vernal keratoconjunctivitis, Th1-mediated inflammation is predomit in atopic keratoconjunctivitis. Blocking the Th2 pathway with dupilumab therapy might result in a shift towards Th1, causing the ocular findings associated with dupilumab. In addition, blockage of IL-13 might have implications with regards to mucin production and ocular surface health. This review highlights the clinical manifestations, reviews treatment options and offers explanations for pathogenesis of this ocular surface diseases associated with dupilumab treatment.

Describe ReactomeGSA

ReactiveomeGSA is a novel resource for comparative pathway analyses of multi-omics datasets. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAt Atlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. reactomegSA greatly reduces the technical barrier for multi-CSF, cross-species, and Comparative Pathways analysis.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

What is the role of phenylbutyrate–taurursodiol for amyotrophic lateral sclerosis?

Treatment of amyotrophic lateral sclerosis patients with phenylbutyrate–taurursodiol was associated with both functional and survival benefits.

Conflict of interest statement: S. Paganoni reports grants from Amylyx Pharmaceuticals, Inc, The ALS Association, and ALS Finding A Cure during the conduct of the study, and grants from Revalesio, Ra Pharma, Biohaven, Clene Nanomedicine, and Prilenia, outside the submitted work. S. Hendrix reports other personal fees from Pentara Corporation, outside the submitted work. S.P. Dickson reports other personal fees from Pentara outside the submitted work. E.A. Macklin reports grants from Amylyx, The ALS Association, and ALS Finding A Cure Foundation during the conduct of the study. He is also a data and safety monitoring board member (DSMB) and reports grants from Acorda Therapeutics; steering committee membership for Biogen; consultant work for Cerevance; grants from GlaxoSmithKline; consultant work for Inventram, Lavin Consulting, and Myolex; grants from Mitsubishi Tanabe Pharmaceuticals; and DSMB membership for Novartis Pharmaceuticals and Shire Human Genetic Therapies, outside the submitted work. J.D. Berry reports grants from ALS Finding A Cure, The ALS Association, and Amylyx during the conduct of the study; personal fees from Biogen and Clene Nanomedicine; and grants from Alexion, Biogen, MT Pharma of America, Anelixis Therapeutics, Brainstorm Cell Therapeutics, Genentech, nQ Medical, National Institute of Neurological Disorders and Stroke, and the Muscular Dystrophy Association, outside the submitted work. M.A. Elliott has received personal fees from Amylyx and personal fees from Biogen. C. Karam reports grants and personal fees from Akcea, Alnylam, and Genzyme, and personal fees from Acceleron, Biogen, Alexion, Argenx, Cytokinetics, and CSL Behring, outside the submitted work. J.B. Caress reports grants from Amylyx during the conduct of the study, and grants from Orion Pharmaceuticals, MTB Pharma, and Cytokinetics, outside the submitted work. J. Wymer reports grants from Amylyx during the conduct of the study. S.A. Goutman reports grants from The ALS Association during the conduct of the study; grants from the National Institutes of Health/National Institutes of Environmental Health Sciences, The ALS Association, and Target ALS, outside the submitted work; consultant work for Biogen and ITF Pharma, outside the submitted work; and personal fees from Biogen, ITF Pharma, Watermark Research Partners, and for expert testimony, outside the submitted work. T.D. Heiman‐Patterson reports grants from Mitsubishi Tanabe Pharma America, Amylyx Pharmaceuticals, The ALS Association, and Orion Pharma, and personal fees from Cytokinetics, ITF, and Biohaven, outside the submitted work. C.E. Jackson reports grants from Amylyx during the conduct of the study; grants and personal fees from Cytokinetics, personal fees from CSL Behring, grants and personal fees from Mitsubishi Tanabe Pharma America, DSMB membership, and personal fees from Brainstorm and Mallinckrodt during the conduct of this study; and personal fees from ITF Pharma, outside the submitted work. C. Quinn received personal fees for serving on an advisory board of Amylyx, outside the submitted work. J.D. Rothstein reports licensing agreement and nonficial support from Ionis Pharmaceuticals; nonficial support from Calico, Biogen, and IBM Watson; research grant support from the National Institute of Neurological Disorders and Stroke, National Institute on Aging, Department of Defense, the Chan Zuckerberg Initiative, Microsoft, The ALS Association, the Muscular Dystrophy Association, Target ALS, F Prime, ALS Finding A Cure, Answer ALS, Robert Packard Center for ALS Research, GlaxoSmithKline, Travelers Insurance, American Airlines, Caterpillar, and the National Football League; and personal consulting fees from Expansion Therapeutics and Team Gleason. He also reports that his institution was a trial site and thus had a contract with Amylyx to participate in the study. J. Katz reports personal fees from MT Pharma America, Denali Pharmaceuticals, Genentech, and Calico, outside the submitted work. S. Ladha reports grants from Amylyx, Biogen, and MT Pharma, and personal fees from Amylyx and Biogen. T.M. Miller reports licensing agreement and nonficial support from Ionis Pharmaceuticals, a licensing agreement with C2N, grants and personal fees from Biogen, and personal fees from Cytokinetics and Disarm Therapeutics, outside the submitted work. S.N. Scelsa reports grants from Amylyx during the conduct of the study, and grants from Orion Pharma, outside the submitted work. T.H. Vu reports personal fees from the speakers bureau of Mitsubishi Tanabe Pharmaceuticals, and has participated in clinical trials sponsored by Amylyx, Orion, Biogen, Mallinckrodt, and Cytokinetics during the conduct of the study. J.D. Glass reports that his institution was a trial site and thus had a contract from Amylyx to participate in the study. A. Swenson reports research support from Amylyx, The ALS Association, Massachusetts General Hospital, the National Institutes of Health/National Institute of Neurological Disorders and Stroke, and serving on an independent data monitoring committee for Alexion. P.L. Andres reports personal fees from Amylyx for consulting during the conduct of the study and has an isometric strength testing apparatus (US Patent 7493812B2) held by the Hospital Corporation. S. Babu reports research support from the American Academy of Neurology, the AANEM Foundation, The ALS Association, the Muscular Dystrophy Association, Biogen, Orion, Voyager Therapeutics, and Novartis. M. Chase reports grants to the Massachusetts General Hospital (MGH) from The ALS Association, grants to the MGH from ALS Finding A Cure, and fee for service from Amylyx during the conduct of the study. M. Hall reports grants for funding for clinical trial monitoring and outcomes training support from The ALS Association and Amylyx during the conduct of the study. G. Kittle reports grants from The ALS Association and Amylyx during the conduct of the study. J.M. Shefner reports grants and personal fees from Amylyx during the conduct of the study; personal fees for consulting work from Cytokinetics and Brainstorm; grants and personal fees for outcomes training and study design from Mitsubishi Pharma America, outside the submitted work; personal fees for consulting from Neurosense and Otsuka, outside the submitted work; and grants for outcomes training from Alexion, Medicinova, and Biogen, outside the submitted work. He is also Neuromuscular section editor for UpToDate. J. Wittes and Z.‐F. Yu report payments from Amylyx to their employer during the conduct of the study. J. Cohen and J. Klee report a relationship with Amylyx during the conduct of the study, and they serve as co‐CEOs of Amylyx, outside the submitted work, with multiple patents issued to Amylyx. K. Leslie reports being full‐time employee of Amylyx during the conduct of the study, and personal fees from Amylyx, outside the submitted work. R.E. Tanzi reports personal fees from Amylyx outside the submitted work; has helped with inception and design of the clinical trial but was not involved with running the trial and had no contact with the trial subjects; and owns founding equity in Amylyx and serves as head of the company's scientific advisory board. W. Gilbert was director of Amylyx during the conduct of the study and a company shareholder. P.D. Yeramian reports full‐time employment at Amylyx during the conduct of the study. D. Schoenfeld reports grants from The ALS Association, during the conduct of the study, and personal fees from Immunitypharma and Alexion, outside the submitted work. M.E. Cudkowicz reports grants from Massachusetts General Hospital during the conduct of the study; grants from Clene Nanomedicine, Ra Pharma, Biohaven, and Prilenia, outside the submitted work; and personal fees from Takeda, Biogen, Sunovian, Cytokinetics, and Immunity Pharma, outside the submitted work. The remaining authors declare no potential conflicts of interest.

What is known about growth arrest-specific 6 protein?

Growth arrest-specific 6 (Gas6) is a vitamin K-dependent protein secreted by immune cells, endothelial cells, vascular smooth muscle cells, and adipocytes. Recent studies indicate that Gas6 and receptors of the TAM (Tyro3, Axl, and Mer) family may be involved in the pathogenesis of obesity, systemic inflammation, and insulin resistance.

BACKGROUND: Growth arrest-specific 6 (Gas6) is a vitamin K-dependent protein secreted by immune cells, endothelial cells, vascular smooth muscle cells, and adipocytes. Recent studies indicate that Gas6 and receptors of the TAM (Tyro3, Axl, and Mer) family may be involved in the pathogenesis of obesity, systemic inflammation, and insulin resistance. The aim of this study was to investigate the association between plasma Gas6 protein and the c.843 + 7G>A Gas6 polymorphism in metabolic syndrome (MetS). METHODS: Two hundred five adults (88 men and 117 women) were recruited in this study. Plasma Gas6 concentration, general, and biochemical data were measured. All subjects were genotyped for the c.843 + 7G>A Gas6 polymorphism. RESULTS: Plasma Gas6 concentrations decreased in parallel with various MetS components in all groups (P = 0.017 for trend). Patients in the second and third tertiles of Gas6 level had higher high-density lipoprotein cholesterol (HDL-C) levels than those in the first tertile overall and in the female group. Plasma Gas6 levels were significantly positively correlated with HDL-C level and negatively with fasting glucose level in the female patients. The A allele and genotype AA in single nucleotide polymorphism c.843 + 7G>A were less frequent in the subjects with MetS compared to those without MetS. CONCLUSIONS: Our results demonstrated a positive correlation between Gas6 protein values and HDL-C and reinforce the association with fasting glucose. In addition, the presence of c.843 + 7G>A Gas6 polymorphisms, especially the AA genotype, had an association with MetS. The potential role of the Gas6/TAM system in MetS deserves further investigation. AIMS/INTRODUCTION: Obesity is characterized by disturbed adipocytokine expression and insulin resistance in adipocytes. Growth arrest-specific 6 (GAS6) is a gene encoding the Gas6 protein, which is expressed in fibroblasts, and its related signaling might be associated with adipose tissue inflammation, glucose intolerance and insulin resistance. The aim of this study was to investigate the associations among Gas6, adipocytokines and insulin resistance in adipocytes. MATERIALS AND METHODS: Mature Simpson Golabi Behmel Syndrome adipocytes were treated with high levels of insulin to mimic insulin resistance, and were examined for the expressions of Gas6, cytokines and adipocytokines from preadipocytes in differentiation. In an animal study, high-fat diet-induced obese mice were used to verify the Gas6 expression in vitro. RESULTS: During the differentiation of adipocytes, the expression of Gas6 gradually decreased, and was obviously downregulated with adipocyte inflammation and insulin resistance. Gas6 levels were found to be in proportion to the expression of adiponectin, which has been regarded as closely relevant to improved insulin sensitivity after metformin treatment. Similar results were also confirmed in the animal study. CONCLUSIONS: Our results suggest that Gas6 might modulate the expression of adiponectin, and might therefore be associated with insulin resistance in adipose tissues.

What is Leptomeningeal disease?

Neoplastic leptomeningeal disease (LMD) represents infiltration of the leptomeninges by tumor cells.

What percent of Rheumatoid Arthritis (RA) patients are not responding to anti-TNF therapy?

These therapies are, however, expensive and 30% of patients fail to respond.

BACKGROUND: Anti-tumour necrosis factor-alpha (TNF-alpha) therapies represent an important advancement in therapy for rheumatoid arthritis (RA). However, there remains a proportion of patients who do not improve despite therapy. These drugs are expensive and have the potential of serious toxicity. Therefore, it would be ideal to predict the patients who will respond, so that the use of these drugs can be targetted. OBJECTIVE: To identify the clinical factors present at the start of anti-TNF-alpha therapy that are associated with response at 6 months in patients with RA. METHODS: The British Society for Rheumatology (BSR) Biologics Register collects detailed data on all patients with a rheumatic disease receiving biologic therapy in the UK. We studied all patients with RA who had started etanercept (ETA) or infliximab (INF) and had achieved a minimum 6 months follow-up by 1 October, 2004. The disease status at the baseline and at 6 months was assessed using the Disease Activity Score (DAS28). The response was classified according to the European League against Rheumatism (EULAR) improvement criteria. The effect of baseline characteristics on response was studied using multivariate ordinal logistic regression. RESULTS: 2879 patients were included in this analysis (1267 ETA, 1612 INF). At the start of therapy, the mean age was 55 yrs, disease duration 14 yrs, baseline DAS28 6.7 and health assessment questionnaire (HAQ) 2.1. In all, 28% of ETA and 86% of INF patients were receiving methotrexate. After 6 months, 18% had a good EULAR response, of whom 9% were considered to be in remission and 50% had a moderate response. There was no overall difference in response rate between the two anti-TNF-alpha therapies. A higher baseline HAQ score correlated with a lower response rate while a better response was associated with the current use of NSAIDs and the use of methotrexate (MTX), although the latter only reached statistical significance with ETA [OR 1.82 (95% CI 1.38-2.40)]. There was a lower response rate among current smokers, particularly in patients receiving INF [OR 0.77 (95% CI 0.60-0.99)]. Age, disease duration, rheumatoid factor and the previous number of disease-modifying antirheumatic drugs (DMARDs) did not predict response to either drug. However, females were less likely to achieve remission. CONCLUSIONS: These data support an improved outcome among patients receiving MTX in combination with anti-TNF-alpha therapies. However, the most disabled patients were less likely to respond, despite concurrent MTX. The benefits of NSAIDs may reflect the relative absence of comorbidities in patients who can tolerate these drugs or the continuing presence of reversible inflammatory symptoms. The association of smoking and poor outcome with INF is a novel finding and may reflect alterations in pharmacokinetics. The inability of other baseline disease characteristics to predict the outcome suggests that other factors, including potential genetic differences in drug metabolism, may be influencing the response to anti-TNF-alpha therapies. Anti-TNF treatments have given patients with rheumatoid arthritis considerable hope and relief. However, 20-30% of patients do not respond sufficiently to a given anti-TNF drug. In this situation, current strategies include switching to an alternative agent, increasing the dose of the current agent or to return to conventional DMARDs. The arrival of new biologics, which target different molecules than TNF, opens the perspective to other pathways of immunomodulation in RA. These drugs include rituximab (anti-CD20), abatacept (CTLA4Ig) and tocilizumab (anti- IL6R). Comparative studies are urgently needed to assess the efficacy of the different treatment options in order to provide the best therapeutic strategy for our patients. OBJECTIVE: To evaluate the effectiveness and safety of anti-tumor necrosis factor (anti-TNF) therapies in rheumatoid arthritis (RA), and to identify the factors involved in this response. METHODS: Dynamic prospective cohort study of patients with RA treated with anti-TNF under clinical practice conditions. Effectiveness was evaluated using Disease Activity Score (DAS) 28, European League Against Rheumatism (EULAR) response, Health Assessment Questionnaire (HAQ), and time to treatment failure. Prior adherence was evaluated retrospectively and safety was evaluated by adverse events (AE). The analysis was restricted to anti-TNF-naive patients. RESULTS: The study included 161 patients treated for RA during 6 years (60 infliximab, 79 etanercept, and 22 adalimumab). At 6 months, 15% reached a good EULAR response and 38% a moderate response. A mean decrease of -1.5 (p < 0.0001) was observed in the DAS28 and of -0.34 in the HAQ (p < 0.0001); however, women showed poorer progress in terms of DAS and HAQ. In the first year, 64.3% did not experience treatment failure and this figure was 50.5% after 2 years. In one-third, glucocorticoids were withdrawn and in the remainder the dose was reduced by 50%. Adherence to treatment, selection of etanercept, and intensification of infliximab were associated with a lower probability of premature failure in the multivariate model. AE were similar to other those in studies and no outstanding differences in safety were found between the 3 anti-TNF therapies. CONCLUSIONS: Anti-TNF treatments are effective and safe, reducing the activity of the disease, disability, and the need for corticosteroids. Patients who displayed good adherence prior to the anti-TNF treatment and were treated with etanercept or with increasing doses of infliximab had the best chance of displaying a response. OBJECTIVE: To identify the clinical factors predicting failure or a good clinical response in the cohort of RA patients entered in the Lombardy Rheumatology Network (LORHEN) registry after 3 years of treatment with anti-TNF agents. METHODS: We studied the patients who had received anti-TNF agents and been followed up for a minimum of 6 months. Disease activity at baseline and after 6 months was assessed using the DAS28, and response was evaluated according to the EULAR improvement criteria. RESULTS: 1005 patients (55.72 years) were included in the analysis. at baseline the DAS-28 was 5.91+/-0.95 and a HAQ score was 1.46+/-0.61. At mean of 14.57 months, 29.9% of the patients achieved a DAS-28 of <or=2.6 (remission). A higher RR for remission was associated with male gender (AHR 1.51, 95% CI 1.14-2.00; p: 0.004) and a lower RR for remission with: prior treatment with >3 DMARDs (AHR 0.077, 95% CI 0.58-1.03; p: 0.074), a high ESR (AHR 0.86, 95% CI 0.81-0.92; p: 0.000), Steinbrocker's functional class III/IV (AHR 0.66, 95% CI 0.48-0.90; p: 0.010), a high TJC (AHR 0.97, 95% CI 0.94-0.99; p: 0.011). A 12-month EULAR non-response was observed in 153/821 (18.6%) associated with a higher baseline HAQ score (AOR 1.51, 95% CI 1.03-2.20, p: 0.033), prior treatment with >3 DMARDs (AOR 1.76, 95% CI 1.09-2.85; p: 0.021) and corticosteroid >5 mg/day (AOR 2.05, 95% CI 1.06-3.97; p: 0.034). CONCLUSION: We found that only a minority of patients with long-standing RA treated with anti-TNF agents achieve a good clinical response or remission. Although TNF inhibitors have dramatically improved the outcome of patients with rheumatoid arthritis, 30-40% of patients do not respond well to them and treatment needs to be changed. In an effort to discriminate good and poor responders, we focused on the change in serum and synovial fluid levels of interleukin (IL-) 33 before and after treatment with TNF inhibitors. They were also measured in synovial fluids from 17 TNF inhibitor-naïve patients, and fibroblast-like synoviocytes (FLS) in-culture from 6 patients and correlated with various pro-inflammatory cytokines. Serum levels of IL-33 at 6 months after treatment decreased significantly in responders, while they did not change in non-responders. Synovial fluid levels of IL-33 in 6 patients under treatment with TNF inhibitors stayed high in 3 who were refractory and slightly elevated in 2 moderate responders, while they were undetectable in one patient under remission. Among inflammatory cytokines measured in 17 synovial fluids from TNF inhibitor-naïve patients, levels of IL-33 showed a significant positive correlation only to those of IL-1β. IL-1β increased IL-33 expression markedly in FLS in vitro, compared to TNF-α. IL-1β might be inducing RA inflammation through producing pro-inflammatory IL-33 in TNF inhibitor-hypo-responders. Sustained elevation of serum and/or synovial levels of IL-33 may account for a poor response to TNF inhibitors, although how TNF inhibitors affect the level of IL-33 remains to be elucidated. Approximately 30% of rheumatoid arthritis patients achieve inadequate response to anti-TNF biologics. Attempts to identify molecular biomarkers predicting response have met with mixed success. This may be attributable, in part, to the variable and subjective disease assessment endpoints with large placebo effects typically used to classify patient response. Sixty-one patients with active RA despite methotrexate treatment, and with MRI-documented synovitis, were randomized to receive infliximab or placebo. Blood was collected at baseline and genome-wide transcription in whole blood was measured using microarrays. The primary endpoint in this study was determined by measuring the transfer rate constant (Ktrans) of a gadolinium-based contrast agent from plasma to synovium using MRI. Secondary endpoints included repeated clinical assessments with DAS28(CRP), and assessments of osteitis and synovitis by the RAMRIS method. Infliximab showed greater decrease from baseline in DCE-MRI Ktrans of wrist and MCP at all visits compared with placebo (P<0.001). Statistical analysis was performed to identify genes associated with treatment-specific 14-week change in Ktrans. The 256 genes identified were used to derive a gene signature score by averaging their log expression within each patient. The resulting score correlated with improvement of Ktrans in infliximab-treated patients and with deterioration of Ktrans in placebo-treated subjects. Poor responders showed high expression of activated B-cell genes whereas good responders exhibited a gene expression pattern consistent with mobilization of neutrophils and monocytes and high levels of reticulated platelets. This gene signature was significantly associated with clinical response in two previously published whole blood gene expression studies using anti-TNF therapies. These data provide support for the hypothesis that anti-TNF inadequate responders comprise a distinct molecular subtype of RA characterized by differences in pre-treatment blood mRNA expression. They also highlight the importance of placebo controls and robust, objective endpoints in biomarker discovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01313520. Although anti-TNF drugs have changed the clinical course of rheumatoid arthritis (RA), survival rates and resistance-to-therapy data confirm that about 30% of RA patients fail to respond. The aim of this study was to evaluate the correlations between the development of antidrug antibodies, specific IgG4 antibodies against TNF inhibitors, and resistance to therapy in RA patients. This retrospective study involved 129 patients with established RA naïve to biological agents (98 females and 32 males, mean age 56.7±12.3 years, disease duration 6.3±1.2 years, baseline Disease Activity Score [DAS]-28 3.2-5.6) who received treatment with anti-TNF agents after the failure of conventional disease-modifying antirheumatic drugs (32 received infliximab [IFX], 58 etanercept [ETN], and 39 adalimumab [ADA]). After 6 months of treatment, the patients were classified as being in remission (DAS28 <2.6), having low disease activity (LDA; DAS28 2.6-3.2), or not responding (NR: DAS28 >3.2). The patients were also tested for serum antidrug antibodies and IgG4 antibodies against TNF inhibitors. After 24 weeks of treatment, 38% of the ETN-treated patients and 28% of those treated with ADA had injection-site reactions; the rate of systemic reactions in the IFX group was 25%. The differences among the three groups were not statistically significant (P=0.382; ETN versus ADA P=0.319). The percentages of patients with adverse events stratified by drug response were: LDA 8% and NR 18% in the ADA group; in remission 3%, LDA 22%, and NR 10% in the ETN group; and LDA 6% and NR 16% in the IFX group (P=0.051). The percentages of patients with antidrug antibodies were: ADA 33.3%, ETN 11.5%, and IFX 10.3% (P=0.025; ADA versus ETN P=0.015). The percentages of patients with IgG4 antibodies were: ADA 6%, ETN 13%, and IFX 26% (P=0.017; ADA versus ETN P=0.437). Associations between antidrug antibodies, specific IgG4 antibodies, and adverse reactions were not significant for any of the three drugs. IgG4 levels were higher in the ADA group than in the other two groups, and higher in the patients with worse DAS28 (NR) and in those experiencing adverse events. These data suggest a possible association between IgG4 levels and worse DAS28 (r (2)=5.8%, P=0.011). The presence of specific IgG4 antibodies against TNF blockers in patients with RA might affect the drugs' activity. Patients with injection-site reactions and IgG4 against ETN may show a decreased response. OBJECTIVES: Rheumatoid arthritis (RA) patients with moderate disease activity show progression of joint damage and have impaired quality of life, physical function, work and daily activities. Little is known about management of patients with moderate RA. The aim of the study was to assess the 1-year response to anti-TNF in biologic-naïve RA patients with moderate (3.2 <DAS28 ≤5.1) disease activity despite DMARD treatment, in the Italian clinical practice. METHODS: The MODERATE study is a multicentre prospective, cohort non-interventional study, conducted in 19 Italian rheumatology sites. Patients with moderate RA, diagnosed according to the 2010 American College of Rheumatology (ACR)/EULAR criteria, were enrolled if they also were aged ≥18 years, had disease onset after 16 years old, moderate disease at baseline (DAS28 score >3.2 and ≤5.1), and were naïve to anti-TNF treatment. RESULTS: Among 157 RA patients, 93 (59%) underwent etanercept, 43 (22%) adalimumab, 26 (17%) certolizumab, 10 golimumab and 2 infliximab; 80% of patients were still in treatment after 12-month observation. One-year clinical remission was achieved by 27 RA patients (21%), reduction of DAS28 score greater than 1.2 was observed in 75 (58%) patients. Moderate and good response according to EULAR criteria was observed in 59 (46%) and 45 (35%) patients, respectively. CONCLUSIONS: Results confirm the efficacy of anti-TNF alpha also in moderate RA patients, who may achieve a substantial decrease of disease activity, and improve their quality of life. The low rate of patients achieving remission may suggest that therapeutic strategies should be more timely and aggressive.

Which recombinant antibody therapeutics were granted marketing approval in 2014?

Six recombinant antibody therapeutics (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted their first marketing approvals in 2014.

The commercial pipeline of recombit antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 7 antibody therapeutics (secukinumab, evolocumab, mepolizumab, dinutuximab, nivolumab, blinatumomab, necitumumab) are undergoing a first regulatory review in the EU or US. Of the 39 novel mAbs currently in Phase 3 studies, a marketing application for one (alirocumab) may be submitted in late 2014, and marketing application submissions for at least 4 (reslizumab, ixekizumab, ocrelizumab, obiltoxaximab) are expected in 2015. Other 'antibodies to watch' are those in Phase 3 studies with estimated primary completion dates in late 2014 or 2015, which includes 13 for non-cancer indications (brodalumab, bimagrumab, bococizumab, MABp1, gevokizumab, dupilumab, sirukumab, sarilumab, tildrakizumab, guselkumab, epratuzumab, combination of actoxumab + bezlotoxumab, romosozumab) and 2 (racotumomab and clivatuzumab tetraxetan) undergoing evaluation as treatments for cancer. In addition to the novel antibody therapeutics mentioned, biosimilar infliximab and biosimilar trastuzumab are 'antibodies to watch' in 2015 because of their potential for entry into the US market and regulatory review, respectively.

What is caused by BACH2-related immunodeficiency?

Affected subjects of a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation.

Author information: (1)Lymphocyte Cell Biology Section (Molecular Immunology and Inflammation Branch), Biodata Mining and Discovery Section and Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA. (2)MRC Centre for Transplantation, King's College London, London, UK. (3)Molecular Development of the Immune System Section, NIAID Clinical Genomics Program, Biological Imaging Section (Research Technologies Branch) and Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. (4)Molecular Neuroscience, Institute of Neurology, Faculty of Brain Sciences, University College London, London, UK. (5)Department of Medicine, Imperial College London, London, UK. (6)Laboratory of Lymphocyte Signaling and Development, Babraham Institute, Cambridge, UK. (7)Translational Gastroenterology Unit, Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, UK. (8)Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK. (9)Department of Biochemistry and Department of Computer Science, Purdue University, West Lafayette, Indiana, USA. (10)Imperial BRC Genomics Facility, Hammersmith Hospital, London, UK. (11)Merck Research Laboratories, Merck &Co. Inc., Boston, Massachusetts, USA. (12)Clinical Research Directorate/CMRP, Leidos Biomedical Research Inc., NCI at Frederick, Frederick, Maryland, USA. (13)National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. (14)Department of Paediatrics, University of Oxford, Oxford, UK. (15)Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK. (16)Department of Haematology, Northern Centre for Cancer Care, Newcastle upon Tyne, UK.

Should cerebrolysin be used for aneurysmal subarachnoid hemorrhage?

No. Randomized clinical trial did not find any superior effects of cerebrolysin for patients with aneurysmal subarachnoid hemorrhage.

INTRODUCTION: Cerebrolysin is a neuroprotective drug used in the treatment of acute ischemic stroke. To our knowledge, this drug has never been evaluated in patients with aneurysmal subarachnoid hemorrhage (SAH). The aim of this study was to evaluate the effect of Cerebrolysin in patients with aneurysmal SAH. METHODS: Aneurysmal SAH patients who had their aneurysm obliterated at our institution from 2007 to 2016 were retrospectively studied. Patients received Cerebrolysin treatment or standard care only (control group). Subgroup analyses were performed according to Hunt and Hess grade (good grade ≤ 2, N = 216; poor grade ≥ 3, N = 246) and treatment procedure (clip or coil). RESULTS: In good-grade patients (N = 216), clinical outcomes and mortality did not differ significantly between the control and Cerebrolysin groups. In poor-grade patients (N = 246), the mortality rate was significantly lower in the Cerebrolysin group (8.7%) than in the control group (25.4%, p = 0.006). In patients who received microsurgical clipping (N = 328), the mortality rate was significantly lower in the Cerebrolysin group (7.3%) than in the control group (18.5%, p = 0.016). CONCLUSION: Cerebrolysin injection during the acute period of SAH appeared to reduce the mortality rate, especially in poor-grade patients. This study suggests the potential of Cerebrolysin for treating aneurysmal SAH. Further studies are needed to confirm our results.

Do bacteria release extracellular vesicles?

Yes, Bacterial extracellular vesicles (EVs) are bilayered lipid membrane structures, bearing integral proteins and able to carry diverse cargo outside the cell to distant sites.

Bacterial extracellular vesicles (EVs) are bilayered lipid membrane structures, bearing integral proteins and able to carry diverse cargo outside the cell to distant sites. In microorganisms, EVs carry several types of molecules: proteins, glycoproteins, mRNAs and small RNA species, as mammalian EVs do, but also carbohydrates. Studying EVs opens a whole new world of possibilities to better understand the interplay between host and bacteria crosstalks, although there are still many questions to be answered in the field, especially when it comes to microbiota-derived EVs. In this review, we propose to summarize and analyse the current literature about bacterial EVs and possible clinical applications, through answering three main questions: (a) What are bacterial EVs? (b) What are EV impacts on skin inflammatory disease physiopathology? (iii) What are the possible and expected clinical applications of EVs to treat inflammatory skin diseases? Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have been less thoroughly investigated most probably due to the methodological challenges in separating BEVs from their matrix and host-derived eukaryotic extracellular vesicles (EEVs) such as exosomes and microvesicles. Here, we present a step-by-step procedure to separate and characterize BEVs from human body fluids. BEVs are separated through the orthogonal implementation of ultrafiltration, size-exclusion chromatography (SEC) and density-gradient centrifugation. Size separates BEVs from bacteria, flagella and cell debris in stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, maintains the integrity of BEVs and ensures reproducibility through the use of automated liquid handlers. Post-separation BEVs are characterized using orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in combination with proteomics, electron microscopy and oparticle tracking analysis (NTA) to evaluate BEV quality, abundance, structure and molecular cargo. Separation and characterization of BEVs from body fluids can be done within 72 h, is compatible with EEV analysis and can be readily adopted by researchers experienced in basic molecular biology and extracellular vesicle analysis. We anticipate that this protocol will expand our knowledge on the biological heterogeneity, molecular cargo and function of BEVs in human body fluids and steer the development of laboratory research tools and clinical diagnostic kits.

What is the effect of the venom of the cone snail, Conus tulipa?

Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins.

Cotokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Cotokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like cotokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) cotokin-T has NMDA antagonist activity. A sequence comparison of cotokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide. This study investigated the mode of action of cotokin-T, a 21 amino acid peptide toxin isolated from the venom of the fish-hunting cone snail Conus tulipa, on excitatory synaptic transmission in rat hippocampal slices using intracellular recording techniques. Superfusion of cotokin-T (1-500 nM) specifically and irreversibly decreased the pharmacologically isolated N-methyl-D-aspartate receptor (NMDA)-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner but had no effect on normal excitatory synaptic transmission (EPSP). The sensitivity of postsynaptic neurons to NMDA but not to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid was also antagonized by cotokin-T pretreatment. In addition, the cotokin-T-induced depression of EPSPNMDA could be antagonized by prior treatment of hippocampal slices with either DL-2-amino-5-phosphonovaleate (10 microM) or ifenprodil (20 microM). However, 7-chlorokynurenic acid (1 microM) had no effect on the action of cotokin-T. These findings indicated that cotokin-T modulates the NMDA receptor by an interaction with its glutamate binding site and polyamine recognition site. Subunit non-selective N-methyl-D-aspartate (NMDA) receptor antagonists reduce injury-induced pain behavior, but generally produce unacceptable side effects. In this study, we examined the antinociceptive and motor effects of cone snail venom-derived peptides, cotokins G and T (conG and conT), which are selective inhibitors of the NR2B or NR2A and NR2B subtypes of the NMDA receptor, respectively. We tested the effects of conG and conT in models of tissue (formalin test), nerve injury (partial sciatic nerve ligation) and inflammation-induced (intraplantar Complete Freund's Adjuvant; CFA) pain in mice. In the formalin test, intrathecal (i.t.) conG or conT suppressed the ongoing pain behavior (ED(50) and 95% confidence intervals (CI), 11 (7-19) and 19 (11-33), respectively) at doses that were 17-27 times lower than those required to impair motor function (accelerating rotarod treadmill test: ED(50) and 95% CI, 300 (120-730) and 320 (190-540) pmol, respectively). By comparison, SNX-111, an N-type voltage-sensitive calcium channel antagonist that is also derived from cone snail venom, produced significant motor impairment at a dose (3.0 pmol, i.t.) that was only partially efficacious in the formalin test. Furthermore, conG reversed the allodynia produced by nerve injury, with greater potency on thermal (ED50 and 95% CI, 24 (10-55) pmol) than on mechanical allodynia (59 (33-105) pmol). Finally, a single dose of conG (100 pmol, i.t.) also reduced CFA-evoked thermal and mechanical allodynia. Taken together, these results demonstrate that cotokins exhibit potent antinociceptive effects in several models of injury-induced pain. The study supports the notion that drugs directed against subtypes of the NMDA receptor, by virtue of their reduced side-effect profile, hold promise as novel therapeutic agents for the control of pain. Author information: (1)Department of Biology, University of Utah, Salt Lake City, UT 84112; Department of Biology, Copenhagen Biocenter, DK-2200 Copenhagen, Denmark; [email protected] [email protected]. (2)Department of Biology, University of Utah, Salt Lake City, UT 84112; (3)University of Utah Molecular Medicine Program, University of Utah School of Medicine, Salt Lake City, UT 84112; Department of Internal Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Utah School of Medicine, Salt Lake City, UT 84112; (4)Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia; (5)Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, NY 10016; (6)Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84112; (7)University of Utah Molecular Medicine Program, University of Utah School of Medicine, Salt Lake City, UT 84112; Department of Internal Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Utah School of Medicine, Salt Lake City, UT 84112; Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112; (8)Eccles institute of Human Genetics, University of Utah, Salt Lake City, UT 84112; The Utah Science Technology and Research Initiative Center for Genetic Discovery, University of Utah, Salt Lake City, UT 84112; and. (9)Eccles institute of Human Genetics, University of Utah, Salt Lake City, UT 84112; (10)Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia. (11)Department of Biology, Copenhagen Biocenter, DK-2200 Copenhagen, Denmark; (12)Department of Biology, University of Utah, Salt Lake City, UT 84112; [email protected] [email protected]. Plain Language Summary: Insulin is a hormone critical for maintaining healthy blood sugar levels in humans. When the insulin system becomes faulty, blood sugar levels become too high, which can lead to diabetes. At the moment, the only effective treatment for one of the major types of diabetes are daily insulin injections. However, designing fast-acting insulin drugs has remained a challenge. Insulin molecules form clusters (so-called hexamers) that first have to dissolve in the body to activate the insulin receptor, which plays a key role in regulating the blood sugar levels throughout the body. This can take time and can therefore delay the blood-sugar control. In 2015, researchers discovered that the fish-hunting cone snail Conus geographus uses a specific type of insulin to capture its prey – fish. The cone snail releases insulin into the surrounding water and then engulfs its victim with its mouth. This induces dangerously low blood sugar levels in the fish and so makes them an easy target. Unlike the human version, the snail insulin does not cluster, and despite structural differences, can bind to the human insulin receptor. Now, Ahorukomeye, Disotuar et al. – including some of the authors involved in the previous study – wanted to find out whether other fish-hunting cone snails also make insulins and if they differed from the one previously discovered in C. geographus. The insulin molecules were extracted and analyzed, and the results showed that the three cone snail species had different versions of insulin – but none of them formed clusters. Ahorukomeye, Disotuar et al. further revealed that the snail insulins could bind to the human insulin receptors and could also reverse high blood sugar levels in fish and mouse models of the disease. This research may help guide future studies looking into developing fast-acting insulin drugs for diabetic patients. A next step will be to fully understand how snail insulins can be active at the human receptor without forming clusters. Cone snails solved this problem millions of years ago and by understanding how they have done this, researchers are hoping to redesign current diabetic therapeutics. Since the snail insulins do not form clusters and should act faster than currently available insulin drugs, they may lead to better or new diabetes treatments. Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the cotokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or cotokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

Which component of the Influenza A Virus affects mRNA transcription termination?

Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs).

Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir, NSC 614846) is an anti-retroviral agent that may be useful in the treatment of AIDS. We have examined the ability of (-)-etiomeric carbovir triphosphate to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49). A comparison of inhibition kinetics was made with 3'-azido-2',3'-dideoxythymidine triphosphate and phosphonoformate. Inhibition of the reverse transcriptase was evaluated using poly(rA).oligo(dT)12-18, poly(rC).oligo(dG)12-18, or influenza virion RNA template with a specific oligodeoxynucleotide as primer. (-)-Carbovir 5'-triphosphate was shown to be a potent inhibitor of HIV-1 reverse transcriptase with an apparent Ki similar to that of 3'-azido-2',3'-dideoxythymidine triphosphate. Chain elongation studies utilizing an MS2 RNA template showed that (-)-carbovir 5'-triphosphate terminated transcription at positions identical to those where dideoxy-GTP terminated. This indicates that (-)-carbovir 5'-monophosphate is incorporated into the newly synthesized DNA and terminates transcription at that point. We conclude that (-)-carbovir 5'-triphosphate is a potent inhibitor of the HIV-1 reverse transcriptase enzyme and that (-)-carbovir most likely inhibits HIV by activity at the triphosphate level by a combination of direct competition for binding of the natural deoxynucleoside triphosphates to the reverse transcriptase and chain termination. Influenza virus gene 8 codes for two nonstructural proteins (NS1 and NS2) which are translated, respectively, from a colinear and an interrupted mRNA. To investigate the mechanism of transcription processing by splicing, cloned full-length NS DNA was inserted into the late region of an SV40 vector. In this manner, transcription of NS RNA from the recombit initiates and terminates using SV40 control sequences. RNA mapping and sequence analysis showed that the RNA transcripts are processed to produce both the spliced and unspliced NS mRNAs in approximately equal abundance. The junction sequence of the spliced mRNA is identical to that of NS2 mRNA found in influenza virus-infected cells. In addition, another mRNA species was found to contain a chimera splice between SV40 and NS sequences. Both NS1 and NS2 polypeptides are produced in the recombit-infected cells as predicted from sequence analysis. These studies establish that during influenza virus infections processing of the NS1 mRNA transcript undergoes a mechanism of splicing similar to that occurring with DNA-directed RNA transcription. Viruses have evolved a number of translational control mechanisms to regulate the levels of expression of viral proteins on polycistronic mRNAs, including programmed ribosomal frameshifting and stop codon readthrough. More recently, another unusual mechanism has been described, that of termination-dependent re-initiation (also known as stop-start). Here, the AUG start codon of a 3' ORF (open reading frame) is proximal to the termination codon of a uORF (upstream ORF), and expression of the two ORFs is coupled. For example, segment 7 mRNA of influenza B is bicistronic, and the stop codon of the M1 ORF and the start codon of the BM2 ORF overlap in the pentanucleotide UAAUG (stop codon of M1 is shown in boldface and start codon of BM2 is underlined). This short review aims to provide some insights into how this translational coupling process is regulated within different viral systems and to highlight some of the differences in the mechanism of re-initiation on prokaryotic, eukaryotic and viral mRNAs. Viruses utilize a number of translational control mechanisms to regulate the relative expression levels of viral proteins on polycistronic mRNAs. One such mechanism, that of termination-dependent reinitiation, has been described in a number of both negative- and positive-strand RNA viruses. Dicistronic RNAs which exhibit termination-reinitiation typically have a start codon of the 3'-ORF (open reading frame) proximal to the stop codon of the upstream ORF. For example, the segment 7 RNA of influenza B is dicistronic, and the stop codon of the M1 ORF and the start codon of the BM2 ORF overlap in the pentanucleotide UAAUG (the stop codon of M1 is shown in bold and the start codon of BM2 is underlined). Recent evidence has highlighted the potential importance of mRNA-rRNA interactions in reinitiation on caliciviral and influenza B viral RNAs, probably used to tether 40S ribosomal subunits to the RNA after termination in time for initiation factors to be recruited to the AUG of the downstream ORF. The present review summarizes how such interactions regulate reinitiation in an array of RNA viruses, and discusses what is known about reinitiation in viruses that do not rely on apparent mRNA-rRNA interactions. Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription. Author information: (1)Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (2)Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (3)Department of Obstetrics and Gynecology, Stanford University, Stanford, CA, USA. (4)Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Stanford, CA, USA. (5)Department of Medicine (Infectious Diseases), Northwestern University Feinberg School of Medicine, Chicago, IL, USA. (6)Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. (7)Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (8)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore. (9)Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (10)Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic. (11)Epinova Epigenetics Discovery Performance Unit, Immuno-Inflammation Therapy Area, GlaxoSmithKline, Medicines Research Centre, Stevenage, UK. (12)Laboratory of Immune Cell Epigenetics and Signaling, The Rockefeller University, New York, NY, USA. (13)Cancer Cell Biology Programme, Centro Nacional de Investigaciones Oncológicas, CNIO, Madrid, Spain. (14)Department of Psychiatry, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (15)Department of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (16)Department of Medicine, Clinical Immunology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (17)Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. (18)Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (19)Division of Hematology and Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (20)Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (21)Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (22)Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. [email protected]. (23)Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. [email protected].

Which two genes are predominantly considered by warfarin initial dosing algorithms?

Polymorphisms in CYP2C9 and VKORC1 are taken into consideration by warfarin initial dosing algorithms.

Author information: (1)Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, ON, Canada. (2)Department of Pediatrics, Yokohama City University, School of Medicine, Yokohama, Kanagawa, Japan. (3)Division of Clinical Research Planning, Department of Development Strategy, Center for Clinical Research and Development, National Center for Child Health and Development, Tokyo, Japan. (4)Institute of Cellular Medicine, Newcastle University and Newcastle upon Tyne Hospitals, NHS Foundation Trust, Newcastle upon Tyne, UK. (5)Department of Haematology, The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. (6)Division of Pediatric Hematology/Oncology/BMT, Nationwide Children's Hospital, Columbus, OH, US. (7)Division of Pediatric Hematology/Oncology, Department of Pediatrics, Monroe Carell Jr. Children's Hospital at Vanderbilt, Vanderbilt University Medical Center, Nashville, TN, US. (8)Assistance Publique Hôpitaux de Paris, Hôpital Necker Enfants Malades, Unité Médico-Chirurgicale de Cardiologie Congénitale et Pédiatrique, Centre de référence M3C, Paris, France. (9)University Paris Descartes, Paris, France. (10)Université Paris Descartes, Inserm Unité Mixte de Recherche (UMR)-S, Paris, France. (11)University Paris Descartes, INSERM UMR 1147, Paris, France. (12)Influenza and Emerging Respiratory Pathogens, BC Centre for Disease Control, Vancouver, BC, Canada. (13)Division of Translational Therapeutics, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada. (14)Department of Medical Sciences, Clinical Pharmacology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. (15)Department of Pediatrics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. (16)Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. (17)Department of Cardiology, Kanagawa Children's Medical Center, Yokohama, Japan. (18)Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan. (19)Department of Clinical Pharmacology & Genetics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan. (20)Division of Pediatric Hematology/Oncology, The Hospital for Sick Children, Toronto, ON, Canada. (21)Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, ON, Canada. [email protected]. WHAT IS KNOWN AND OBJECTIVE: Warfarin is an oral anticoagulant which has been widely used to treat and prevent thromboembolic events. Managing warfarin therapy requires careful monitoring and dose titration. This randomized controlled study was designed to assess the effect of genotype-guided warfarin anticoagulation in Chinese elderly patients with nonvalvular atrial fibrillation. METHODS: 507 adults were randomized to receive initial dosing as determined by an algorithm containing genetic (VKORC1 and CYP2C9) plus clinical information or only clinical information. The primary endpoint was the time in therapeutic range (TTR) over 90 days. Secondary end points included haemorrhagic events, thrombotic events and mortality. RESULTS: The TTR was significantly different between genetic group and control group. The average TTR was (70.80 ± 24.39) % in the genotype-guided group as compared with (53.44 ± 26.73) % in the control group. This represents a difference of 17.36% (95% CI, 11.82 to 22.89, P < .001). The cumulative incidence of total haemorrhagic events, minor haemorrhagic events, gastrointestinal bleeding and intracerebral bleeding events was not significantly different between two groups (P > .05). Follow-up showed that the cumulative incidence of ischaemic stroke events occurred in the genetic group was significantly lower than that in the control group (2.39% vs 6.82%), and the genetic group had a significant lower risk than control group in cumulative incidence of ischaemic stroke events [HR 0.22, (95% CI 0.065 to 0.77), P < .05]. WHAT IS NEW AND CONCLUSION: Genotype-guided dosing could improve the average TTR, and follow-up result showed that genotype-guided therapy resulted in a significantly lower risk of ischaemic stroke events. Further research is required to focus on the clinical benefit of genotype-guided dosing.

Describe MSstatsTMT

MSstatsTMT is a general statistical approach for relative protein quantification in MS- based experiments with TMT labeling.

Tandem mass tag (TMT) is a multiplexing technology widely-used in proteomic research. It enables relative quantification of proteins from multiple biological samples in a single MS run with high efficiency and high throughput. However, experiments often require more biological replicates or conditions than can be accommodated by a single run, and involve multiple TMT mixtures and multiple runs. Such larger-scale experiments combine sources of biological and technical variation in patterns that are complex, unique to TMT-based workflows, and challenging for the downstream statistical analysis. These patterns cannot be adequately characterized by statistical methods designed for other technologies, such as label-free proteomics or transcriptomics. This manuscript proposes a general statistical approach for relative protein quantification in MS- based experiments with TMT labeling. It is applicable to experiments with multiple conditions, multiple biological replicate runs and multiple technical replicate runs, and unbalanced designs. It is based on a flexible family of linear mixed-effects models that handle complex patterns of technical artifacts and missing values. The approach is implemented in MSstatsTMT, a freely available open-source R/Bioconductor package compatible with data processing tools such as Proteome Discoverer, MaxQuant, OpenMS, and SpectroMine. Evaluation on a controlled mixture, simulated datasets, and three biological investigations with diverse designs demonstrated that MSstatsTMT balanced the sensitivity and the specificity of detecting differentially abundant proteins, in large-scale experiments with multiple biological mixtures.

Is Tranexamic acid effective for intracerebral haemorrhage?

No. According to clinical trial data tranexamic acid does not improve outcomes of patients with intracerebral haemorrhage.

Author information: (1)Stroke Trials Unit, Division of Clinical Neuroscience, University of Nottingham, City Hospital Campus, Nottingham, UK; Stroke, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, UK. Electronic address: [email protected]. (2)Stroke Trials Unit, Division of Clinical Neuroscience, University of Nottingham, City Hospital Campus, Nottingham, UK. (3)Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK. (4)Department of Neurology, Semmelweis University, Budapest, Hungary. (5)The First University Clinic of Tbilisi State Medical University, Tbilisi, Georgia. (6)Department of Neurology, Bispebjerg and Frederiksberg Hospital, University of Copenhagen, Copenhagen, Denmark. (7)Neurology Unit, Azienda Socio Sanitaria Territoriale di Mantova, Mantua, Italy. (8)Stroke Service, Adelaide and Meath Hospital, Tallaght, Ireland. (9)2nd Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland. (10)Radiological Sciences, Division of Clinical Neuroscience, University of Nottingham, Queens Medical Centre Campus, Nottingham, UK; NIHR Nottingham Biomedical Research Centre, Nottingham, UK. (11)Nottingham Clinical Trials Unit, University of Nottingham, Queen's Medical Centre, Nottingham, UK. (12)UGC de Medicina Intensiva, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Seville, Spain. (13)Vascular Medicine, Division of Medical Sciences and Graduate Entry Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, UK. (14)Stroke Trials Unit, Division of Clinical Neuroscience, University of Nottingham, City Hospital Campus, Nottingham, UK; Stroke, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, UK. (15)Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden. (16)Stroke Trials Unit, Division of Clinical Neuroscience, University of Nottingham, City Hospital Campus, Nottingham, UK; Stroke, Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, UK; Department of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia. (17)Department of Neurology, Selcuk University Medical Faculty, Konya, Turkey. (18)Department of Medical Statistics, London School of Hygiene & Tropical Medicine, London, UK. (19)Clinical Trials Unit, London School of Hygiene & Tropical Medicine, London, UK. (20)Department of Cardiovascular Sciences and NIHR Leicester Biomedical Research Centre, University of Leicester, Leicester, UK. (21)Stroke Research, Faculty of Medicine and Health Sciences, Keele University, Staffordshire, UK. (22)Stroke Center, Neurology and Department of Clinical Research, University Hospital, University Basel, Basel, Switzerland. (23)Division of Neurosurgery, Department of Surgery, National University of Malaysia, Kuala Lumpur, Malaysia. (24)Stroke Research Centre, UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery, University College London, London, UK. (25)School of Economics, University of Nottingham, University Park, Nottingham, UK. PURPOSE: Haematoma expansion is a devastating complication of intracerebral haemorrhage (ICH) with no established treatment. Tranexamic acid had been an effective haemostatic agent in reducing post-operative and traumatic bleeding. We review current evidence examining the efficacy of tranexamic acid in improving clinical outcome after ICH. METHOD: We searched MEDLINE, EMBASE, CENTRAL and clinical trial registers for studies using search strategies incorporating the terms 'intracerebral haemorrhage', 'tranexamic acid' and 'antifibrinolytic'. Authors of ongoing clinical trials were contacted for further details. FINDINGS: We screened 268 publications and retrieved 17 articles after screening. Unpublished information from three ongoing clinical trials was obtained. We found five completed studies. Of these, two randomised controlled trials (RCTs) comparing intravenous tranexamic acid to placebo (n = 54) reported no significant difference in death or dependency. Three observational studies (n = 281) suggested less haematoma growth with rapid tranexamic acid infusion. There are six ongoing RCTs (n = 3089) with different clinical exclusions, imaging selection criteria (spot sign and haematoma volume), time window for recruitment and dosing of tranexamic acid. DISCUSSION: Despite their heterogeneity, the ongoing trials will provide key evidence on the effects of tranexamic acid on ICH. There are uncertainties of whether patients with negative spot sign, large haematoma, intraventricular haemorrhage, or poor Glasgow Coma Scale should be recruited. The time window for optimal effect of haemostatic therapy in ICH is yet to be established. CONCLUSION: Tranexamic acid is a promising haemostatic agent for ICH. We await the results of the trials before definite conclusions can be drawn. INTRODUCTION: Seizures are common after intracerebral haemorrhage. Tranexamic acid increases the risk of seizures in non-intracerebral haemorrhage population but its effect on post-intracerebral haemorrhage seizures is unknown. We explored the risk factors and outcomes of seizures after intracerebral haemorrhage and if tranexamic acid increased the risk of seizures in the Tranexamic acid for IntraCerebral Haemorrhage-2 trial. PATIENTS AND METHODS: Seizures were reported prospectively up to day 90. Cox regression analyses were used to determine the predictors of seizures within 90 days and early seizures (≤7 days). We explored the effect of early seizures on day 90 outcomes. RESULTS: Of 2325 patients recruited, 193 (8.3%) had seizures including 163 (84.5%) early seizures and 30 (15.5%) late seizures (>7 days). Younger age (adjusted hazard ratio (aHR) 0.98 per year increase, 95% confidence interval (CI) 0.97-0.99; p = 0.008), lobar haematoma (aHR 5.84, 95%CI 3.58-9.52; p < 0.001), higher National Institute of Health Stroke Scale (aHR 1.03, 95%CI 1.01-1.06; p = 0.014) and previous stroke (aHR 1.66, 95%CI 1.11-2.47; p = 0.013) were associated with early seizures. Tranexamic acid did not increase the risk of seizure within 90 days. Early seizures were associated with worse modified Rankin Scale (adjusted odds ratio (aOR) 1.79, 95%CI 1.12-2.86, p = 0.015) and increased risk of death (aOR 3.26, 95%CI 1.98-5.39; p < 0.001) at day 90.Discussion and conclusion: Lobar haematoma was the strongest independent predictor of early seizures after intracerebral haemorrhage. Tranexamic acid did not increase the risk of post-intracerebral haemorrhage seizures in the first 90 days. Early seizures resulted in worse functional outcome and increased risk of death.

Are interferons defensive proteins?

Yes, The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections.

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish. In response to viral infections, various pattern recognition receptors (PRRs) are activated for the production of type I interferon (IFN I). As a center of these receptor responses, TANK binding kinase-1 (TBK1) activates interferon regulatory factor 3 (IRF3). SRC is a member of Src family kinases (SFK) which participates in TBK1-mediated IFN I signaling pathway. In mammals, the immunological function of SRC is depended on its interaction with TBK1. To date, SRC has not been studied in fish. In this paper, we cloned the ORF of grass carp (Ctenopharyngodon idellus) SRC (CiSRC). CiSRC has a closer relationship with Sinocyclocheilus rhinocerous SRC (SrSRC). The expression level of CiSRC was significantly up-regulated following poly (I:C) stimulation in grass carp tissues and cells. Subcellular localization results showed that CiSRC is located both in the cytoplasm and nucleus, while CiTBK1 is only located in the cytoplasm of CIK cells. When GFP-CiSRC and FLAG-CiTBK1 were co-transfected into CIK cells, we found that they were co-localized in the cytoplasm. GST-pulldown and Co-immunoprecipitation analysis revealed that CiSRC and CiSRC tyrosine kinase domain deletion mutant (SRC-ΔTyrkc) can interact with CiTBK1, respectively. CiSRC promotes the phosphorylation of CiTBK1. Furthermore, the phosphorylation of TBK1 is more strongly under poly (I:C) stimulation. We also demonstrated that SRC can up-regulate IFN I expression. These results above unraveled that CiSRC initiates innate immune response by binding to and then up-regulating the phosphorylation of TBK1.

What effect does Methylsulfonylmethane (MSM) have on inflammation?

Methylsulfonylmethane (MSM) is a sulfur-based nutritional supplement that is purported to have pain and inflammation-reducing effects.

Methylsulfonylmethane (MSM) is a popular dietary supplement used in a variety of conditions including pain, inflammation, allergies, arthritis, parasitic infections and the maintece of normal keratin levels in hair, skin and nails. Despite its popularity, there is little published toxicology data on MSM. The objective of this study was to evaluate the acute and subchronic toxicity of MSM in rats at a dose five to seven times the maximum recommended dose in humans. MSM administered in a single gavage dose of 2 g/kg resulted in no adverse events or mortality. MSM administered as a daily dose of 1.5 g/kg for 90 days by gavage resulted in no adverse events or mortality. Necropsy did not reveal any gross pathological lesions or changes in organ weights. Renal histology of treated animals was normal. It is concluded that MSM is well tolerated in rats at an acute dose of 2 g/kg and at a subacute chronic dose of 1.5 g/kg. OBJECTIVE: Glucosamine, classified as a slow-acting drug in osteoarthritis (SADOA), is an efficacious chondroprotective agent. Methylsulfonylmethane (MSM), the isoxidised form of dimethyl-sulfoxide (DSMO), is an effective natural analgesic and anti-inflammatory agent. The aim of this study was to compare the efficacy and safety of oral glucosamine (Glu), methylsulfonylmethane (MSM), their combination and placebo in osteoarthritis of the knee. PATIENTS AND DESIGN: A total of 118 patients of either sex with mild to moderate osteoarthritis were included in the study and randomised to receive either Glu 500mg, MSM 500mg, Glu and MSM or placebo capsules three times daily for 12 weeks. Patients were evaluated at 0 (before drug administration), 2, 4, 8 and 12 weeks post-treatment for efficacy and safety. The efficacy parameters studied were the pain index, the swelling index, visual analogue scale pain intensity, 15m walking time, the Lequesne index, and consumption of rescue medicine. RESULTS: Glu, MSM and their combination significantly improved signs and symptoms of osteoarthritis compared with placebo. There was a statistically significant decrease in mean (+/- SD) pain index from 1.74 +/- 0.47 at baseline to 0.65 +/- 0.71 at week 12 with Glu (p < 0.001). MSM significantly decreased the mean pain index from 1.53 +/- 0.51 to 0.74 +/- 0.65, and combination treatment resulted in a more significant decrease in the mean pain index (1.7 +/- 0.47 to 0.36 +/- 0.33; p < 0.001). After 12 weeks, the mean swelling index significantly decreased with Glu and MSM, while the decrease in swelling index with combination therapy was greater (1.43 +/- 0.63 to 0.14 +/- 0.35; p < 0.05) after 12 weeks. The combination produced a statistically significant decrease in the Lequesne index. All treatments were well tolerated. CONCLUSION: Glu, MSM and their combination produced an analgesic and anti-inflammatory effect in osteoarthritis. Combination therapy showed better efficacy in reducing pain and swelling and in improving the functional ability of joints than the individual agents. All the treatments were well tolerated. The onset of analgesic and anti-inflammatory activity was found to be more rapid with the combination than with Glu. It can be concluded that the combination of MSM with Glu provides better and more rapid improvement in patients with osteoarthritis. Methylsulfonylmethane (MSM), also known as dimethyl sulfone and methyl sulfone, is an organic sulfur-containing compound that occurs naturally in a variety of fruits, vegetables, grains, and animals, including humans. In the present study, we demonstrated the anti-inflammatory effects of MSM in lipopolysaccharide (LPS)-stimulated murine macrophages, RAW264.7 cells. MSM significantly inhibited the release of nitric oxide and prostaglandin E(2) by alleviating the expression of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-stimulated RAW264.7 cells. Furthermore, the levels of interleukin-6 and tumor necrosis factor-alpha were decreased by MSM treatment in cell culture supernatants. Further study indicated that the translocation of the p65 subunit of nuclear factor (NF)-kappaB to the nucleus was inhibited by MSM treatment in LPS-stimulated RAW264.7 cells, in which it helped block degradation of inhibitor of NF-kappaB. In addition, in vivo studies demonstrated that topical administration of MSM at 500-1250 microg/ear resulted in similar inhibitory activities in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema. Collectively, theses results indicate that MSM inhibits LPS-induced release of pro-inflammatory mediators in murine macrophages through downregulation of NF-kappaB signaling. Methylsulfonylmethane (MSM), which is one of the popular ingredients of so-called health foods in Japan, is expected to relieve inflammation in arthritis and allergies. However, there is no scientific evidence to confirm the efficacy and safety of MSM in detail. In this study, we examined the effects of MSM on cartilage formation in growing rats (G) and cartilage degradation in STR/Ort mice (A), an accepted human osteoarthritis (OA) model. For cartilage formation study, 6-week-old growing male Wister rats were assigned to four groups to receive a control or MSM-containing diet. To examine the efficacy of MSM on the cartilage of OA model mouse, 10-week-old male STR/OrtCrlj mice were assigned to three groups to receive a control or MSM-containing diet. The dosages used were amounts equal to the recommended supplements for humans [0.06 g/kg body weight (BW)/day: MSM1G and MSM1A], 10 fold higher (0.6 g/kg BW/day: MSM10G and MSM10A), and 100 fold higher (6 g/kg BW/day: MSM100G). Intake of MSM for 4 weeks did not affect cartilage formation in the knee joint in growing rats. Body, liver, and spleen weight in the MSM100G group were significantly lower than those in the control group. Intake of MSM for 13 weeks decreased degeneration of the cartilage at the joint surface in the knee joints in STR/Ort mice in a dose-dependent manner. These results suggest that appropriate intake of MSM is possibly effective in OA model mice; however, intake of large amounts of MSM induced atrophy of several organs. BACKGROUND: Methylsulfonylmethane (MSM) has been reported to provide anti-inflammatory and antioxidant effects in both animal and man. Strenuous resistance exercise has the potential to induce both inflammation and oxidative stress. Using a pilot (proof of concept) study design, we determined the influence of MSM on markers of exercise recovery and performance in healthy men. METHODS: Eight, healthy men (27.1 ± 6.9 yrs old) who were considered to be moderately exercise-trained (exercising <150 minutes per week) were randomly assigned to ingest MSM at either 1.5 grams per day or 3.0 grams per day for 30 days (28 days before and 2 days following exercise). Before and after the 28 day intervention period, subjects performed 18 sets of knee extension exercise in an attempt to induce muscle damage (and to be used partly as a measure of exercise performance). Sets 1-15 were performed at a predetermined weight for 10 repetitions each, while sets 16-18 were performed to muscular failure. Muscle soreness (using a 5-point Likert scale), fatigue (using the fatigue-inertia subset of the Profile of Mood States), blood antioxidant status (glutathione and Trolox Equivalent Antioxidant Capacity [TEAC]), and blood homocysteine were measured before and after exercise, pre and post intervention. Exercise performance (total work performed during sets 16-18 of knee extension testing) was also measured pre and post intervention. RESULTS: Muscle soreness increased following exercise and a trend was noted for a reduction in muscle soreness with 3.0 grams versus 1.5 grams of MSM (p = 0.080), with a 1.0 point difference between dosages. Fatigue was slightly reduced with MSM (p = 0.073 with 3.0 grams; p = 0.087 for both dosages combined). TEAC increased significantly following exercise with 3.0 grams of MSM (p = 0.035), while homocysteine decreased following exercise for both dosages combined (p = 0.007). No significant effects were noted for glutathione or total work performed during knee extension testing (p > 0.05). CONCLUSION: MSM, especially when provided at 3.0 grams per day, may favorably influence selected markers of exercise recovery. More work is needed to extend these findings, in particular using a larger sample of subjects and the inclusion of additional markers of exercise recovery and performance. Methylsulfonylmethane (MSM) is a natural organosulfur compound that exhibits antioxidative and anti-inflammatory effects. This study was carried out to investigate the effect of MSM on paraquat (PQ)-induced acute lung and liver injury in mice. A single dose of PQ (50 mg/kg, i.p.) induced acute lung and liver toxicity. Mice were treated with MSM (500 mg/kg/day, i.p.) for 5 days. At the end of the experiment, animals were euthanized, and lung and liver tissues were collected for histological and biochemical analysis. Tissue samples were used to determine malondialdehyde (MDA), myeloperoxidase (MPO), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and tumor necrosis factor-α (TNF-α) levels. Blood samples were used to measure plasma alanine transaminase (ALT), γ-glutamyl transferase (GGT), and alkaline phosphatase (ALP). Histological examination indicated that MSM decreased lung and liver damage caused by PQ. Biochemical results showed that MSM treatment significantly reduced tissue levels of MDA, MPO, and TNF-α, while increased the levels of SOD, CAT, and GSH compared with PQ group. MSM treatment also significantly reduced plasma levels of ALT, GGT, and ALP. These findings suggest that MSM as a natural product attenuates PQ-induced pulmonary and hepatic oxidative injury. Methylsulfonylmethane (MSM) is an organosulfur compound and the health benefits associated with MSM include inflammation. Although MSM has been shown to have various physiological effects, no study has yet focused on inflammasome activation. The inflammasome is a multiprotein complex that serves as a platform for caspase 1-dependent proteolytic maturation and secretion of interleukin-1β (IL-1β). In this study, we tested the effect of MSM on inflammasome activation using mouse and human macrophages. In our results, MSM significantly attenuated NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, although it had no effect on NLCR4 or AIM2 inflammasome activation. Extracts of MSM-enriched vegetables presented the same inhibitory effect on NLRP3 inflammasome activation as MSM. MSM also attenuated the transcriptional expression of IL-1α, IL-1β, IL-6, and NLRP3. Taken together, these results show that MSM has anti-inflammatory characteristics, interrupts NLRP3 inflammasome activation, and inhibits pro-cytokine expression. We further confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. Both chemicals showed a synergic effect on anti-NLRP3 activation and attenuated production of mitochondrial reactive oxygen species (ROS). Thus, MSM is a selective inhibitor of NLRP3 inflammasome activation and can be developed as a supplement to control several metabolic disorders. Until now glucosamine sulfate (GS) has been the most widely used supplement and has been shown to be efficacious in the treatment of osteoarthritis (OA). Methylsulfonylmethane (MSM) and boswellic acids (BA) are new effective supplements for the management of inflammation and joint degeneration, according to previous experimental studies. The aim of our study is to test the effectiveness of association of MSM and BA in comparison with GS in knee arthritis.In this prospective randomized clinical trial, MEBAGA (Methylsulfonylmethane and Boswellic Acids versus Glucosamine sulfate in the treatment of knee Arthritis), 120 participants affected by arthritis of the knee were randomly assigned to an experimental group (MB group) or a control group (GS group) treated for 60 days with 5 g of MSM and 7.2 mg of BA or with 1500 mg of GS daily, respectively. At the 2-month (T1) and 6-months (T2) follow-up , the efficacy of these two nutraceuticals was assessed using the visual analog pain scale (VAS) and the Lequesne Index (LI) for joint function, along with the use of anti-inflammatory drugs (non-steroidal anti-inflammatory drugs and anti-cyclooxygenase-2).The repeated measures ANOVA analysis shows that for VAS, LI, and the use of anti-inflammatory drugs scores there are improvements due to the time in the two groups (respectively, F=26.0; P<0.0001; F=4.15; P=0.02; F=3.38; P=0.04), with a tendency to better values for the MB group at T2.On the basis of these preliminary data, we could support the efficacy of the MSM in association with BA in the treatment of OA. These results are consistent with the anti-inflammatory and chondroprotective effects previously occurred in experimental studies. This new combination of integration (MSM and BS) has presented good results and satisfactory in comparison with GS, until now the cornerstone of the treatment of arthritis in according to guidelines. Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3's involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that STAT3 plays a pivotal role in RANKL-induced osteoclast formation, and that MSM can attenuate RANKL-induced osteoclastogenesis by blocking both NF-kB and STAT3 activity. Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties. Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise. Ex vivo and in vitro testing consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, while in vivo testing included the evaluation of cytokines before and through 72 hours after exercise. Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM. Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise. BACKGROUND: Oxidative stress and muscle damage occur during exhaustive bouts of exercise, and many runners report pain and soreness as major influences on changes or breaks in training regimens, creating a barrier to training persistence. Methylsulfonylmethane (MSM) is a sulfur-based nutritional supplement that is purported to have pain and inflammation-reducing effects. To investigate the effects of MSM in attenuating damage associated with physical exertion, this randomized, double-blind, placebo-controlled study evaluated the effects of MSM supplementation on exercise-induced pain, oxidative stress and muscle damage. METHODS: Twenty-two healthy females (n = 17) and males (n = 5) (age 33.7 ± 6.9 yrs.) were recruited from the 2014 Portland Half-Marathon registrant pool. Participants were randomized to take either MSM (OptiMSM®) (n = 11), or a placebo (n = 11) at 3 g/day for 21 days prior to the race and for two days after (23 total). Participants provided blood samples for measurement of markers of oxidative stress, and completed VAS surveys for pain approximately one month prior to the race (T0), and at 15 min (T1), 90 min (T2), 1 Day (T3), and 2 days (T4) after race finish. The primary outcome measure 8-hydroxy-2-deoxyguanine (8-OHdG) measured oxidative stress. Secondary outcomes included malondialdehyde (MDA) for oxidative stress, creatine kinase (CK) and lactate dehydrogenase (LDH) as measures of muscle damage, and muscle (MP) and joint pain (JP) recorded using a 100 mm Visual Analogue Scale (VAS). Data were analyzed using repeated and multivariate ANOVAs, and simple contrasts compared post-race time points to baseline, presented as mean (SD) or mean change (95% CI) where appropriate. RESULTS: Running a half-marathon induced significant increases in all outcome measures (p < 0.001). From baseline, 8-OHdG increased significantly at T1 by 1.53 ng/mL (0.86-2.20 ng/mL CI, p < 0.001) and T2 by 1.19 ng/mL (0.37-2.01 ng/mL CI, p < 0.01), and fell below baseline levels at T3 by -0.46 ng/mL (-1.18-0.26 CI, p > 0.05) and T4 by -0.57 ng/mL (-1.27-0.13 CI, p > 0.05). MDA increased significantly at T1 by 7.3 μM (3.9-10.7 CI, p < 0.001). Muscle damage markers CK and LDH saw significant increases from baseline at all time-points (p < 0.01). Muscle and joint pain increased significantly from baseline at T1, T2, and T3 (p < 0.01) and returned to baseline levels at T4. Time-by-treatment results did not reach statistical significance for any outcome measure, however, the MSM group saw clinically significant (Δ > 10 mm) reductions in both muscle and joint pain. CONCLUSION: Participation in a half-marathon was associated with increased markers of oxidative stress, muscle damage, and pain. MSM supplementation was not associated with a decrease from pre-training levels of oxidative stress or muscle damage associated with an acute bout of exercise. MSM supplementation attenuated post-exercise muscle and joint pain at clinically, but not statistically significant levels. The development of various cardiovascular diseases (CVDs) are associated with chronic inflammation. Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine that activates the nuclear factor-κB (NF-κB) signaling pathway, leading to increased inflammatory cytokine expression, such as interleukin-6 (IL-6). Interventions to reduce each of these factors have been demonstrated to reduce the development of CVD. Methylsulfonylmethane (MSM) is a naturally occurring compound that demonstrates anti-inflammatory effects in humans and various animal and cell culture models. The effects of MSM include decreased NF-κB activation, decreased expression of TNF-α, and IL-6. However, the effects of MSM within the heart have not yet been examined. Therefore, the purpose of this investigation was to determine whether MSM protects cardiac cells from inflammation that occurs in response to pro-inflammatory stimuli. A novel immortalized human ventricular cardiomyocyte cell line, designated Ac16, developed and characterized in the laboratory of Dr. Mercy Davidson, Columbia Invention Report No. 823, U.S. patent No. 7,223,599 were utilized. Cells were treated with TNF-α, alone or in combination with MSM. To confirm an appropriate dosage of MSM, the effect of various concentrations on cell viability, and IL-6 production were examined. The effect of MSM on transcript expression of pro-inflammatory markers and activation of NF-κB were examined with the established dose by real-time quantitative PCR and western blot, respectively. MSM treatment combined with TNF-α significantly decreased IL-6 production and transcript expression compared to TNF-α alone. These findings indicate that MSM may protect against inflammation in the heart, and thereby protect against inflammation-linked CVDs. Further study is warranted to determine the effect of MSM on cardiovascular health outcomes. High glucose-induced inflammation leads to atherosclerosis, which is considered a major cause of death in type 1 and type 2 diabetic patients. Nuclear factor-kappa B (NF-κB) plays a central role in high glucose-induced inflammation and is activated through toll-like receptors (TLRs) as well as canonical and protein kinase C-dependent (PKC) pathways. Non-toxic sulfur (NTS) and methylsulfonylmethane (MSM) are two sulfur-containing natural compounds that can induce anti-inflammation. Using Western blotting, real-time polymerase chain reaction, and flow cytometry, we found that high glucose-induced inflammation occurs through activation of TLRs. An effect of NTS and MSM on canonical and PKC-dependent NF-κB pathways was also demonstrated by western blotting. The effects of proinflammatory cytokines were investigated using a chromatin immunoprecipitation assay and enzyme-linked immunosorbent assay. Our results showed inhibition of the glucose-induced expression of TLR2 and TLR4 by NTS and MSM. These sulfur compounds also inhibited NF-κB activity through reactive oxygen species (ROS)-mediated canonical and PKC-dependent pathways. Finally, NTS and MSM inhibited the high glucose-induced expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and binding of NF-κB protein to the DNA of proinflammatory cytokines. Together, these results suggest that NTS and MSM may be potential drug candidates for anti-inflammation therapy. BACKGROUND: Methylsulfonylmethane (MSM) is a commonly used diet supplement believed to decrease the inflammation in joints and fastens recovery in osteoarthritis, gastric mucosal injury, or obesity-related disorders. It was also suggested that MSM might play a beneficial role in cancer treatment. PURPOSE: So far, the MSM might have a potentially beneficial effect in endometrial cancer (EC) treatment. STUDY DESIGN: This study evaluated the effect and usefulness of MSM in combinatory therapy with known drug doxorubicin (DOX). METHODS: The effect of combinational treatment of MSM and DOX on the induction of apoptosis was evaluated in EC cell lines (ISHIKAWA, MFE-296, MFE-280). RESULTS: We observed that MSM itself induces apoptosis in EC cell lines, and pre-treatment with MSM for 24 h increases the sensitivity of EC cells to DOX-induced apoptosis and DNA damage and that effect might be regulated by p42/44 (Erk1/2) MAPK and Akt (protein kinase B). CONCLUSION: These results for the first time show that MSM might act as a sensitizer of EC cells to known drugs, for which EC cells quickly acquire resistance. Graphical abstract. Rotator cuff tears (RCTs) and rotator cuff disease (RCD) are important causes of disability in middle-aged individuals affected by nontraumatic shoulder dysfunctions. Our previous studies have demonstrated that four different hyaluronic acid preparations (HAPs), including Artrosulfur® hyaluronic acid (HA) (Alfakjn S.r.l., Garlasco, Italy), may exert a protective effect in human RCT-derived tendon cells undergoing oxidative stress damage. Recently, methylsulfonylmethane (MSM) (Barentz, Paderno Dugo, Italy) has proven to have anti-inflammatory properties and to cause pain relief in patients affected by tendinopathies. This study aims at evaluating three preparations (Artrosulfur® HA, MSM, and Artrosulfur® MSM + HA) in the recovery from hydrogen peroxide-induced oxidative stress damage in human tenocyte. Cell proliferation, Lactate Dehydrogenase (LDH) release, and inducible nitric oxide synthases (iNOS) and prostaglandin E2 (PGE2) modulation were investigated. In parallel, expression of metalloproteinases 2 (MMP2) and 14 (MMP14) and collagen types I and III were also examined. Results demonstrate that Artrosulfur® MSM + HA improves cell escape from oxidative stress by decreasing cytotoxicity and by reducing iNOS and PGE2 secretion. Furthermore, it differentially modulates MMP2 and MMP14 levels and enhances collagen III expression after 24 h, proteins globally related to rapid acceleration of the extracellular matrix (ECM) remodelling and thus tendon healing. By improving the anti-cytotoxic effect of HA, the supplementation of MSM may represent a feasible strategy to ameliorate cuff tendinopathies.

What is particular about the 3D structure of the inactive X chromosome?

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. This specific bipartite organization of the inactive X chromosome that probably plays an important role in maintenance of gene silencing.

The 3D folding structure formed by different genomic regions of a chromosome is still poorly understood. So far, only relatively simple geometric features, like distances and angles between different genomic regions, have been evaluated. This work is concerned with more complex geometric properties, i.e., the complete shape formed by genomic regions. Our work is based on statistical shape theory and we use different approaches to analyze the considered structures, e.g., shape uniformity test, 3D point-based registration, Fisher distribution, and 3D non-rigid image registration for shape normalization. We have applied these approaches to analyze 3D microscopy images of the X-chromosome where four consecutive genomic regions (BACs) have been simultaneously labeled by multicolor FISH. We have acquired two sets of four consecutive genomic regions with an overlap of three regions. From the experimental results, it turned out that for all data sets the complete structure is non-random. In addition, we found that the shapes of active and inactive X-chromosomal genomic regions are statistically independent. Moreover, we reconstructed the average 3D structure of chromatin in a small genomic region (below 4 Mb) based on five BACs resulting from two overlapping four BAC regions. We found that geometric normalization with respect to the nucleus shape based on non-rigid image registration has a significant influence on the location of the genomic regions. BACKGROUND: In mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems. RESULTS: We find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele. CONCLUSIONS: By applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintece of gene silencing. The long non-coding RNA Xist directs a remarkable instance of developmentally regulated, epigenetic change known as X Chromosome Inactivation (XCI). By spreading in cis across the X chromosome from which it is expressed, Xist RNA facilitates the creation of a heritably silent, heterochromatic nuclear territory that displays a three-dimensional structure distinct from that of the active X chromosome. How Xist RNA attaches to and propagates across a chromosome and its influence over the three-dimensional (3D) structure of the inactive X are aspects of XCI that have remained largely unclear. Here, we discuss studies that have made significant contributions towards answering these open questions. The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. Using Hi-C in edited mouse cells with allelic deletions or inversions within the hinge, here we show that the conserved Dxz4 locus is necessary to maintain this bipartite structure. Dxz4 orientation controls the distribution of contacts on the Xi, as shown by a massive reversal in long-range contacts after Dxz4 inversion. Despite an increase in CTCF binding and chromatin accessibility on the Xi in Dxz4-edited cells, only minor changes in TAD structure and gene expression were detected, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of its bank of CTCF motifs, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

Why mothers with a CYP2D6 ultrarapid metabolizer phenotype may expose their infants to risk of adverse events when taking codeine while breastfeeding?

Mothers with a CYP2D6 ultrarapid metabolizer phenotype may expose their infants to risk of adverse events when taking codeine while breastfeeding, by producing more of the active metabolite, morphine.

Which R package can infer protein-protein interactions via thermal proximity coaggregation (TPCA)?

Rtpca is an R package implemented methods for inferring protein-protein interactions (PPIs) based on thermal proteome profiling experiments of a single condition or in a differential setting via an approach called thermal proximity coaggregation. It offers user-friendly tools to explore datasets for their PPI predictive performance and easily integrates with available R packages.

SUMMARY: Rtpca is an R package implementing methods for inferring protein-protein interactions (PPIs) based on thermal proteome profiling experiments of a single condition or in a differential setting via an approach called thermal proximity coaggregation. It offers user-friendly tools to explore datasets for their PPI predictive performance and easily integrates with available R packages. AVAILABILITY AND IMPLEMENTATION: Rtpca is available from Bioconductor (https://bioconductor.org/packages/Rtpca). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

What is the mechanisms of action of Evinacumab?

Evinacumab is angiopoietin-like protein 3 (ANGPTL3) monoclonal antibody that was shown to substantially reduce low-density lipoprotein cholesterol concentration.

Angiopoietin-like 3 protein (ANGPTL3) is an inhibitor of both lipoprotein lipase and endothelial lipase in humans. Population studies indicate a relationship between loss of function mutations in ANGPTL3 and favorable reductions in triglycerides and non- high-density lipoprotein cholesterol. In addition, loss of function mutations is associated with a reduced risk of coronary artery disease. Whereas ANGPTL3's role in human lipid metabolism has yet to be fully clarified, it is unlikely that ANGPTL3 impacts cholesterol uptake via the low-density lipoprotein-receptor, unlike the proprotein convertase subtilisin/kexin9 inhibitors. In contrast to other forms of lipid-lowering therapy, ANGPTL3 inhibition may improve insulin sensitivity. The promise of this new therapy, particularly its independence from the low-density lipoprotein-receptor, has prompted the creation of a monoclonal antibody inhibitor; evinacumab. Evinacumab has shown favorable lipid-lowering action in both human and mouse models. Efficacy trials are currently ongoing and will be completed in the near future. In addition, ANGPTL3 inhibition via an antisense oligonucleotide was performed in healthy human subjects, which resulted in a dose-dependent reduction in circulating ANGPTL3 levels and an antiatherogenic lipid profile. When tested in mouse models, administration of the antisense oligonucleotide caused a reduction in progression of atherosclerosis. Further investigation is required to evaluate the efficacy, safety and net benefit of clinical ANGPTL3 inhibition before it can be accepted into clinical practice. INTRODUCTION: Cardiovascular (CV) diseases are the leading cause of death and disability in the developed countries. Lipid-lowering therapy is a cornerstone of the CV risk modification strategy. The first line treatment for hyperlipidemia is statins, which decrease low-density lipoprotein cholesterol (LDL-C) by 30-50% and proportionally reduce the CV events. However, they are not always enough to achieve LDL-C goals in many patients, and some patients are statin intolerant. For this reason, new powerful injectable lipid-lowering drugs have been developed. AREAS COVERED: The aim of this narrative review was to summarize the more recent clinical data on safety and tolerability of injectable lipid-lowering drugs. After an attentive literature search, the authors resumed here information on proprotein convertase subtilisin/kexin 9 inhibitors (evolocumab and alirocumab), small interfering RNA molecule inclisiran, antisense oligonucleotides (mipomersen, volanesorsen, ISIS 681257), and drugs targeting angiopoietin-like protein 3 (evinacumab, IONIS-ANGPTL3Rx). EXPERT OPINION: Injectable lipid-lowering therapy for patients at high risk for CV disease complications or with severe inherited hypercholesterolemias can be an important element of the available therapeutic armamentarium. Clinical data prove the favorable risk-benefit profile of evolocumab, alirocumab, and inclisiran. Mipomersen, volanesorsen, ISIS 681257, evinacumab, and IONIS-ANGPTL3Rx safety is currently less extensively studied, especially in patients with comorbidities and polypharmacotherapy. BACKGROUND: Hypertriglyceridemia is associated with increased cardiovascular risk and may be caused by impaired lipoprotein clearance. Angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity, increasing triglycerides and other lipids. Evinacumab, an ANGPTL3 inhibitor, reduced triglycerides in healthy human volunteers and in homozygous familial hypercholesterolemic individuals. Results from 2 Phase 1 studies in hypertriglyceridemic subjects are reported here. METHODS: Subjects with triglycerides >150 but ≤450 mg/dL and low-density lipoprotein cholesterol ≥100 mg/dL (n=83 for single ascending dose study [SAD]; n=56 for multiple ascending dose study [MAD]) were randomized 3:1 to evinacumab:placebo. SAD subjects received evinacumab subcutaneously at 75/150/250 mg, or intravenously at 5/10/20 mg/kg, monitored up to day 126. MAD subjects received evinacumab subcutaneously at 150/300/450 mg once weekly, 300/450 mg every 2 weeks, or intravenously at 20 mg/kg once every 4 weeks up to day 56 with 6 months of follow-up. The primary outcomes were incidence and severity of treatment-emergent adverse events. Efficacy analyses included changes in triglycerides and other lipids over time. RESULTS: In the SAD, 32 (51.6%) versus 9 (42.9%) subjects on evinacumab versus placebo reported treatment-emergent adverse events. In the MAD, 21 (67.7%) versus 9 (75.0%) subjects on subcutaneously evinacumab versus placebo and 6 (85.7%) versus 1 (50.0%) on intravenously evinacumab versus placebo reported treatment-emergent adverse events. No serious treatment-emergent adverse events or events leading to death or treatment discontinuation were reported. Elevations in alanine aminotransferase (7 [11.3%] SAD), aspartate aminotransferase (4 [6.5%] SAD), and creatinine phosphokinase (2 [3.2%) SAD, 1 [14.3%] MAD) were observed with evinacumab (none in the placebo groups), which were single elevations and were not dose-related. Dose-dependent reductions in triglycerides were observed in both studies, with maximum reduction of 76.9% at day 3 with 10 mg/kg intravenously (P<0.0001) in the SAD and of 83.1% at day 2 with 20 mg/kg intravenously once every 4 weeks (P=0.0003) in the MAD. Significant reductions in other lipids were observed with most evinacumab doses versus placebo. CONCLUSION: Evinacumab was well-tolerated in 2 Phase 1 studies. Lipid changes in hypertriglyceridemic subjects were similar to those observed with ANGPTL3 loss-of-function mutations. Because the latter is associated with reduced cardiovascular risk, ANGPTL3 inhibition may improve clinical outcomes. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov. Unique identifiers: NCT01749878 and NCT02107872. PURPOSE OF REVIEW: Homozygous familial hypercholesterolemia (HoFH) is a rare disorder associated with early atherosclerotic disease due to impairment of the LDL receptor (LDLR) pathway. Because of their molecular defect, current treatment options have limited success in bringing HoFH patient to LDL-C target and morbidity and mortality remain high. We review current and upcoming therapies directed at HoFH, including gene therapy. RECENT FINDINGS: Recent real-world studies have confirmed the strength in lomitapide as a treatment adjunct to statins and other lipid-lowering therapies in HoFH patients. The approval of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor monoclonal antibodies has also been a welcome addition to the treatment armamentarium offering an additional average reduction in LDL-C levels of 24% when added to background lipid-lowering therapies in this population. Although achieving adequate LDL-C levels in this population is difficult, there are several therapies on the horizon that may help more patients reach goal. Evinacumab, a monoclonal antibody against ANGPTL3, has been shown to substantially reduce LDL-C of an average of 49%, independently of residual LDLR activity. RNA interference targeting PCSK9 and ANGPTL3 shows promise in clinical trials. Adeno-associated virus-mediated gene transfer and gene editing techniques are in early clinical and preclinical development. SUMMARY: LDL-C lowering in HoFH patients remains very challenging. However, novel treatment options are emerging. Upcoming therapies directed at PCSK9 and ANPTL3 may offer additional LDL-C reduction, to help patients achieve adequate LDL-C levels. Gene therapy and gene editing techniques, if proven effective, may offer a unique opportunity to treat patients with a one-time treatment. 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. PURPOSE OF REVIEW: Homozygous familial hypercholesterolemia (HoFH) is an orphan disease caused by biallelic mutations at the LDL receptor (LDLR) gene, with a prevalence estimated at 1 : 250 000 to 1 : 630 000. HoFH is characterized by extremely elevated plasma levels of LDL-C greater than 10 mmol/l (>387 mg/dl), tendinous and cutaneous xanthomas in youth and premature atherosclerotic cardiovascular disease (ASCVD). The expected prevalence varies from country to country depending on the presence of founder effects, genetic probability and life expectancy. Untreated, HoFH is a fatal condition before age 30. Plasma levels of LDL-C are the major cause of mortality and the therapeutic target. Statin therapy led to a remarkable improvement in survival but is of limited use in loss-of-function LDLR gene variants or 'null' mutations. Inhibitors of PCSK9 are a useful adjunct in patients with LDLR mutations with residual activity. Extracorporeal LDL filtration has improved survival since its introduction three decades ago. RECENT FINDINGS: Novel therapies, not dependent on a functioning LDLR include lomitapide and mipomersen, which decrease hepatic apolipoprotein B secretion, and evinacumab, directed at the angiopoietin like-3 protein (ANGPLT-3). SUMMARY: Over the past 3-4 decades, the survival of patients with HoFH has increased markedly. New therapeutic options offer new hope. BACKGROUND: Homozygous familial hypercholesterolemia is characterized by premature cardiovascular disease caused by markedly elevated levels of low-density lipoprotein (LDL) cholesterol. This disorder is associated with genetic variants that result in virtually absent (null-null) or impaired (non-null) LDL-receptor activity. Loss-of-function variants in the gene encoding angiopoietin-like 3 (ANGPTL3) are associated with hypolipidemia and protection against atherosclerotic cardiovascular disease. Evinacumab, a monoclonal antibody against ANGPTL3, has shown potential benefit in patients with homozygous familial hypercholesterolemia. METHODS: In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned in a 2:1 ratio 65 patients with homozygous familial hypercholesterolemia who were receiving stable lipid-lowering therapy to receive an intravenous infusion of evinacumab (at a dose of 15 mg per kilogram of body weight) every 4 weeks or placebo. The primary outcome was the percent change from baseline in the LDL cholesterol level at week 24. RESULTS: The mean baseline LDL cholesterol level in the two groups was 255.1 mg per deciliter, despite the receipt of maximum doses of background lipid-lowering therapy. At week 24, patients in the evinacumab group had a relative reduction from baseline in the LDL cholesterol level of 47.1%, as compared with an increase of 1.9% in the placebo group, for a between-group least-squares mean difference of -49.0 percentage points (95% confidence interval [CI], -65.0 to -33.1; P<0.001); the between-group least-squares mean absolute difference in the LDL cholesterol level was -132.1 mg per deciliter (95% CI, -175.3 to -88.9; P<0.001). The LDL cholesterol level was lower in the evinacumab group than in the placebo group in patients with null-null variants (-43.4% vs. +16.2%) and in those with non-null variants (-49.1% vs. -3.8%). Adverse events were similar in the two groups. CONCLUSIONS: In patients with homozygous familial hypercholesterolemia receiving maximum doses of lipid-lowering therapy, the reduction from baseline in the LDL cholesterol level in the evinacumab group, as compared with the small increase in the placebo group, resulted in a between-group difference of 49.0 percentage points at 24 weeks. (Funded by Regeneron Pharmaceuticals; ELIPSE HoFH ClinicalTrials.gov number, NCT03399786.). Publisher: La quilomicronemia familiar es una condición en que una mutación genética altera la capacidad de metabolizar los triglicéridos que viajan en las lipoproteínas, causando elevación extrema de triglicéridos plasmáticos y complicaciones asociadas. La complicación más frecuente es la pancreatitis, que puede llevar a falla multiorgánica o insuficiencia pancreática. La quilomicronemia familiar también afecta la calidad de vida, las relaciones sociales y el desarrollo profesional. El gen más frecuentemente afectado en la quilomicronemia familiar es el de lipoproteína lipasa-1 (LPL), enzima que hidroliza triglicéridos circulantes para su captación tisular. Mutaciones en genes (como APOC2, APOAV, LMF-1, GPIHBP-1) que codifican para proteínas que regulan la maduración, transporte o polimerización de lipoproteína lipasa-1, también pueden estar involucradas. Sin embargo, en cerca del 30% de los pacientes no se encuentra la variante causal. La quilomicronemia familiar debe sospecharse en casos de hipertrigliceridemia extrema, resistente al tratamiento convencional, o que se acompaña de xantomas eruptivos, lipemia retinalis o dolor abdominal. La disponibilidad de escalas de riesgo y pruebas genéticas deben promover la detección oportuna. La nutrición se basa en una dieta muy baja en grasa con adecuada suplencia de vitaminas liposolubles y ácidos grasos esenciales, además de evitar el consumo de alcohol. Si bien el tratamiento farmacológico incluye fibratos y ácidos grasos omega 3, el enfoque actual privilegia agentes biotecnológicos dirigidos a los defectos moleculares propios de la enfermedad. Ello incluye un oligonucleótido antisentido dirigido contra apoC-III (volanesorsen), un anticuerpo monoclonal contra la proteína similar a angiopoietina tipo 3 (evinacumab), y otros compuestos en desarrollo. BACKGROUND: Patients with refractory hypercholesterolemia, who have high low-density lipoprotein (LDL) cholesterol levels despite treatment with lipid-lowering therapies at maximum tolerated doses, have an increased risk of atherosclerosis. In such patients, the efficacy and safety of subcutaneous and intravenous evinacumab, a fully human monoclonal antibody against angiopoietin-like 3, are not known. METHODS: In this double-blind, placebo-controlled, phase 2 trial, we enrolled patients with or without heterozygous familial hypercholesterolemia who had refractory hypercholesterolemia, with a screening LDL cholesterol level of 70 mg per deciliter or higher with atherosclerosis or of 100 mg per deciliter or higher without atherosclerosis. Patients were randomly assigned to receive subcutaneous or intravenous evinacumab or placebo. The primary end point was the percent change from baseline in the LDL cholesterol level at week 16 with evinacumab as compared with placebo. RESULTS: In total, 272 patients were randomly assigned to the following groups: subcutaneous evinacumab at a dose of 450 mg weekly (40 patients), 300 mg weekly (43 patients), or 300 mg every 2 weeks (39 patients) or placebo (41 patients); or intravenous evinacumab at a dose of 15 mg per kilogram of body weight every 4 weeks (39 patients) or 5 mg per kilogram every 4 weeks (36 patients) or placebo (34 patients). At week 16, the differences in the least-squares mean change from baseline in the LDL cholesterol level between the groups assigned to receive subcutaneous evinacumab at a dose of 450 mg weekly, 300 mg weekly, and 300 mg every 2 weeks and the placebo group were -56.0, -52.9, and -38.5 percentage points, respectively (P<0.001 for all comparisons). The differences between the groups assigned to receive intravenous evinacumab at a dose of 15 mg per kilogram and 5 mg per kilogram and the placebo group were -50.5 percentage points (P<0.001) and -24.2 percentage points, respectively. The incidence of serious adverse events during the treatment period ranged from 3 to 16% across trial groups. CONCLUSIONS: In patients with refractory hypercholesterolemia, the use of evinacumab significantly reduced the LDL cholesterol level, by more than 50% at the maximum dose. (Funded by Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT03175367.). Introduction: Homozygous Familial Hypercholesterolemia (HoFH) is a very severe genetic form of hypercholesterolemia. Lacking LDL receptors in the liver, subjects with HoFH have raised plasma levels of LDL cholesterol, and up to 100 times higher risk of premature atherosclerotic cardiovascular disease than the general population.Areas covered: This evaluation is of a phase 3 trial of evinacumab; Evinacumab Lipid Studies in Patients with Homozygous Familial Hypercholesterolemia (ELIPSE HoFH). Evinacumab is a human monoclonal antibody inhibitor of angiopoietin-like protein 3 (ANGPTL3). In ELIPSE HoFH, evinacumab reduced LDL cholesterol by 47.1 ± 4.6%, HDL cholesterol by 30.4%, and triglycerides by 50.4 ± 7.7%.Expert opinion: Evinacumab is not the ideal treatment for HoFH as it does not reduce LDL cholesterol levels to treatment targets while increasing HDL cholesterol. Although the incidence of adverse effects with evinacumab was low in ELIPSE HoFH, further studies are necessary to clarify its effects on liver enzymes and clinical cardiovascular outcomes. Evinacumab is a candidate to become the standard treatment for HoFH, as it may be better tolerated and/or more efficacious than the presently available specific treatment (lomitapide). However, the widespread use of evinacumab to treat high triglycerides or LDL cholesterol is unlikely due to evinacumab decreasing HDL cholesterol.

Do honey contain diastases/amylases?

Yes honey contain the protein amylase.

Previous experiments to separate the amylase and "invertase" of honey by chromatography on sephadex-gels were unsuccesful. It was shown that honey-amylase, -like amylases form other sources -- has hydrophobic properties. Therefore it was possible to separate amylase-activity from other activities by means of hydrophobic affinity chromatography on phenyl-butylamine-Sepharose 4B. The major alpha-amylase in honey was characterized. The optimum pH range and temperature were determined for the enzyme as 4.6 to 5.3 and 55 degrees C, respectively. The enzyme was stable at pH values from 7 to 8. The half-lives of the purified enzyme at different temperatures were determined. The activation energy for heat inactivation of honey amylase was 114.6 kJ/mol. The enzyme exhibited Michaelis-Menten kinetics with soluble starch and gave KM and Vmax values of 0.72 mg/mL and 0.018 units/mL, respectively. The enzyme was inhibited by CuCl (34.3%), MgCl2 (22.4%), and HgCl2 (13.4%), while CaCl2, MnCl2, and ZnSO4 did not have any effect. Starch had a protective effect on thermal stability of honey amylase. Therefore, it might be critical to process or control the amylase in honey before incorporation into starch-containing foods to aid in the preservation of starch functionality. One step could involve heat treating honey with other ingredients, especially those that dilute and acidify the honey environment. A new rapid method for the determination of honey diastase activity using direct potentiometric principles has been proposed. A platinum redox sensor has been used to quantify the amount of free triiodide released from a starch triiodide complex after starch hydrolysis by honey diastase. The method was tested on honey samples with varying diastase activities. The first 5 min of data for each sample were used for linear regression analysis in order to calculate diastase activity. The new method was compared with classical Schade and commercial Phadebas procedures. The results showed good correlations with both methods and offered a simple method for unit conversion to DN units for diastase activity, making the method suitable for routine analysis. This study aimed to screen alpha-amylase producing microorganisms from honey as a low water activity medium, a suitable source for selecting stable and cost-beneficial bacterial enzyme production systems. Plackett-Burman method was used to select twelve effective factors including pH, inoculum size, temperature, time, corn starch, KH2PO4, peptone, MgSO4, CaCl2, NaCl, glycerin, and yeast extract concentrations on bacterial alpha-amylases production yield. The Box-Behnken method was utilized to optimize the level of selected significant factors. The stability of bacterial alpha-amylases was also determined in low pH and high-temperature conditions. In addition, in silico study was used to create the alpha-amylase structure and study the stability in high-temperature and low water available condition. Among all isolated and characterized microorganisms, Bacillus megaterium produced the highest amount of alpha-amylases. The in silico data showed the enzyme 3D structure similarity to alpha-amylase from Halothermothrix orenii and highly negative charge amino acids on its surface caused the enzyme activity and stability in low water conditions. Based on Box-Behnken results, the temperature 35 °C, pH 6 and starch 40 g/l were determined as the optimum level of significant factors to achieve the highest alpha-amylases unit (101.44 U/ml). This bacterial alpha-amylases enzyme showed stability at pH 5 and a range of temperatures from 40 to 60 °C that indicates this enzyme may possess the potential for using in industrial processes.

What year was the first successful human heart transplant performed?

The first human heart transplant in 1967 was performed using a deceased donor heart,

The world's first human-to-human heart transplant was performed at Groote Schuur Hospital on the 2nd December 1967. Between 1967 and 1973, 10 patients underwent orthotopic heart transplantation. Four lived for more than 1 year. The longest survivor died after 12 1/2 years, and one patient remains alive and fully employed 11 1/2 years after transplantation. Since 1974, 44 patients have undergone heterotopic heart transplantation, whereby the donor heart is inserted in parallel with the recipient's own heart. Four of these patients have undergone retransplantation for acute or chronic rejection. Survival has been almost 60% for 1 year, falling to 21% by 5 years. The major complications of heart transplantation have been early acute and late chronic rejection; immunosuppression has been complicated by a high incidence of infection, particularly during the first year, and by a 10% incidence of the development of maligt tumors. A portable hypothermic perfusion system has been developed to store and transport donor hearts for periods of up to 24 hours. For centuries, the medical community has known that human cadavers and human organs are invaluable to medical science for transplantations. On December 3, 1967, in Cape Town, South Africa, Dr. Christian Barnard revolutionized organ transplantation with the first successful human heart transplant. With it came the dawning of a new era--life for one preserved through the death of another. But with this advancement came great demands. Patients waited, hoping sometimes in vain, that a kidney transplant would end their long hours of suffering on dialysis, or that a heart transplant would prolong their otherwise shortened existence. Expectations grew, but the supply of organs did not. It became clear that the full and continued benefit of such medical advances depended on an increased supply of human organs for transplantation. This article focuses on the issues surrounding the procurement of human organs for transplantation. A brief history of transplantation and the benefits of transplantation are presented, as well as the present means of supplying organs for transplantation and how this supply could be enhanced by alternate procurement methods. The field of heart transplantation was built upon the discoveries of immunity and tolerance by Landsteiner, Medawar, Burnet, and others, as well as technical advancements in surgical technique by Carrel. Since the first successful human heart transplant performed by Christiaan Barnard in 1967, there has been substantial progress in the field of heart transplantation, especially over the last several decades. With advances in immunosuppression and surgical techniques, the rates of acute rejection and infection leading to graft failure have declined. However, the detection of acute and chronic allograft rejection remains one of the most important yet unsettled matters. As such, many new horizons exist for further advancement of the field of heart transplantation and for improving the outcomes of the patients we serve. It has been 40 years since the first human-to-human heart transplant performed in South Africa by Christiaan Barnard in December 1967. This achievement did not come as a surprise to the medical community but was the result of many years of early pioneering experimental work by Alexis Carrel, Frank Mann, Norman Shumway, and Richard Lower. Since then, refinement of donor and recipient selection methods, better donor heart management, and advances in immunosuppression have significantly improved survival. In this article, we hope to give a perspective on the changing face of heart transplantation. Topics that will be covered in this review include the changing patient population as well as recent advances in transplantation immunology, organ preservation, allograft vasculopathy, and immune tolerance. Christiaan (Chris) Barnard was born in 1922 and qualified in medicine at the University of Cape Town in 1946. Following surgical training in South Africa and the USA, Barnard established a successful open-heart surgery programme at Groote Schuur Hospital and the University of Cape Town in 1958. In 1967, he led the team that performed the world's first human-to-human heart transplant. The article describing this remarkable achievement was published in the South African Medical Journal just three weeks after the event and is one of the most cited articles in the cardiovascular field. In the lay media as well, this first transplant remains the most publicised event in world medical history. Although the first heart transplant patient survived only 18 days, four of Groote Schuur Hospital's first 10 patients survived for more than one year, two living for 13 and 23 years, respectively. This relative success amid many failures worldwide did much to generate guarded optimism that heart transplantation would eventually become a viable therapeutic option. This first heart transplant and subsequent ongoing research in cardiac transplantation at the University of Cape Town and in a few other dedicated centres over the subsequent 15 years laid the foundation for heart transplantation to become a well-established form of therapy for end-stage cardiac disease. During this period from 1968 to 1983, Chris Barnard and his team continued to make major contributions to organ transplantation, notably the development of the heterotopic ( 'piggy-back') heart transplants; advancing the concept of brain death, organ donation and other related ethical issues; better preservation and protection of the donor heart (including hypothermic perfusion storage of the heart; studies on the haemodynamic and metabolic effects of brain death; and even early attempts at xenotransplantation. Article on the first heart transplant, performed at Groote Schuur Hospital, Cape Town, on 3 December 1967. Reprinted from the SAMJ of 30 December 1967 to commemorate the 50th anniversary of the transplant. The first human-to-human heart transplant was performed 50 years ago in 1967. Heart transplantation has now entered an era of tremendous growth and innovation. The future of heart transplantation is bright with the advent of newer immunosuppressive medications and strategies that may even result in tolerance. Much of this progress in heart transplant medicine is predicated on a better understanding of acute and chronic rejection pathways through basic science studies. The future will also include personalized medicine where genomics and molecular science will dictate customized treatment for optimal outcomes. The introduction of mechanical circulatory support (MCS) devices has changed the landscape for patients with severe heart failure to stabilize the most ill patient and make them better candidates for heart transplant. As ex vivo preservation takes hold, we may witness an expansion of the donor pool through the use of donation after cardiac death (DCD) donors. In addition, further geographical donor heart sharing through ex vivo preservation may further decrease waitlist mortality by enabling longer distance donor hearts to be allocated for the sickest waitlist patient. It is no doubt an exciting time to be involved in the field of heart transplantation. In this perspective, we will summarize the present state of heart transplantation and discuss various innovations that are being pursued. 50 years have passed since the first human to human heart transplantation, performed by Christiaan Barnard in Cape Town December 3rd 1967. Over the years there has been a dramatic improvement in postoperative survival, mainly due to numerous diagnostic and therapeutic advances. Today, heart transplantation constitutes the treatment of choice among suitable patients with severe heart failure who worsen despite medical and surgical optimization. The world average 10-year survival has now reached more than 58 %. This text summarizes the past, present and future. In 2017, we celebrated the 50th anniversary of the first human heart transplant that had been carried out by the South African surgeon, Christiaan ('Chris') Barnard at Groote Schuur Hospital in Cape Town on December 3rd, 1967. The daring operation and the charismatic surgeon received immense public attention around the world. The patient's progress was covered by the world's media on an almost hourly basis. Although the patient, Mr. Louis Washansky, died after only 18 days, Barnard soon carried out a second transplant, and this patient led an active life for almost 19 months. Remarkably, Barnard's fifth and sixth patients lived for almost 13 and 24 years, respectively. Barnard subsequently introduced the operation of heterotopic heart transplantation in which the donor heart acted as an auxiliary pump, with some advantages in that early era. It took great courage to carry out the first heart transplant, and this is why Barnard is remembered as a pioneer in cardiac surgery. The gold standard and sole curative therapy for advanced stage heart failure is cardiac transplantation. As the population ages, the number of patients diagnosed with advanced heart failure and listed for transplant steadily increases annually. However, there remains a paucity of eligible donation after brain death (DBD) donor hearts which severely limits access to cardiac transplantation and leads to increasing wait-list times and avoidable patient mortalities. Though the first human heart transplant in 1967 was performed using a deceased donor heart, the advent of brain death criteria and the ability to avoid long warm ischemic times led donation after cardiac death (DCD) transplantation to fall out of favor. Due the current state of cardiac transplantation, there has been a resurgence in interest in DCD heart transplantation leading to the development of DCD heart transplantation programs in the UK and Australia after positive reports of successful DCD cardiac transplantation in the pediatric literature. These programs have demonstrated favorable post-transplantation outcomes equivalent to matched traditional DBD transplants with current techniques and strict donor criteria. This technique has been proven safe with favorable outcomes and has been demonstrated to significantly increase transplant volumes and decrease patient mortality. Given these outcomes and the high patient benefit to risk ratio, DCD donor heart transplantation is necessary to expand the donor pool and decrease patient mortality and should be developed in high volume experienced cardiac transplant centers. The first successful human heart transplantation was reported on 3 December 1967, by Christiaan Barnard in South Africa. Since then this life-saving procedure has been performed in over 120 000 patients. A limitation to the performance of this procedure is the availability of donor hearts with as many as 20% of patients dying before a donor's heart is available for transplant. Today, hearts for transplantation are procured from individuals experiencing donation after brain death (DBD). Interestingly, this, however, was not always the case as the first heart transplants occurred after circulatory death. Revisiting the availability of hearts for transplant from those experiencing donation after circulatory death (DCD) could further expand the number of hearts suitable for transplantation. There are several considerations pertinent to transplanting hearts from those undergoing circulatory death. In this review, we summarize the main distinctions between DBD and DCD heart donation and discuss the research relevant to increasing the number of hearts available for transplantation by including individual's hearts that experience circulatory death.

Which disease is monitored in the BIOCURA cohort?

Rheumatoid Arthritis (RA) is one of the diseases that is monitored in the BIOCURA cohort. There are other diseases that are monitored as well, such as breast cancer, ovarian cancer, and thyroid cancer.

BACKGROUND: In rheumatoid arthritis, prediction of response to TNF-alpha inhibitor (TNFi) treatment would be of clinical value. This study aims to discover miRNAs that predict response and aims to replicate results of two previous studies addressing this topic. METHODS: From the observational BiOCURA cohort, 40 adalimumab- (ADA) and 40 etanercept- (ETN) treated patients were selected to enter the discovery cohort and baseline serum profiling on 758 miRNAs was performed. The added value of univariately selected miRNAs (p < 0.05) over clinical parameters in prediction of response was determined by means of the area under the receiver operating characteristic curve (AUC-ROC). Validation was performed by TaqMan single qPCR assays in 40 new patients. RESULTS: Expression of miR-99a and miR-143 predicted response to ADA, and miR-23a and miR-197 predicted response to ETN. The addition of miRNAs increased the AUC-ROC of a model containing only clinical parameters for ADA (0.75 to 0.97) and ETN (0.68 to 0.78). In validation, none of the selected miRNAs significantly predicted response. miR-23a was the only overlapping miRNA compared to the two previous studies, however inversely related with response in one of these studies. The reasons for the inability to replicate previously proposed miRNAs predicting response to TNFi and replicate those from the discovery cohort were investigated and discussed. CONCLUSIONS: To date, no miRNA consistently predicting response to TNFi therapy in RA has been identified. Future studies on this topic should meet a minimum of standards in design that are addressed in this study, in order to increase the reproducibility. OBJECTIVE: In rheumatoid arthritis (RA), it is of major importance to identify non-responders to tumour necrosis factor-α inhibitors (TNFi) before starting treatment, to prevent a delay in effective treatment. We developed a protein score for the response to TNFi treatment in RA and investigated its predictive value. METHOD: In RA patients eligible for biological treatment included in the BiOCURA registry, 53 inflammatory proteins were measured using xMAP® technology. A supervised cluster analysis method, partial least squares (PLS), was used to select the best combination of proteins. Using logistic regression, a predictive model containing readily available clinical parameters was developed and the potential of this model with and without the protein score to predict European League Against Rheumatism (EULAR) response was assessed using the area under the receiving operating characteristics curve (AUC-ROC) and the net reclassification index (NRI). RESULTS: For the development step (n = 65 patient), PLS revealed 12 important proteins: CCL3 (macrophage inflammatory protein, MIP1a), CCL17 (thymus and activation-regulated chemokine), CCL19 (MIP3b), CCL22 (macrophage-derived chemokine), interleukin-4 (IL-4), IL-6, IL-7, IL-15, soluble cluster of differentiation 14 (sCD14), sCD74 (macrophage migration inhibitory factor), soluble IL-1 receptor I, and soluble tumour necrosis factor receptor II. The protein score scarcely improved the AUC-ROC (0.72 to 0.77) and the ability to improve classification and reclassification (NRI = 0.05). In validation (n = 185), the model including protein score did not improve the AUC-ROC (0.71 to 0.67) or the reclassification (NRI = -0.11). CONCLUSION: No proteomic predictors were identified that were more suitable than clinical parameters in distinguishing TNFi non-responders from responders before the start of treatment. As the results of previous studies and this study are disparate, we currently have no proteomic predictors for the response to TNFi. BACKGROUND: Several studies have employed microarray-based profiling to predict response to tumor necrosis factor-alpha inhibitors (TNFi) in rheumatoid arthritis (RA); yet efforts to validate these targets have failed to show predictive abilities acceptable for clinical practice. METHODS: The eighty most extreme responders and nonresponders to TNFi therapy were selected from the observational BiOCURA cohort. RNA sequencing was performed on mRNA from peripheral blood mononuclear cells (PBMCs) collected before initiation of treatment. The expression of pathways as well as individual gene transcripts between responders and nonresponders was investigated. Promising targets were technically replicated and validated in n = 40 new patients using qPCR assays. RESULTS: Before therapy initiation, nonresponders had lower expression of pathways related to interferon and cytokine signaling, while also showing higher levels of two genes, GPR15 and SEMA6B (p = 0.02). The two targets could be validated, however, additional analyses revealed that GPR15 and SEMA6B did not independently predict response, but were rather dose-dependent markers of smoking (p < 0.0001). CONCLUSIONS: The study did not identify new transcripts ready to use in clinical practice, yet GPR15 and SEMA6B were recognized as candidate explanatory markers for the reduced treatment success in RA smokers.

Which gene is associated with response to abacavir?

Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir.

Many pharmacogenomic biomarkers (PGBM) were identified and translated into clinical practice, affecting the usage of drugs via label updates. In this context, abacavir is one of the most brilliant examples of pharmacogenetic studies translated into clinical practice. Pharmacogenetic studies have revealed that abacavir HSRs are highly associated with the major histocompatibility complex class I. Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results the abacavir label has been modified: the European Medicines Agency (EMA) and the FDA recommend/suggested that the administration of abacavir must be preceded by a specific genotyping test. The HLA locus is extremely polymorphic, exhibiting many closely related alleles, making it difficult to discriminate HLA-B*57:01 from other related alleles, and a number of different molecular techniques have been developed recently to detect the presence of HLA-B*57:01. In this review, we provide a summary of the available techniques used by laboratories to genotype HLA-B*57:01, outlining the scientific and pharmacoeconomics pros and cons.

Is adenosine signaling prognostic for cancer outcome?

Yes, adenosine signaling has been shown to be prognostic for cancer outcome.

Which drugs are included in the CNIC polypill?

CNIC polypill includes atorvastatin 40mg, ramipril 10mg and aspirin 100mg.

Aim: To determine the effectiveness of Centro Nacional de Investigaciones Cardiovasculares (CNIC)-polypill (acetylsalicylic acid 100 mg, ramipril 5/10 mg, simvastatin 40 mg) in achieving blood pressure (BP) goals. Patients & methods: A multicenter, observational, one cohort, prospective study. BP targets were analyzed in patients with cardiovascular disease after 12-months treatment with the CNIC polypill. Results: A total of 572 patients (59.4 ± 13.9 years, 57.3% men) were analyzed. At baseline, BP was 147.1 ± 18.1/88.3 ± 10.6 mmHg, 97.1% of patients were taken renin-angiotensin system inhibitors, 5.4% calcium antagonists, 1.9% diuretics and 13.1% β-blockers. The proportion of patients who achieved BP targets increased from 20.1 to 55.4% (p < 0.001). Conclusion: In routine practice, switching from usual care to the CNIC-polypill in patients with cardiovascular disease could facilitate achieving BP goals. INTRODUCTION AND OBJECTIVES: To compare the pharmacodynamics of the CNIC polypill (atorvastatin 40mg/ramipril 10mg/aspirin 100mg) in terms of low-density lipoprotein cholesterol (LDL-C) and systolic blood pressure (SBP), with the corresponding reference products (atorvastatin and ramipril). METHODS: This was a multicenter, randomized, open-label, and parallel 3-arm study comparing the effect of the CNIC polypill vs ramipril 10mg and atorvastatin 40mg on SBP and LDL-C. The coprimary endpoints were differences in the adjusted mean 24-hour SBP (using ambulatory BP measurement) and LDL-C during the study period estimated using an ANCOVA model. RESULTS: Of the 241 patients included in the per protocol population, 84 received the CNIC polypill (group A), 84 atorvastatin (group B), and 73 ramipril (group C). SBP decreased from 139.3±12.5 to 133.2±12.9mmHg in group A and from 138.1±11.9 to 134.0±12.8mmHg in group C (baseline adjusted mean difference for the decrease in SBP was 1.77mmHg (90%CI, -0.5 to 4.0) in favor of group A, without reaching statistical significance. LDL-C was reduced by 33.9±21.6 and 29.2±25.8mg/dL in groups A and B, respectively (baseline adjusted mean difference for the decrease in LDL-C was 7.0% (90%CI, 1.5-12.4), a significantly greater decrease with the polypill). The 3 treatments were well tolerated. CONCLUSIONS: The results of this study rule out a negative effect on blood pressure of the interaction between the components of the CNIC polypill. The reduction in LDL-C was greater in the CNIC polypill group, suggesting a synergistic effect of the components.

Which human tissue synthesize CRP?

CRP is predominantly produced in the liver in a native pentameric form (nCRP).

In conditions of acute and chronic inflammation hepatic detoxification capacity is severely impaired due to coordinated downregulation of drug metabolizing enzymes and transporters. Using global transcriptome analysis of liver tissue from donors with pathologically elevated C-reactive protein (CRP), we observed comparable extent of positive and negative acute phase response, where the top upregulated gene sets included immune response and defense pathways while downregulation occurred mostly in metabolic and catabolic pathways including many important drug metabolizing enzymes and transporters. We hypothesized that microRNAs (miRNA), which usually act as negative regulators of gene expression, contribute to this process. Microarray and quantitative real-time PCR analyses identified differentially expressed miRNAs in liver tissues from donors with elevated CRP, cholestasis, steatosis, or non-alcoholic steatohepatitis. Using luciferase reporter constructs harboring native and mutated 3'-untranslated gene regions, several predicted miRNA binding sites on RXRα (miR-130b-3p), CYP2C8 (miR-452-5p), CYP2C9 (miR-155-5p), CYP2C19 (miR-155-5p, miR-6807-5p), and CYP3A4 (miR-224-5p) were validated. HepaRG cells transfected with miRNA mimics showed coordinate reductions in mRNA levels and several cytochrome P450 enzyme activities particularly for miR-155-5p, miR-452-5p, and miR-6807-5p, the only miRNA that was deregulated in all four pathological conditions. Furthermore we observed strong negative correlations between liver tissue miRNA levels and hepatic CYP phenotypes. Since miR-155 is well known for its multifunctional roles in immunity, inflammation, and cancer, our data suggest that this and other miRNAs contribute to coordinated downregulation of drug metabolizing enzymes and transporters in inflammatory conditions.

What is the relationship between the X chromosome and a neutrophil drumstick?

In particular, up to 17% of neutrophil nuclei of healthy women exhibit a drumstick-shaped appendage that contains the inactive X chromosome.

The sex chromosomal constitution has been determined in various types of human leukocytes at interphase by use of fluorescence in situ hybridization with X- and/or Y-specific DNA probes. It is found that during aging and differentiation of myelocytes into polymorphs there is no significant change in the relative frequency of various types of male and female cells with a specific type of sex chromosomal constitution. Non-random variability of the relative proximity between the X chromosomes within the nuclei is also observed in female cells. Moreover, we are the first to determine that sex-specific "drumsticks" and "sessile nodules" in female polymorphs originate from the X chromosomes and that non-sex-specific "drumstick-like" bodies in male polymorphs are of Y chromosomal origin. An X chromosome specific nucleic acid probe was used to study the positions of the X chromosomes in leukocyte nuclei by in situ hybridization to smears of peripheral blood. This autoradiographic approach allowed the first direct demonstration of the presence of X chromosomal material in the drumstick-like structures of female polymorphonuclear leukocytes. An individual with normal male habitus, body proportions, and secondary sexual characteristics was admitted to the hospital with head trauma. A routine blood smear demonstrated that 36% of the granulocytes had "drumsticks". Chromosomal analysis revealed a 46,XYqh+ karyotype. the extremely large Y chromosome was located by quinacrine fluorescence in the "drumstick" of the polymorphonuclear granulocytes. The presence of a large Y chromosome may thus produce pseudo-drumsticks. Fluorescent staining can distinguish between true drumsticks bearing the inactive X of normal females and the pseudo-drumsticks in a normal male produced by a large Y chromosome. The nuclei of human neutrophils typically consist of a linear array of three or four lobes joined by DNA-containing filaments. Terminal lobes are connected to internal lobes via a single filament, while internal lobes have two filaments, each to an adjacent lobe. Some lobes also have appendages of various shapes and sizes. In particular, up to 17% of neutrophil nuclei of healthy women exhibit a drumstick-shaped appendage that contains the inactive X chromosome. This report provides a detailed analysis of the relationship between nuclear morphology and the location of the X and Y chromosomes in human neutrophils. Fluorescent in situ hybridization analysis revealed that the X and the Y chromosomes of male neutrophil nuclei are randomly distributed among nuclear lobes. Similarly, in female neutrophil nuclei with a drumstick appendage, the active X chromosome is also randomly distributed among lobes. In contrast, the inactive X chromosome is preferentially located in a terminal lobe in over 90% nuclei with drumsticks. Within the terminal lobe of nuclei with drumsticks, the inactive X chromosome lies distal to the point of filament attachment in 80% of the nuclei. The inactive X chromosome also exhibits a specific orientation within the drumstick appendage, with over 95% of nuclei having the X centromere located toward the tip of the appendage. Female nuclei without a drumstick appendage also have one of the X chromosomes (presumably the inactive chromosome) preferentially situated in a terminal lobe. Nonrandom distribution of the inactive X chromosome is discussed in the context of a model that considers chromosomes as determits of neutrophil nuclear morphology. Granulocytic early progenitors and terminally differentiated - mature granulocytes with segmented nuclei were studied using computer-assisted diameter and heterochromatin optical image densitometry to provide more information on the nuclear size and heterochromatin condensation state. Bone marrow smears of patients suffering from chronic myeloid leukaemia untreated as well as treated with "specific" anti-leukaemic therapy with imatinib mesylate are a convenient model for such study because they possess a satisfactory number of cells for diameter and optical density measurements. In addition, the identification of developmental stages of granulocytes is very easy and the morphology is not different from that in not-leukaemic persons. As it was expected, the mean diameter of nuclear segments in fully differentiated and mature granulocytes was much smaller than that in non-segmented nuclei of early granulocytic precursors. Therefore, no wonder that the heterochromatin condensation state in nuclear segments of mature granulocytes was much larger than in non-segmented nuclei of granulocytic progenitors. On the other hand, the sum of mean diameters of all nuclear segments per cell was close to the mean nuclear diameter of early granulocytic progenitors. The heterochromatin condensation state in granulocytic progenitors or fully differentiated mature granulocytes exhibited marked stability and did not change after the anti-leukaemic therapy. In addition, Barr bodies of characteristic drumstick appearance bearing inactive X chromosome in interphase nuclei of mature granulocytes in fertile female patients exhibited a heterochromatin condensation state similar to nuclear segments. This heterochromatin condensation state was also stable and constant, and was not apparently influenced by the anti-leukaemic therapy.

The formation of which inflammatory molecule is regulated by MAP3K8 (TPL2)?

MAP3K8 (Tpl2) regulates the formation of inflammatory molecule IL-1β

OBJECTIVE: Activation of extracellular signal-regulated kinase-(ERK)-1/2 by cytokines in adipocytes is involved in the alterations of adipose tissue functions participating in insulin resistance. This study aims at identifying proteins regulating ERK1/2 activity, specifically in response to inflammatory cytokines, to provide new insights into mechanisms leading to abnormal adipose tissue function. RESEARCH DESIGN AND METHODS: Kinase activities were inhibited with pharmacological inhibitors or siRNA. Lipolysis was monitored through glycerol production. Gene expression in adipocytes and adipose tissue of obese mice and subjects was measured by real-time PCR. RESULTS: IkappaB kinase-(IKK)-beta inhibition prevented mitogen-activated protein (MAP) kinase kinase (MEK)/ERK1/2 activation in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha but not insulin in 3T3-L1 and human adipocytes, suggesting that IKKbeta regulated a MAP kinase kinase kinase (MAP3K) involved in ERK1/2 activation induced by inflammatory cytokines. We show that the MAP3K8 called Tpl2 was expressed in adipocytes and that IL-1beta and TNF-alpha activated Tpl2 and regulated its expression through an IKKbeta pathway. Pharmacological inhibition or silencing of Tpl2 prevented MEK/ERK1/2 activation by these cytokines but not by insulin, demonstrating its involvement in ERK1/2 activation specifically in response to inflammatory stimuli. Importantly, Tpl2 was implicated in cytokine-induced lipolysis and in insulin receptor substrate-1 serine phosphorylation. Tpl2 mRNA expression was upregulated in adipose tissue of obese mice and patients and correlated with TNF-alpha expression. CONCLUSIONS: Tpl2 is selectively involved in inflammatory cytokine-induced ERK1/2 activation in adipocytes and is implicated in their deleterious effects on adipocyte functions. The deregulated expression of Tpl2 in adipose tissue suggests that Tpl2 may be a new actor in adipose tissue dysfunction in obesity. Tumor progression locus 2 (Tpl2, also known as Map3k8 and Cot) is a serine-threonine kinase critical in innate immunity, linking toll-like receptors (TLRs) to TNF production through its activation of ERK. Tpl2(-/-) macrophages have abrogated TNF production but overproduce IL-12 in response to TLR ligands. Despite enhanced IL-12 production, Tpl2(-/-) T cells have impaired IFN-gamma production. Therefore, the role of Tpl2 in a bona fide bacterial infection where all of these cytokines are important in host defense is unclear. To address this issue, we infected Tpl2(-/-) mice with the model pathogen Listeria monocytogenes. We found that Tpl2(-/-) mice infected i.v. with L. monocytogenes had increased pathogen burdens compared with wild-type mice and rapidly succumbed to infection. Enhanced susceptibility correlated with impaired signaling through TLR2 and nucleotide-binding oligomerization domain 2, two receptors previously shown to mediate Listeria recognition. Surprisingly, TNF production in response to infection was not significantly impaired, even though Tpl2 has been implicated in the regulation of TNF. We found that the role of Tpl2 has cell-type specific effects in regulating TNF and transduces signals from some, but not all, pattern recognition receptors (PRR). In contrast to the cell-type- and receptor-specific regulation of TNF, we found that Tpl2 is essential for IL-1beta production from both macrophages and dendritic cells. These studies implicate Tpl2 as an important mediator for collaboration of pattern recognition receptors with danger-associated molecular patterns to induce TNF and IL-1beta production and optimal host defense. Cot/tpl2 (also known as MAP3K8) has emerged as a new and potentially interesting therapeutic anti-inflammatory target. Here, we report the first study of Cot/tpl2 involvement in acute peripheral inflammation in vivo. Six hours after an intraplantar injection of zymosan, Cot/tpl2(-/-) mice showed a 47% reduction in myeloperoxidase activity, concomitant with a 46% lower neutrophil recruitment and a 40% decreased luminol-mediated bioluminescence imaging in vivo. Accordingly, Cot/tpl2 deficiency provoked a 25-30% reduction in luminol-mediated bioluminescence and neutrophil recruitment together with a 65% lower macrophage recruitment 4 h following zymosan-induced peritonitis. Significantly impaired levels of G-CSF and GM-CSF and of other cytokines such as TNFα, IL-1β, and IL-6, as well as some chemokines such as MCP-1, MIP-1β, and keratinocyte-derived chemokine, were detected during the acute zymosan-induced intraplantar inflammatory response in Cot/tpl2(-/-) mice. Moreover, Cot/tpl2 deficiency dramatically decreased the production of the hypernociceptive ligand NGF at the inflammatory site during the course of inflammation. Most importantly, Cot/tpl2 deficiency significantly reduced zymosan-induced inflammatory hypernociception in mice, with a most pronounced effect of a 50% decrease compared with wild type (WT) at 24 h following intraplantar injection of zymosan. At this time, Cot/tpl2(-/-) mice showed significantly reduced NGF, TNFα, and prostaglandin E(2) levels compared with WT littermates. In conclusion, our study demonstrates an important role of Cot/tpl2 in the NGF, G-CSF, and GM-CSF production and myeloperoxidase activity in the acute inflammatory response process and its implication in inflammatory hypernociception. Interleukin-22 (IL-22), one of the cytokines secreted by T-helper 17 (Th17) cells, binds to a class II cytokine receptor containing an IL-22 receptor 1 (IL-22R1) and IL-10R2 and influences a variety of immune reactions. IL-22 has also been shown to modulate cell cycle and proliferation mediators such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but little is known about the underlying molecular mechanisms of IL-22 in tumorigenesis. In this paper, we propose that IL-22 has a crucial role to play in controlling epithelial cell proliferation and tumorigenesis in the breast. IL-22 increased MAP3K8 phosphorylation through IL-22R1, followed by the induction of MEK-ERK, JNK-c-Jun, and STAT3 signaling pathways. Furthermore, IL-22-IL-22R1 signaling pathway activated activator protein-1 and HER2 promoter activity. In addition, Pin1 was identified as a key positive regulator for the phosphorylation-dependent MEK, c-Jun and STAT3 activity induced by IL-22. Pin1(-/-) mouse embryonic fibroblasts (MEF) exhibited significantly a decrease in IL-22-induced MEK1/2, c-Jun, and STAT3 phosphorylation compared with Pin1(+/+) MEF. In addition, a knockdown of Pin1 prevented phosphorylation induced by IL-22. The in vivo chorioallantoic membrane assay also showed that IL-22 increased tumor formation of JB6 Cl41 cells. Moreover, the knockdown of MAP3K8 and Pin1 attenuated tumorigenicity of MCF7 cells. Consistent with these observations, IL-22 levels positively correlate with MAP3K8 and Pin1 expression in human breast cancer. Overall, our findings point to a critical role for the IL-22-induced MAP3K8 signaling pathway in promoting cancer-associated inflammation in the tumor microenvironment. Chronic low-grade inflammation in adipose tissue often accompanies obesity, leading to insulin resistance and increasing the risk for metabolic diseases. MAP3K8 (TPL2/COT) is an important signal transductor and activator of pro-inflammatory pathways that has been linked to obesity-induced adipose tissue inflammation. We used human adipose tissue biopsies to study the relationship of MAP3K8 expression with markers of obesity and expression of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8). Moreover, we evaluated obesity-induced adipose tissue inflammation and insulin resistance in mice lacking MAP3K8 and WT mice on a high-fat diet (HFD) for 16 weeks. Individuals with a BMI >30 displayed a higher mRNA expression of MAP3K8 in adipose tissue compared to individuals with a normal BMI. Additionally, high mRNA expression levels of IL-1β, IL-6 and IL-8, but not TNF -α, in human adipose tissue were associated with higher expression of MAP3K8. Moreover, high plasma SAA and CRP did not associate with increased MAP3K8 expression in adipose tissue. Similarly, no association was found for MAP3K8 expression with plasma insulin or glucose levels. Mice lacking MAP3K8 had similar bodyweight gain as WT mice, yet displayed lower mRNA expression levels of IL-1β, IL-6 and CXCL1 in adipose tissue in response to the HFD as compared to WT animals. However, MAP3K8 deficient mice were not protected against HFD-induced adipose tissue macrophage infiltration or the development of insulin resistance. Together, the data in both human and mouse show that MAP3K8 is involved in local adipose tissue inflammation, specifically for IL-1β and its responsive cytokines IL-6 and IL-8, but does not seem to have systemic effects on insulin resistance. OBJECTIVE: IBD is characterised by dysregulated intestinal immune homeostasis and cytokine secretion. In the intestine, properly regulating pattern recognition receptor (PRR)-mediated signalling and cytokines is crucial given the ongoing host-microbial interactions. TPL2 (MAP3K8, COT) contributes to PRR-initiated pathways, yet the mechanisms for TPL2 signalling contributions in primary human myeloid cells are incompletely understood and its role in intestinal myeloid cells is poorly defined. Furthermore, functional consequences for the IBD-risk locus rs1042058 in TPL2 are unknown. METHODS: We analysed protein, cytokine and RNA expression, and signalling in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR and flow cytometry. RESULTS: PRR-induced cytokine secretion was increased in MDMs from rs1042058 TPL2 GG risk individuals. TPL2 activation by the Crohn's disease-associated PRR nucleotide-oligomerisation domain (NOD)2 required PKC, and IKKβ, IKKα and IKKγ signalling. TPL2, in turn, significantly enhanced NOD2-induced ERK, JNK and NFκB signalling. We found that another major mechanism for the TPL2 contribution to NOD2 signalling was through ERK-dependent and JNK-dependent caspase-1 and caspase-8 activation, which in turn, led to early autocrine interleukin (IL)-1β and IL-18 secretion and amplification of long-term cytokines. Importantly, Salmonella typhimurium-induced cytokines from human intestinal myeloid-derived cells required TPL2 as well as autocrine IL-1β and IL-18. Finally, rs1042058 GG risk carrier MDMs from healthy individuals and patients with Crohn's disease had increased TPL2 expression and NOD2-initiated TPL2 phosphorylation, ERK, JNK and NFκB activation, and early autocrine IL-1β and IL-18 secretion. CONCLUSIONS: Taken together, the rs1042058 GG IBD-risk polymorphism in TPL2 results in a gain-of-function by increasing TPL2 expression and signalling, thereby amplifying PRR-initiated outcomes. Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved. Endogenous noradrenaline (NA) has multiple bioactive functions and, in the central nervous system (CNS), has been implicated in modulating neuroinflammation via β-adrenergic receptors (β-ARs). Microglia, resident macrophages in the CNS, have a central role in the brain immune system and have been reported to be activated by NA. However, intracellular signaling mechanisms of the AR-mediated proinflammatory responses of microglia are not fully understood. Using a rapid and stable in vitro reporter assay system to evaluate IL-1β production in microglial BV2 cells, we found that NA and the β-AR agonist isoproterenol upregulated the IL-1β reporter activity. This effect was suppressed by β-AR antagonists. We further examined the involvement of EPAC (exchange protein directly activated by cAMP) and TPL2 (tumor progression locus 2, MAP3K8) and found that inhibitors for EPAC and TPL2 reduced AR agonist-induced IL-1β reporter activity. These inhibitors also suppressed NA-induced endogenous Il1b mRNA expression and IL-1β protein production. Our results suggest that EPAC and TPL2 are involved in β-AR-mediated IL-1β production in microglial cells, and extend our understanding of its intracellular signaling mechanism.

Which company produces the Oncomine Dx target test?

The Oncomine Dx Target Test Panel is produced by Thermo Fisher Scientific.

Author information: (1)HM Sanchinarro University Hospital-CIBERONC, Madrid, Spain. (2)HM Sanchinarro University Hospital, Madrid, Spain. (3)La Paz University Hospital, Madrid, Spain. (4)Ramon y Cajal University Hospital, IRYCIS and CIBERESP, Madrid, Spain. (5)Germans Trias i Pujol University Hospital, Badalona, Spain. (6)Catalan Institute of Oncology-Germans Trias i Pujol University Hospital, Universitat Autònoma Barcelona (UAB), Badalona-Applied Research Group of Oncology (B-ARGO), Badalona, Spain. (7)General University Hospital-ISABIAL, Alicante, Spain. (8)Institute of Health Research-Jimenez Diaz Foundation-CIBERONC, Madrid, Spain. (9)Institute of Health Research-Jimenez Diaz Foundation, Madrid, Spain. (10)Vall d'Hebron University Hospital, Barcelona, Spain. (11)Quironsalud Hospital, Barcelona, Spain. (12)La Fe University Hospital, Valencia, Spain. (13)Clinico San Carlos University Hospital, Madrid, Spain. (14)Puerta del Mar University Hospital, Cadiz, Spain. (15)Clinico de Santiago University Hospital, Santiago De Compostela, Spain. (16)Insular Materno-Infantil University Hospital Complex, Las Palmas de Gran Canaria, Spain. (17)Clinic Hospital, Barcelona, Spain. (18)Alvaro Cunqueiro Hospital, Vigo, Spain. (19)University of Navarra Clinic, Pamplona, Spain. (20)Marques de Valdecilla University Hospital, Santander, Spain. (21)Hospital del Mar, Barcelona, Spain. (22)Clinico University Hospital, Valencia, Spain. (23)Cruces University Hospital, Baracaldo, Spain. (24)Miguel Servet University Hospital, Zaragoza, Spain. (25)University Hospital of Gran Canaria Doctor Negrin, Las Palmas de Gran Canaria, Spain. (26)12 de Octubre University Hospital, Madrid, Spain. (27)Ramon y Cajal University Hospital, Madrid, Spain. (28)12 de Octubre University Hospital-CIBERONC, Madrid, Spain. (29)Ramon y Cajal University Hospital-CIBERONC, Madrid, Spain. (30)HM Sanchinarro University Hospital-CIBERONC, Madrid, Spain. Electronic address: [email protected].