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Describe the INSPEcT R package

INSPEcT is a computational tool to infer mRNA synthesis, processing and degradation dynamics from RNA- and 4sU-seq time course experiments.

MOTIVATION: Cellular mRNA levels originate from the combined action of multiple regulatory processes, which can be recapitulated by the rates of pre-mRNA synthesis, pre-mRNA processing and mRNA degradation. Recent experimental and computational advances set the basis to study these intertwined levels of regulation. Nevertheless, software for the comprehensive quantification of RNA dynamics is still lacking. RESULTS: INSPEcT is an R package for the integrative analysis of RNA- and 4sU-seq data to study the dynamics of transcriptional regulation. INSPEcT provides gene-level quantification of these rates, and a modeling framework to identify which of these regulatory processes are most likely to explain the observed mRNA and pre-mRNA concentrations. Software performance is tested on a synthetic dataset, instrumental to guide the choice of the modeling parameters and the experimental design. AVAILABILITY AND IMPLEMENTATION: INSPEcT is submitted to Bioconductor and is currently available as Supplementary Additional File S1. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Which molecule is targeted by Teprotumumab?

Teprotumumab is a human monoclonal antibody that targets IGF-1R. It can be used for treatment of thyroid eye disease.

PURPOSE OF REVIEW: In recent years, immunosuppressive therapy, as an alternative to corticosteroids, has been proposed as novel agents which target the various antigens involved in the pathogenesis of Graves' ophthalmopathy. Although the lack of randomized and controlled studies suggests caution in generalizing results, some data show interesting results. RECENT FINDINGS: Potential targets for immune therapy in Graves' ophthalmopathy are the antigens expressed on the target organ of inflammation, namely the receptor and the insulin growth factor -1 receptor on fibroblasts, inflammatory cytokines, and B and T cells. The most promising results are observed with small thyroid stimulating hormone receptor molecules interacting with the receptor on thyrocytes and fibroblasts and with the anti-IGF-1 receptor antibody teprotumumab. A recent open study with tocilizumab, an anti-soluble interleukin-6 receptor, has shown inactivation of Graves' ophthalmopathy. Consistent reports on the efficacy of rituximab will have to be confirmed by randomized controlled trials, which are now in progress. SUMMARY: Current clinical practice for Graves' ophthalmopathy will greatly benefit from the availability of immunosuppressors that act as disease-modifying drugs, as compared to steroids, the current standard treatment for Graves' ophthalmopathy. Rituximab seems to be a good candidate, as preliminary results from ongoing randomized trials suggest good efficacy with a relative well tolerated profile. PURPOSE: Fibrocytes (FC) are bone marrow-derived progenitor cells that are more abundant and infiltrate the thyroid and orbit in Graves orbitopathy (GO). FCs express high levels of thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R). These receptors are physically and functionally associated, but their role in GO pathogenesis is not fully delineated. Treatment of FCs with thyroid stimulating hormone (TSH) or M22 (activating antibody to TSHR) induces the production of numerous cytokines, including tumor necrosis factor α (TNFα). Teprotumumab (TMB) is a human monoclonal IGF-1R blocking antibody currently in clinical trial for GO and inhibits TSHR-mediated actions in FCs. AIM: To characterize the molecular mechanisms underlying TSH-induced TNFα production by FCs, and the role of IGF-1R blockade by TMB. DESIGN: FCs from healthy and GD patients were treated with combinations of TSH, M22, MG132 and AKTi (inhibitors of NF-κB and Akt, respectively), and TMB. TNFα protein production was measured by Luminex and flow cytometry. Messenger RNA expression was quantified by real time PCR. RESULTS: Treatment with TSH/M22 induced TNFα protein and mRNA production by FCs, both of which were reduced when FCs were pretreated with MG132 and AKTi (p<0.0001). TMB decreased TSH-induced TNFα protein production in circulating FCs from mean fluorescent index (MFI) value of 2.92 to 1.91, and mRNA expression in cultured FCs from 141- to 52-fold expression (p<0.0001). TMB also decreased M22-induced TNFα protein production from MFI of 1.67 to 1.12, and mRNA expression from 6- to 3-fold expression (p<0.0001). CONCLUSION: TSH/M22 stimulates FC production of TNFα mRNA and protein. This process involves the transcription factor NF-κB and its regulator Akt. Blocking IGF-1R attenuates TSH/M22-induced TNFα production. This further delineates the interaction of TSHR and IGF1-R signaling pathways. By modulating the proinflammatory properties of FCs such as TNFα production, TMB may be a promising therapeutic agent for GO. The medical treatment of Graves' orbitopathy (GO) is usually reserved to moderate to severe disease. Steroids have been widely employed and possess anti-inflammatory activity, but about 20-30% of patients are not responsive and about 20% present with disease recurrence. Immunosuppressive therapy alternative to corticosteroids may target the different antigens involved in pathogenic mechanisms of GO. Some have already been employed in clinical studies and showed interesting results, although the lack of randomized and controlled trials suggests caution for their use in clinical practice. Potential targets for therapy in GO are the TSH receptor and the IGF-1 receptor on the fibroblasts, inflammatory cytokines, B and T cells. Most promising results are obtained by interacting with the PIK3/mTORC1 signaling cascades for adipogenesis and the anti-IGF-1R with the monoclonal antibody teprotumumab. A recent open study has shown that tocilizumab, an anti-sIL-6R antibody, inactivates GO. Consistent reports on the efficacy of rituximab have recently been challenged by randomized controlled trials. Clinical practice will greatly benefit from the use of disease modifying agents in GO, as compared to steroids, currently standard treatment for GO. Among these, rituximab may be useful, especially in patients resistant to steroid or with contraindications to steroids. However, larger randomized controlled trials are needed for definitive data on the potential disease-modifying role of rituximab in GO. Direct targeting of the orbital fibroblast via immunosuppression or nonimmunosuppressive drugs is emerging as a promising alternative. BACKGROUND: Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, remains inadequately treated. Current medical therapies, which primarily consist of glucocorticoids, have limited efficacy and present safety concerns. Inhibition of the insulin-like growth factor I receptor (IGF-IR) is a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy. METHODS: We conducted a multicenter, double-masked, randomized, placebo-controlled trial to determine the efficacy and safety of teprotumumab, a human monoclonal antibody inhibitor of IGF-IR, in patients with active, moderate-to-severe ophthalmopathy. A total of 88 patients were randomly assigned to receive placebo or active drug administered intravenously once every 3 weeks for a total of eight infusions. The primary end point was the response in the study eye. This response was defined as a reduction of 2 points or more in the Clinical Activity Score (scores range from 0 to 7, with a score of ≥3 indicating active thyroid-associated ophthalmopathy) and a reduction of 2 mm or more in proptosis at week 24. Secondary end points, measured as continuous variables, included proptosis, the Clinical Activity Score, and results on the Graves' ophthalmopathy-specific quality-of-life questionnaire. Adverse events were assessed. RESULTS: In the intention-to-treat population, 29 of 42 patients who received teprotumumab (69%), as compared with 9 of 45 patients who received placebo (20%), had a response at week 24 (P<0.001). Therapeutic effects were rapid; at week 6, a total of 18 of 42 patients in the teprotumumab group (43%) and 2 of 45 patients in the placebo group (4%) had a response (P<0.001). Differences between the groups increased at subsequent time points. The only drug-related adverse event was hyperglycemia in patients with diabetes; this event was controlled by adjusting medication for diabetes. CONCLUSIONS: In patients with active ophthalmopathy, teprotumumab was more effective than placebo in reducing proptosis and the Clinical Activity Score. (Funded by River Vision Development and others; ClinicalTrials.gov number, NCT01868997 .). Thyroid-associated ophthalmopathy (TAO) is a vexing and poorly understood autoimmune process involving the upper face and tissues surrounding the eyes. In TAO, the orbit can become inflamed and undergo substantial remodeling that is disfiguring and can lead to loss of vision. There are currently no approved medical therapies for TAO, the consequence of its uncertain pathogenic nature. It usually presents as a component of the syndrome known as Graves' disease where loss of immune tolerance to the thyrotropin receptor (TSHR) results in the generation of activating antibodies against that protein and hyperthyroidism. The role for TSHR and these antibodies in the development of TAO is considerably less well established. We have reported over the past 2 decades evidence that the insulin-like growth factorI receptor (IGF1R) may also participate in the pathogenesis of TAO. Activating antibodies against IGF1R have been detected in patients with GD. The actions of these antibodies initiate signaling in orbital fibroblasts from patients with the disease. Further, we have identified a functional and physical interaction between TSHR and IGF1R. Importantly, it appears that signaling initiated from either receptor can be attenuated by inhibiting the activity of IGF1R. These findings underpin the rationale for therapeutically targeting IGF1R in active TAO. A recently completed therapeutic trial of teprotumumab, a human IGF1R inhibiting antibody, in patients with moderate to severe, active TAO, indicates the potential effectiveness and safety of the drug. It is possible that other autoimmune diseases might also benefit from this treatment strategy. Thyroid-associated ophthalmopathy (TAO) is a complex disease process presumed to emerge from autoimmunity occurring in the thyroid gland, most frequently in Graves disease (GD). It is disfiguring and potentially blinding, culminating in orbital tissue remodeling and disruption of function of structures adjacent to the eye. There are currently no medical therapies proven capable of altering the clinical outcome of TAO in randomized, placebo-controlled multicenter trials. The orbital fibroblast represents the central target for immune reactivity. Recent identification of fibroblasts that putatively originate in the bone marrow as monocyte progenitors provides a plausible explanation for why antigens, the expressions of which were once considered restricted to the thyroid, are detected in the TAO orbit. These cells, known as fibrocytes, express relatively high levels of functional TSH receptor (TSHR) through which they can be activated by TSH and the GD-specific pathogenic antibodies that underpin thyroid overactivity. Fibrocytes also express insulin-like growth factor I receptor (IGF-IR) with which TSHR forms a physical and functional signaling complex. Notably, inhibition of IGF-IR activity results in the attenuation of signaling initiated at either receptor. Some studies suggest that IGF-IR-activating antibodies are generated in GD, whereas others refute this concept. These observations served as the rationale for implementing a recently completed therapeutic trial of teprotumumab, a monoclonal inhibitory antibody targeting IGF-IR in TAO. Results of that trial in active, moderate to severe disease revealed dramatic and rapid reductions in disease activity and severity. The targeting of IGF-IR with specific biologic agents may represent a paradigm shift in the therapy of TAO. Thyroid eye disease is a disabling autoimmune disease associated with orbital inflammation and tissue remodeling which can result in significant proptosis, leading to visual alterations and is potentially sight threatening. Current evidence indicates that autoantibodies to the insulin-like growth factor 1 receptor (IGF-1R), along with the thyroid-stimulating hormone receptor (TSHR), mediate the pathogenesis in susceptible individuals. Teprotumumab, a monoclonal IGF-1R antagonist, has demonstrated previously in a 24 week, randomized, controlled trial to produce significant changes in composite outcomes of proptosis and clinical activity score as compared with placebo. Further examination of the proptosis results reported here, indicate that the proptosis outcome (≥ 2 mm reduction) was met in 71.4% of the teprotumumab-treated patients as compared with 20% of the placebo-treated patients (p < 0.001). Additionally, the proptosis benefit was observed early in the trial (study week 6), and all individual patients demonstrated some benefit at week 24. Improvement was noted among smokers, non-smokers, men and women, and particularly those with higher levels of proptosis at baseline. The level of proptosis reduction with teprotumumab reported here is similar to that seen with decompression surgery. If these results are confirmed in the ongoing Phase 3 trial, teprotumumab will offer an alternative to surgery and its associated complications. PURPOSE OF REVIEW: Thyroid eye disease is a complex autoimmune disorder which causes substantial morbidity. It can result in orbital disfigurement, double vision, and visual loss. Consequently, it has a substantial negative effect on quality of life, mental health, and socioeconomic status. Most signs and symptoms of thyroid eye disease (TED) can be explained by the expansion of the orbital contents. Steroids are the mainstay of treatment in TED. However, recurrence may occur once steroids are withdrawn. Furthermore, in most cases, normal orbital anatomy is not restored, and skilled rehabilitative surgery is required to reduce disfigurement, double vision, and to preserve vision. Therefore, novel, causal, and more efficacious treatment strategies are warranted. RECENT FINDINGS: In the last decade, the pathophysiology of TED has also been revised with the identification of new potential therapeutic targets. Recent clinical trials have shown that considerable benefit may be derived from the addition of antiproliferative agents (e.g., mycophenolate sodium) in preventing deterioration after steroid cessation. In addition, targeted biologic therapies have shown promise, including teprotumumab (anti-IGFR) which appears to substantially reduce proptosis, rituximab (anti-CD20) which reduces inflammation and tocilizumab (anti-IL-6) which potentially benefits both of these parameters. SUMMARY: This short review summarizes the recent research developments in this area. PURPOSE: Improved understanding of thyroid eye disease (TED) pathogenesis has facilitated identification of a targeted molecular approach for TED treatment offering the potential to halt or slow disease progression in a nonsurgical manner. Herein, we provide a summary of the current knowledge of TED management, followed by discussion of a novel insulin-like growth factor-1 receptor (IGF-1R) antagonist antibody and its potential to change the course of the disease. DESIGN: Perspective. METHODS: Review of the literature and authors' experience. RESULTS: Many publications demonstrate IGF-1R overexpression in TED, and its activation as an autoantigen as a critical factor in TED pathogenesis. Several in vitro studies demonstrate that IGF-1R inhibition attenuates downstream molecular events including cytokine and hyaluro production, and cellular differentiation. These observations led to the hypothesis that blocking IGF-1R may abrogate the clinical progression of TED. The recent completion of phase 2 and 3 randomized, placebo-controlled trials demonstrate the efficacy and safety of teprotumumab, a fully human monoclonal IGF-1R antagonist antibody, in patients with moderate-to-severe, active TED. Both the phase 2 and the recent phase 3 study results demonstrate that more patients with active TED receiving teprotumumab experienced a meaningful improvement in proptosis. CONCLUSIONS: Current TED treatment strategies target inflammation and symptoms, but do not modify the disease course. Therefore, proptosis as well as strabismus and its resulting diplopia often remain, impacting patient well-being and quality of life over the long term. Targeted molecular therapy using teprotumumab demonstrates disease-modifying benefits with the potential to shift the paradigm for TED treatment. Thyroid-associated ophthalmopathy (TAO) is an autoimmune component of Graves' disease for which no currently available medical therapy provides reliable and safe benefit. Based on insights generated experimentally over the past several decades, the insulin-like growth factor-I receptor (IGF-IR) has been implicated in the pathogenesis of TAO. Furthermore, an IGF-IR inhibitor, teprotumumab, has emerged from 2 clinical trials as a promising treatment for active, moderate to severe TAO. This brief review intends to provide an overview of the rationale underlying the development of teprotumumab for this disease. It is possible that teprotumumab will soon take its place in our therapeutic armamentarium for active TAO. Author information: (1)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (2)Department of Translational Research and of New Technologies in Medicine and Surgery, University of Pisa, 56126, Pisa, Italy. Electronic address: [email protected]. (3)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (4)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (5)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (6)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (7)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (8)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (9)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. (10)Sapienza University of Rome, 9311, Medicosurgical Sciences and Biotechnologies, C.so della Repubblica 79, Latina, 04100, Italy. Electronic address: [email protected]. (11)Sapienza University of Rome, 9311, Medicosurgical Sciences and Biotechnologies, C.so della Repubblica 79, Latina, 04100, Italy; ASL Latina, 217138, Endocrinology Unit, Latina, Lazio, Italy. Electronic address: [email protected]. (12)Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Israel; Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University), Russia. Electronic address: [email protected]. (13)Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Electronic address: [email protected]. Teprotumumab (teprotumumab-trbw; TEPEZZA™ - Horizon Therapeutics) is a monoclonal antibody insulin-like growth factor-I receptor (IGF-IR) antagonist developed for the treatment of thyroid eye disease (Graves ophthalmopathy/orbitopathy, thyroid-associated ophthalmopathy). Based on positive results from two multinational clinical trials teprotumumab was recently approved for this indication in the US. This article summarizes the milestones in the development of teprotumumab leading to this first approval for thyroid eye disease. INTRODUCTION: Thyroid-associated ophthalmopathy (TAO) is a disfiguring, potentially blinding, and sub-optimally managed autoimmune condition. Current therapy of active TAO consists most frequently of glucocorticoid steroids, orbital radiation, or B-cell depletion; all of which are associated with substantial side effects. Teprotumumab (Tepezza) is a human monoclonal antibody against the insulin-like growth factor type I receptor (IGF-IR), recently evaluated in two clinical trials for active moderate-to-severe TAO that was recently approved by the United States Food and Drug Administration (FDA) for use in TAO. AREAS COVERED: This article reviews phase II and III placebo-controlled, double-masked, prospective, multicenter studies assessing the efficacy and safety of teprotumumab for the treatment of active, moderate-to-severe TAO. EXPERT OPINION: Teprotumumab has demonstrated substantial and rapid improvement in Clinical Activity Score and proptosis reduction in TAO compared to placebo. Subjective diplopia and quality of life were also improved in both clinical trials. Teprotumumab exhibited a favorable safety profile, with transient hyperglycemia, muscle cramps, and auditory side effects being associated with the drug; these were usually transient. The trial findings indicate that teprotumumab is a promising, potential first-line therapy for treating TAO. INTRODUCTION: Thyroid eye disease is a debilitating, disfiguring, and potentially blinding periocular condition. Teprotumumab is a human insulin-like growth factor-I receptor monoclonal inhibitor antibody which indicated for treating thyroid eye disease. AREAS COVERED: The authors performed a systematic review of the literature using the PubMed database, and the following keywords were used: 'teprotumumab,' 'thyroid eye disease,' and 'insulin-like growth factor I receptor.' The chemical property, mechanism of action, pharmacokinetics, clinical efficacy, and safety of teprotumumab were introduced in this paper. EXPERT OPINION: Teprotumumab is a human monoclonal antibody targeting insulin-like growth factor-I receptor. Clinical trials indicated that proptosis response of teprotumumab was 83%, and clinical activity score, diplopia, and quality of life were also better than placebo. Teprotumumab was well tolerated, common adverse reactions included muscle spasm, nausea, alopecia, diarrhea, fatigue, hyperglycemia, hearing impairment, dysgeusia, headache, and dry skin. Adult respiratory distress syndrome (ARDS) is the leading cause of death associated with SARS-CoV-2 infection and COVID-19. IGF-1 has been implicated in ARDS, yet its role in relation to COVID-19-related lung injury has not been investigated. We hypothesize that blockage of the IGF-1 receptor (IGF-1R) mitigates lung injury and decreases the risk of death in patients COVID-19-related ARDS. Patients with fibroproliferative ARDS have been shown to have increased IGF-1 and IGF-1R staining in lung tissue specimens. Rising levels of IGF-1 in bronchioalveolar fluid (BAL) and increased IGF-1 mRNA expression in lung tissues (but declining serum IGF-1 levels) have been found in late stage ARDS compared with early lung injury. Blockage of IGF-1R decreases lung tissue damage and increases survival in bleomycin-induced as well as H1N1 influenza-related lung injury in animal models. Teprotumumab is a monoclonal antibody directed against the IGF-1R that was FDA-approved in 2020 for the treatment of Graves' orbitopathy. In order to determine if teprotumumab may reduce lung injury and death related to ARDS in the setting of COVID-19, preliminary clinical data is needed. IGF-1 levels in serum and BAL fluid must be measured in patients with COVID-19-related ARDS. Histopathology from lung samples from patients with COVID-19-related ARDS must be examined for increased expression of the IGF-1R. Once these are ascertained, and if the data support IGF-1 involvement, a randomized, placebo-controlled phase 2A trial of teprotumumab therapy in the setting of COVID-19-related ARDS and non-COVID-19-related ARDS designed to generate initial data on short-term efficacy, safety, dosing and administration should be performed. PURPOSE: Teprotumumab, a blocking antibody to the insulin like growth factor 1 receptor (IGF-1R) has been shown to significantly reduce proptosis in recent phase 2 and 3 trials in patients with inflammatory thyroid eye disease (TED). Herein, we investigate the impact of teprotumumab on patients with non-inflammatory TED. We also investigate the expression of the IGF-1R on orbital tissues from patients with inflammatory and non-inflammatory TED compared to controls. METHODS: Consecutive patients with non-inflammatory TED (clinical activity score, CAS ≤ 1, for at least 4 months, were treated with teprotumumab. They received a complete course (total eight infusions) of teprotumumab (10 mg/kg for the first infusion and 20 mg/kg for subsequent infusions every 3 weeks). The primary outcome was a proptosis response at week 24. Further, IGF-1R α and β expression was evaluated on orbital tissue from patients with inflammatory and non-inflammatory TED, as well as healthy controls. Non-biased histological analysis of IGF-1R expression was performed using ImageJ. RESULTS: Four patients met eligibility criteria for the clinical study, with a mean (SD) CAS of 0 (0). Following teprotumumab treatment, there was a mean (SD) reduction in proptosis of 2.6 mm (1.2). Five patients were included for each group of the histological study; inflammatory TED, non-inflammatory TED and controls. IGF-1Rα and IGF-1Rβ expression was significantly greater in the orbital tissues of patients with inflammatory TED and non-inflammatory TED, when compared to controls. CONCLUSION: Our findings demonstrate for the first time, that teprotumumab, a blocking antibody to the IGF-1R reduces proptosis in a series of patients with non-inflammatory TED. Overexpression of the IGF-1R in orbital tissue from patients with non-inflammatory disease compared to controls may be an important consideration for effect. BACKGROUND: Thyroid eye disease (TED) is characterized by orbital inflammation and complicated by extraocular muscle fibrosis. Treatment with rapamycin/sirolimus has been reported to improve ocular motility and disease manifestations in TED. Whether this resulted from a primary antifibrotic effect on fibroblasts or was secondary to immune-suppression is unclear. METHODS: In vitro contractility studies of primary orbital fibroblasts. Cells from patients with TED and controls were treated with rapamycin [mechanistic target of rapamycin an (mTOR) inhibitor] and MHY1485 (an mTOR stimulator) as well as inhibitors upstream in the same signaling cascade (saracatinib and befatinib). RESULTS: At concentrations consistent with the therapeutic dosing range in humans, rapamycin/sirolimus significantly reduces fibrosis in orbital fibroblasts from TED patients and controls in vitro. This effect is separate from, and in addition to, its immune suppressive effect. mTOR-driven fibrotic activity is greater in TED-derived fibroblasts and can be blocked also upstream of mTOR by inhibition of src. There was no adverse effect on cell survival. CONCLUSION: The authors present evidence for a direct antifibrotic effect of rapamycin/sirolimus in primary orbital fibroblasts. Targeting mTOR signaling presents a further and adjunctive treatment of TED alongside other immune-suppressive agents. By acting downstream of IGF1-R, sirolimus may offer a cost-effective alternative to teprotumumab therapy. Clinical case reports, now supplemented by this in vitro evidence, support the initiation of a clinical trial to treat the fibrotic sequelae of TED with this already-approved agent. Such an "off-the-shelf" therapy is a welcome prospect for TED treatment, particularly one available at a low price.

Which protein is encoded by the protein APOBEC3C?

The gene APOBEC3C codes for: apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C

Gastric cancer is one of the leading causes of cancer-associated mortality worldwide. Cytotoxin‑associated gene A (CagA) has been reported to be associated with gastric diseases. Phosphatase and tensin homolog (PTEN) and tet methylcytosine dioxygenase 1 (Tet1) are important tumor‑suppressor genes. The present study aimed to investigate the underlying functions of CagA in human gastric cancer, and to explore the associations between CagA, PTEN and Tet1 in gastric cancer. For that purpose, CagA overexpression and Tet1 interference recombit lentiviral plasmids were constructed. Quantitative polymerase chain reaction (qPCR) was utilized to screen gene expression in HGC‑27 human gastric cancer cells overexpressing CagA. qPCR and western blotting were used to detect gene and protein expression, respectively. In addition, the methylation status of PTEN was detected by methylation‑specific PCR. The expression levels of PTEN, Tet1, apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC)3A, APOBEC3C and APOBEC3F were significantly decreased in the CagA overexpression group compared with in the negative control group in HGC‑27 cells. Compared with in the negative control group, the mRNA and protein expression levels of PTEN were markedly decreased in cells with Tet1 interference. The decreased expression of PTEN was associated with increased methylation levels in the cells. In addition, the protein expression levels of PTEN were significantly decreased in HGC‑27 cells when CagA was overexpressed. The expression levels of PTEN and Tet1 were also markedly decreased in CagA+ gastric cancer tissues compared with in non‑cancerous tissues. The decreased expression of PTEN in CagA+ gastric cancer tissues was associated with increased methylation levels. In conclusion, overexpression of CagA significantly decreased the expression of PTEN, Tet1, APOBEC3A, APOBEC3C and APOBEC3F in human gastric cancer. In addition, CagA increased DNA methylation and decreased PTEN expression, which was reversed by Tet1 overexpression. The present study may facilitate future therapeutic approaches targeting human gastric cancer. Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection. The artemisinin class of anticancer compounds is well known for oxidative DNA-damage-mediated growth arrest, followed by cell death. However, the nature of this genotoxic stress for cancer therapeutics remains elusive. Here we show that artesunate (Art), a water-soluble artemisinin analog, triggers inducible anticancer responses directly implicated in the DNA-damage-intended therapy. We observed that the level of the antiviral enzyme APOBEC3C (apolipoprotein-B mRNA-editing catalytic polypeptide-like 3C (A3C)) preferentially increased upon the treatment with Art against tumor xenografts of p53-deficient H1299 cells. Using gain-of-function experiments, A3C could improve the therapeutic efficacy of Art, as determined by cell proliferation and colony formation assays. Furthermore, elevated A3C provoked a minor accumulation of γH2AX foci and the phosphorylation of RPA32 and Chk1, which strongly sensitized H1299 cells to Art. The employment of A3C also caused an increase in the synergistic interaction between Art and Chk1 inhibition. Besides, A3C overexpression delayed the cell cycle at the S phase, accompanied by attenuated G2/M arrest in the presence of Art. The enzymatic inactivation of A3C by the mutation of zinc-coordinating residues (C97S and C100S) indicated that A3C sensitized Art in a deaminase-dependent manner. Furthermore, we showed that using small interfering RNA against A3C can induce the chemoresistance of Art. These studies combine to suggest that upregulated A3C is involved in the Art-induced DNA damage response as a consequent event to improve the overall cytotoxic responses of Art. Allogeneic hematopoietic stem cell transplants can lead to dramatic reductions in human immunodeficiency virus (HIV) reservoirs. This effect is partially mediated by donor T cells recognizing lymphocyte-expressed minor histocompatibility antigens (mHAgs). The potential to mark maligt and latently infected cells for destruction makes mHAgs attractive targets for cellular immunotherapies. However, testing such HIV reservoir reduction strategies will likely require preclinical studies in non-human primates (NHPs). In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify an mHAg in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1∗063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8+ T cells recognized immortalized B cells but not fibroblasts from an mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs.

Does AZD3759 cross the blood brain barrier?

AZD3759 is a novel EGFR tyrosine kinase inhibitor with high capability to penetrate the blood-brain barrier.

Since the discovery of sensitizing EGFR mutations as a predictive marker of sensitivity to EGFR tyrosine kinase inhibitors (TKIs), the field of targeted therapy in non-small cell lung cancer (NSCLC) has been revolutionized. Patients harbouring these sensitizing mutations treated with EGFR TKI have derived significant clinical outcome when compared with standard platinum based chemotherapy doublets. However disease progression invariably occurs at a median of about 9-13 months from initiation treatment, if acquired resistance commonly due to the development of EGFR T790M mutation. A novel class of "third generation" EGFR TKIs have been developed that is sensitising and T790M mutant-specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Early phase clinical data suggest the third generation EGFR TKIs such as osimertinib, rociletinib, and HM61713 are highly efficacious and well tolerated. Another promising class of EGFR TKI such as AZD3759 has been designed to penetrate blood brain barrier to treat brain metastases and leptomeningeal disease and has showed promising responses in patients with brain metastases. Acquired resistance to third generation EGFR TKIs has been reported including EGFR C797S. Given its non-invasive nature, plasma ctDNA is being explored as a possible approach to detect T790M mutation and to also inform on novel molecular mechansims of tertiary resistance to third generation EGFR TKIs. An understanding of the mechanisms of acquired resistance to the third-generation EGFR TKIs will greatly aid in the development of the next generation of EGFR TKIs. Non-small-cell lung cancer patients with activating mutations in epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitor (TKI) treatment. Nevertheless, patients often develop central nervous system (CNS) metastases during treatment, even when their extracranial tumors are still under control. In the absence of effective options, much higher doses of EGFR TKIs have been attempted clinically, with the goal of achieving high enough drug concentrations within the CNS. Although limited tumor responses have been observed with this approach, the toxicities outside the CNS have been too high to tolerate. We report the discovery and early clinical development of AZD3759, a selective EGFR inhibitor that can fully penetrate the blood-brain barrier (BBB), with equal free concentrations in the blood, cerebrospinal fluid, and brain tissue. Treatment with AZD3759 causes tumor regression in subcutaneous xenograft, leptomeningeal metastasis (LM), and brain metastasis (BM) lung cancer models and prevents the development of BM in nude mice. An early clinical study in patients with BM and LM treated with AZD3759 confirms its BBB-penetrant properties and antitumor activities. Our data demonstrate the potential of AZD3759 for the treatment of BM and LM and support its further clinical evaluation in larger trials. With the use of EGFR TKIs, patient survival is now prolonged and as a consequence, a higher chance of development of CNS metastases has been observed during the course of the disease. CNS metastases remains a therapeutically challenging subset of patient to treat owing to the blood-brain barrier (BBB). Prior to routine EGFR mutation testing, surgical resection, stereotactic radiosurgery and/or whole brain radiation therapy (WBRT) were the main treatment options whereas treatment options for patients with leptomeningeal metastases (LM) included intra-thecal chemotherapy, WBRT, and ventriculo-peritoneal shunting. Unfortunately outcome for both BM and LM remains poor with median survival between 3 and 6 months. Systemic treatment with EGFR TKIs had been effective in the treatment of intracranial metastases but efficacy of early generation TKIs were hampered by its limited BBB penetration. The next generation EGFR TKIs osimertinib and AZD3759 have improved BBB penetration and the BLOOM study of osimertinib and AZD3759 has reported highly promising intracranial efficacy and may herald a new frontier to treat this therapeutically challenging subset of advanced EGFR mutant patients. BACKGROUND: CNS metastases-including brain and leptomeningeal metastases-from epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) are associated with poor prognosis. AZD3759 is a novel EGFR tyrosine kinase inhibitor with high capability to penetrate the blood-brain barrier. We aimed to assess the safety, tolerability, pharmacokinetics, and efficacy of AZD3759 in patients with EGFR-mutant NSCLC with brain and leptomeningeal metastases. METHODS: This open-label, multicentre, phase 1 study was undertaken at 11 centres and hospitals in Australia, South Korea, Taiwan, and the USA. Eligible patients included those with histologically confirmed, advanced-stage, EGFR-mutant NSCLC. The study was done in two parts, with dose-escalation and dose-expansion phases. In the dose-escalation phase, patients who had progressed after treatment with an EGFR tyrosine kinase inhibitor received AZD3759 at 50 mg, 100 mg, 200 mg, 300 mg, or 500 mg twice a day. In the dose-expansion phase, AZD3759 at 200 mg or 300 mg twice a day was administered to patients with either brain or leptomeningeal metastases who had never received an EGFR tyrosine kinase inhibitor and patients with leptomeningeal metastases who had been pretreated with an EGFR tyrosine kinase inhibitor. The primary objective was safety and tolerability, with severity of adverse events assessed with the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 4.03. This trial is registered with ClinicalTrials.gov, number NCT02228369. FINDINGS: Between Nov 18, 2014, and Sept 7, 2016, 67 patients with NSCLC were enrolled into the study, 29 to the dose-escalation phase and 38 to the dose-expansion phase. At data cutoff (Dec 12, 2016), three (10%) patients in the dose-escalation phase and 20 (53%) in the dose-expansion phase were still receiving treatment. Dose-limiting toxic effects occurred in two (67%) of three patients who received 500 mg twice a day in the dose-escalation phase (grade 3 acne [n=1] and intolerable grade 2 mucosal inflammation [n=1]); hence, doses of 200 mg and 300 mg twice a day were selected for further assessment in the dose-expansion phase. Drug-related skin and gastrointestinal disorders of any grade occurred in 35 (92%) and 29 (76%) patients in the dose-expansion phase, respectively, and led to treatment discontinuation in one (4%) patient treated with 200 mg twice a day (grade 3 increase of alanine aminotransferase and aspartate aminotransferase) and two (13%) patients given 300 mg twice a day (grade 3 diarrhoea [n=1] and grade 3 skin rash [n=1]). Grade 3 skin and gastrointestinal disorders occurred in four (17%) and two (9%) patients, respectively, at a dose of 200 mg twice a day, and in six (40%) and four (27%) patients, respectively, at a dose of 300 mg twice a day. No grade 4 disorders arose. Other grade 3 disorders included hepatobiliary and renal disorders (three [13%] at 200 mg twice a day), asthenia (one [7%] at 300 mg twice a day), infections and infestations (one [7%] at 300 mg twice a day), and metabolism and nutrition disorders (one [4%] at 200 mg twice a day and one [7%] at 300 mg twice a day). INTERPRETATION: AZD3759 at a dose of 200 mg twice daily showed a tolerable safety profile in patients with NSCLC and CNS metastases who had either never received a tyrosine kinase inhibitor or who had been pretreated with a tyrosine kinase inhibitor. The good penetration of the blood-brain barrier by AZD3759, and its promising clinical activity, support further assessment of this compound in studies. FUNDING: AstraZeneca. The prognosis of patients with brain metastasis (BM) is poor. In our study, we demonstrated that AZD3759, an EGFR tyrosine kinase inhibitors (TKIs) with excellent blood-brain barrier (BBB) penetration, combined with radiation enhanced the antitumor efficacy in BM model from EGFR mutant (EGFRm) NSCLC. Besides, the antitumor activity displayed no difference between radiation concurrently with AZD3759 and radiation sequentially with AZD3759. Mechanistically, we found that two factors determined the enhanced efficacy: cells with EGFRm which were sensitive to AZD3759, and a relative high concentration of AZD3759. We have validated mechanisms underlying the radiosensitizing effect of AZD3759, which were involved in decreased cell proliferation and survival, and suppressed repair of DNA damage. Moreover, our study found that AZD3759 inhibited both the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA double-strand breaks (DSBs) repair pathway, and abrogated the G2/M checkpoint to suppress DNA damage repair. We also detected the BBB penetration of AZD3759 when combined with cranial radiation. The results showed the BBB penetration of AZD3759 was decreased within 24 hr after radiation, however, the free concentration of AZD3759 in brain kept at a high level in the context of radiation. In conclusion, our findings suggest that AZD3759 combined with radiation enhances the antitumor activity in BM from EGFRm NSCLC, this combination therapy may be an effective treatment option for BM from EGFRm NSCLC.

What is the major sequence determinant for nucleosome positioning?

G+C content is the primary determinant of MNase-derived nucleosome occupancy.

We assess the role of intrinsic histone-DNA interactions by mapping nucleosomes assembled in vitro on genomic DNA. Nucleosomes strongly prefer yeast DNA over Escherichia coli DNA, indicating that the yeast genome evolved to favor nucleosome formation. Many yeast promoter and terminator regions intrinsically disfavor nucleosome formation, and nucleosomes assembled in vitro show strong rotational positioning. Nucleosome arrays generated by the ACF assembly factor have fewer nucleosome-free regions, reduced rotational positioning and less translational positioning than obtained by intrinsic histone-DNA interactions. Notably, nucleosomes assembled in vitro have only a limited preference for specific translational positions and do not show the pattern observed in vivo. Our results argue against a genomic code for nucleosome positioning, and they suggest that the nucleosomal pattern in coding regions arises primarily from statistical positioning from a barrier near the promoter that involves some aspect of transcriptional initiation by RNA polymerase II. Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determits of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken beta-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the '601' nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp - an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning 'rules' they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL+/- 3.5 and +/- 5.5, which is similar to the alpha-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the '601' clone) the same YR/YYRR motifs occur predomitly at the sites SHL +/- 1.5. The interaction between the H3/H4 tetramer and the central part of the nucleosomal DNA is likely to be responsible for the positioning of nucleosomes of this group, and the DNA trajectory in these nucleosomes may differ in detail from the published structures. Thus, from the stereochemical perspective, the in vitro nucleosomes studied here follow either an X-ray-like pattern (with strong deformations in the terminal parts of nucleosomal DNA), or an alternative pattern (with the deformations occurring predomitly in the central part of the nucleosomal DNA). The results presented here may be useful for genome-wide classification of nucleosomes, linking together structural and thermodynamic characteristics of nucleosomes with the underlying DNA sequence patterns guiding their positions. MOTIVATION: The intrinsic DNA sequence is an important determit of nucleosome positioning. Some DNA sequence patterns can facilitate nucleosome formation, while others can inhibit nucleosome formation. Nucleosome positioning influences the overall rate of sequence evolution. However, its impacts on specific patterns of sequence evolution are still poorly understood. RESULTS: Here, we examined whether nucleosomal DNA and nucleosome-depleted DNA show distinct polymorphism patterns to maintain adequate nucleosome architecture on a genome scale in yeast. We found that sequence polymorphisms in nucleosomal DNA tend to facilitate nucleosome formation, whereas polymorphisms in nucleosome-depleted DNA tend to inhibit nucleosome formation, which is especially evident at nucleosome-disfavored sequences in nucleosome-free regions at both ends of genes. Sequence polymorphisms facilitating nucleosome positioning correspond to stable nucleosome positioning. These results reveal that sequence polymorphisms are under selective constraints to maintain nucleosome positioning. CONTACT: [email protected]; [email protected] A major question in chromatin biology is to what extent the sequence of DNA directly determines the genetic and chromatin organization of a eukaryotic genome? We consider two aspects to this question: the DNA sequence-specified positioning of nucleosomes and the determination of NDRs (nucleosome-depleted regions) or barriers. We argue that, in budding yeast, while DNA sequence-specified nucleosome positioning may contribute to positions flanking the regions lacking nucleosomes, DNA thermodynamic stability is a major component determit of the genetic organization of this organism. The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determits of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determit of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome organization alone is sufficient to elucidate much of the circuitry of pluripotency. Our results, suggest that nucleosome organization is associated with numerous genomic and epigenomic processes and can be used to elucidate cellular identity.

Which is the primary enzyme metabolizing esomeprazole?

Esomeprazole is primarily metabolized by CYP2C19.

Which factors contribute to the risk of very-early-onset inflammatory bowel disease?

Somalatic mosaicism and common genetic variation contribute to the risk of very-early-onset inflammatory bowel disease, particularly atrial fibrillation and dysregulation of transcphenne muscular dystrophy, as well as other genetic variation, in the early phase of disease.

Author information: (1)MOE Key Laboratory of Gene Function and Regulation, Department of Gastroenterology and Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. (2)MOE Key Laboratory of Gene Function and Regulation, Department of Gastroenterology and Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China; Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China. (3)Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China. (4)the Department of Pediatrics & Adolescent Medicine, The University of Hong Kong, Hong Kong, China. (5)Walter and Eliza Hall Institute of Medical Research and Departments of Medical Biology and Microbiology & Immunology, University of Melbourne, Parkville, Melbourne, Australia. (6)the Department of Pediatrics & Adolescent Medicine, The University of Hong Kong, Hong Kong, China. Electronic address: [email protected]. (7)MOE Key Laboratory of Gene Function and Regulation, Department of Gastroenterology and Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. Electronic address: [email protected]. (8)MOE Key Laboratory of Gene Function and Regulation, Department of Gastroenterology and Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. Electronic address: [email protected]. (9)MOE Key Laboratory of Gene Function and Regulation, Department of Gastroenterology and Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. Electronic address: [email protected].

What disease is treated with BIVV001?

BIVV001 fusion protein has been developed as Factor VIII replacement therapy for hemophilia A

BACKGROUND: Factor VIII replacement products have improved the care of patients with hemophilia A, but the short half-life of these products affects the patients' quality of life. The half-life of recombit factor VIII ranges from 15 to 19 hours because of the von Willebrand factor chaperone effect. BIVV001 (rFVIIIFc-VWF-XTEN) is a novel fusion protein designed to overcome this half-life ceiling and maintain high sustained factor VIII activity levels. Data are lacking on the safety and pharmacokinetics of single-dose BIVV001. METHODS: In this phase 1-2a open-label trial, we consecutively assigned 16 previously treated men (18 to 65 years of age) with severe hemophilia A (factor VIII activity, <1%) to receive a single intravenous injection of recombit factor VIII at a dose of 25 IU per kilogram of body weight (lower-dose group) or 65 IU per kilogram (higher-dose group). This injection was followed by a washout period of at least 3 days. The patients then received a single intravenous injection of BIVV001 at the same corresponding dose of either 25 IU or 65 IU per kilogram. Adverse events and pharmacokinetic measurements were assessed. RESULTS: No inhibitors to factor VIII were detected and no hypersensitivity or anaphylaxis events were reported up to 28 days after the injection of single-dose BIVV001. The geometric mean half-life of BIVV001 was three to four times as long as that of recombit factor VIII (37.6 hours vs. 9.1 hours in the lower-dose group and 42.5 vs. 13.2 hours in the higher-dose group); the area under the curve (AUC) for product exposure was six to seven times as great in the two dose groups (4470 hours vs. 638 hours × IU per deciliter in the lower-dose group and 12,800 hours vs. 1960 hours × IU per deciliter in the higher-dose group). After the injection of BIVV001 in the higher-dose group, the mean factor VIII level was in the normal range (≥51%) for 4 days and 17% at day 7, which suggested the possibility of a weekly interval between treatments. CONCLUSIONS: In a small, early-phase study involving men with severe hemophilia A, a single intravenous injection of BIVV001 resulted in high sustained factor VIII activity levels, with a half-life that was up to four times the half-life associated with recombit factor VIII, an increase that could signal a new class of factor VIII replacement therapy with a weekly treatment interval. No safety concerns were reported during the 28-day period after administration. (Funded by Sanofi and Sobi; ClinicalTrials.gov number, NCT03205163.).

What is the mode of action of Thiazovivin?

Thiazovivin is a selective small molecule that directly targets Rho-associated kinase (ROCK) and increases expression of pluripotency factors.

Today, there is a need for a platform to efficiently generate and maintain a feeder free culture of pluripotent stem cells by small molecules or pharmacological agents. Induced pluripotent stem cell (iPSC) is considered a promising resource for restorative cell therapy in clinical areas. While fully reprogrammed iPSCs are similar to embryonic stem cells, iPSCs could be derived from the patient's own cells (autologous), which avoids the immune rejection activities. Recent advances have demonstrated that iPSCs could be generated from human fibroblasts using only four transcription factors: OCT4, SOX2, CMYC, and KLF4. However, the limitations of reprogramming technologies include low efficiency, slow kinetics, transgene integration and residual expression. Surprisingly, adult stem cells from human endometrium (endometrial stem cells; EnSCs) express OCT4 and KLF4 pluripotency factors. On the other hand, small molecule inhibitors of specific signaling pathways such as thiazovivin have been used in various aspects of iPSC generation and maintece. Thiazovivin is a selective small molecule that directly targets Rho-associated kinase (ROCK) and increases expression of pluripotency factors. The process using thiazovivin could be easier, faster and more cost effective than transgene integration into somatic cells. So reprogramming of OCT4 and KLF4 expressing EnSCs by a ROCK inhibitor, thiazovivin, could result in producing more efficient iPSCs compared with fibroblasts or conventional somatic cells without integration any transgene and retroviral vector. Corneal diseases exhibit a high prevalence and are prone to cause blindness; furthermore, maintaining the morphology and ionic transporter expression in corneal endothelial cells (CECs) is crucial for treatment of these diseases. This study aimed to investigate the effects of the novel Rho associated coiled-coil containing protein kinase (ROCK) inhibitor, thiazovivin (2,4‑disubstituted thiazole, TZV), on human corneal endothelial‑to‑mesenchymal transition/epithelial‑to‑mesenchymal transition (EndMT/EMT), cell morphology, junction proteins and ionic transporter expression in human CECs (HCECs) in vitro and then to clarify the mechanisms of action of TZV. In the present study, primary HCECs were cultured in vitro and passaged. The expression levels of adhesion proteins (E‑cadherin and N‑cadherin), the EndMT/EMT marker, α smooth muscle  actin (α‑SMA), the tight junction protein, Zonula occludens-1 (ZO‑1), and the ionic transporter, Na+/K+‑ATPase, were detected by immunofluorescence. The proliferative ability of the HCECs was determined by CCK-8 assay. The mRNA expression of the EndMT/EMT‑inducing gene, Snail, was examined by RT‑PCR. The protein expression levels of ROCK1/2 were evaluated by western blot analysis. The HCECs were cultured with TZV at various concentrations (2, 4, or 6 µM) for different periods of time (24 or 48 h). We found that the the cell states of the HCECs co‑cultured with 4 µM TZV for 48 h reached the optimum, and corneal EndMT/EMT was inhibited, as evidenced by the significantly upregulated expression of ZO‑1 and E‑cadherin, and the markedly downregulated expression of N‑cadherin and α‑SMA. Furthermore, the cells exhibited a normal, tightly connected hexagonal or pentagonal morphology. Additionally, the protein expression of ROCK1/2 and the mRNA expression of Snail were significantly decreased. However, there was no significant difference between the TZV‑treated and the control groups as regards HCEC proliferative ability. These findings suggested that the ROCK inhibitor, TZV (4 µM), was effective in improving the morphology, cell junctions and ionic transporter expression of HCECs by inhibiting EndMT/EMT, but had no effect on HCEC proliferation.

Explain the association between Barr bodies (nuclear inclusions) and the X chromosome?

Barr body is an inactivated X chromosome in the normal female somatic cell.

Addition of exogen DB-c AMP in medium of human fibroblasts culture, with 46 XX karyotypes, enables us to observe a high frequency of Barr bodies, about 80 or 85%. This observation suggests that the condensation of the X chromosome in the form of Barr bodies depends on the level of endogen cyclic AMP. The chromosome in situ hybridization with human X chromosome alpha satellite DNA probe (pBamX7) on human lymphocyte metaphases and interphase nuclei was performed for interphase cytogenetic studies. The individuals with numerical or structural abnormalities of X chromosome were studied. The results showed that the probe hybridized specifically to the centromeric region (p11----q11) of X chromosome. The number of silver grain clusters in interphase nuclei was correlated with that of X chromosome. Most of the clusters located near the nuclear membrane where inactive X chromatins (Barr-bodies) were usually found. The method of ascertaining the number of X chromosomes by in situ hybridization was much more reliable than that by counting the number of Barr-bodies. The modified R-banding technique was introduced and the significance of this work was also discussed. Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence in situ hybridization with a probe against XIST RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with XIST RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation. The Barr body has long been recognized as the cytological manifestation of the inactive X chromosome (Xi) in interphase nuclei. Despite being known for over 50 years, relatively few components of the Barr body have been identified. In this study, we have screened over 30 histone variants, modified histones and non-histone proteins for their association with or exclusion from the Barr body. We demonstrate that, similar to the histone variant macroH2A, heterochromatin protein-1 (HP1), histone H1 and the high mobility group protein HMG-I/Y are elevated at the territory of the Xi in interphase in human cell lines, but only when the Xi chromatin is heteropycnotic, implicating each as a component of the Barr body. Surprisingly, however, virtually all other candidate proteins involved in establishing heterochromatin and gene silencing are notably absent from the Barr body despite being localized generally elsewhere throughout the nucleus, indicating that the Barr body represents a discrete subnuclear compartment that is not freely accessible to most chromatin proteins. A similar dichotomous pattern of association or exclusion describes the spatial relationship of a number of specific histone methylation patterns in relation to the Barr body. Notably, though, several methylated forms of histone H3 that are deficient in Xi chromatin generally are present at a region near the macrosatellite repeat DXZ4, as are the chromatin proteins CTCF and SAP30, indicating a distinctive chromatin state in this region of the Xi. Taken together, our data imply that the Xi adopts a distinct chromatin configuration in interphase nuclei and are consistent with a mechanism by which HP1, through histone H3 lysine-9 methylation, recognizes and assists in maintaining heterochromatin and gene silencing at the human Xi. Barr body is an inactivated X chromosome in the normal female somatic cell. Inactivation of these chromosomes is known as Lyonization. Lyonization has both genetic and clinical significance. Barr body can be easily identified with ordinary stains. It also helps in identifying the sex of an individual when used judiciously. A review is made on the lyonization of Barr body and its utility in sex determination. The radial positions of the centromeric regions of chromosomes 1 and X were determined in normal male fibroblasts (XY) and in fibroblasts from a patient with a rare case of XXXXY polysomy. The centromeric regions and presumably the whole territories of active X chromosomes were demonstrated to occupy similar, although not identical, positions in XY and XXXXY cells. The centromeres of inactive X chromosomes (Barr bodies) were located closer to the nuclear periphery as compared with the centromeres of active X chromosomes. In addition, it was established that the nuclear radial position of gene-rich chromosome 1 was changed in XXXXY cells as compared to normal XY cells. The data are discussed in the context of the hypothesis postulating that changes in nuclear positioning of chromosomal territories induced by the presence of extra copies of individual chromosomes may contribute to the development of diseases related to different polysomies. Interest has recently reawakened in whether loss of the heterochromatic X chromosome (Barr body) is prevalent in certain breast and ovarian cancers, and new insights into the mechanisms involved have emerged. Mitotic segregation errors commonly explain the loss of the inactive X chromosome (Xi), but compromise of Xi heterochromatin in some cancers may signal broader deficits of nuclear heterochromatin. The debated link between BRCA1 and Xi might reflect a general relationship between BRCA1 and heterochromatin, which could connect BRCA1 to both epigenetic and genetic instability. We suggest that heterochromatic instability is a common but largely unexplored mechanism, leading to widespread genomic misregulation and the evolution of some cancers. The Barr body is the inactive X chromosome in a female somatic cell. It is readily identified as plano-convex structure of 2-3 micron in diameter on the periphery of the nuclear membrane. The aim of this study is to evaluate the significance of Barr body count in maligt ovarian tumors on fine needle aspiration cytology (FNAC) smears. In this retrospective study, Barr body was counted in FNAC smears of 20 successive maligt ovarian lesions and expressed as percentage. Mean (±SD) Barr body score was 2.4 ± 2.58. Minimum Barr body count was 1 and maximum was 9. The gross reduction of Barr body in ovarian neoplasms is an interesting cytomorphologic finding. Human inactive X chromosome (Xi) forms a compact structure called the Barr body, which is enriched in repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3) and Lys27 (H3K27me3). These two histone marks are distributed in distinct domains, and X-inactive specific transcript (XIST) preferentially colocalizes with H3K27me3 domains. Here we show that Xi compaction requires HBiX1, a heterochromatin protein 1 (HP1)-binding protein, and structural maintece of chromosomes hinge domain-containing protein 1 (SMCHD1), both of which are enriched throughout the Xi chromosome. HBiX1 localization to H3K9me3 and XIST-associated H3K27me3 (XIST-H3K27me3) domains was mediated through interactions with HP1 and SMCHD1, respectively. Furthermore, HBiX1 was required for SMCHD1 localization to H3K9me3 domains. Depletion of HBiX1 or SMCHD1, but not Polycomb repressive complex 2 (PRC2), resulted in Xi decompaction, similarly to XIST depletion. Thus, the molecular network involving HBiX1 and SMCHD1 links the H3K9me3 and XIST-H3K27me3 domains to organize the compact Xi structure. BACKGROUND: Cytogenetic studies from past decades have shown that interphase cells of female cats contain a densely stained chromatin mass in their nuclei called as Barr bodies (BBs) named after the scientist Murray Barr. BBs are unique chromatin structures formed due to the condensation of the X-chromosome. Many psychopathic disorders originate from defective genes including the multiple X syndromes. Males with extra X-chromosome generally present with severe personality disorder. The present study was conducted to determine the presence of extra X-chromosome in male jail inmates through the detection of BB in peripheral blood and buccal smear. MATERIALS AND METHODS: Study included 100 male subjects (fifty jail inmates and fifty controls), after obtaining the consent, peripheral blood smears (PBS) and buccal smears (BS) were prepared and stained using Leishman's and cresyl violet stain respectively. One hundred neutrophils in PBS and epithelial cells in BS were screened for detection of the BB; accumulated data were tabulated and statistically analyzed using t-test and Chi-square test. RESULTS: 60% of cases in PBS and 36% in BS showed positivity for the presence of BB in jail inmates as compared to 14% of cases in PBS and none in BS were positive for BB in controls. CONCLUSION: Presence of BB in male suggests increased likelihood of criminal tendencies. Further studies are to be carried out to compare the results with karyotyping. 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.

Is the zelda transcription factor a chromatin remodeller?

During developmental transition, the zygotic genome is largely transcriptionally quiescent and undergoes significant chromatin remodeling. In Drosophila, the DNA-binding protein Zelda (also known as Vielfaltig) is required for this transition and for transcriptional activation of the zygotic genome. Zelda is differentially required for chromatin accessibility, transcription factor binding, and gene expression in the early Drosophila embryo. Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation. Early enhancers are characterized by an intrinsically high nucleosome barrier. Zelda tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of zelda(zld) motifs.

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting. Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. Here we tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA. In this review, we discuss a novel on-site remodeling function that is mediated by the H2A-ubiquitin binding protein ZRF1. ZRF1 facilitates the remodeling of multiprotein complexes at chromatin and lies at the heart of signaling processes that occur at DNA damage sites and during transcriptional activation. In nucleotide excision repair ZRF1 remodels E3 ubiquitin ligase complexes at the damage site. During embryonic stem cell differentiation, it contributes to retinoic acid-mediated gene activation by altering the subunit composition of the Mediator complex. We postulate that ZRF1 operates in conjunction with cellular remodeling machines and suggest that on-site remodeling might be a hallmark of many chromatin-associated signaling pathways. We discuss yet unexplored functions of ZRF1-mediated remodeling in replication and double strand break repair. In conclusion, we postulate that on-site remodeling of multiprotein complexes is essential for the timing of chromatin signaling processes. In the endopterygote Drosophila melanogaster, Zelda is an activator of the zygotic genome during the maternal-to-zygotic transition (MZT). Zelda binds cis-regulatory elements (TAGteam heptamers), making chromatin accessible for gene transcription. Zelda has been studied in other endopterygotes: Apis mellifera and Tribolium castaneum, and the paraneopteran Rhodnius prolixus. We studied Zelda in the cockroach Blattella germanica, a hemimetabolan, short germ-band, and polyneopteran species. B. germanica Zelda has the complete set of functional domains, which is typical of species displaying ancestral features concerning embryogenesis. Interestingly, we found D. melanogaster TAGteam heptamers in the B. germanica genome. The canonical one, CAGGTAG, is present at a similar proportion in the genome of these two species and in the genome of other insects, suggesting that the genome admits as many CAGGTAG motifs as its length allows. Zelda-depleted embryos of B. germanica show defects involving blastoderm formation and abdomen development, and genes contributing to these processes are down-regulated. We conclude that in B. germanica, Zelda strictly activates the zygotic genome, within the MZT, a role conserved in more derived endopterygote insects. In B. germanica, zelda is expressed during MZT, whereas in D. melanogaster and T. castaneum it is expressed beyond this transition. In these species and A. mellifera, Zelda has functions even in postembryonic development. The expansion of zelda expression beyond the MZT in endopterygotes might be related with the evolutionary innovation of holometabolan metamorphosis. DATABASES: The RNA-seq datasets of B. germanica, D. melanogaster, and T. castaneum are accessible at the GEO databases GSE99785, GSE18068, GSE63770, and GSE84253. In addition, the RNA-seq library from T. castaneum adult females is available at SRA: SRX021963. The B. germanica reference genome is available as BioProject PRJNA203136.

What is the effect of the alleles CYP2C19*2 and CYP2C19*3 on CYP2C19 function?

The CYP2C19*2 and CYP2C19*3 alleles of CYP2C19 are associated with loss-of-function (LOF).

BACKGROUND: The CYP2C19*2 or *3 loss-of-function (LOF) allele is associated with high platelet reactivity (HPR) on clopidogrel treatment. East Asians may benefit from a lower dose of prasugrel due to their more potent platelet inhibitory response. The impact of LOF alleles on the pharmacodynamic response to half-dose prasugrel in patients with non-ST-elevation acute coronary syndrome (NSTE-ACS) undergoing percutaneous coronary intervention (PCI) is unknown. METHODS: Seventy patients with the LOF alleles were assigned to half-dose prasugrel (n = 35, 30-mg load followed by 5 mg daily) or clopidogrel (n = 35, 600-mg load followed by 75 mg daily). The primary endpoint was the rate of HPR, defined as VerifyNow-P2Y12 reaction unit (PRU) >235, at 24 h post loading. RESULTS: Prasugrel achieved a lower PRU compared to clopidogrel after loading (119 [56-175] vs. 245 [189-299]), at 24 h (34 [8-58] vs. 196 [122-244]), and at 30 days (134 [98-189] vs. 203 [144-248]). Prasugrel had a lower rate of HPR after loading (5.7% vs. 57.1%, p <0.001), at 24 h (2.9% vs. 28.6%, p=0.006), and at 30 days (11.4% vs. 34.3%, p=0.004). Prasugrel had a similar rate of optimal platelet reactivity at 30 days (71.4% vs. 60.0%, p=0.450). There was no significant difference in the occurrence of periprocedural myonecrosis within 48 h after PCI with clopidogrel and prasugrel (22.9% vs. 17.1%, p>0.960). CONCLUSIONS: Half-dose prasugrel provided potent platelet inhibition in NSTE-ACS patients that were carriers of the CYP2C19*2 or *3 allele, with a lower rate of HPR. Periprocedural myonecrosis was not significantly different in the 2 groups.

Describe Full Spectrum of Intolerance to Loss-of-function (FUSIL)

Full Spectrum of Intolerance to Loss-of-function (FUSIL) is a cross-species gene classification across the full spectrum of mutations in genes of unknown function. FUSIL has been proposed as a method to identify potentially pathogenic variants in genes not previously associated with rare diseases.

Author information: (1)Clinical Pharmacology, William Harvey Research Institute, School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK. (2)European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK. (3)The Jackson Laboratory, Bar Harbor, ME, 4609, USA. (4)Departments of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, 77030, USA. (5)Departments of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA. (6)Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. (7)The Centre for Phenogenomics, The Hospital for Sick Children, Toronto, ON, M5T 3H7, Canada. (8)The Centre for Phenogenomics, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5T 3H7, Canada. (9)Medical Research Council Harwell Institute (Mammalian Genetics Unit and Mary Lyon Centre), Harwell, Oxfordshire, OX11 0RD, UK. (10)German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764, Neuherberg, Germany. (11)Université de Strasbourg, CNRS, INSERM, Institut Clinique de la Souris, PHENOMIN-ICS, 67404, Illkirch, France. (12)Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Vestec, 252 50, Prague, Czech Republic. (13)Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK. (14)Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany. (15)Department of Developmental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, 85764, Neuherberg, Germany. (16)Deutsches Institut für Neurodegenerative Erkrankungen (DZNE) Site Munich, Munich Cluster for Systems Neurology (SyNergy), Adolf-Butedt-Institut, Ludwig-Maximilians-Universität München, 80336, Munich, Germany. (17)Monterotondo Mouse Clinic, Italian National Research Council (CNR), Institute of Cell Biology and Neurobiology, 00015, Monterotondo Scalo, Italy. (18)Translational Medicine, The Hospital for Sick Children, Toronto, ON, M5T 3H7, Canada. (19)Université de Strasbourg, CNRS, INSERM, Institut de Génétique, Biologie Moléculaire et Cellulaire, Institut Clinique de la Souris, IGBMC, PHENOMIN-ICS, 67404, Illkirch, France. (20)Department of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, 85354, Freising-Weihenstephan, Germany. (21)German Center for Diabetes Research (DZD), 85764, Neuherberg, Germany. (22)Mouse Biology Program, University of California, Davis, CA, 95618, USA. (23)Clinical Pharmacology, William Harvey Research Institute, School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK. [email protected]. (#)Contributed equally

Is capmatinib effective for glioblastoma?

No. Combination of capmatinib buparlisib resulted in no clear activity in patients with recurrent PTEN-deficient glioblastoma.

Conflict of interest statement: In relation to this presentation, we declare the following, real or perceived conflicts of interest: M. van den Bent has received grants from Abbvie, and honoraria from Cellgene, BMS, Boehringer, AGIOS and VaXIMM. A. Azaro has received consulting fees from Orion Pharmaceuticals and Amcure GmbH. F. De Vos has received ficial support for conducting clinical trials from Novartis, BMS, AbbVie and Bioclin. J.M. Sepulveda has received consulting fees from Celgene, Pfizer and Abbvie; he has received research grants from Pfizer and Catalysis. W.K.A. Yung holds stocks in DNATrix; he has received honoraria from DNATrix and Boehringer Ingelheim; he holds patents, royalties and/or intellectual property in, and has participated in a consulting or advisory role for DNATrix; he has received travel and/or accommodation expenses from Boehringer Ingelheim. P. Wen has received grants/research support from Lilly USA, Agios, AstraZeneca, Beigene, Eli Lily, Immunocellular Therapeutics, Kazai, Kadmon, Karyopharm, Merck, Novartis, Oncoceutics, Vascular Biogenics and Vaccines; he has received speaker’s bureau fees from Merck; he has received consultant/advisory board fees from Genentech/Roche, Taiho Oncology, Novartis, Agios Pharmaceuticals Inc, Merck, Puma, Abbvie, AstraZeneca, Eli Lilly, GW Pharmaceuticals, Immunomic Therapeutics, Kadmon, Vascular Biogenics, Ziopharm, Monteris and Tocagen. A. Lassman reports grants and non-ficial support from Novartis, during the conduct of the study; personal fees and non-ficial support from Orbus, grants, personal fees and non-ficial support from Karyopharm, personal fees and non-ficial support from NW Biotherapeutics, grants and non-ficial support from Oncoceutics, personal fees and non-ficial support from Agios, personal fees and non-ficial support from Celgene, personal fees and non-ficial support from Novocure, non-ficial support from Tocagen, non-ficial support from BMS, grants, personal fees and non-ficial support from Kadmon, grants and non-ficial support from Genentech/Roche, grants and non-ficial support from Amgen, grants and non-ficial support from Millenium, non-ficial support from Celldex, grants and non-ficial support from Pfizer, non-ficial support from Keryx/Aeterna Zentaris, grants and non-ficial support from VBI Vaccines, grants and non-ficial support from Beigene, personal fees from Bioclinica as an expert blinded independent reviewer of clinical and imaging data for a BMS-sponsored trial, personal fees from prIME Oncology, personal fees and non-ficial support from Sapience, personal fees from WebMD, personal fees and non-ficial support from Physicians' Education Resource, personal fees from Cortice, grants, personal fees and non-ficial support from AbbVie, personal fees and non-ficial support from Forma, personal fees and non-ficial support from Bayer, grants and non-ficial support from Global Coalition for Adaptive Research, personal fees and non-ficial support from American Society of Clinical Oncology, grants and non-ficial support from QED, grants, personal fees and non-ficial support from NCI, non-ficial support from New York University, grants and non-ficial support from NRG Oncology/RTOG-Foundations, grants from UCLA, grants from Northwestern University, grants from James S. McDonnell Foundation, non-ficial support from Yale University, non-ficial support from Radiological Society of North America, non-ficial support from FDA, personal fees from Italian Foundation for Cancer Research, personal fees and non-ficial support from Abbott Molecular, and personal fees from Elsevier, outside the submitted work M. Joerger has received grants from BMS and AstraZeneca. G. Tabatabai has served on Advisory Boards for AbbVie and BMS, has received research/travel grants from Medac, Novocure and Roche Diagnostics, and has received speaker`s fees from Meda and Novocure. J. Rodon reports non-ficial support and reasonable reimbursement for travel from European Journal of Cancer, Vall d'Hebron Institut of Oncology, Chinese University of Hong Kong, SOLTI, Elsevier, GlaxoSmithKline; receiving consulting and travel fees from Novartis, Eli Lilly, Orion Pharmaceuticals, Servier Pharmaceuticals, Peptomyc, Merck Sharp & Dohme, Kelun Pharmaceutical/Klus Pharma, Spectrum Pharmaceuticals Inc, Pfizer, Roche Pharmaceuticals, Ellipses Pharma (including serving on the scientific advisory board from 2015-present), receiving research funding from Bayer and Novartis, and serving as investigator in clinical trials with Spectrum Pharmaceuticals, Tocagen, Symphogen, BioAtla, Pfizer, GenMab, CytomX, Kelun-Biotech, Takeda-Millenium, GlaxoSmithKline, IPSEN and travel fees from ESMO, US Department of Defense, Louissiana State University, Hunstman Cancer Institute, Cancer Core Europe, Karolinska Cancer Institute and King Abdullah International Medical Research Center (KAIMRC). R. Tiedt, T. Kirsilae and S. Vicente are employees of Novartis Pharma AG. S. Zhao is an employee of Novartis Institutes for Biomedical Research (China). A. Balbin is an employee of Novartis Institutes for Biomedical Research (US). H. Zhang is an employee of Novartis and holds shares with Novartis. W. Wick receives study support to the institution from Apogenix, Pfizer and Roche.

Are there antimicrobial proteins in royal jelly?

Yes, Jelleines, isolated as novel antibacterial peptides from the Royal Jelly (RJ) of bees, exhibit broad-spectrum protection against microbial infections.

Typically, antimicrobial peptides (AMPs) are short positive charged peptides serving a key role in innate immunity as well as antimicrobial activity. Discovering novel therapeutic agents is considered as an undeniable demand due to increasing microbial species with antibiotic resistance. In this direction, the unique ability of AMPs to modulate immune responses highlighted them as novel drug candidates in the field of microbiology. Patients affected by leishmaniasis; a neglected tropical disease, confront serious problems for their treatment including resistance to common drugs as well as toxicity and high cost of therapy. So, there is a need for development of new drug candidates to control the diseases. Jellein, a peptide derived from royal jelly of honeybee has been shown to have promising effect against several bacterial and fungal species. In current study, anti-leishmanial effect of Jellein and its lauric acid conjugated form was investigated against two forms of Leishmania major (L. major) parasite. Moreover, cytotoxic effect of these peptides was studied in THP1 cell line and human Red Blood Cells (RBCs). Furthermore, the mechanism of action of peptides on L. major promastigotes was assessed through different methods. The results demonstrated that, conjugation of lauric acid to Jellein not only had no effect on the elevation of antimicrobial activity but also halted it completely. Moreover, Jellein caused a limitation in the number of L. major promastigotes by pore formation as well as changing the membrane potential rather than induction of apoptosis or activation of caspases. Royal jelly (RJ) is a yellowish-white and acidic secretion of hypopharyngeal and mandibular glands of nurse bees used to feed young worker larvae during the first three days and the entire life of queen bees. RJ is one of the most appreciated and valued natural product which has been mainly used in traditional medicines, health foods, and cosmetics for a long time in different parts of the world. It is also the most studied bee product, aimed at unravelling its bioactivities, such as antimicrobial, antioxidant, anti-aging, immunomodulatory, and general tonic action against laboratory animals, microbial organisms, farm animals, and clinical trials. It is commonly used to supplement various diseases, including cancer, diabetes, cardiovascular, and Alzheimer's disease. Here, we highlight the recent research advances on the main bioactive compounds of RJ, such as proteins, peptides, fatty acids, and phenolics, for a comprehensive understanding of the biochemistry, biological, and pharmaceutical responses to human health promotion and life benefits. This is potentially important to gain novel insight into the biological and pharmaceutical properties of RJ.

What are the years of the initiation and completion of the Human Genome project?

The Human Genome Project was initiated in 1990 and completed in 2003.

Information obtained from the Human Genome Project, initiated in 1990 and targeted for completion in 2005, will influence both health care and nursing practice. It will substantially revise our understanding of disease susceptibility and causation. Additional genetic tests will be developed and gene therapies explored. The project has implications for both nursing research and nursing practice. This article reviews the establishment of the Human Genome Project, reports on current progress of the project, and identifies some implications of the project for health care generally and nursing specifically. The Human Genome Project (HGP) was initiated in 1990 and completed in 2003. It aimed to sequence the whole human genome. Although it represented an advance in understanding the human genome and its complexity, many questions remained uswered. Other projects were launched in order to unravel the mysteries of our genome, including the ENCyclopedia of DNA Elements (ENCODE). This review aims to analyze the evolution of scientific knowledge related to both the HGP and ENCODE projects. Data were retrieved from scientific articles published in 1990-2014, a period comprising the development and the 10 years following the HGP completion. The fact that only 20,000 genes are protein and RNA-coding is one of the most striking HGP results. A new concept about the organization of genome arose. The ENCODE project was initiated in 2003 and targeted to map the functional elements of the human genome. This project revealed that the human genome is pervasively transcribed. Therefore, it was determined that a large part of the non-protein coding regions are functional. Finally, a more sophisticated view of chromatin structure emerged. The mechanistic functioning of the genome has been redrafted, revealing a much more complex picture. Besides, a gene-centric conception of the organism has to be reviewed. A number of criticisms have emerged against the ENCODE project approaches, raising the question of whether non-conserved but biochemically active regions are truly functional. Thus, HGP and ENCODE projects accomplished a great map of the human genome, but the data generated still requires further in depth analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:215-223, 2016. Since the completion of the Human Genome Project in 2003 and the announcement of the Precision Medicine Initiative by U.S. President Barack Obama in January 2015, human beings have initially completed the " three steps" of " genomics to biology, genomics to health as well as genomics to society". As a new inter-discipline, the emergence and development of precision medicine have relied on the support and promotion from biological science, basic medicine, clinical medicine, epidemiology, statistics, sociology and information science, etc. Meanwhile, molecular epidemiology is considered to be the core power to promote precision medical as a cross discipline of epidemiology and molecular biology. This article is based on the characteristics and research progress of medicine and molecular epidemiology respectively, focusing on the contribution and significance of molecular epidemiology to precision medicine, and exploring the possible opportunities and challenges in the future. Lately, the term "genomics" has become ubiquitous in many scientific articles. It is a rapidly growing aspect of the biomedical sciences that studies the genome. The human genome contains a torrent of information that gives clues about human origin, evolution, biological function, and diseases. In a bid to demystify the workings of the genome, the Human Genome Project (HGP) was initiated in 1990, with the chief goal of sequencing the approximately 3 billion nucleotide base pairs of the human DNA. Since its completion in 2003, the HGP has opened new avenues for the application of genomics in clinical practice. This review attempts to overview some milestone discoveries that paved way for the initiation of the HGP, remarkable revelations from the HGP, and how genomics is influencing a paradigm shift in routine clinical practice. It further highlights the challenges facing the implementation of genomic medicine, particularly in Africa. Possible solutions are also discussed.

Which methods infer 3D genome structure without proximity ligation?

SPRITE is a method that enables genome-wide detection of higher-order interactions within the nucleus. Transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. Genome architecture mapping (GAM) can be used for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections.

Since Jacob and Monod's characterization of the role of DNA elements in gene control, it has been recognized that the linear organization of genome structure is important for the regulation of gene transcription and hence the manifestation of phenotypes. Similarly, it has long been hypothesized that the spatial organization (in three dimensions evolving through time), as part of the epigenome, makes a significant contribution to the genotype-phenotype transition. Proximity ligation assays commonly known as chromosome conformation capture (3C) and 3C based methodologies (e.g., GCC, HiC and ChIA-Pet) are increasingly being incorporated into empirical studies to investigate the role that three-dimensional genome structure plays in the regulation of phenotype. The apparent simplicity of these methodologies-crosslink chromatin, digest, dilute, ligate, detect interactions-belies the complexity of the data and the considerations that should be taken into account to ensure the generation and accurate interpretation of reliable data. Here we discuss the probabilistic nature of these methodologies and how this contributes to their endogenous limitations. BACKGROUND: Several recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication. RESULTS: We describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines. CONCLUSION: TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin. High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains. The packaging of DNA inside a nucleus shows complex structure stabilized by a host of DNA-bound factors. Both the distribution of these factors and the contacts between different genomic locations of the DNA can now be measured on a genome-wide scale. This has advanced the development of models aimed at predicting the conformation of DNA given only the locations of bound factors-the chromatin folding problem. Here we present a maximum-entropy model that is able to predict a contact map representation of structure given a sequence of bound factors. Non-local effects due to the sequence neighborhood around contacting sites are found to be important for making accurate predictions. Lastly, we show that the model can be used to infer a sequence of bound factors given only a measurement of structure. This opens up the possibility for efficiently predicting sequence regions that may play a role in generating cell-type specific structural differences. Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus. Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4+ T cells revealed substantial reorganization of enhancer-promoter interactions associated with changes in gene expression after T cell receptor stimulation.

How many genes are screened by the FoundationOne companion diagnostic?

FoundationOne CDx comprises a 324-gene panel.

Interaction of WDR5 with which gene has a critical role in pancreatic cancer?

Mechanistically, WDR5 functions to sustain proper execution of DNA replication in pancreatic ductal adenocarcinoma (PDAC) cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. It was indeed demonstrated that interaction with c-Myc is critical for this function.

Author information: (1)Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Molecular and Cellular Oncology, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy. Electronic address: [email protected]. (2)Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Molecular and Cellular Oncology, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (3)Institute for Applied Cancer Science, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (4)Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy. (5)Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (6)Sheikh Ahmed Bin Zayed Al Nahyan Center for Pancreatic Cancer Research, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (7)Department of Epigenetics and Molecular Carcinogenesis, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (8)Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy; Department of Oncology and Hemato-oncology, University of Milan, Milan 20139, Italy. (9)Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan 20139, Italy. (10)Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Institute for Applied Cancer Science, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (11)Department of Surgical Oncology, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (12)Department of Pathology, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (13)Department of Cancer Biology, UT MD Anderson Cancer Center, Houston, TX 77030, USA. (14)Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy. Electronic address: [email protected]. (15)C-4 Therapeutics, Cambridge, MA 02142, USA. Electronic address: [email protected]. (16)Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Institute for Applied Cancer Science, UT MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Molecular and Cellular Oncology, UT MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: [email protected].

Can thiotepa be recommended for treatment of osteosarcoma?

No. Thiotepa did not improve survival of patients with osteosarcoma and therefore can not be recommended for treatment of osteosarcoma.

Introduction. Osteosarcoma relapse has a poor prognosis, with less than 25% survival at 5 years. We describe the experience of the French Society of Paediatric Oncology (SFCE) with high dose (HD) thiotepa and autologous stem cell transplantation (ASCT) in 45 children with relapsed osteosarcoma. Patients and Methods. Between 1992 and 2004, 53 patients received HD thiotepa (900 mg/m(2)) followed by ASCT in 6 centres. Eight patients were excluded from analysis, and we retrospectively reviewed the clinical radiological and anatomopathological patterns of the 45 remaining patients. Results. Sixteen girls and 29 boys (median age, 15.9 years) received HD thiotepa after initial progression of metastatic disease (2), first relapse (26), and second or third relapse (17). We report 12 radiological partial responses and 9 of 31 histological complete responses. Thirty-two patients experienced further relapses, and 13 continued in complete remission after surgical resection of the residual disease. Three-year overall survival was 40%, and 3-year progression-free survival was 24%. Delay of relapse (+/- 2 years from diagnosis) was a prognostic factor (P = 0.011). No acute toxic serious adverse event occurred. Conclusion. The use of HD thiotepa and ASCT is feasible in patients with relapsed osteosarcoma. A randomized study for recurrent osteosarcoma between standard salvage chemotherapy and high dose thiotepa with stem cell rescue is ongoing.

What is known about aberrant proteolytic activity in disease?

Research into Alzheimer's disease pathology and treatment has often focused on presenilin proteins. These proteins provide the key catalytic activity of the γ-secretase complex in the cleavage of amyloid-β precursor protein and resultant amyloid tangle deposition. Over the last 25 years, screening novel drugs to control this aberrant proteolytic activity has yet to identify effective treatments for the disease. Others are human skin disorders where proteolytic pathways are dysregulated.

INTRODUCTION: In the nonobese diabetic (NOD) mouse model of Sjögren's syndrome, lymphocytic infiltration is preceded by an accumulation of dendritic cells in the submandibular glands (SMGs). NOD mice also exhibit an increased frequency of mature, fractalkine receptor (CX3C chemokine receptor [CX3CR]1) expressing monocytes, which are considered to be precursors for tissue dendritic cells. To unravel further the role played by fractalkine-CX3CR1 interactions in the salivary gland inflammation, we studied the expression of fractalkine in NOD SMGs. METHODS: We studied protein expression using Western blot analysis of whole tissue lysates. Protease activity was measured in salivary gland tissue lysates using fluorimetric substrates. Digestive capacity of enzymes was determined by in vitro incubation of recombit enzyme and fractalkine, followed by protein staining and Western blot. RESULTS: Fractalkine was detected in salivary glands of both NOD and control mice at all ages. Western blot analysis showed fractalkine cleavage with increasing age, which was more pronounced in NOD mice. This cleavage resulted in a decrease in the 31 kDa form of the protein, and the generation of an approximately 19 kDa band. Furthermore, in NOD animals older than 15 weeks, we noted the presence of a unique approximately 17 kDa fragment. This cleavage was organ specific, because it did not occur in brain or pancreas. Increased gelatinase and alpha-secretase activity were detected in NOD SMG and contributed to cleavage of the 31 kDa protein. Because aberrant cleavage products may induce autoimmunity, we studied the presence of autoantibodies against fractalkine. Indeed, NOD mice exhibited significantly more antibodies against fractalkine than did control animals. CONCLUSION: These data indicate that aberrant proteolytic activity in the NOD SMG results in increased fractalkine cleavage and generation of a unique fractalkine fragment. This specific cleavage may contribute to autoimmunity. Recent findings from the clinic and the laboratory have transformed the way proteases and their inhibitors are perceived in the outermost layer of the skin, the epidermis. It now appears that an integrated proteolytic network operates within the epidermis, comprising more than 30 enzymes that carry out a growing list of essential functions. Equally, defective regulation or execution of protease-mediated processes is emerging as a key contributor to diverse human skin pathologies, and in recent years the number of diseases attributable to aberrant proteolytic activity has more than doubled. Here, we survey the different roles of proteases in epidermal homeostasis (from processing enzymes to signalling molecules) and explore the spectrum of rare and common human skin disorders where proteolytic pathways are dysregulated. Research into Alzheimer's disease pathology and treatment has often focused on presenilin proteins. These proteins provide the key catalytic activity of the γ-secretase complex in the cleavage of amyloid-β precursor protein and resultant amyloid tangle deposition. Over the last 25 years, screening novel drugs to control this aberrant proteolytic activity has yet to identify effective treatments for the disease. In the search for other mechanisms of presenilin pathology, several studies have demonstrated that mammalian presenilin proteins also act in a non-proteolytic role as a scaffold to co-localize key signaling proteins. This role is likely to represent an ancestral presenilin function, as it has been described in genetically distant species including non-mammalian animals, plants, and a simple eukaryotic amoeba Dictyostelium that diverged from the human lineage over a billion years ago. Here, we review the non-catalytic scaffold role of presenilin, from mammalian models to other biomedical models, and include recent insights using Dictyostelium, to suggest that this role may provide an early evolutionary function of presenilin proteins.

Does a comet assay measure radiation induced mutations?

Yes. The comet assay is frequently used to measure DNA damage in individual cells.

The comet assay is frequently used to measure DNA damage in individual cells. In order to better understand the mechanisms behind the technique, we have studied the behaviour of DNA under different electrophoresis conditions in mammalian cells exposed to gamma radiation. The comet tails obtained after neutral electrophoresis seem to consist of DNA loops which are attached to structures in the nucleus, since the DNA cannot move in the second direction after two-dimensional electrophoresis. When the DNA is labelled by a short pulse, microautoradiography reveals that all label appear in the head of the comets when neutral electrophoresis is applied. After chase incubation, the label moves out into the tails. This gives further support to the view that the DNA loops are fixed to some structure in the nucleus where also the DNA synthesis takes place. Under alkaline electrophoresis conditions, however, the entire comet tails move in the new electrophoresis direction. Thus, it appears that the alkaline comet tails consist of free DNA fragments. Further, the effects of alkaline concentration and sodium chloride during unwinding and electrophoresis are discussed. Throughout the study, a protocol for drying and fixation of the comets has been used. The single cell gel test (SCG-test or comet assay) is a rapid and sensitive method for measuring DNA damage and repair in individual cells. A wide variety of mutagens have been shown to cause DNA alterations detectable with the comet assay, but it is not yet clear whether a relationship exists between the DNA effects and the induction of mutations. We are therefore investigating in a cell culture system with human cells (MRC5CV1) the induction of DNA damage by environmental mutagens and the formation of mutations at the HPRT gene. In the present study we investigated benzo[a]pyrene (BP), an environmental mutagenic and carcinogenic polycyclic aromatic hydrocarbon, and its reactive metabolite (+)-anti-benzo[a]pyrene-7,8-diol 9, 10-oxide ((+)-anti-BPDE). S9 mix activated BP and the direct acting mutagen (+)-anti-BPDE caused a concentration-related increase in DNA migration in the comet assay. Postincubation experiments indicated that induced DNA effects are eliminated by DNA repair within 24 h. BP-treatment caused a strong genotoxic effect in the comet assay but had only a marginal effect on the frequency of gene mutations. When cells were treated with BP in the presence of cadmium sulphate, a clear increase in genotoxicity was observed while the effect on mutations was unchanged. Our results indicate that DNA alterations detected with the comet assay do not necessarily relate to mutagenesis. The absence of a close relationship between DNA migration in the comet assay and mutagenesis may be explained by the fact that some effects seen in the comet assay occur as a consequence of an error free DNA repair process. The alkaline single-cell gel electrophoresis (SCGE or Comet) assay appears to be a promising tool for measuring DNA damage at the individual cell level in both in vitro and in vivo studies. To provide further data on the possible applicability of this assay in human biomonitoring studies, we have evaluated the eventual genetic damage induced by therapeutic exposure to 131I, by measuring the Comet length and the amount of DNA damage in peripheral blood leukocytes from a group of 28 thyroid cancer patients who received 131I sodium iodide via oral administration. Blood samples were taken just before the treatment and 1 week after it. From the results obtained after radioiodine therapy, a small increase in the Comet length and in the grade of DNA damage is observed; however, this increase is not statistically significant because of inter-individual variability and the variable responses before and after 131I treatment. Considering our previous studies showing significant increases in the frequency of cytogenetic damage (when measured as micronuclei) in patients treated with relatively low doses of 131I, the results obtained in the present work by using the Comet assay could indicate that 1 week after the exposure most of the radioiodine-induced DNA lesions, that can be detected with this assay, have already been repaired. BACKGROUND: In clinical radiotherapy most patients tolerate the applied dosage with no or moderate side effects. However, 5 to 10% of all individuals show increased acute and/or late reactions. In-vitro test systems are investigated for their suitability for predictive purposes. This paper attempts a correlation between the induction and repair of DNA damage measured in the comet assay and the clinical observed reaction in order to evaluate the suitability of the comet assay for prediction of radiation sensitivity. PATIENTS AND METHODS: Skin fibroblasts of 30 patients with average tissue reactions or acute and/or late increased side effects and cell lines of 4 individuals carrying the heritable disease ataxia telangiectasia (AT) were irradiated in vitro. The induction and repair of DNA damage was measured at different time points after irradiation in the comet assay (single cell gel electrophoresis). These results were compared to the acute and late clinical reactions classified according to the RTOG grading system. RESULTS: The radiation induced DNA damage decreased over time reflecting DNA repair. Cells of the AT individuals showed an elevated damage induction and a reduced repair capacity compared to patients with average tissue reactions. Fibroblasts of patients with increased acute and late side effects exhibited slower DNA repair. In addition to the known lack of cell cycle control, our results indicate that AT cells show reduced DNA repair capacity. CONCLUSIONS: The comet assay seems to be able to detect some types of increased individual radiation sensitivity. In contrast to other predictive in-vitro tests, the comet assay needs less time and fewer cells, which would be useful in a clinical setting. The single-cell electrophoresis (comet) assay is an established method for measuring radiation-induced strand breaks in DNA. The extent of damage is quantified in individual cells by assessing the intensity and distribution of a fluorescent DNA-binding dye using image analysis software. Using the Kinetic Imaging Komet3 system, it is demonstrated that the fluorochrome SYBR Green I improves the resolution and sensitivity of the comet assay, particularly for measuring radiation-induced double strand breaks. Recent in vivo and in vitro data of patients analyzed for genetic susceptibility to radiation during cancer therapy have shown structural changes in the chromosomes to be prevalent both in the patients being treated and in their immediate family members. As structural changes in chromosomes frequently lead to activation of proto-oncogenes and elimination of tumor-suppressor genes, they represent important mechanisms for the initiation of DNA repair processes and tumorigenesis. With the exception of rare genetic syndromes such as AT (Ataxia telangiectasia) or NBS (Nijmegen Breakage Syndrome), the background for the inheritance of genetic susceptibility to radiation is unknown. Recently, a large-scale genetic screen of mouse mutants has been established within the German Human Genome Project (Hrabè de Angelis and Balling 1998). The goal of this ENU (ENU: ethylnitrosourea) mutagenesis screen is the generation of mutant mice that will serve as animal models for human diseases and genetic susceptibility. In order to fully utilize the potential of a genetic screen of this magnitude, in which exploration for genes responsible for genomic instability and radiation sensitivity is to occur, it is necessary to establish a simple assay system that is amenable to automation. Hence, we are using the single-cell gel electrophoresis (comet assay) to detect mouse mutants that display a genetic susceptibility to ionizing radiation. We have established the analysis parameters in the comet assay which are currently used to detect radiation-sensitive mouse mutants and to control the variance within the mouse population in the ENU screen. The assay can be used to isolate genes that are responsible for DNA repair and radiation sensitivity in mouse and human. The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125-1 Gy) and compared with the effects resulting from irradiation with (60)Co gamma. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by (60)Co gamma. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation. The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented. Lymphocytes of healthy volunteers (n = 24) and of tumour patients (n = 30, 18 of whom had experienced severe side-effects) were irradiated with x-rays in vitro. DNA damage was analysed after 0.25-2 Gy and DNA repair after 2 Gy, and quantification of both endpoints was done by the comet assay. The individual differences in radiation-induced DNA damage as well as in the repair kinetics were observed to be striking for both healthy donors and tumour patients. After a repair time of 3 h, following 2 Gy x-irradiation, some of the healthy volunteers showed no residual DNA damage at all in their lymphocytes, whereas others revealed about 30%. There was no indication that our results were affected by either age, gender or smoking habits. Slow repair kinetics and high amounts of residual damage were characteristic for many but not all tumour patients who had experienced severe side-effects in their normal tissues during or after radiotherapy (n = 18). Our conclusion is that those individuals showing poor DNA repair characteristics in the lymphocytes following in vitro irradiation, have a high probability of being radiosensitive. The opposite conclusion is not necessarily true: if repair is effective, this does not mean that the individual is radioresistant, because factors other than impaired repair may cause radiosensitivity. BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway. The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by gamma-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells. Lymphoblastoid cell lines (LCL) with a heterozygous mutation in the breast cancer susceptibility gene BRCA1 have been repeatedly used to elucidate the biological consequences of such a mutation with respect to radiation sensitivity and DNA repair deficiency. Our previous results indicated that LCL with a BRCA1 mutation do not generally show the same chromosomal mutagen sensitivity in the micronucleus test as lymphocytes with the same BRCA1 mutation. To further study the radiosensitivity of LCL with a BRCA1 mutation, we now performed comparative investigations with the alkaline (pH 13) and the neutral (pH 8.3) comet assay and pulsed field gel electrophoresis (PFGE). These tests are commonly used to determine the repair capacity for DNA double strand breaks (DNA-DSB). Six LCL (three established from women with a heterozygous BRCA1 mutation and three from healthy controls) were investigated. Induction (2 and 5 Gy) of gamma-ray-induced DNA damage and its repair (during 60 min after irradiation) was measured with the alkaline and neutral comet assay. Comparative experiments were performed with PFGE determining the induction of DNA-DSB by 10-50 Gy gamma-irradiation and their repair during 6 h. There was no significant difference between LCL with and without BRCA1 mutation in any of these experiments. Therefore, using these methods, no indication for a delayed repair of DNA-DSB in LCL with a BRCA1 mutation was found. However, these results do not generally exclude DNA-DSB repair deficiency in these cell lines because the methods applied have limited sensitivity and only measure the speed but not the fidelity of the repair process. The alkaline version of single cell gel electrophoresis (comet) assay is widely used for evaluating DNA damage at the individual cell level. The standard alkaline method of the comet assay involves deproteinization of cells embedded in agarose gel using a high salt-detergent lysis buffer, followed by denaturation of DNA and electrophoresis using a strong alkali at pH>13 [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184-191]. However, a recent report showed that a strong alkali treatment results in simultaneous deproteinization of cells and denaturation of genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single cell-level, Mutat. Res. 607 (2006) 205-214]. This study was carried out to test whether the strong alkali deproteinization of cells could replace the high salt-detergent lysis step used in the standard method of the alkaline comet assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the comet assay was performed according to the standard alkaline method (pH>13) and a modified method. In the modified method, agarose embedded cells were treated with a strong alkali (0.3M NaOH, 0.02 M Trizma and 1mM EDTA, pH>13) for 20 min to allow deproteinization of cells and denaturation of DNA. This was followed by electrophoresis using the same alkali solution to obtain comets. DNA damage expressed in terms of comet tail length, percentage of DNA in comet tail and tail moment obtained by the standard alkaline method and the modified method were compared. In both methods, DNA damage showed a good correlation with the dose of gamma ray. The results indicate a satisfactory sensitivity of the modified method in detecting radiation-induced DNA damage in human peripheral blood lymphocytes. Phototherapy (PT) is the most widely used form of treatment for unconjugated hyperbilirubinemia. One possible harmful consequence of PT is of a genetic nature. High levels of bilirubin may lead to oxidative damage in newborns: photochemical reactions may produce toxic photoproducts, probably peroxides. In order to investigate this hypothesis further under in vivo conditions, DNA strand-break frequency was examined by means of the comet assay in peripheral lymphocytes of icteric newborns undergoing PT treatment, and the levels of catalase, an antioxidant enzyme, were determined. We analyzed 20 term non-hemolitic hyperbilirubinemic jaundiced neonates before PT ('before PT' group) and just prior to ending PT ('after PT' group) and compared comet scores of these patients with those of 20 healthy term neonates who all had bilirubin levels in the physiological range. Comet scores (tail length, tail moment and %DNA in tail) of the group 'before phototherapy' were 23.5 +/- 16.3, 7.41 (0.97-40.7), 33.0 +/- 12.1, respectively and scores of after phototherapy group were 3.2 +/- 1.8, 0.29 (0.3-3.2), 10.7 +/- 3.7, respectively. Comet scores of the control group were 3.0 +/- 2.9, 0.25 (0.03-3.22), 10.9 +/- 4.5, respectively. Comet scores and plasma catalase activities in hyperbilirubinemic newborns were significantly higher before phototherapy, compared with the values after phototherapy and in the control groups (p < 0.001). There was no statistical difference between the 'after phototherapy' group and the controls (p > 0.05). These results indicate that high serum bilirubin level has genotoxic effects as is evident from the high rate of DNA strand-breaks in jaundiced newborns. Also PT does not cause an increase in DNA oxidation or induce the genotoxic effects of bilirubin. The counteracting effect of higher catalase activities in hyperbilirubinemic newborns may be responsible for the inactivating toxic and DNA-damaging effects of PT. The COMET assay is recognized as a rapid and sensitive method in quantifying radiation induced DNA damage. We investigated the distorting influence of endogenous, assay-inherent factors onto base (single cell level) and primary outcome measures (experimental/slide level), such as olive tail moment (OTM) and percentage DNA in the tail (%tail-DNA). From 2003 to 2008, we performed the assay on lymphocytes isolated from the blood samples of 355 lung cancer patients, 170 controls, and 610 relatives, as well as one single reference individual, repeated 170 times. In total, the data from 10,016 single experiments containing around 1,750,000 cells have been included in this study. This is the first time that the endogenous variability of the COMET assay has been validated systematically on such a huge data set over a 5 year period. Assuming that the reference sample reflects assay specific white noise, we estimated a proportion of 7-95% of the variability of the outcome measures due to assay variation (white noise) depending on parameter, exposure level, and study group. The proportion of white noise was largest for the initial radiation damage. The specific endogenous factors considered attribute to 14.8% of the total variability in the primary outcome measurements of the OTM and 6.9% of the %tail-DNA. OTM turns out to be a sensitive parameter to detect variation, but is also more susceptible to disturbance caused by endogenous factors than %tail-DNA. To reduce the experimental variability in COMET assays, we recommend a highly standardized operation protocol as well as inspecting and/or adjusting the primary outcome measures according to endogenous factors before calculating secondary outcome measures, e.g. DNA repair capacity (DRC) or testing statistical inference. A human reference (HR) sample is also useful to inspect homogeneity in the temporal progression of long lasting investigations, but not for calibrating primary outcome measurements. Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of 'Comet assay' for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in both bivalve species. This showed a dose-dependent increase of genetic damage induced in bivalves by EMS as well as gamma radiation. Further, the highest DNA damage was observed at 24h. The damage gradually decreased with time, i.e. was smaller at 48 and 72 h than at 24h post irradiation in both species of bivalves. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the post irradiation time advanced. The present study reveals that gamma radiation induces single strand breaks in DNA as measured by alkaline comet assay in bivalves and comet assay serves as a sensitive and rapid method to detect genotoxicity of gamma radiation. This study further indicates that both M. casta and P. malabarica exhibit almost identical sensitivity to gamma radiation as measured by DNA damage. Evaluation of primary DNA-damage is one way to identify potential genotoxic agents and for this purpose the Comet assay has, for the last decades, been used to monitor DNA single strand and double strand breaks in individual cells. Various attempts have been made to modify the different steps in the in vitro protocol for the Comet assay in order to improve its sensitivity. However, to the best of our knowledge, nobody has tried to replace the traditionally used NaOH-based electrophoresis solution (pH > 13), with another type of solution. In the present paper, using TK-6 cells exposed to different concentrations of H2O2 or ionizing radiation, we present evidence clearly showing that a low-conductive LiOH-based electrophoresis solution at pH 12.5, and a more gentle lysis procedure, significantly improved both the speed and sensitivity of the assay. The new approach, which we call the Flash-comet, is based on a lysis buffer at pH 8.5, an unwinding time of 2.5 min in a LiOH solution without EDTA at pH 12.5, and an electrophoresis time of 1 min at 150 V (5 V/cm) using the same solution.

What is formative pluripotency?

Formative pluripotency features a gene regulatory network switch from the nave state. Two phases of pluripotency, called nave and primed, have previously been described.

In the mouse blastocyst, epiblast cells are newly formed shortly before implantation. They possess a unique developmental plasticity, termed naive pluripotency. For development to proceed, this naive state must be subsumed by multi-lineage differentiation within 72 h following implantation. In vitro differentiation of naive embryonic stem cells (ESCs) cultured in controlled conditions provides a tractable system to dissect and understand the process of exit from naive pluripotency and entry into lineage specification. Exploitation of this system in recent large-scale RNAi and mutagenesis screens has uncovered multiple new factors and modules that drive or facilitate progression out of the naive state. Notably, these studies show that the transcription factor network that governs the naive state is rapidly dismantled prior to upregulation of lineage specification markers, creating an intermediate state that we term formative pluripotency. Here, we summarize these findings and propose a road map for state transitions in ESC differentiation that reflects the orderly dynamics of epiblast progression in the embryo. The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification.

Has the companion diagnostic HercepTest received FDA approval?

Yes, the HercepTest has received FDA approval.

Which R/Bioconductor package has been developed for visualizing differential amino acid group usage in proteomics?

DagLogo is an R/Bioconductor package for identifying and visualizing differential amino acid group usage in proteomics data. DagLogo allows various formats for input sets and provides comprehensive options to build optimal background models. It implements different reduced AA alphabets to group AAs of similar properties. Furthermore, dagLogo provides statistical and visual solutions for differential AA (or AA group) usage analysis of both large and small data sets. Case studies showed that dagLogo can better identify and visualize conserved protein sequence patterns from different types of inputs and can potentially reveal the biological patterns that could be missed by other logo generators.

Dasatinib and Blinatumomab are used for treatment of which disease?

Dasatinib and Blinatumomab produces molecular responses in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Liposomal vincristine is designed to reduce neurotoxicity and increase dose intensity delivery, and has been approved as salvage therapy in relapsed/refractory acute lymphoblastic leukemia (ALL). Our aim was to evaluate the response rate, toxicities, and outcome of adults with newly diagnosed ALL who received liposomal vincristine, rather than regular vincristine in combination with intensive chemotherapy (Hyper-CMAD). In a single-center, phase 2 study, patients ≥18 years with newly-diagnosed B-cell ALL were eligible to receive hyper-CMAD alternating with high-dose methotrexate and cytarabine. Rituximab was administered in CD20 positive ALL. Tyrosine kinase inhibitors (imatinib or dasatinib) were added in Philadelphia chromosome-positive (Ph-positive) ALL. Thirty-one patients were enrolled, median follow-up of 59 months (0.3-70). Thirteen patients (42%) had CD20 positive ALL, and 21 (68%) had Ph-positive ALL. Thirty (97%) achieved complete remission (CR). All 26 patients with abnormal karyotype achieved complete cytogenetic response (CCyR), and 27/30 (90%) achieved negative minimal residual disease status by multicolor flow cytometry. Of 20 evaluable Ph-positive ALL patients, major molecular response (MMR) was achieved in 19 patients (95%); complete molecular response (CMR) in 14 (70%). Grade 3/4 peripheral neuropathy was observed in five (16%) with all grade peripheral neuropathy in 21 (68%). With a median follow-up of 59 months, 21 (68%) patients are alive. The 5-year CR duration and survival rates were 73% and 61%, respectively. Ten (32%) patients died: one, sepsis on C1D10; four, unknown; one, post-transplant complications; four, relapse. Hyper-CMAD with liposomal vincristine is safe and demonstrated high response and survival rates in newly diagnosed ALL. Improving survival outcomes in adult B-cell acute lymphoblastic leukemia (B-ALL) remains a clinical challenge. Relapsed disease has a poor prognosis despite the use of tyrosine kinase inhibitors (TKIs) for Philadelphia chromosome positive (Ph+ ALL) cases and immunotherapeutic approaches, including blinatumomab and chimeric antigen receptor T cells. Targeting aberrant cell survival pathways with selective small molecule BH3-mimetic inhibitors of BCL-2 (venetoclax, S55746), BCL-XL (A1331852), or MCL1 (S63845) is an emerging therapeutic option. We report that combined targeting of BCL-2 and MCL1 is synergistic in B-ALL in vitro. The combination demonstrated greater efficacy than standard chemotherapeutics and TKIs in primary samples from adult B-ALL with Ph+ ALL, Ph-like ALL, and other B-ALL. Moreover, combined BCL-2 or MCL1 inhibition with dasatinib showed potent killing in primary Ph+ B-ALL cases, but the BH3-mimetic combination appeared superior in vitro in a variety of Ph-like ALL samples. In PDX models, combined BCL-2 and MCL1 targeting eradicated ALL from Ph- and Ph+ B-ALL cases, although fatal tumor lysis was observed in some instances of high tumor burden. We conclude that a dual BH3-mimetic approach is highly effective in diverse models of high-risk human B-ALL and warrants assessment in clinical trials that incorporate tumor lysis precautions. BACKGROUND: Outcomes in patients with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) have improved with the use of tyrosine kinase inhibitors. Molecular remission is a primary goal of treatment. METHODS: We conducted a phase 2 single-group trial of first-line therapy in adults with newly diagnosed Ph-positive ALL (with no upper age limit). Dasatinib plus glucocorticoids were administered, followed by two cycles of blinatumomab. The primary end point was a sustained molecular response in the bone marrow after this treatment. RESULTS: Of the 63 patients (median age, 54 years; range, 24 to 82) who were enrolled, a complete remission was observed in 98%. At the end of dasatinib induction therapy (day 85), 29% of the patients had a molecular response, and this percentage increased to 60% after two cycles of blinatumomab; the percentage of patients with a molecular response increased further after additional blinatumomab cycles. At a median follow-up of 18 months, overall survival was 95% and disease-free survival was 88%; disease-free survival was lower among patients who had an IKZF1 deletion plus additional genetic aberrations (CDKN2A or CDKN2B, PAX5, or both [i.e., IKZF1 plus]). ABL1 mutations were detected in 6 patients who had increased minimal residual disease during induction therapy, and all these mutations were cleared by blinatumomab. Six relapses occurred. Overall, 21 adverse events of grade 3 or higher were recorded. A total of 24 patients received a stem-cell allograft, and 1 death was related to transplantation (4%). CONCLUSIONS: A chemotherapy-free induction and consolidation first-line treatment with dasatinib and blinatumomab that was based on a targeted and immunotherapeutic strategy was associated with high incidences of molecular response and survival and few toxic effects of grade 3 or higher in adults with Ph-positive ALL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others; GIMEMA LAL2116 D-ALBA EudraCT number, 2016-001083-11; ClinicalTrials.gov number, NCT02744768.). A phase II trial shows that a chemotherapy-free regimen of dasatinib and blinatumomab produces molecular responses in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. The study found that 60% of patients had molecular responses after two cycles of blinatumomab and 71% had molecular responses after five cycles.

Is phosphoenolpyruvate carboxykinase 1 (PCK1) the rate-limiting enzyme in gluconeogenesis?

Yes, Pck1 is a rate-limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure.

Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at conditions of glucose deprivation. Pck1 is a rate-limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a target for proteasomal degradation through its proline residue at the penultimate position, recognized by Gid4 E3 ligase, but with a lack of informative structural details. In this study, we delineate the localized sequence motif, degron, that specifically interact with Gid4 ligase and unravel the binding mode of Pck1 to the Gid4 ligase by using molecular docking and molecular dynamics. The peptide/protein docking HPEPDOCK web server along with molecular dynamic simulations are applied to demonstrate the binding mode and interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently solved structure of Gid4 ligase. Results unveil a distinct binding mode of the mutated peptide compared with the wild type despite having comparable binding affinities to Gid4. Moreover, the four-residue peptide is found insufficient for Gid4 binding, while the seven-residue peptide suffices for binding to Gid4. The amino acids S134, K135, and N137 in the loop L1 (between β1 and β2) of the Gid4 are essential for the stabilization of the seven-residue peptide in the binding site of the ligase. The presence of Val4 instead of Lys4 smashes the H-bonds that are formed between Lys4 and Gid4 in the wild type peptide, making the peptide prone to bind with the other side of the binding pocket (L4 loop of Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant Pck1 peptide is introduced. This opens the door to explore the mutation effects on the binding mode and smooth the path to target protein degradation by design competitive and non-competitive inhibitors. BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. METHODS: Key residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. RESULTS: Mutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. CONCLUSIONS: Our results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. GENERAL SIGNIFICANCE: In higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus. The proteolytic cleavage of Fibronectin type III domain-containing 5 (FNDC5) generates soluble irisin. Initially described as being mainly produced in muscle during physical exercise, irisin mediates adipose tissue thermogenesis and also regulates carbohydrate and lipid metabolism. The aim of this study was to evaluate the hepatic expression of FNDC5 and its role in hepatocytes in Non-Alcoholic Fatty Liver (NAFL). Here we report that hepatic expression of FNDC5 increased with hepatic steatosis and liver injury without impacting the systemic level of irisin in mouse models of NAFLD (HFD and MCDD) and in obese patients. The increased Fndc5 expression in fatty liver resulted from its upregulation in hepatocytes and non-parenchymal cells in mice. The local production of Fndc5 in hepatocytes was influenced by genotoxic stress and p53-dependent pathways. The down-regulation of FNDC5 in human HepG2 cells and in primary mouse hepatocytes increased the expression of PEPCK, a key enzyme involved in gluconeogenesis associated with a decrease in the expression of master genes involved in the VLDL synthesis (CIDEB and APOB). These alterations in FNDC5-silenced cells resulted to increased steatosis and insulin resistance in response to oleic acid and N-acetyl glucosamine, respectively. The downregulation of Fndc5 also sensitized primary hepatocytes to apoptosis in response to TNFα, which has been associated with decreased hepatoprotective autophagic flux. In conclusion, our human and experimental data strongly suggest that the hepatic expression of FNDC5 increased with hepatic steatosis and its upregulation in hepatocytes could dampen the development of NAFLD by negatively regulating steatogenesis and hepatocyte death. Author information: (1)Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Electronic address: [email protected]. (2)Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Electronic address: [email protected]. (3)Biomarker & Translational Research Department, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-0005, Japan. Electronic address: [email protected]. (4)Biomarker & Translational Research Department, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-0005, Japan. Electronic address: [email protected]. (5)Oncology Research Laboratories I, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-0005, Japan. Electronic address: [email protected]. (6)Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Electronic address: [email protected]. (7)Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Electronic address: [email protected]. (8)Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Electronic address: [email protected]. Author information: (1)School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics (Theragnostic), Tianjin, People's Republic of China. (2)School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics (Theragnostic), Tianjin, People's Republic of China; Department of Pharmacy, Tianjin Children's Hospital, People's Republic of China. (3)Department of Immunology, Key Laboratory of Immune Microenvironment and Disease of the Educational Ministry of China, People's Republic of China. (4)School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics (Theragnostic), Tianjin, People's Republic of China; Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, People's Republic of China. Electronic address: [email protected]. (5)School of Pharmacy, Tianjin Medical University, Tianjin Key Laboratory on Technologies Enabling Development Clinical Therapeutics and Diagnostics (Theragnostic), Tianjin, People's Republic of China. Electronic address: [email protected].

What conditions are diagnosed using the scratch collapse test?

Scouring collapse test is used for the diagnosis of cts, cubital tunnel syndrome and carpal tunnel syndrome.

BACKGROUND: The scratch collapse test is a recently described provocative test for diagnosis of peripheral nerve compression. METHODS: The scratch collapse test was studied prospectively in 24 consecutive patients with a diagnosis of common peroneal nerve compression neuropathy. The diagnosis was confirmed by history, physical examination, and electrodiagnostic testing. Provocative testing by the scratch collapse test and Tinel's sign was performed. RESULTS: The scratch collapse test showed a sensitivity of 0.77 and a specificity of 0.99, while the Tinel's sign showed 0.65 and 0.99, respectively. CONCLUSION: The scratch collapse test is a sensitive and specific provocative test that compares favorably to existing clinical tests and aids in the diagnosis of common peroneal neuropathy. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, II. The utility of the scratch collapse test has been demonstrated in examination of patients with carpal and cubital tunnel syndromes and long thoracic and peroneal nerve compressions. In the authors' clinic, this lesser known test plays a key role in peripheral nerve examination where localization of the nerve irritation or injury is not fully understood. Test utility and accuracy in patients with more challenging presentations likely correlate with tester understanding and experience. This article offers a clear outline of all stages of the test to improve interrater reliability. The nuances of test performance are described, including a description of situations where the scratch collapse test is deemed inappropriate. Four clinical scenarios where the scratch collapse test may be useful are included. Corresponding video content is provided to improve performance and interpretation of the scratch collapse test. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, V. BACKGROUND: Despite the fact that carpal tunnel syndrome (CTS) is the most common entrapment neuropathy, the diagnostic accuracy of clinical screening examinations for CTS is controversial. The scratch collapse test (SCT) is a novel test that may be of diagnostic advantage. The purpose of our study was to determine the diagnostic accuracy of the SCT for CTS. METHODS: A literature search was performed using PubMed (1966 to April 2018); Ovid MEDLINE (1966 to April 2018); EMBASE (1988 to April 2018); and Cochrane Central Register of Controlled Trials (The Cochrane Library, to April 2018). We examined the studies for the pooled sensitivity, specificity, and likelihood ratios of the SCT. This review has been registered with PROSPERO (CRD42018077115). RESULTS: The literature search generated 13 unique articles. Seven articles were included for full text screening and 3 articles met our inclusion criteria, all of which were level II evidence with low risk of bias (165 patients). Pooled sensitivities, specificities, positive likelihood ratio, and negative likelihood ratios were 0.32 [95% CI (0.24-0.41)], 0.62 [95% CI (0.45-0.78)], 0.75 [95% CI (0.33-1.67)], and 1.03 [95% CI (0.61-1.74)], respectively. The calculated area under the summary receiver operating characteristic (AUSROC) curve was 0.25, indicating a low diagnostic accuracy. CONCLUSION: The SCT has poor sensitivity; however, it is moderately specific. Based on the current literature and their variable quality of the evidence, we conclude that the SCT is not an adequate screening test for detecting CTS. PURPOSE: To clarify the sensitivity, specificity, and interrater reliability of the scratch collapse test for carpal tunnel syndrome (CTS) and cubital tunnel syndrome, using blinded observers in a general patient population. METHODS: Ninety-two subjects were recruited from all patients referred for electrodiagnostic studies for upper extremity symptoms that were thought to be related to an entrapment mononeuropathy. The scratch collapse test was performed twice on each patient, once by the resident and once by a nerve conduction technician. Both observers were blinded to all aspects of the patient's presentation. Sensitivity and specificity for the scratch collapse test were calculated twice, once using electrodiagnostic testing results and a second time using a validated clinical tool (the CTS-6) as the reference standard. The interrater reliability was also calculated. RESULTS: Using electrodiagnostic criteria as a reference standard, the scratch collapse test had a sensitivity of 7% and a specificity of 78% for CTS. Using clinical criteria as a reference standard, the test had a sensitivity of 15% and a specificity of 87%. For cubital tunnel syndrome, the sensitivity was 10% and the specificity was 90%. For the resident/technician 1, kappa was -0.025 (worse than chance alone). For the resident/technician 2, kappa was 0.211 (fair strength of agreement). CONCLUSIONS: The sensitivity of the scratch collapse test for CTS and cubital tunnel syndrome was lower than that found in other studies, regardless of whether a clinical or an electrodiagnostic reference standard was used. The specificity was high. Overall interrater agreement was lower than previously reported. These results call into question the sensitivity and interrater reliability of the scratch collapse test for CTS and cubital tunnel syndrome. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic II. Background: The scratch collapse test (SCT) is a clinical examination maneuver that has been previously reported as a reliable and reproducible test to diagnose carpal tunnel syndrome (CTS). The initial study by Cheng et al in 2008 showed a simple test with high sensitivity. However, subsequent attempts to reproduce those findings have resulted in lower accuracy. Our goal was to evaluate the use of the SCT for patients presenting with symptoms of pain, numbness, or weakness in an upper extremity. Methods: Forty patients were referred to the electrodiagnostic (EDX) lab for evaluation of an upper extremity. One blinded examiner who was familiar with the maneuver performed the SCT on all 40 patients. Another physician or technician performed the nerve conduction study and electromyography. Patient history and accompanying physical examination findings were not revealed to the SCT examiner. Results: The relationship between the SCT performed by a blinded examiner and the EDX performed by blinded examiners was nonsignificant (P = .676) and showed a sensitivity of 0.48, specificity of 0.59, positive predictive value of 0.61, and negative predictive value of 0.45. Conclusion: Based on this study and previous findings by other authors, we would advise against the use of the SCT in CTS for important patient-care decisions, such as surgical decision-making, until future research is done. It is possible that the SCT, in combination with other physical examination maneuvers, could increase diagnostic accuracy and enhance patient management. The diagnosis of nerve compression relies on collecting diagnostic clues from the history, physical examination, imaging and diagnostic testing. There are several provocative tests to aid in the diagnosis of nerve compression. The 'Scratch Collapse Test' (SCT) has emerged as a new provocative test to assist in the localisation of peripheral nerve compression. This study aims to perform a systematic review of literature to assess the data on the reliability of the SCT as a diagnostic test for entrapment neuropathy. Ten articles were reviewed. Five articles had sufficient numerical data for analysis, and in these five articles, the positive predictive values and specificity were high, i.e. between 0.71 and 0.99 and 0.6 and 0.99, respectively, whereas other values were very variable, i.e. individual negative predictive values ranged from 0.15 to 0.92 and the sensitivity values ranged from 0.24 to 0.77. Another main finding was the versatility of the test in that it can be used for various nerve entrapments and to localise the exact level of compression. Literature suggests that SCT has potential to be used as a clinical diagnostic tool for entrapment neuropathy. However, wide variations in early literature suggest that SCT should not be used as a sole diagnostic tool but as an adjunct to a surgeon's diagnostic repertoire.

Which is the main gene signature in Systemic Lupus Erythematosus (SLE)?

Systemic Lupus Erythematosus (SLE) has a type I interferon (IFN) gene signature.

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity. Using oligonucleotide microarrays, we now show that active SLE can be distinguished by a remarkably homogeneous gene expression pattern with overexpression of granulopoiesis-related and interferon (IFN)-induced genes. Using the most stringent statistical analysis (Bonferroni correction), 15 genes were found highly up-regulated in SLE patients, 14 of which are targets of IFN and one, defensin DEFA-3, a major product of immature granulocytes. A more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN-regulated and 4 granulocyte-specific. Indeed immature neutrophils were identified in a large fraction of SLE patients white blood cells. High dose glucocorticoids, a standard treatment of disease flares, shuts down the interferon signature, further supporting the role of this cytokine in SLE. The expression of 10 genes correlated with disease activity according to the SLEDAI. The most striking correlation (P < 0.001, r = 0.55) was found with the formyl peptide receptor-like 1 protein that mediates chemotactic activities of defensins. Therefore, while the IFN signature confirms the central role of this cytokine in SLE, microarray analysis of blood cells reveals that immature granulocytes may be involved in SLE pathogenesis. Recent studies of patients with systemic lupus erythematosus, together with data from lupus-prone mice, suggest that inappropriate activation of type I interferon might have a role in disease pathogenesis. Serum levels of IFN-alpha are elevated in SLE patients, and gene expression profiling of peripheral blood cells shows that most lupus cases demonstrate an upregulation of IFN-responsive genes. Of interest, the IFN gene 'signature' correlates with more severe disease. The available data support a model whereby chromatin-containing immune complexes circulating in the blood of lupus patients stimulate leukocytes to produce IFN, which perpetuates disease. These emerging insights into the connection between IFN and lupus provide a host of new diagnostic and therapeutic opportunities. To date, the pathogenesis of systemic lupus erythematosus (SLE) is only partially understood. A characteristic feature of this disease is the occurrence of antibodies against nuclear antigens. Further, SLE is characterized by a type-I interferon gene signature. A dysregulation of apoptosis and phagocytosis of apoptotic cells is discussed as a central pathogenetic mechanism in the development of SLE. Several publications in recent years have described an accumulation of autoantigens within apoptotic cells, as well as an exposition of these antigens on the surface of apoptotic cells. During late stages of apoptosis, autoantigens are posttranslationally modified, leading to the formation of neoantigens. Subcellular fragments such as apoptotic microparticles also seem to be involved in the pathogenesis of SLE. It has been shown that microparticles contain relevant antigens and stimulate B- and T-lymphocytes. Moreover, apoptotic microparticles have the ability to stimulate plasmacytoid dendritic cells, causing a secretion of interferon alpha. These observations provide a link between dysregulation of apoptosis and phagocytosis and the type-I interferon signature observed in SLE patients. OBJECTIVE: Type I interferons (IFNs) play an important role in the pathogenesis of systemic lupus erythematosus (SLE). This phase Ia trial was undertaken to evaluate the safety, pharmacokinetics, and immunogenicity of anti-IFNalpha monoclonal antibody (mAb) therapy in SLE. During the trial, we also examined whether overexpression of an IFNalpha/beta-inducible gene signature in whole blood could serve as a pharmacodynamic biomarker to evaluate IFNalpha neutralization and investigated downstream effects of neutralizing IFNalpha on BAFF and other key signaling pathways, i.e., granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), tumor necrosis factor alpha (TNFalpha), and IL-1beta, in SLE. METHODS: Affymetrix Human Genome U133 Plus 2.0 microarrays were used to profile whole blood and lesional skin of patients receiving standard therapy for mild to moderate SLE. Selected IFNalpha/beta-inducible proteins were analyzed by immunohistochemistry. RESULTS: With the study treatment, we observed anti-IFNalpha mAb-specific and dose-dependent inhibition of overexpression of IFNalpha/beta-inducible genes in whole blood and skin lesions from SLE patients, at both the transcript and the protein levels. In SLE patients with overexpression of messenger RNA for BAFF, TNFalpha, IL-10, IL-1beta, GM-CSF, and their respective inducible gene signatures in whole blood and/or skin lesions, we observed a general trend toward suppression of the expression of these genes and/or gene signatures upon treatment with anti-IFNalpha mAb. CONCLUSION: IFNalpha/beta-inducible gene signatures in whole blood are effective pharmacodynamic biomarkers to evaluate anti-IFNalpha mAb therapy in SLE. Anti-IFNalpha mAb can neutralize overexpression of IFNalpha/beta-inducible genes in whole blood and lesional skin from SLE patients and has profound effects on signaling pathways that may be downstream of IFNalpha in SLE. Interferon (IFN) signature genes have been shown to be expressed highly in peripheral blood of patients with systemic lupus erythematosus (SLE), especially in the presence of active disease. However, the expression of this gene signature in individuals with incomplete forms of lupus and the pathogenic relationship between IFN signature genes and autoantibody production have not been explored fully. In the present study, we examined the gene expression and autoantibody profiles of patients diagnosed with incomplete lupus erythematosus (ILE) to determine correlations of the gene expression signature with autoantibody production. Gene expression analysis was carried out on the 24K Illumina Human Refseq-8 arrays using blood samples from 84 subjects, including patients with SLE (n = 27) or ILE (n = 24), first-degree relatives (FDR) of these patients (n = 22) and non-autoimmune control (NC) individuals (n = 11). Autoantibody expression was measured using standard immunoassays and autoantigen proteomic arrays. Up-regulation of a set of 63 IFN signature genes was seen in 83% of SLE patients and 50% of ILE patients. High levels of IFN gene expression in ILE and SLE showed significant correlations with the expression of a subset of IgG autoantibodies, including chromatin, dsDNA, dsRNA, U1snRNP, Ro/SSA, La/SSB, topoisomerase I and Scl 70, while low IFN levels were correlated with immunoglobulin (Ig)M autoreactivity. These studies suggest that in patients with ILE the IFN gene expression signature may identify a subset of these individuals who are at risk for disease progression. Furthermore, high levels of alpha IFN may promote autoantibody class-switch from IgM to the more pathogenic IgG class. Patients with systemic lupus erythematosus (SLE) have a markedly increased risk to develop cardiovascular disease, and traditional cardiovascular risk factors fail to account for this increased risk. We used microarray to probe the platelet transcriptome in patients with SLE and healthy controls, and the gene and protein expression of a subset of differentially expressed genes was further investigated and correlated to platelet activation status. Real-time PCR was used to confirm a type I interferon (IFN) gene signature in patients with SLE, and the IFN-regulated proteins PRKRA, IFITM1 and CD69 (P < .0001) were found to be up-regulated in platelets from SLE patients compared with healthy volunteers. Notably, patients with a history of vascular disease had increased expression of type I IFN-regulated proteins as well as more activated platelets compared with patients without vascular disease. We suggest that interferogenic immune complexes stimulate production of IFNα that up-regulates the megakaryocytic type I IFN-regulated genes and proteins. This could affect platelet activation and contribute to development of vascular disease in SLE. In addition, platelets with type I IFN signature could be a novel marker for vascular disease in SLE. OBJECTIVE: To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues. METHODS: Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements. RESULTS: A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc. CONCLUSIONS: The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy. BACKGROUND: There is increased expression of type I interferon (IFN)-regulated proteins in the blood and target tissues of patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE). Patients with SLE have increased IFN-regulated gene expression pointing towards a possible underlying genetic defect. OBJECTIVES: To determine expression levels of five type I IFN-regulated genes that are highly expressed in SLE in the peripheral blood of patients with CLE and to correlate the expression levels with cutaneous disease activity. METHODS: Peripheral blood was obtained from 10 healthy controls and 30 patients with CLE, including eight with concomitant SLE. Total RNA was extracted and reverse transcribed into complementary DNA. Gene expression levels were measured by real-time polymerase chain reaction. Gene expression was normalized to GAPDH, standardized to healthy controls and then summed to calculate an IFN score for each patient. Disease activity was assessed with the Cutaneous Lupus Area and Severity Index (CLASI). RESULTS: Patients with subacute CLE (SCLE) and discoid lupus erythematosus (DLE) had elevated IFN scores compared with healthy controls regardless of concomitant SLE (P < 0·01 with SLE and P < 0·05 without SLE). There was no difference between patients with tumid lupus erythematosus (TLE) and healthy controls. The IFN score correlated with CLASI scores (Spearman's rho = 0·55, P = 0·0017). CONCLUSIONS: Patients with SCLE and DLE have an IFN signature, as seen in SLE. The level of gene expression correlates with cutaneous disease activity. These findings support a shared pathogenesis between SLE and some subtypes of CLE. Systemic lupus erythematosus (SLE) is a generalized autoimmune disease characterized by abnormal B cell activation and the occurrence of increased frequencies of circulating plasma cells (PC). The molecular characteristics and nature of circulating PC and B cells in SLE have not been completely characterized. Microarray analysis of gene expression was used to characterize circulating PC in subjects with active SLE. Flow cytometry was used to sort PC and comparator B cell populations from active SLE blood, normal blood and normal tonsil. The gene expression profiles of the sorted B cell populations were then compared. SLE PC exhibited a similar gene expression signature as tonsil PC. The differences in gene expression between SLE PC and normal tonsil PC and tonsil plasmablasts (PB) suggest a mature Ig secreting cell phenotype in the former population. Despite this, SLE PC differed in expression of about half the genes from previously published gene expression profiles of normal bone marrow PC, indicating that these cells had not achieved a fully mature status. Abnormal expression of several genes, including CXCR4 and S1P(1), suggests a mechanism for the persistence of SLE PC in the circulation. All SLE B cell populations revealed an interferon (IFN) gene signature previously only reported in unseparated SLE peripheral blood mononuclear cells. These data indicate that SLE PC are a unique population of Ig secreting cells with a gene expression profile indicative of a mature, but not fully differentiated phenotype. In recent years, biomarkers have shown significant promise in helping decision-making in drug development. Systemic lupus erythematosus (SLE) is a complicated and highly heterogeneous disease that involves all organs. Only one drug, belimumab, has been approved by the US Food and Drug Administration to treat SLE during the last 50 years and there remains a high unmet medical need to develop new and effective therapies to benefit different patient populations in SLE. Due to the extreme heterogeneity of the disease and the complex and rigorous process to validate individual biomarkers, there is currently a very limited number of consensus biomarkers to aid the treatment decision-making in SLE. This review provides a snapshot of some biomarkers in the field that have the potential to make a big impact on drug development and/or treatment decisions by physicians. These include: type I interferon (IFN) gene signature as a pharmacodynamic marker and potential predictive marker for anti-type I IFN therapy; anti-double stranded DNA as a disease marker and potential predictive marker for flares; the complements and neutrophil signatures as disease marker of SLE; and TWEAK (a tumor necrosis factor family member produced by macrophages) and MCP-1 as potential markers to predict renal flares. Most of these markers need carefully planned and prospective studies with high statistical power to confirm their respective utilities. With the development and application of powerful new technologies, more successful biomarkers will emerge in SLE. This could improve the management of patients in the clinic and facilitate the development of novel and more effective therapeutics for this difficult-to-treat disease. OBJECTIVE: To evaluate the safety and tolerability of multiple intravenous (IV) doses of sifalimumab in adults with moderate-to-severe systemic lupus erythematosus (SLE). METHODS: In this multicenter, double-blind, placebo-controlled, sequential dose-escalation study, patients were randomized 3:1 to receive IV sifalimumab (0.3, 1.0, 3.0, or 10.0 mg/kg) or placebo every 2 weeks to week 26, then followed up for 24 weeks. Safety assessment included recording of treatment-emergent adverse events (AEs) and serious AEs. Pharmacokinetics, immunogenicity, and pharmacodynamics were evaluated, and disease activity was assessed. RESULTS: Of 161 patients, 121 received sifalimumab (26 received 0.3 mg/kg; 25, 1.0 mg/kg; 27, 3.0 mg/kg; and 43, 10 mg/kg) and 40 received placebo. Patients were predomitly female (95.7%). At baseline, patients had moderate-to-severe disease activity (mean SLE Disease Activity Index score 11.0), and most (75.2%) had a high type I interferon (IFN) gene signature. In the sifalimumab group versus the placebo group, the incidence of ≥1 treatment-emergent AE was 92.6% versus 95.0%, ≥1 serious AE was 22.3% versus 27.5%, and ≥1 infection was 67.8% versus 62.5%; discontinuations due to AEs occurred in 9.1% versus 7.5%, and death occurred in 3.3% (n=4) versus 2.5% (n=1). Serum sifalimumab concentrations increased in a linear and dose-proportional manner. Inhibition of the type I IFN gene signature was sustained during treatment in patients with a high baseline signature. No statistically significant differences in clinical activity (SLEDAI and British Isles Lupus Assessment Group score) between sifalimumab and placebo were observed. However, when adjusted for excess burst steroids, SLEDAI change from baseline showed a positive trend over time. A trend toward normal complement C3 or C4 level at week 26 was seen in the sifalimumab groups compared with baseline. CONCLUSION: The observed safety/tolerability and clinical activity profile of sifalimumab support its continued clinical development for SLE. BACKGROUND AND OBJECTIVES: Sifalimumab is a fully human immunoglobulin G1κ monoclonal antibody that binds to and neutralizes a majority of the subtypes of human interferon-α. Sifalimumab is being evaluated as a treatment for systemic lupus erythematosus (SLE). The primary objectives of this analysis were (a) to develop a population pharmacokinetic model for sifalimumab in SLE; (b) to identify and quantitate the impact of patient/disease characteristics on pharmacokinetic variability; and (c) to evaluate fixed versus body weight (WT)-based dosing regimens. METHODS: Sifalimumab serum concentration-time data were collected from a phase Ib study (MI-CP152) designed to evaluate the safety and tolerability of sifalimumab in adult patients with SLE. Sifalimumab was administered every 14 days as a 30- to 60-minute intravenous infusion with escalating doses of 0.3, 1.0, 3.0, and 10 mg/kg and serum concentrations were collected over 350 days. A total of 120 patients provided evaluable pharmacokinetic data with a total of 2,370 serum concentrations. Sifalimumab serum concentrations were determined using a validated colorimetric enzyme-linked immunosorbent assay (ELISA) with a lower limit of quantitation of 1.25 μg/mL. Population pharmacokinetic modeling of sifalimumab was performed using a non-linear mixed effects modeling approach with NONMEM VII software. Impact of patient demographics, clinical indices, and biomarkers on pharmacokinetic parameters were explored using a stepwise forward selection and backward elimination approach. The appropriateness of the final model was tested using visual predictive check (VPC). The impact of body WT-based and fixed dosing of sifalimumab was evaluated using a simulation approach. The final population model was utilized for phase IIb dosing projections. RESULTS: Sifalimumab pharmacokinetics were best described using a two-compartment linear model with first order elimination. Following intravenous dosing, the typical clearance (CL) and central volume of distribution (V 1) were estimated to be 176 mL/day and 2.9 L, respectively. The estimates (coefficient of variation) of between-subject variability for CL and V 1 were 28 and 31 %, respectively. Patient baseline body WT, interferon gene signature from 21 genes, steroid use, and sifalimumab dose were identified as significant covariates for CL, whereas only baseline body WT was a significant covariate for V 1 and peripheral volume of distribution (V 2). Although the above-mentioned covariates were statistically significant, they did not explain variability in pharmacokinetic parameters to any relevant extent (<7 %). Thus, no dosing adjustments are necessary. VPC confirmed good predictability of the final population pharmacokinetic model. Simulation results demonstrate that both fixed and body WT-based dosing regimens yield similar median steady state concentrations and overall variability. Fixed sifalimumab doses of 200, 600, and 1,200 mg monthly (with a loading dose at Day 14) were selected for a phase IIb clinical trial. CONCLUSION: A two-compartment population pharmacokinetic model adequately described sifalimumab pharmacokinetics. The estimated typical pharmacokinetic parameters were similar to other monoclonal antibodies without target mediated elimination. Although the population pharmacokinetic analysis identified some statistically significant covariates, they explained <7 % between-subject variability in pharmacokinetic parameters indicating that these covariates are not clinically relevant. The population pharmacokinetic analysis also demonstrated the feasibility of switching to fixed doses in phase IIb clinical trials of sifalimumab. INTRODUCTION: A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature. METHODS: In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN⁻) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN⁻ patients and HCs. RESULTS: Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN⁻ patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)-a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells. CONCLUSIONS: We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis. Type I interferons (IFN) are primarily regarded as an inhibitor of viral replication. However, type I IFN, mainly IFNalpha, plays a major role in activation of both the innate and adaptive immune systems. Systemic lupus erythematosus (SLE) is a chronic, multi-systemic, inflammatory autoimmune disease with undefined etiology. SLE is characterized by dysregulation of both the innate and the adaptive immune systems. An increased expression of type I IFN-regulated genes, termed IFN signature, has been reported in patients with SLE. We review here the role of IFNalpha in the pathogenesis and course of SLE and the possible role of IFNalpha inhibition as a novel treatment for lupus patients. Blood gene expression profiling has led to major advances in the field of rheumatology over the last few decades. Specifically, DNA microarray technology has been integral in increasing our knowledge of key players in the pathogenesis of some rare pediatric rheumatic diseases. Our group, using microarray analysis, identified the interferon (IFN) gene signature in pediatric systemic lupus erythematosus (SLE) and has published data that suggest high doses of intravenous corticosteroid treatment may have benefit over strictly oral regimens. Additionally, DNA microarray technology led to our discovery that the interleukin (IL)-1 gene signature is associated with systemic juvenile idiopathic arthritis (sJIA) and to the use of IL-1 blockade with anakinra in this disease. We also reported the biologic rationale for use of anakinra early in the disease course. Anakinra is now being used as first-line treatment in sJIA in multiple centers. Herein, we review how information obtained from blood gene expression profiling has changed our clinical practice. OBJECTIVES: The interferon (IFN) signature (IS) in patients with systemic lupus erythematosus (SLE) includes over 100 genes induced by type I IFN pathway activation. We developed a method to quantify the IS using three genes-the IS metric (ISM)-and characterised the clinical characteristics of patients with SLE with different ISM status from multiple clinical trials. METHODS: Blood microarray expression data from a training cohort of patients with SLE confirmed the presence of the IS and identified surrogate genes. We assayed these genes in a quantitative PCR (qPCR) assay, yielding an ISM from the IS. The association of ISM status with clinical disease characteristics was assessed in patients with extrarenal lupus and lupus nephritis from four clinical trials. RESULTS: Three genes, HERC5, EPSTI and CMPK2, correlated well with the IS (p>0.96), and composed the ISM qPCR assay. Using the 95th centile for healthy control data, patients with SLE from different studies were classified into two ISM subsets-ISM-Low and ISM-High-that are longitudinally stable over 36 weeks. Significant associations were identified between ISM-High status and higher titres of anti-dsDNA antibodies, presence of anti extractable nuclear antigen autoantibodies, elevated serum B cell activating factor of the tumour necrosis factor family (BAFF) levels, and hypocomplementaemia. However, measures of overall clinical disease activity were similar for ISM-High and ISM-Low groups. CONCLUSIONS: The ISM is an IS biomarker that divides patients with SLE into two subpopulations-ISM-High and ISM-Low-with differing serological manifestations. The ISM does not distinguish between high and low disease activity, but may have utility in identifying patients more likely to respond to treatment(s) targeting IFN-α. CLINICALTRIALSGOV REGISTRATION NUMBER: NCT00962832. OBJECTIVES: The efficacy and safety of sifalimumab were assessed in a phase IIb, randomised, double-blind, placebo-controlled study (NCT01283139) of adults with moderate to severe active systemic lupus erythematosus (SLE). METHODS: 431 patients were randomised and received monthly intravenous sifalimumab (200 mg, 600 mg or 1200 mg) or placebo in addition to standard-of-care medications. Patients were stratified by disease activity, interferon gene-signature test (high vs low based on the expression of four genes) and geographical region. The primary efficacy end point was the percentage of patients achieving an SLE responder index response at week 52. RESULTS: Compared with placebo, a greater percentage of patients who received sifalimumab (all dosages) met the primary end point (placebo: 45.4%; 200 mg: 58.3%; 600 mg: 56.5%; 1200 mg 59.8%). Other improvements were seen in Cutaneous Lupus Erythematosus Disease Area and Severity Index score (200 mg and 1200 mg monthly), Physician's Global Assessment (600 mg and 1200 mg monthly), British Isles Lupus Assessment Group-based Composite Lupus Assessment (1200 mg monthly), 4-point reductions in the SLE Disease Activity Index-2000 score and reductions in counts of swollen joints and tender joints. Serious adverse events occurred in 17.6% of patients on placebo and 18.3% of patients on sifalimumab. Herpes zoster infections were more frequent with sifalimumab treatment. CONCLUSIONS: Sifalimumab is a promising treatment for adults with SLE. Improvement was consistent across various clinical end points, including global and organ-specific measures of disease activity. TRIAL REGISTRATION NUMBER: NCT01283139; Results. OBJECTIVE: To characterize baseline gene expression and pharmacodynamically induced changes in whole blood gene expression in 1,760 systemic lupus erythematosus (SLE) patients from 2 phase III, 52-week, randomized, placebo-controlled, double-blind studies in which patients were treated with the BAFF-blocking IgG4 monoclonal antibody tabalumab. METHODS: Patient samples were obtained from SLE patients from the ILLUMINATE-1 and ILLUMINATE-2 studies, and control samples were obtained from healthy donors. Blood was collected in Tempus tubes at baseline, week 16, and week 52. RNA was analyzed using Affymetrix Human Transcriptome Array 2.0 and NanoString. RESULTS: At baseline, expression of the interferon (IFN) response gene was elevated in patients compared with controls, with 75% of patients being positive for this IFN response gene signature. There was, however, substantial heterogeneity of IFN response gene expression and complex relationships among gene networks. The IFN response gene signature was a predictor of time to disease flare, independent of anti-double-stranded DNA (anti-dsDNA) antibody and C3 and C4 levels, and overall disease activity. Pharmacodynamically induced changes in gene expression following tabalumab treatment were extensive, occurring predomitly in B cell-related and immunoglobulin genes, and were consistent with other pharmacodynamic changes including anti-dsDNA antibody, C3, and immunoglobulin levels. CONCLUSION: SLE patients demonstrated increased expression of an IFN response gene signature (75% of patients had an elevated IFN response gene signature) at baseline in ILLUMINATE-1 and ILLUMINATE-2. Substantial heterogeneity of gene expression was detected among individual patients and in gene networks. The IFN response gene signature was an independent risk factor for future disease flares. Pharmacodynamic changes in gene expression were consistent with the mechanism of BAFF blockade by tabalumab. OBJECTIVE: To assess the efficacy and safety of anifrolumab, a type I interferon (IFN) receptor antagonist, in a phase IIb, randomized, double-blind, placebo-controlled study of adults with moderate-to-severe systemic lupus erythematosus (SLE). METHODS: Patients (n = 305) were randomized to receive intravenous anifrolumab (300 mg or 1,000 mg) or placebo, in addition to standard therapy, every 4 weeks for 48 weeks. Randomization was stratified by SLE Disease Activity Index 2000 score (<10 or ≥10), oral corticosteroid dosage (<10 or ≥10 mg/day), and type I IFN gene signature test status (high or low) based on a 4-gene expression assay. The primary end point was the percentage of patients achieving an SLE Responder Index (SRI[4]) response at week 24 with sustained reduction of oral corticosteroids (<10 mg/day and less than or equal to the dose at week 1 from week 12 through 24). Other end points (including SRI[4], British Isles Lupus Assessment Group [BILAG]-based Composite Lupus Assessment [BICLA], modified SRI[6], and major clinical response) were assessed at week 52. The primary end point was analyzed in the modified intent-to-treat (ITT) population and type I IFN-high subpopulation. The study result was considered positive if the primary end point was met in either of the 2 study populations. The Type I error rate was controlled at 0.10 (2-sided), within each of the 2 study populations for the primary end point analysis. RESULTS: The primary end point was met by more patients treated with anifrolumab (34.3% of 99 for 300 mg and 28.8% of 104 for 1,000 mg) than placebo (17.6% of 102) (P = 0.014 for 300 mg and P = 0.063 for 1,000 mg, versus placebo), with greater effect size in patients with a high IFN signature at baseline (13.2% in placebo-treated patients versus 36.0% [P = 0.004] and 28.2% [P = 0.029]) in patients treated with anifrolumab 300 mg and 1,000 mg, respectively. At week 52, patients treated with anifrolumab achieved greater responses in SRI(4) (40.2% versus 62.6% [P < 0.001] and 53.8% [P = 0.043] with placebo, anifrolumab 300 mg, and anifrolumab 1,000 mg, respectively), BICLA (25.7% versus 53.5% [P < 0.001] and 41.2% [P = 0.018], respectively), modified SRI(6) (28.4% versus 49.5% [P = 0.002] and 44.7% [P = 0.015], respectively), major clinical response (BILAG 2004 C or better in all organ domains from week 24 through week 52) (6.9% versus 19.2% [P = 0.012] and 17.3% [P = 0.025], respectively), and several other global and organ-specific end points. Herpes zoster was more frequent in the anifrolumab-treated patients (2.0% with placebo treatment versus 5.1% and 9.5% with anifrolumab 300 mg and 1,000 mg, respectively), as were cases reported as influenza (2.0% versus 6.1% and 7.6%, respectively), in the anifrolumab treatment groups. Incidence of serious adverse events was similar between groups (18.8% versus 16.2% and 17.1%, respectively). CONCLUSION: Anifrolumab substantially reduced disease activity compared with placebo across multiple clinical end points in the patients with moderate-to-severe SLE. OBJECTIVE: We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. METHODS: IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element-luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. RESULTS: Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombit and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. CONCLUSIONS: Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling. OBJECTIVE: The interferon (IFN) signature is related to disease activity and vascular disease in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and represents a promising therapeutic target. Quantification of the IFN signature is currently performed by gene expression analysis, limiting its current applicability in clinical practice. Therefore, the objective of this study was to establish an easy to measure biomarker for the IFN signature. METHODS: Serum levels of galectin-9, CXCL-10 (IP-10) and tumour necrosis factor receptor type II (TNF-RII) were measured in patients with SLE, SLE+APS and primary APS (PAPS) and healthy controls (n=148) after an initial screening of serum analytes in a smaller cohort (n=43). Analytes were correlated to measures of disease activity and the IFN signature. The performance of galectin-9, CXCL-10 and TNF-RII as biomarkers to detect the IFN signature was assessed by receiver operating characteristic curves. RESULTS: Galectin-9, CXCL-10 and TNF-RII were elevated in patients with SLE, SLE+APS and PAPS (p<0.05) and correlated with disease activity and tissue factor expression. Galectin-9 correlated stronger than CXCL-10 or TNF-RII with the IFN score (r=0.70, p<0.001) and was superior to CXCL-10 or TNF-RII in detecting the IFN signature (area under the curve (AUC) 0.86). Importantly, in patients with SLE(±APS), galectin-9 was also superior to anti-dsDNA antibody (AUC 0.70), or complement C3 (AUC 0.70) and C4 (AUC 0.78) levels in detecting the IFN signature. CONCLUSION: Galectin-9 is a novel, easy to measure hence clinically applicable biomarker to detect the IFN signature in patients with systemic autoimmune diseases such as SLE and APS. Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which 70% of patients experience disfiguring skin inflammation (grouped under the rubric of cutaneous lupus erythematosus [CLE]). There are limited treatment options for SLE and no Food and Drug Administration-approved therapies for CLE. Studies have revealed that IFNs are important mediators for SLE and CLE, but the mechanisms by which IFNs lead to disease are still poorly understood. We aimed to investigate how IFN responses in SLE keratinocytes contribute to development of CLE. A cohort of 72 RNA sequencing samples from 14 individuals (seven SLE and seven healthy controls) were analyzed to study the transcriptomic effects of type I and type II IFNs on SLE versus control keratinocytes. In-depth analysis of the IFN responses was conducted. Bioinformatics and functional assays were conducted to provide implications for the change of IFN response. A significant hypersensitive response to IFNs was identified in lupus keratinocytes, including genes (IFIH1, STAT1, and IRF7) encompassed in SLE susceptibility loci. Binding sites for the transcription factor PITX1 were enriched in genes that exhibit IFN-sensitive responses. PITX1 expression was increased in CLE lesions based on immunohistochemistry, and by using small interfering RNA knockdown, we illustrated that PITX1 was required for upregulation of IFN-regulated genes in vitro. SLE patients exhibit increased IFN signatures in their skin secondary to increased production and a robust, skewed IFN response that is regulated by PITX1. Targeting these exaggerated pathways may prove to be beneficial to prevent and treat hyperinflammatory responses in SLE skin. Systemic lupus erythematosus (SLE) is a complex autoimmune disease which affects multiple organs. The type I interferon (IFN) gene signature and circulating autoantibodies are hallmarks of SLE. Plasmacytoid dendritic cells (pDCs) are considered the main producers of type I IFN and production is modulated by multiple other immune cell types. In SLE, essentially every immune cell type is dysregulated and aberrant deregulation is thought to be due, in part, to direct or indirect exposure to IFN. Genetic variants within or around the transcription factor interferon regulatory factor 5 (IRF5) associate with SLE risk. Elevated IFNα activity was detected in the sera of SLE patients carrying IRF5 risk polymorphisms who were positive for either anti-RNA binding protein (anti-RBP) or anti-double-stranded DNA (anti-dsDNA) autoantibodies. Neutrophils are also an important source of type I IFNs and are found in abundance in human blood. Neutrophil extracellular traps (NETs) are considered a potential source of antigenic trigger in SLE that can lead to type I IFN gene induction, as well as increased autoantibody production. In this review, we will focus on immune cell types that produce type I IFNs and/or are affected by type I IFN in SLE. In addition, we will discuss potential inducers of endogenous type I IFN production in SLE. Last, we will postulate how the different immune cell populations may be affected by an IRF5-SLE risk haplotype. OBJECTIVES: Type I interferon (IFN) is implicated in systemic lupus erythematosus (SLE) pathogenesis. We aimed to identify type I IFN signaling-dependent and -independent molecular pathways in a large population of patients with SLE. METHODS: Baseline blood samples from adult patients with moderate to severe SLE from two Phase IIb studies (NCT01438489, n = 265; NCT01283139, n = 416) were profiled using whole transcriptome array analyses. Type I IFN gene signature (IFNGS) test status (high or low) was determined using a validated qualitative polymerase chain reaction-based test. IFN-type-specific signatures were developed by stimulating healthy blood with IFN-β, IFN-γ, IFN-λ, IFN-ω, or pooled IFN-α. These, and multiple literature-derived cell type and cytokine pathway signatures, were evaluated in individual and pooled study populations. A Fisher's exact test was used for associations, adjusted for false discovery rate. RESULTS: Whole blood samples from IFNGS test-high patients were enriched versus IFNGS test-low patients for CD40L signaling (Q < 0.001), CXC cytokine (Q < 0.001), TLR8-mediated monocyte activation (Q < 0.001), IgG (Q < 0.001), major histocompatibility complex class I (Q < 0.001), and plasma cell (Q < 0.001) gene expression signatures. IFNGS test-low patients had significant enrichment of eosinophil (Q < 0.001), IFN-γ-specific (Q = 0.005), and T-cell or B-cell (Q < 0.001) signatures. Similar enrichment profiles were demonstrated in patients with primary Sjögren's syndrome, systemic sclerosis, and dermatomyositis. CONCLUSIONS: IFNGS test-high patients overexpressed many gene signatures associated with SLE pathogenesis compared with IFNGS test-low patients, reflecting broad immune activation. These results provide new insights into the molecular heterogeneity underlying SLE pathogenesis, highlighting shared mechanisms beyond type I IFN, across several autoimmune diseases. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01438489 and NCT01283139. OBJECTIVES: Plasmacytoid dendritic cells (pDCs), through the production of type 1 interferons (IFNs) and other cytokines, are major contributors to systemic lupus erythematosus (SLE) pathogenesis. IL-3 promotes pDC survival, but its role in SLE is not well characterised. This study investigated serum IL-3 and IFN levels, and a whole blood 'IL-3 gene signature', in human SLE. METHODS: Serum cytokine levels were measured by ELISA in n = 42 SLE patients, and n = 44 healthy donors. IL-3-regulated genes were determined by RNASeq of healthy donor whole blood cells (WBCs) stimulated in vitro with IL-3 for 6 or 24 h. Whole blood cell RNASeq analysis was undertaken in a separate cohort of n = 31 SLE patients, and n = 28 healthy donors. RESULTS: Serum IL-3 levels correlated with IFNα (r = 0.612, 95% CI 0.455-0.733, P < 0.001) and type III IFN (r = 0.585, 95% CI 0.406-0.720, P < 0.0001). IL-3 stimulation of WBC in vitro altered 794 genes (-1 ≥ logFC ≥ 1, FDR < 0.05), of which 35 overlapped with genes differentially expressed between SLE and healthy donors. These 35 genes were expressed in 27/31 SLE donors, revealing the presence of an 'IL-3 gene signature'. There was strong correlation between the IL-3 signature and an IFN signature, as determined by hierarchical clustering of the 500 most variable genes in SLE donors (r = 0.939, 95% CI 0.898-0.964, P < 0.0001). CONCLUSION: A dual IL-3/IFN gene signature is a feature of SLE. An association between IL-3 and IFN raises the possibility that dual blockade of IL-3 and IFN may be especially useful for SLE patients with this dual cytokine gene signature. BACKGROUND: Systemic lupus erythematosus (SLE) is a complex multi-system disease, characterized by both autoimmune and autoinflammatory clinical and laboratory features. The role of type I interferon (IFN) in SLE has been demonstrated from the 2000s, by gene expression analyses showing significant over-expression of genes related to type I IFN signalling pathway (IFN signature). However, several studies questioned the role of measuring the intensity of IFN signature (IFN score) to chase SLE activity. We would assess if the IFN signature can help the clinical and therapeutic stratification of patients with pediatric SLE. METHODS: We measured the IFN score in peripheral whole blood from a series of subjects with childhood-onset SLE and correlated the results with clinical and laboratory parameters. RESULTS: Thirty-one subjects were included in the study, among which the 87% displayed a positive IFN score. The only significant relation was found for high IFN score in subjects with normocomplementemia. No correlation was observed between IFN score and SLEDAI-2K, BILAG-2004 and SLICC. Patients with high IFN score and normal complement levels also presented lower anti-dsDNA antibodies. CONCLUSIONS: The integration between IFN signature analysis and complement levels may easily distinguish two groups of subjects, in which the autoimmune or autoinflammatory component of the disease seems to be prevalent.

Has FTY720 been considered for the treatment of stroke?

Yes, FTY720 is a strong candidate for stroke treatment.

FTY720 (Fingolimod) is a known sphingosine-1-phosphate (S1P) receptor agonist that exerts strong anti-inflammatory effects and was approved as the first oral drug for the treatment of multiple sclerosis by the US Food and Drug Administration (FDA) in 2010. FTY720 is mainly associated with unique functional "antagonist" and "agonist" mechanisms. The functional antagonistic mechanism is mediated by the transient down-regulation and degradation of S1P receptors on lymphocytes, which prevents lymphocytes from entering the blood stream from the lymph node. This subsequently results in the development of lymphopenia and reduces lymphocytic inflammation. Functional agonistic mechanisms are executed through S1P receptors expressed on the surface of various cells including neurons, astrocytes, microglia, and blood vessel endothelial cells. These functions might play important roles in regulating anti-apoptotic systems, modulating brain immune and phagocytic activities, preserving the Blood-Brain-Barrier (BBB), and the proliferation of neural precursor cells. Recently, FTY720 have shown receptor-independent effects, including intracellular target bindings and epigenetic modulations. Many researchers have recognized the positive effects of FTY720 and launched basic and clinical experiments to test the use of this agent against stroke. Although the mechanism of FTY720 has not been fully elucidated, its efficacy against cerebral stroke is becoming clear, not only in animal models, but also in ischemic stroke patients through clinical trials. In this article, we review the data obtained from laboratory findings and preliminary clinical trials using FTY720 for stroke treatment.

List R packages for lipidomics

R packages for lipidomics: lipidomics, masspix, lipidms, lipidr and lipid mini-on.

INTRODUCTION: Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools. OBJECTIVES: We have developed massPix-an R package for analysing and interpreting data from MSI of lipids in tissue. METHODS: massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries. RESULTS: Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering. CONCLUSION: massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications. High resolution LC-MS untargeted lipidomics using data independent acquisition (DIA) has the potential to increase lipidome coverage, as it enables the continuous and unbiased acquisition of all eluting ions. However, the loss of the link between the precursor and the product ions combined with the high dimensionality of DIA data sets hinder accurate feature annotation. Here, we present LipidMS, an R package aimed to confidently identify lipid species in untargeted LC-DIA-MS. To this end, LipidMS combines a coelution score, which links precursor and fragment ions with fragmentation and intensity rules. Depending on the MS evidence reached by the identification function survey, LipidMS provides three levels of structural annotations: (i) "subclass level", e.g., PG(34:1); (ii) "fatty acyl level", e.g., PG(16:0_18:1); and (iii) "fatty acyl position level", e.g., PG(16:0/18:1). The comparison of LipidMS with freely available data dependent acquisition (DDA) and DIA identification tools showed that LipidMS provides significantly more accurate and structural informative lipid identifications. Finally, to exemplify the utility of LipidMS, we investigated the lipidomic serum profile of patients diagnosed with nonalcoholic steatohepatitis (NASH), which is the progressive form of nonalcoholic fatty liver disease, a disorder underlying a strong lipid dysregulation. As previously published, a significant decrease in lysophosphatidylcholines, phosphatidylcholines and cholesterol esters and an increase in phosphatidylethanolamines were observed in NASH patients. Remarkably, LipidMS allowed the identification of a new set of lipids that may be used for NASH diagnosis. Altogether, LipidMS has been validated as a tool to assist lipid identification in the LC-DIA-MS untargeted analysis of complex biological samples. SUMMARY: Here we introduce Lipid Mini-On, an open-source tool that performs lipid enrichment analyses and visualizations of lipidomics data. Lipid Mini-On uses a text-mining process to bin individual lipid names into multiple lipid ontology groups based on the classification (e.g. LipidMaps) and other characteristics, such as chain length. Lipid Mini-On provides users with the capability to conduct enrichment analysis of the lipid ontology terms using a Shiny app with options of five statistical approaches. Lipid classes can be added to customize the user's database and remain updated as new lipid classes are discovered. Visualization of results is available for all classification options (e.g. lipid subclass and individual fatty acid chains). Results are also visualized through an editable network of relationships between the individual lipids and their associated lipid ontology terms. The utility of the tool is demonstrated using biological (e.g. human lung endothelial cells) and environmental (e.g. peat soil) samples. AVAILABILITY AND IMPLEMENTATION: Rodin (R package: https://github.com/PNNL-Comp-Mass-Spec/Rodin), Lipid Mini-On Shiny app (https://github.com/PNNL-Comp-Mass-Spec/LipidMiniOn) and Lipid Mini-On online tool (https://omicstools.pnnl.gov/shiny/lipid-mini-on/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. The rapid evolution of mass spectrometry (MS)-based lipidomics has enabled the simultaneous measurement of numerous lipid classes. With lipidomics datasets becoming increasingly available, lipidomic-focused software tools are required to facilitate data analysis as well as mining of public datasets, integrating lipidomics-unique molecular information such as lipid class, chain length, and unsaturation. To address this need, we developed lipidr, an open-source R/Bioconductor package for data mining and analysis of lipidomics datasets. lipidr implements a comprehensive lipidomic-focused analysis workflow for targeted and untargeted lipidomics. lipidr imports numerical matrices, Skyline exports, and Metabolomics Workbench files directly into R, automatically inferring lipid class and chain information from lipid names. Through integration with the Metabolomics Workbench API, users can search, download, and reanalyze public lipidomics datasets seamlessly. lipidr allows thorough data inspection, normalization, and uni- and multivariate analyses, displaying results as interactive visualizations. To enable interpretation of lipid class, chain length, and total unsaturation data, we also developed and implemented a novel lipid set enrichment analysis. A companion online guide with two live example datasets is presented at https://www.lipidr.org/. We expect that the ease of use and innovative features of lipidr will allow the lipidomics research community to gain novel detailed insights from lipidomics data.

Which disease can be treated with Relugolix.

Relugolix has a role in treatment of prostate cancer, uterine fibroids, endometriosis and uterine myomas.

OBJECTIVE: To investigate the noninferiority of relugolix compared with leuprorelin acetate in reducing heavy menstrual bleeding associated with uterine leiomyomas. METHODS: In a double-blind, double-dummy trial, premenopausal women with uterine leiomyomas and heavy menstrual bleeding defined as a pictorial blood loss assessment chart score of at least 120 were randomized in a 1:1 ratio to relugolix (40 mg, oral, once daily) or leuprorelin acetate (1.88 mg or 3.75 mg, monthly injection) for 24 weeks. The primary endpoint was the proportion of patients with a total pictorial blood loss assessment chart score of less than 10 for weeks 6-12. Secondary endpoints included myoma and uterine volumes, and hemoglobin levels. A sample size of 144 patients per group (n=288) was estimated to provide at least 90% power to demonstrate noninferiority (prespecified noninferiority margin -15%; one-sided 0.025 level of significance). RESULTS: From March 2016 to September 2017, 281 patients were randomized (relugolix, n=139, leuprorelin n=142). Demographic and baseline characteristics were well balanced; mean pictorial blood loss assessment chart score was 254.3 in the relugolix group and 263.7 in the leuprorelin group. The proportion of patients with total pictorial blood loss assessment chart score of less than 10 for weeks 6-12 was 82.2% in the relugolix group and 83.1% in the leuprorelin group, demonstrating noninferiority of relugolix compared with leuprorelin (relugolix-leuprorelin difference -0.9%; 95% CI: -10.10 to 8.35; prespecified noninferiority margin -15%; P=.001). Reductions in myoma and uterine volumes and increases in hemoglobin levels were comparable in the two groups. Relugolix was associated with an earlier effect on menstrual bleeding than leuprorelin (pictorial blood loss assessment chart score of less than 10, 64.2% vs 31.7% [relugolix-leuprorelin difference 32.5%; 95% CI: 20.95-44.13%] for weeks 2-6 and pictorial blood loss assessment chart score of 0, 52.6% vs 21.8% [30.7%; 95% CI: 19.45-42.00%] for weeks 2-6) and faster recovery of menses after treatment discontinuation (relugolix median [Q1, Q3], 37 days [32.0, 46.0]; leuprorelin median, 65 days [54.0, 77.0]). Adverse events and bone mineral density loss were similar between relugolix and leuprorelin treatment groups. CONCLUSION: In women with uterine leiomyomas, once-daily treatment with relugolix, an oral gonadotropin-releasing hormone antagonist, demonstrated noninferiority to monthly leuprorelin for improvement of heavy menstrual bleeding at 6-12 weeks of treatment, had a more rapid effect on menstrual bleeding, and was generally well tolerated. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT02655237; JAPIC Clinical Trial Information, JapicCTI-163128. FUNDING SOURCE: Takeda Pharmaceutical Company Limited and an affiliate of NovaQuest Capital Management LLC. The orally active nonpeptide gonadotropin-releasing hormone (GnRH)-receptor antagonist relugolix (Relumina) is being developed by Takeda and ASKA Pharmaceutical as a treatment for various sex hormone related disorders. Relugolix was recently approved for marketing in Japan as a treatment for symptoms associated with uterine fibroids, and studies evaluating the efficacy of the drug as treatment for endometriosis-associated pain and prostate cancer are currently underway. This article summarizes the milestones in the development of relugolix leading to this first approval for the treatment of symptoms associated with uterine fibroids. Uterine leiomyomas represent the most common form of benign gynecological tumors affecting 20-40% of women during their life. Several therapeutic options are available for treating these patients. The use of medical treatment for myomas has largely grown in the last years, in particular for women who would refuse, postpone or are not candidates for surgery. In the last years, the clinical investigation of gonadotropin-releasing hormone (GnRH) antagonists (GnRH-ants) has emerged. This class of drugs exerts pure competitive antagonistic activity on the GnRH receptor at the pituitary gland, producing an immediate stop in the release of gonadotropins and sex steroids. Relugolix is an orally active nonpeptide GnRH-ant, recently licensed for marketing in Japan for the treatment of symptoms related to uterine myomas. Currently, several phase III clinical trials are ongoing to evaluate this molecule in this setting in the U.S. and Europe. OBJECTIVE: To investigate the efficacy and safety of the oral gonadotropin-releasing hormone receptor antagonist, relugolix, in patients experiencing uterine fibroid-associated pain. DESIGN: Phase 3, multicenter, randomized, double-blind, placebo-controlled study. SETTING: Medical centers. PATIENT(S): Premenopausal Japanese women (N = 65) experiencing moderate-to-severe uterine fibroid-associated pain with a maximum Numerical Rating Scale (NRS) score of ≥4 were randomized and completed the study. INTERVENTION(S): Once-daily 40 mg relugolix (n = 33) or placebo (n = 32) for 12 weeks. MAIN OUTCOME MEASURE(S): Primary end point: proportion of patients with a maximum NRS score of ≤1 during the 28-day period before the final dose of study drug. Secondary end points: proportion of patients with no pain (NRS = 0) and percentage of days without pain during the 28-day period before the final dose of study drug; adverse events. RESULT(S): More patients receiving relugolix versus placebo achieved a maximum NRS score of ≤1 during the 28-day period before the final dose of study drug (57.6% vs. 3.1%). Similarly, more patients receiving relugolix versus placebo achieved a maximum NRS score of 0 (48.5% vs. 3.1%) and experienced more days without pain (96.4% vs. 71.4%). More patients receiving relugolix versus placebo experienced treatment-emergent adverse events (TEAEs; 87.9% vs. 56.3%); however, the rate of treatment discontinuation was low and not different between groups. Most TEAEs were mild to moderate in intensity. TEAEs (≥10%) included hot flush, metrorrhagia, hyperhidrosis, and menorrhagia, consistent with relugolix's mechanism of action, and viral upper respiratory tract infection. CONCLUSION(S): Relugolix improved uterine fibroid-associated pain and was well tolerated. CLINICAL TRIAL REGISTRATION NUMBERS: NCT02655224. JAPIC CLINICAL TRIAL INFORMATION: JapicCTI-163127. BACKGROUND: External beam radiotherapy (EBRT) with neoadjuvant/adjuvant androgen deprivation therapy (ADT) is an established treatment option to prolong survival for patients with intermediate- and high-risk prostate cancer (PCa). Relugolix, an oral gonadotropin-releasing hormone (GnRH) receptor antagonist, was evaluated in this clinical setting in comparison with degarelix, an injectable GnRH antagonist. OBJECTIVE: To evaluate the safety and efficacy of relugolix to achieve and maintain castration. DESIGN, SETTING, AND PARTICIPANTS: A phase 2 open-label study was conducted in 103 intermediate-risk PCa patients undergoing primary EBRT and neoadjuvant/adjuvant ADT between June 2014 and December 2015. INTERVENTION: Patients randomly assigned (3:2) to 24-wk treatment with either daily oral relugolix or 4-wk subcutaneous depot degarelix (reference control). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was the rate of effective castration (testosterone <1.73nmol/l) in relugolix patients between 4 and 24 wk of treatment. Secondary endpoints included rate of profound castration (testosterone <0.7nmol/l), prostate-specific antigen (PSA) levels, prostate volume, quality of life (QoL) assessed using the Aging Males' Symptoms scale, and the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life (30-item EORTC core questionnaire [EORTC QLQ-C30] and 25-item EORTC prostate cancer module [EORTC QLQ-PR25]) questionnaires, and safety. No formal statistical comparisons with degarelix were planned. RESULTS AND LIMITATIONS: Castration rates during treatment were 95% and 82% with relugolix and 89% and 68% with degarelix for 1.73 and 0.7nmol/l thresholds, respectively. Median time to castration in the relugolix arm was 4 d. During treatment, PSA levels and prostate volumes were reduced in both groups. Three months after discontinuing treatment, 52% of men on relugolix and 16% on degarelix experienced testosterone recovery (statistical significance of differences not tested). Mean and median QoL scores improved following treatment discontinuation. The most common adverse event was hot flush (relugolix 57%; degarelix 61%). Lack of blinding was a potential limitation. CONCLUSIONS: Relugolix achieved testosterone suppression to castrate levels within days and maintained it over 24 wk with a safety profile consistent with its mechanism of action. PATIENT SUMMARY: Oral once-daily relugolix may be a novel oral alternative to injectable androgen deprivation therapies. Results from the phase 3 HERO trial(NCT03085095), presented during the 2020 American Society of Clinical Oncology Virtual Scientific Program, indicated that relugolix (Relumina) demonstrated superiority over leuprolide (Lupron) in sustained testosterone suppression through 48 weeks, fast testosterone recovery after discontinuation, and a 50% reduction in major adverse cardiovascular events (MACE) in patients with advanced prostate cancer. INTRODUCTION: Uterine fibroids (UF) are benign tumors common in premenopausal women, with strong impact on the health-care systems. For many years, surgery represented the only therapy for symptomatic fibroids. However, clinicians are observing a switch from surgery to noninvasive methods; in particular, medical treatment has been shown to be efficacious in obtaining a bleeding reduction and in ameliorating patient conditions. AREAS COVERED: The authors review the current options available for the treatment of women with UF, with a special focus on the newest one, relugolix. It is an orally active non-peptide Gonadotropin-releasing hormone (GnRH)-receptor antagonist recently licensed for women with symptomatic fibroids. Relugolix is a well-tolerated safe drug; it is effective in inducing a dose-dependent decrease in menstrual blood loss, with faster reduction of heavy menstrual bleeding (HMB) and a greater shrinkage in fibroid volume compared to the current standard of GnRH agonist treatment. EXPERT OPINION: Relugolix is a promising drug for the non-surgical treatment of women with UF. To date, the only published data come from a well-selected Japanese female population study while results from worldwide ongoing studies are ongoing in order to confirm the efficacy of this GnRH agonist receptor. We here describe a case of the prolapse of pedunculated submucosal leiomyoma through the cervix during the treatment of a gonadotropin-releasing hormone (GnRH) antagonist relugolix. We also present the literature review of the cases of leiomyoma prolapse during GnRH modulators. A 55-year-old woman with atypical vaginal bleeding diagnosed submucosal uterine fibroid 6 cm in diameter, and daily oral administration of relugolix was conducted. On the 35th day of the administration, heavy vaginal bleeding suddenly occurred due to leiomyoma prolapse. Finally, she underwent abdominal hysterectomy to treat heavy bleeding. To date, six cases of leiomyoma prolapse during GnRH modulators have been reported, in which all the previous cases were treated with GnRH agonists. This is the first case report of leiomyoma prolapse during GnRH antagonist treatment. Notably, leiomyoma prolapse is a possible common adverse effect of GnRH modulators for the treatment of submucosal leiomyoma, which is caused by rapid decrease in its volume. OBJECTIVE: To evaluate the efficacy and safety of three dose levels of relugolix, a gonadotropin-releasing hormone receptor antagonist, compared with placebo and leuprorelin in women with endometriosis-associated pain. DESIGN: Phase 2, multicenter, randomized, double-blind, placebo-controlled study. SETTING: Hospitals and clinics. PATIENT(S): Adult premenopausal women with endometriosis who had dysmenorrhea and endometriosis-associated pelvic pain. INTERVENTION(S): During a 12-week treatment period, patients received relugolix 10 mg (n = 103), 20 mg (n = 100), or 40 mg (n = 103) as a daily oral dose; placebo (n = 97) as a daily oral dose; or leuprorelin 3.75 mg (n = 80) as a monthly subcutaneous injection. MAIN OUTCOME MEASURE(S): Primary endpoint was the change from baseline in mean visual analog scale score for pelvic pain during 28 days before the end of treatment. RESULT(S): The mean changes in mean visual analog scale score for pelvic pain were -3.8 mm in the placebo group; -6.2, -8.1, and -10.4 mm in the relugolix 10-mg, 20-mg, and 40-mg groups; respectively; and -10.6 mm in the leuprorelin group. The major adverse events with relugolix were hot flush, metrorrhagia, menorrhagia, and irregular menstruation, and bone mineral density decrease in a dose-response manner, which were also observed in the leuprorelin group with a frequency comparable with that in the relugolix 40-mg group. CONCLUSION(S): Oral administration of relugolix alleviated endometriosis-associated pain in a dose-response manner and was generally well tolerated. Relugolix 40 mg demonstrated efficacy and safety comparable with those of leuprorelin. CLINICAL TRIAL REGISTRATION NUMBER: NCT01458301.

What is the effect of Carbendazim on bees?

Carbendazim is an environmental risk factor that likely weakens bee colonies, partially due to reduced expression of major royal jelly proteins, which may be potential causes of colony collapse disorder.

Publisher: Los abejorros están constantemente expuestos a una amplia gama de agentes estresantes bióticos y abióticos de los que deben defenderse para sobrevivir. Los patógenos y los pesticidas son importantes factores estresantes que influyen en la salud de los abejorros, tanto cuando actúan solos como en combinación. Para tener un mejor conocimiento sobre la salud de los abejorros, debemos investigar cómo interactúan estos factores estresantes, pero los estudios experimentales hasta la fecha generalmente se centran en estudiar solo uno o dos factores. El objetivo de nuestro estudio es evaluar cómo los efectos combinados de cuatro importantes factores estresantes (el parásito intestinal Nosema ceranae, el insecticida neonicotinoide tiametoxam, el insecticida piretroide cipermetrina y el fungicida EBI tebuconazol) interactúan para afectar a los abejorros a nivel individual y de colonia. Establecimos siete grupos de tratamiento de colonias de abejorros que expusimos a diferentes combinaciones de estos factores estresantes durante dos semanas en condiciones de laboratorio, y posteriormente se colocaron en el campo durante siete semanas, para evaluar el efecto de los tratamientos sobre la prevalencia de N. ceranae en abejorros inoculados, los niveles de expresión de genes relacionados con la inmunidad y la desintoxicación, la recolección de alimentos, el aumento de peso, el número de obreras y machos, y la producción de cría de obreras, machos y reinas. La exposición a mezclas de pesticidas redujo la recolección de alimentos por parte de los abejorros. Todos los genes relacionados con la inmunidad se sobre-expresaron en los abejorros inoculados con N. ceranae cuando no habían estado expuestos a mezclas de pesticidas, y los abejorros expuestos al fungicida y al piretroide presentaron menos probabilidades de tener N. ceranae. La exposición combinada a la mezcla de tres pesticidas y N. ceranae redujo el crecimiento de la colonia de abejorros y todos los tratamientos tuvieron efectos perjudiciales en la producción de crías. Los grupos expuestos al insecticida neonicotinoide produjeron entre un 40 y un 76% menos de reinas que las colonias control. Nuestros hallazgos muestran que la exposición a combinaciones de factores estresantes con los que los abejorros entran frecuentemente en contacto tiene efectos perjudiciales sobre la salud y el rendimiento de la colonia y, por lo tanto, podría tener un impacto a nivel poblacional. Estos resultados también tienen importantes implicaciones para las prácticas y políticas actuales de evaluación de riesgos y uso de plaguicidas, ya que las combinaciones probadas aquí se aplican con frecuencia simultáneamente en el campo. Comprender las interacciones entre los diferentes factores de estrés es fundamental para mejorar nuestra capacidad de gestión de las poblaciones de abejas y así garantizar los servicios de polinización en el futuro. Studying the sublethal effects of agrochemical pesticides on nontarget honeybees (Apis mellifera) is important for agricultural development. Carbendazim is a widely used broad-spectrum fungicide that inhibits mitotic microtubule formation and cell division. However, the impact of carbendazim on bee health and development has not been fully elucidated. Here, using proteomics approaches, we assessed in vitro the changes in the expression of functional proteins in the head of newly emerged adults following treatment with field concentration of carbendazim during the larval stage. Treatment with carbendazim severely altered 266 protein expression patterns in the heads of adults and 218 of them showed downregulation after carbendazim exposure. Notably, major royal jelly proteins, a crucial multifunctional protein family with irreplaceable function in sustaining the development of colonies, were significantly suppressed in carbendazim-treated bees. This result was verified in both head and hypopharyngeal gland of nurse bees. Moreover, visual and olfactory loss, immune functions, muscular activity, social behavior, neural and brain development, protein synthesis and modification, and metabolism-related proteins were likely inhibited by carbendazim treatment. Together, these results suggest that carbendazim is an environmental risk factor that likely weakens bee colonies, partially due to reduced expression of major royal jelly proteins, which may be potential causes of colony collapse disorder.

What 3 organs are the sphincter of Oddi associated with?

Sphincter of Oddi is associated with the pancreatic duct, duodenum and gallbladder.

The sphincter of Oddi is a small sphincter which is strategically placed at the junction of the bile duct and pancreatic duct with the duodenum. It regulates the flow of bile and pancreatic juice into the duodenum and prevents reflux of duodenal contents into the ducts. The structure of the sphincter of Oddi differs from species to species and consequently its physiological action varies in different species. Anatomical and immunohistochemical investigations have demonstrated that the sphincter of Oddi is richly innervated by cholinergic, adrenergic and peptidergic neurons. In addition, neural connections exist between the sphincter, gallbladder and proximal gastrointestinal tract. These nerves in addition to hormones are important in the control of sphincter of Oddi motility and function. The normal human sphincter of Oddi is characterized by prominent phasic contractions which are superimposed on a modest basal pressure. These contractions are present throughout the interdigestive period. The contractions and basal pressure are inhibited by ingestion of a meal or infusion of cholecystokinin octapeptide, thus enhancing the flow of bile and pancreatic juice into the duodenum. Sphincter of Oddi dysfunction has been described in patients who present with recurrent biliary type pain and no evidence of a structural cause for the pain. Motility disorders characterized as an elevated basal pressure, rapid contraction frequency, paradoxical response to cholecystokinin octapeptide or excess of retrograde contractions have been identified. A number of pharmacologically active substances have been used in an attempt to treat these patients. Such pharmaceuticals include nitrites, Ca2+ channel blockers and smooth muscle relaxants. Their effect is transient and side effects relating to cardiovascular actions preclude their longterm use. Division of the sphincter either endoscopically or by open operation has been demonstrated by prospective clinical trials to be the most efficacious treatment for patients with a stenosed sphincter manometrically demonstrated by a high basal pressure. Improved understanding of the controlling mechanisms of sphincter of Oddi motility and the pathophysiology of sphincter of Oddi dysfunction should assist in the development of effective pharmacotherapy for these disorders. Ever since its description approximately 100 years ago, the sphincter of Oddi has been surrounded by controversy. First, whether it indeed existed, second, whether it had a significant physiological role in man and more recently whether abnormalities in its function give rise to a clinical syndrome. Data from animal and human studies, using sensitive techniques, have helped define the physiological role of the sphincter of Oddi, and more recent studies are determining the factors which control sphincter of Oddi function. These studies support Oddi's original description that the sphincter has a major role in the control of flow of bile and pancreatic juice into the duodenum, and equally importantly helps prevent the reflux of duodenal contents into the biliary and pancreatic ductal systems. The controversy of whether abnormalities in sphincter of Oddi motility result in clinical syndromes has not been totally resolved. Part of the difficulty has been inability to document normal and hence abnormal function of the sphincter. With the emergence of endoscopic biliary manometry as a sensitive and reproducible technique, however, the motility of the human sphincter of Oddi has come under closer scrutiny and allowed definition of possible disorders. We have used the term sphincter of Oddi dysfunction to define manometric abnormalities in patients who present with signs and symptoms consistent with a biliary or pancreatic ductal origin. Based on the manometry, we have subdivided the dysfunction into two groups; a group characterised by a stenotic pattern - that is, raised sphincter basal pressure - and a second group having a dyskinetic pattern - that is, paradoxical response to cholecystokinin injection, rapid contraction frequency, high percentage of retrograde contractions, or short periods of raised basal percentage of retrograde contractions, or short periods of raised basal pressure. It is apparent from the mamometry but also from the clinical data that the patients are a heterogeneous group and thus any therapy would need to be tailored for each patient and abnormality. The most recent therapeutic data suggest that patients with the stenotic pattern on manometry respond to division of the sphincter, however, those patients with the dyskinetic manometric pattern show no significant effect after sphincterotomy. Further prospective trials evaluating therapeutic options are under way and their results are eagerly awaited. The sphincter of Oddi is the smooth muscle connection between the bile duct and the duodenum. Its physiological function is associated with a regular motility characterized by phasic contractions superimposed on the sphincter of Oddi baseline pressure. Recently introduced ERCP-manometry permits further studies of sphincter of Oddi pharmacology. A number of drugs have so far been studied. Sedatives of the diazepam type had no effect on the sphincter, while butylscopolaminium bromide, a typical neurotropic agent, brings about cessation of the sphincter motility for 3-8 minutes. Hymecromon lowered the sphincter baseline pressure from 9.8 to 7.8 mmHg. A 1.2 mg sublingual dose of nitroglycerin, a typical musculotropic agent, caused significant relaxation of the sphincter, and decreased baseline pressure from 8.9 mmHg to 2.9 mmHg; Sphincter motility was not affected. Morphine-like analgetics, in particular pentazocine, elevated sphincter baseline pressure, but buprenorphine and tramadol did not. Pharmacological doses of gastrointestinal hormones also affect the sphincter; CCK octapeptide, glucagon and secretin are able to decrease sphincter of Oddi baseline pressure, and CCK octapeptide abolishes sphincter motility. Sphincter of Oddi pharmacology is of clinical interest. The administration of sphincter-relaxing agents, in particular nitroglycerin and butylscopolaminium bromide, enables the endoscopist to extract small common bile duct stones without previous papillotomy. Analgetics that induce sphincter contraction and thus hinder the flow of bile and pancreatic juice, may be helpful for the treatment of pain in patients with pancreatico-biliary disease. Investigations into the effect of CCK on the healthy and diseased sphincter permit us to identify patients with sphincter dysfunction using a special CCK-provocation test. The sphincter of Oddi (SO) is critically located at the junction of the common bile duct (CBD), main pancreatic duct, and the duodenum. It is a high-pressure zone with phasic contractions that regulate bile and pancreatic juice flow. The SO is probably regulated by several gastrointestinal hormones, and its basal pressure and phasic contractions can be elevated or decreased significantly by exogenous drugs. Its role in gallstone formation is probably negligible, but severing the SO allows one to extract CBD stones with an endoscope. Abnormal function of the SO can cause biliarylike pain. Of patients with persistent pain after cholecystectomy, 14% have abnormal SO manometric findings. Endoscopic or surgical sphincterotomy can cure these patients of their pain. The SO may play a significant role in the development of pancreatitis in certain patients, either because of the relationship of the CBD orifice to the pancreatic duct orifice created by the SO or because of the response of the SO to exogenous agents, such as alcohol. Flow through the sphincter of Oddi was studied by a constant-pressure perfusion apparatus, and by using a constant rate of infusion and measuring pressure changes just proximal to the sphincter. Pressure changes from balloons placed in the gastric antrum and duodenum were recorded. Despite considerable variation in activity and behaviour, the following conclusions were reached.1. The sphincter of Oddi appears to be partially autonomous but is readily affected by changes in duodenal tone.2. Reflex inhibition of the sphincter of Oddi is seen on electrical and mechanical stimulation of the gall-bladder but not with passive pressure changes within the gall-bladder.3. This reflex is relayed through the coeliac ganglion.4. There is a definite relationship between gastric antral activity and flow through the sphincter of Oddi. This is more obvious when food is present in the stomach.5. Mechanical or electrical stimulation of the stomach, duodenum, or upper small bowel influences the tone of the sphincter of Oddi. It has been reported that interdigestive motor activities occur in the gallbladder and sphincter of Oddi as well as in the gastroduodenal tract and truncal vagus nerves modulate the gastroduodenal motility pattern. In the present study, the vagal branches which influence on the interdigestive motor activity of these organs were determined in conscious dogs. In the normal dog, interdigestive motor activities closely related to interdigestive migrating motor complex (IMC) in the gastric antrum and descending duodenum were recorded in the gallbladder and sphincter of Oddi. In dogs whose celiac branches of the vagus nerve were chronically resected, cycle periods of interdigestive motor activity in the stomach, duodenum, gallbladder and sphincter of Oddi significantly prolonged as compared with those in the normal dog but the interrelation of them persisted. Quiescent period in motility prolonged and the active period shortened. Resection of the pyloric branches had no influence on the interdigestive motor activities in all organs. The results imply that celiac branches of the vagus nerve modulate the interdigestive motor activity in the stomach, descending duodenum, gallbladder and sphincter of Oddi. We studied the role of the L-arginine-nitric oxide (NO) pathway in non-adrenergic, non-cholinergic (NANC) relaxation of the rabbit sphincter of Oddi by recording changes in isometric tension in response to electrical field stimulation in two series of experiments. In a first set of experiments, biliary sphincters of Oddi removed from New Zealand white rabbits were placed horizontally in an organ bath containing oxygenized, buffered (pH 7.4) Krebs solution. Contractile responses of the whole sphincter to field stimulation were determined. In the second set of experiments, sphincter of Oddi was divided into two parts and the effects of field stimulation were studied separately on areas close to the duodenal papilla (area I) and areas close to the common bile duct (area II). In the whole sphincter of Oddi, field stimulation induced an initial twitch-like contraction followed by relaxation proportional to the number of stimuli (3 and 10 stimuli at 20 Hz, 50 V, 0.1 ms). The magnitude of the contractile responses was considerably reduced by 1 microM atropine, phentolamine and oxprenolol (NANC solution). Field stimulation produced dose-dependent contractions of both segments of sphincter of Oddi in response to the same protocol as used with whole sphincter of Oddi. However, preincubation with NANC solution produced monophasic relaxations in response to field stimulation in area I, whereas area II preparations such as the whole sphincter of Oddi responded with contractions followed by minimal relaxations. Field stimulation failed to induce either contractions or relaxations in the presence of 1 microM tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS) BACKGROUND: This study investigated the existence of direct neural connections between the duodenum and the biliary tract in the Australian possum. METHODS: Retrogradely transported neuronal dyes, Fast Blue and Dil, were injected into the wall of the gallbladder and the sphincter of Oddi. The duodenum, biliary tract, and sympathetic and sensory ganglia were examined for the presence of labeled cell bodies. RESULTS: Two to 3 weeks after gallbladder injection, labeled nerve cell bodies were found in the myenteric plexus of the proximal duodenum but were rare in the duodenum distal to the sphincter of Oddi. No neurons were found in the submucous plexus. Labeled nerve cells were also found in the sphincter of Oddi. After injection of the sphincter, labeled neurons were in both the submucous and myenteric plexuses of the duodenum, on either side. Approximately one third of labeled myenteric neurons were immunoreactive for enkephalin. Labeled cell bodies were also in the coeliaco-mesenteric, nodose, and dorsal root ganglia after both gallbladder and sphincter injection. After a myotomy on the proximal duodenum, no neurons were labeled on the pyloric side of the lesion by subsequent sphincter injection of dye. CONCLUSIONS: Direct neural pathways connect the duodenum with the gallbladder and the sphincter of Oddi, and the sphincter with the gallbladder; this implies that enteric nerve circuits participate in coordinating duodenal and biliary functions. We have investigated the existence of neural connections between the duodenum and the sphincter of Oddi (SO). Stimulation of duodenal myenteric fiber bundles elicited synaptic responses in SO neurons, which included nicotinic fast excitatory postsynaptic potentials (EPSPs), slow EPSPs, and alpha(2)-adrenoreceptor-mediated inhibitory postsynaptic potentials. After 48 h in organ culture, when extrinsic fibers had diminished, only the fast EPSPs persisted. Duodenal mucosal stimulation also elicited nicotinic fast EPSPs in SO neurons. There was no association between the SO neurons that received duodenal input and their chemical coding. A reciprocal projection also exists from the SO to the duodenum. In acute and cultured preparations, duodenal myenteric stimulation caused antidromic responses in 20% of SO neurons. Furthermore, 45.6 +/- 10.5 neurons in SO ganglia were retrogradely labeled from dye application sites in the duodenum. It is proposed that bidirectional neural communication occurs between the duodenum and the SO and that duodenal neurons provide excitatory fast synaptic input to SO neurons through a reflex that can be activated at the duodenal mucosa. Magnetic resoce (MR) cholangiopancreatography has proved a robust and noninvasive imaging modality for evaluating the biliary and pancreatic ducts without the use of ionizing radiation. Although MR cholangiopancreatography reliably depicts the main extrahepatic and intrahepatic bile ducts, it does not depict the segmental intrahepatic ducts unless they are dilated. The segmental ducts are difficult to visualize with MR cholangiopancreatography because of their small caliber and the limited spatial resolution and signal-to-noise ratio achievable with standard MR pulse sequences. However, visualization of the normal (ie, nondistended) biliary system is necessary for the evaluation of donor candidates for living related liver transplantation. Because of the prevalence of variant biliary anatomy, MR cholangiopancreatography is often used for preoperative evaluation of prospective liver donors. Intravenous morphine administered prior to MR cholangiopancreatography can improve image quality by causing the sphincter of Oddi to contract, which increases pressure in and distention of the biliary and pancreatic ducts. Morphine administration may also be particularly helpful for the evaluation of patients with primary sclerosing cholangitis, maligt neoplasms such as cholangiocarcinoma, or cystic and non-organ-deforming benign pancreatic neoplasms. The most common functional disorder of the biliary tract and pancreas relates to the activity of the Sphincter of Oddi. The Sphincter of Oddi is a small smooth muscle sphincter strategically placed at the junction of the bile duct, pancreatic duct, and duodenum. The sphincter controls flow of bile and pancreatic juices into the duodenum and prevents reflux of duodenal content into the ducts. Disorder in its motility is called Sphincter of Oddi dysfunction. Clinically this presents either with recurrent abdominal biliary type pain or episodes of recurrent pancreatitis. Manometry may identify the motility abnormalities, the most clinically significant being an abnormally elevated basal pressure. The most effective treatment once an abnormal basal pressure is identified is division of the sphincter. This is associated with good long-term results.

List clinical phenotypes and molecular genetic features of patients with KMT2B-related disorders

Atypical patterns of dystonia evolution and a subgroup of patients presents with a non-dystonic neurodevelopmental phenotype. In addition to the previously reported systemic features there seem to be co-morbidities, including the risk of status dystonicus, intrauterine growth retardation, and endocrinopathies.

What is the use of Atogepant?

Atogepant is used for preventive treatment of migraine.

The incidence of migraine is higher among women than men and peaks during the reproductive years, when contraceptive medication use is common. Atogepant, a potent, selective antagonist of the calcitonin gene-related peptide receptor-in development for migraine prevention-is thus likely to be used by women taking oral contraceptives. This phase 1, open-label, single-center, 2-period, fixed-sequence study examined the effect of multiple-dose atogepant 60 mg once daily on the single-dose pharmacokinetics of a combination oral contraceptive, ethinyl estradiol 0.03 mg and levonorgestrel 0.15 mg (EE/LNG), in healthy postmenopausal or oophorectomized women. For participants in period 1, a single dose of EE/LNG was followed by a 7-day washout. In period 2, atogepant was given once daily on days 1-17; an oral dose of EE/LNG was coadministered with atogepant on day 14. Plasma pharmacokinetic parameters for EE and LNG were assessed following administration with and without atogepant. Twenty-six participants aged 45-64 years enrolled; 22 completed the study in accordance with the protocol. The area under the concentration-time curve extrapolated to infinity (AUC0-∞ ) of LNG was increased by 19% when administered with atogepant. Coadministration of atogepant and a single dose of EE/LNG did not substantially alter the pharmacokinetics of EE; the ∼19% increase in plasma AUC0-∞ of LNG is not anticipated to be clinically significant. Overall, atogepant alone and in combination with EE/LNG was generally well tolerated, with no new safety signals identified. Introduction: Migraine is one of the most common neurological disorders. Nowadays, the 5-HT1B/1D receptor agonists, namely triptans, are considered as the standard of care for migraine acute treatment. However, triptans have limitations in some patients, such as incomplete pain relief, headache recurrence, and cardiovascular contraindications. New 5-HT1F receptor agonists, namely ditans, and calcitonin gene-related peptide receptor antagonists, namely gepants, have been developed as migraine-specific treatments.Areas covered: This paper reviews the available data from RCTs to assess the clinical efficacy, safety, and tolerability profile of lasmiditan, rimegepant, and ubrogepant for the acute treatment of migraine and atogepant for the prevention of migraine.Expert opinion: Available data suggest that lasmiditan, rimegepant, and ubrogepant might not have a clinical efficacy similar to triptans. Lasmiditan did not cause the typical triptan side effects but was associated with central nervous system side effects, causing temporary driving impairment. On the contrary, the new generation of gepants showed a placebo-like tolerability profile and the absence of a specific pattern of side effects. Future studies on lasmiditan and gepants with respect to established effective comparators are mandatory to support phase III results and to help clinicians to balance the benefit/risk profiles of the various acute and preventive medications. BACKGROUND: Atogepant is an oral, small-molecule, calcitonin gene-related peptide receptor antagonist that is being investigated for the preventive treatment of migraine. METHODS: In a phase 3, double-blind trial, we randomly assigned adults with 4 to 14 migraine days per month in a 1:1:1:1 ratio to receive a once-daily dose of oral atogepant (10 mg, 30 mg, or 60 mg) or placebo for 12 weeks. The primary end point was the change from baseline in the mean number of migraine days per month across the 12 weeks. Secondary end points included headache days per month, a reduction from baseline of at least 50% in the 3-month average of migraine days per month, quality of life, and scores on the Activity Impairment in Migraine-Diary (AIM-D). RESULTS: A total of 2270 participants were screened, 910 were enrolled, and 873 were included in the efficacy analysis; 214 were assigned to the 10-mg atogepant group, 223 to the 30-mg atogepant group, 222 to the 60-mg atogepant group, and 214 to the placebo group. The mean number of migraine days per month at baseline ranged from 7.5 to 7.9 in the four groups. The changes from baseline across 12 weeks were -3.7 days with 10-mg atogepant, -3.9 days with 30-mg atogepant, -4.2 days with 60-mg atogepant, and -2.5 days with placebo. The mean differences from placebo in the change from baseline were -1.2 days with 10-mg atogepant (95% confidence interval [CI], -1.8 to -0.6), -1.4 days with 30-mg atogepant (95% CI, -1.9 to -0.8), and -1.7 days with 60-mg atogepant (95% CI, -2.3 to -1.2) (P<0.001 for all comparisons with placebo). Results for the secondary end points favored atogepant over placebo with the exceptions of the AIM-D Performance of Daily Activities score and the AIM-D Physical Impairment score for the 10-mg dose. The most common adverse events were constipation (6.9 to 7.7% across atogepant doses) and nausea (4.4 to 6.1% across atogepant doses). Serious adverse events included one case each of asthma and optic neuritis in the 10-mg atogepant group. CONCLUSIONS: Oral atogepant once daily was effective in reducing the number of migraine days and headache days over a period of 12 weeks. Adverse events included constipation and nausea. Longer and larger trials are needed to determine the effect and safety of atogepant for migraine prevention. (Funded by Allergan; ADVANCE ClinicalTrials.gov number, NCT03777059.). Conflict of interest statement: Declaration of conflicting interests: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Todd J Schwedt serves on the Board of Directors for the American Headache Society and the International Headache Society. He has received research support from the American Migraine Foundation, Amgen, Henry Jackson Foundation, National Institutes of Health, Patient-Centered Outcomes Research Institute, and US Department of Defense. Within the past 12 months, he has received personal compensation for serving as a consultant or advisory board member for AbbVie/Allergan, Biohaven, Eli Lilly, Equinox, Lundbeck, and Novartis. He has received royalties from UpToDate. He holds stock options in Aural Analytics and Nocira. Richard B Lipton has received research support from the National Institutes of Health, the FDA, and the National Headache Foundation. He serves as consultant, advisory board member, or has received honoraria or research support from AbbVie/Allergan, Amgen, Biohaven, Dr. Reddy’s Laboratories (Promius), electroCore, Eli Lilly, GlaxoSmithKline, Lundbeck, Merck, Novartis, Teva, Vector, and Vedanta Research. He receives royalties from Wolff’s Headache, 8th edition (Oxford University Press, 2009), and Informa. He holds stock/options in Biohaven and Ctrl M. Jessica Ailani has served as a consultant for AbbVie, Amgen, Axsome, Ctrl M, Eli Lilly and Company, Lundbeck, Impel, Satsuma, Theranica, Teva, and Vorso; has served as a speaker for AbbVie, Amgen, Eli Lilly and Company, Lundbeck, and Teva; has received honoraria from Medscape; and has provided editorial services to Current Pain and Headache Reports, NeurologyLive, and SELF magazine. Her institute has received clinical trial support from AbbVie, the American Migraine Foundation, Biohaven, Eli Lilly and Company, Satsuma, and Zosano. Stephen D Silberstein is a consultant and/or advisory panel member for and has received honoraria from AbbVie, Alder Biopharmaceuticals, Amgen, Avanir, eNeura, electroCore Medical, Labrys Biologics, Medscape, Medtronic, Neuralieve, NINDS, Pfizer, and Teva. His employer receives research support from AbbVie, Amgen, Cumberland Pharmaceuticals, electroCore Medical, Labrys Biologics, Eli Lilly, Mars, and Troy Healthcare. Cristina Tassorelli has participated in advisory boards for AbbVie, electroCore, Eli Lilly, Novartis, and Teva. She has lectured at symposia sponsored by AbbVie, Eli Lilly, Novartis, and Teva. She is principal investigator or collaborator in clinical trials sponsored by Alder, Eli Lilly, IBSA, Novartis, and Teva. She has received research grants from the European Commission, the Italian Ministry of Health, the Italian Ministry of University, the Migraine Research Foundation, and the Italian Multiple Sclerosis Foundation. Hua Guo, Kaifeng Lu, Brett Dabruzzo, Rosa Miceli, Lawrence Severt, Michelle Finnegan and Joel M Trugman are employees of AbbVie and may hold AbbVie stock. In this issue of JNO Drs. M. Tariq Bhatti and Mark L. Moster discuss the following 6 articles: Dinah Zur, Michaella Goldstein, Barequet D, Oron Y, Elkayam O, Karni A, Wilf-Yarkoni A, Regev K, Habot-Wilner Z. Susac's syndrome-A new ocular finding and disease outcome. Eye (Lond). 2021. doi: 10.1038/s41433-021-01464-7. Epub ahead of print.Durbant E, Radoi C, Garcia T, Denoyer A, Arndt C. Intravitreal triamcinolone injections in non-arteritic anterior ischemic optic neuropathy-A retrospective report. J Fr Ophtalmol. 2021;44:777-785.Cavanaugh MR, Blanchard LM, McDermott M, Lam BL, Tamhankar M, Feldon SE. Efficacy of Visual Retraining in the Hemianopic Field after Stroke: Results of a Randomized Clinical Trial. Ophthalmology. 2021;128:1091-1101.Czihal M, Lottspeich C, Köhler A, Prearo I, Hoffmann U, Priglinger SG, Mackert MJ. Transocular sonography in acute arterial occlusions of the eye in elderly patients: Diagnostic value of the spot sign. PLoS One. 2021;16:e0247072.Ailani J, Lipton RB, Goadsby PJ, Guo H, Miceli R, Severt L, Finnegan M, Trugman JM; ADVANCE Study Group. Atogepant for the Preventive Treatment of Migraine. N Engl J Med. 2021;385:695-706.Engelter ST, Traenka C, Gensicke H, Schaedelin SA, Luft AR, Simonetti BG, Fischer U, Michel P, Sirimarco G, Kägi G, Vehoff J, Nedeltchev K, Kahles T, Kellert L, Rosenbaum S, von Rennenberg R, Sztajzel R, Leib SL, Jung S, Gralla J, Bruni N, Seiffge D, Feil K, Polymeris AA, Steiner L, Hamann J, Bonati LH, Brehm A, De Marchis GM, Peters N, Stippich C, Nolte CH, Christensen H, Wegener S, Psychogios MN, Arnold M, Lyrer P; TREAT-CAD investigators. Aspirin vs. anticoagulation in cervical artery dissection (TREAT-CAD): an open-label, randomised, non-inferiority trial. Lancet Neurol. 2021;20:341-350. Atogepant (Qulipta™) is an orally administered, small-molecule, calcitonin gene-related peptide (CGRP) receptor antagonist being developed by AbbVie for the prophylaxis of migraine. In September 2021, atogepant was approved in the USA for the preventive treatment of episodic migraine in adults. The drug is also in phase 3 clinical development for the preventive treatment of migraine in various other countries. This article summarizes the milestones in the development of atogepant leading to this first approval for the preventive treatment of episodic migraine in adults.

Is SMOC2 expressed during wound healing?

Yes, SMOC2 response to wounding.

Author information: (1)Developmental Biology and Stem Cells Department, Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, BP 10142, 67404, Illkirch, France. (2)Centre National de la Recherche Scientifique, UMR7104, Illkirch, France. (3)Institut National de la Santé et de la Recherche Médicale, INSERM U1258, Illkirch, France. (4)Université de Strasbourg, Illkirch, France. (5)Faculty of Dentistry, Pediatrics Division, Preventive Department, Khon Kaen University, Khon Kaen, Thailand. (6)Faculté de Chirurgie Dentaire, Université de Strasbourg, 8 rue Ste Elisabeth, 67000, Strasbourg, France. (7)Hôpitaux Universitaires de Strasbourg, Pôle de Médecine et Chirurgie Bucco-Dentaires, Centre de Référence des Maladies Rares Orales et Dentaires, CRMR O Rares, Filière TETECOU, ERN CRANIO, 1 place de l'Hôpital, 67000, Strasbourg, France. (8)Regenerative NanoMedicine, INSERM UMR1260, FMTS, Hôpitaux Universitaires de Strasbourg, 11 rue Humann, 67000, Strasbourg, France. (9)Biomaterials and Bioengineering, Université de Strasbourg, INSERM UMR1121, 11 rue Humann, 67000, Strasbourg, France. (10)Hubrecht Institute, University Medical Center Utrecht, and University Utrecht, Utrecht, The Netherlands. (11)Faculté de Médecine, Université de Strasbourg, FMTS, 4 Rue Kirschleger, 67000, Strasbourg, France. (12)Developmental Biology and Stem Cells Department, Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, BP 10142, 67404, Illkirch, France. [email protected]. (13)Centre National de la Recherche Scientifique, UMR7104, Illkirch, France. [email protected]. (14)Institut National de la Santé et de la Recherche Médicale, INSERM U1258, Illkirch, France. [email protected]. (15)Université de Strasbourg, Illkirch, France. [email protected]. (16)Faculté de Chirurgie Dentaire, Université de Strasbourg, 8 rue Ste Elisabeth, 67000, Strasbourg, France. [email protected]. (17)Hôpitaux Universitaires de Strasbourg, Pôle de Médecine et Chirurgie Bucco-Dentaires, Centre de Référence des Maladies Rares Orales et Dentaires, CRMR O Rares, Filière TETECOU, ERN CRANIO, 1 place de l'Hôpital, 67000, Strasbourg, France. [email protected]. (18)Eastman Dental Institute, University College London, London, UK. [email protected]. (19)Developmental Biology and Stem Cells Department, Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, BP 10142, 67404, Illkirch, France. [email protected]. (20)Centre National de la Recherche Scientifique, UMR7104, Illkirch, France. [email protected]. (21)Institut National de la Santé et de la Recherche Médicale, INSERM U1258, Illkirch, France. [email protected]. (22)Université de Strasbourg, Illkirch, France. [email protected]. (23)Faculté de Chirurgie Dentaire, Université de Strasbourg, 8 rue Ste Elisabeth, 67000, Strasbourg, France. [email protected]. The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single-cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRβ + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA-seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis. BACKGROUND: Asthma is a common chronic respiratory disease that influences 300 million people all over the world. However, the pathogenesis of asthma has not been fully elucidated. It has been reported that transforming growth factor-β (TGF-β) can activate myofibroblasts. Moreover, the fibroblast to myofibroblast transformation (FMT) can be triggered by TGF-β, which is a major mediator of subepithelial fibrosis. Secreted modular calcium-binding protein 2 (SMOC2) is a member of cysteine (SPARC) family and is involved in the progression of multiple diseases. However, its role in asthma remains poorly understood. RT-qPCR evaluated the expression of SMOC2. Bromodeoxyuridine assay and wound-healing assay detected the proliferation and migration of lung fibroblasts, respectively. IF staining was performed to assess the expression of α-smooth muscle actin (α-SMA). Western blot analysis detected the levels of proteins. Flow cytometry was utilized for determination of the number of myofibroblasts. RESULTS: We found the expression of SMOC2 was upregulated by the treatment of TGF-β1 in lung fibroblasts. In addition, SMOC2 promoted the proliferation and migration of lung fibroblasts. More importantly, SMOC2 accelerated FMT of lung fibroblasts. Furthermore, SMOC2 was verified to control the activation of AKT and ERK. Rescue assays showed that the inhibition of AKT and ERK pathway reversed the promoting effect of SMOC2 overexpression on proliferation, migration and FMT in lung fibroblasts. CONCLUSIONS: This work demonstrated that SMOC2 modulated TGF-β1-induced proliferation, migration and FMT in lung fibroblasts and may promote asthma, which potentially provided a novel therapeutic target for the management of asthma.

What is a potential alternate uses(repositioning) for Primaquine

Primaquine Diphosphate, a Known Antimalarial Drug, Blocks Vascular Leakage Acting Through Junction Stabilization. PD could be used as a novel drug for vascular leakage by maintaining endothelial integrity

Drug repositioning identifies new indications for known drugs. Here we report repositioning of the malaria drug amodiaquine as a potential anti-cancer agent. While most repositioning efforts emerge through serendipity, we have devised a computational approach, which exploits interaction patterns shared between compounds. As a test case, we took the anti-viral drug brivudine (BVDU), which also has anti-cancer activity, and defined ten interaction patterns using our tool PLIP. These patterns characterise BVDU's interaction with its target s. Using PLIP we performed an in silico screen of all structural data currently available and identified the FDA approved malaria drug amodiaquine as a promising repositioning candidate. We validated our prediction by showing that amodiaquine suppresses chemoresistance in a multiple myeloma cancer cell line by inhibiting the chaperone function of the cancer target Hsp27. This work proves that PLIP interaction patterns are viable tools for computational repositioning and can provide search query information from a given drug and its target to identify structurally unrelated candidates, including drugs approved by the FDA, with a known safety and pharmacology profile. This approach has the potential to reduce costs and risks in drug development by predicting novel indications for known drugs and drug candidates. As a subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) is characterized by a chromosomal translocation, most of which result in the production of a PML-RAR alpha fusion protein. Although the overall survival rate of APL patients has improved dramatically due to all-trans retinoic acid (ATRA) treatment, ATRA-resistance remains a clinical challenge in the management of APL. Therefore, alternative agents should be considered for ATRA-resistant APL patients. Here, we report that antimalaria drug primaquine phosphate (PRQ) exhibits an anti-leukemia effect on both ATRA-sensitive cell line NB4 and ATRA-resistant APL cell lines, NB4-LR2, NB4-LR1, and NB4-MR2. Moreover, PRQ significantly inhibited primary colony formation of untreated or relapsed APL patients. Further study showed that PRQ could induce the apoptosis of APL cells by inhibiting NF-κB signaling pathway. The in vivo study showed that PRQ significantly inhibited NB4-LR2 xenograft tumors growth. These results suggest that PRQ is a potential therapeutic agent for ATRA-resistant APL patients. Endothelial barrier integrity is important for vascular homeostasis, and hyperpermeability participates in the progression of many pathological states, such as diabetic retinopathy, ischemic stroke, chronic bowel disease, and inflammatory disease. Here, using drug repositioning, we discovered that primaquine diphosphate (PD), previously known as an antimalarial drug, was a potential blocker of vascular leakage. PD inhibited the linear pattern of vascular endothelial growth factors (VEGF)-induced disruption at the cell boundaries, blocked the formation of VEGF-induced actin stress fibers, and stabilized the cortactin actin rings in endothelial cells. PD significantly reduced leakage in the Miles assay and mouse model of streptozotocin (STZ)-induced diabetic retinopathy. Targeted prediction programs and deubiquitinating enzyme activity assays identified a potential mechanism of action for PD and demonstrated that this operates via ubiquitin specific protease 1 (USP1). USP1 inhibition demonstrated a conserved barrier function by inhibiting VEGF-induced leakage in endothelial permeability assays. Taken together, these findings suggest that PD could be used as a novel drug for vascular leakage by maintaining endothelial integrity. Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.

Does daily atemoya juice intake change the pharmacokinetics of CYP1A2 substrates?

No, atemoya juice does not change the pharmacokinetics of CYP1A2 substrates.

BACKGROUND AND OBJECTIVES: Atemoya (Annona atemoya) is increasingly being consumed worldwide because of its pleasant taste. However, only limited information is available concerning possible atemoya-drug interactions. In the present study, the issue of whether atemoya shows food-drug interactions with substrate drugs of the major drug-metabolizing cytochrome P450s (i.e., CYP1A2, CYP2C9, and CYP3A) is addressed. METHODS: The ability of atemoya juice to inhibit the activities of phenacetin O-deethylase (CYP1A2), diclofenac 4'-hydroxylase (CYP2C9), and midazolam 1'-hydroxylase (CYP3A) was examined in vitro using human and rat liver microsomes. The in vivo pharmacokinetics of phenacetin and metabolites derived from it in rats when atemoya juice or fluvoxamine (a CYP1A2 inhibitor) was preadministered were also investigated. RESULTS: Atemoya juice significantly inhibited CYP1A2 activity in human liver microsomes, but not the activities of CYP2C9 and CYP3A. In spite of this inhibition, preadministration of atemoya had no effect on the pharmacokinetics of phenacetin, a CYP1A2 substrate, in rats. Meanwhile, preadministration of fluvoxamine significantly extended the time needed for the elimination of phenacetin, possibly due to the inhibition of CYP1A2. This suggests that the intake of an excess amount of atemoya juice is necessary to cause a change in the pharmacokinetics of phenacetin when the IC50 values for CYP1A2 inhibition by atemoya and fluvoxamine are taken into account. CONCLUSION: The results indicate that a daily intake of atemoya would not change the pharmacokinetics of CYP1A2 substrates such as phenacetin as well as CYP2C9- and CYP3A-substrate drugs.

What is caused by gain-of-function variants in SYK?

Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice.

Is Cabotegravir effective for HIV prevention?

Yes, Cabotegravir is effective for HIV prevention.

PURPOSE OF REVIEW: Preexposure prophylaxis for HIV prevention is highly effective when taken as prescribed. Adherence to required dosing regimens for protection may pose challenges. Long-acting agents for HIV prevention may have the potential to improve adherence via favorable pharmacokinetics supportive of infrequent dosing. This review focuses on the potential benefits and considerations for the study and use of 2 long-acting injectable agents, cabotegravir (GSK1265744LA, CAB LA) and rilpivirine (TMC278LA, RPV LA), for use as chemoprophylaxis for HIV prevention. RECENT FINDINGS: Oral RPV is United States Food and Drug Administration approved for HIV treatment (in combination with other antiretrovirals). Both CAB LA and RPV LA are currently in phase 2a safety/tolerability/pharmacokinetic studies in anticipation and support of future efficacy evaluation. Both agents have favorable pharmacokinetics, and use is complicated by injection site reactions. SUMMARY: Long-acting injectable formulations, if safe and well tolerated, may improve pharmacokinetic coverage of exposures to HIV infection. Complexities around safety, tolerability, and starting/stopping protocols require careful consideration. Modern antiretroviral therapy has demonstrated effectiveness in preexposure prophylaxis (PrEP) and treatment of HIV infection. There is a demand for prevention and treatment regimens that could overcome challenges of improving adherence, toxicity, and dosing convenience. Cabotegravir is an integrase strand transfer inhibitor and an analog of dolutegravir. Unlike dolutegravir, cabotegravir has a long half-life and can be formulated into a long-acting osuspension for parenteral administration. Initial pharmokinetic studies in humans have demonstrated adequate drug levels with intramuscular (IM) administration at 4 weekly and 8 weekly intervals, with few interactions with commonly used concomitant medications. Preliminary animal PrEP studies have shown that IM cabotegravir can prevent simian/HIV acquisition from rectal, vaginal, and intravenous challenge. Currently, there are two ongoing Phase II studies assessing cabotegravir as a PrEP agent in humans: ÉCLAIR and HPTN077. Cabotegravir has been studied in combination with rilpivirine as long-acting IM maintece therapy. The Long-Acting Antiretroviral Treatment Enabling study demonstrated that those switching to oral cabotegravir/rilpivirine once virologically suppressed were more likely to maintain suppression than those continuing standard efavirenz-based therapy (82% vs 71% at 24 weeks). Initial results of the Long-Acting Antiretroviral Treatment Enabling-2 study of parenteral regimens found that 12 weeks after randomization to parenteral or oral regimens, there was no difference in proportions virologically suppressed on cabotegravir/rilpivirine daily orally vs IM every 4 weeks or 8 weeks (91% vs 94% vs 95%). The injections were well tolerated as, although they caused injection site pain in most recipients, most participants reported satisfaction with parenteral therapy. Cabotegravir offers a new member of the integrase strand transfer inhibitor class with potential for alternative mode of delivery. We await Phase III studies to define its efficacy and real-world experience to learn which patient groups stand to benefit most from the novel mode of delivery of treatment and PrEP. Human Immunodeficiency Virus (HIV) is a chronic infection that depletes the immune system of essential components causing those infected to be at risk for multiple life-threatening infections. Worldwide, millions live with this infection, the vast majority attributable to HIV-1. Transmission persists with hundreds of thousands of new infections reported yearly. Implementation of combination antiretroviral therapy (cART) has been effective in improving outcomes and decreasing transmission. Newer co-formulated agents have provided simpler medication regimens, fewer side effects, and, in some cases, a higher barrier to the emergence of medication resistance. Areas covered: Here, we review trials of cabotegravir (CAB) as treatment of HIV-1 infection and its potential use as pre-exposure prophylaxis (PrEP) in high risk individuals, including issues around oral lead in and potential resistance emergence. Expert opinion: CAB is efficacious when used in combination therapy orally or given intramuscularly every 4 to 8 weeks. Its availability in a long-acting injectable formulation (CAB-LA) makes it a valuable, novel drug to treat HIV-1 infection when combined with long-acting injectable rilpivirine (RPV-LA). Moreover, pre-clinical and early Phase 2a studies support its testing as monotherapy as PrEP. Studies are underway comparing the efficacy of every 8 week CAB-LA to tenofovir disoproxil fumarate/emtricitabine (TDF/FTC). BACKGROUND: Cabotegravir (GSK1265744) is an integrase strand transfer inhibitor in development as a long-acting (LA) intramuscular injectable suspension for HIV-1 pre-exposure prophylaxis (PrEP). OBJECTIVE: We report participant outcomes from the phase IIa ECLAIR study related to tolerability, acceptability, and satisfaction of cabotegravir LA. METHODS: The ECLAIR study (ClinicalTrials.gov identifier, NCT02076178) was a randomized, placebo-controlled study in healthy men not at high risk of acquiring HIV-1. Participants were randomized (5:1) to once-daily oral cabotegravir 30 mg or placebo tablets for 4 weeks, followed by gluteal intramuscular injections of cabotegravir LA 800 mg or saline placebo every 12 weeks. The primary objective was to evaluate the safety of cabotegravir LA over three injection cycles (to Week 41). Secondary objectives assessed the tolerability, satisfaction, and acceptability of cabotegravir LA. RESULTS: Among 115 participants who received injections in the cabotegravir (n = 94) and placebo (n = 21) groups, 93% (n = 87) and 95% (n = 20) completed the injection phase, respectively. Injection intolerability led to withdrawal in 4 participants (4%) receiving cabotegravir LA. The most frequently reported Grade ≥2 adverse event was injection-site pain. Most participants (74% [n = 67]) receiving consecutive injections favored cabotegravir LA vs oral cabotegravir. Most participants were satisfied with cabotegravir LA (75% [n = 64]), were willing to continue (79% [n = 68]), and would recommend (87% [n = 75]) the therapy. CONCLUSIONS: While Grade ≥2 injection-site pain was common, most participants reported overall satisfaction with and preference for cabotegravir LA, with few discontinuations due to injection intolerance. These findings support investigation of cabotegravir LA as an alternative to daily oral PrEP regimens. Cabotegravir is an investigational integrase inhibitor in development for the treatment and pre-exposure prophylaxis of HIV-1 infection. Liver disease is a major cause of morbidity and mortality in HIV-infected individuals and can impact the pharmacokinetics (PK) of HIV medications. This phase 1 study evaluated the PK of cabotegravir in individuals with moderate hepatic impairment (n = 8) versus healthy controls (n = 8). Participants received a single oral cabotegravir 30-mg tablet and underwent PK sampling to determine total and unbound plasma cabotegravir concentrations. Calculated geometric least-squares mean ratios (90% confidence intervals) for individuals with hepatic impairment versus healthy controls were 0.73 (0.50-1.06) for AUC0-∞ , 0.69 (0.51-0.93) for Cmax , 1.40 (0.80-2.46) for unbound concentration (CU) 2 hours postdose, 1.55 (0.82-2.94) for CU at 24 hours, 2.14 (1.57-2.90) for unbound fraction (FU) at 2 hours, and 1.90 (1.14-3.18) for FU at 24 hours. Adverse events (AEs) occurred in 2 individuals with hepatic impairment and 3 healthy controls and were grade 1/2 in severity. No participant discontinued because of AEs. Increased FU resulted in a modest decrease in total plasma exposure not considered clinically relevant. We conclude that cabotegravir may be administered without dose adjustment in patients with mild to moderate hepatic impairment. PURPOSE OF REVIEW: This review highlights the development of long-acting injectable cabotegravir (CAB LA) for HIV preexposure prophylaxis (PrEP), with a focus on phase 2 studies and later development. RECENT FINDINGS: Early studies of CAB LA for HIV prevention offered promising pharmacokinetic data and paved the way for phase 2 studies, which have now been completed. On the basis of phase 2 data, dosing of CAB LA at 8-week intervals consistently delivers target trough concentrations in both men and women. Recent studies have shown no required dose adjustments for hepatic or renal disease and minimal drug--drug interactions. Additionally, injectable PrEP is desired by potential PrEP candidates. Still, gaps in knowledge remain with respect to implementation and delivery, the clinical significance of the pharmacologic tail, and dosing in key populations. Phase 3 trials are underway that are anticipated to inform some of these questions and provide efficacy and safety data to support regulatory submissions for CAB LA as a potential PrEP agent. SUMMARY: Recent studies have defined an appropriate CAB LA dosing interval and offered insight into its safety profile. Phase 3 studies will provide much-anticipated efficacy data. If efficacious, CAB LA may provide a desirable PrEP option for those who face challenges to daily pill adherence. A more complete understanding of how to best integrate LA PrEP into service delivery models will be critical for success. Bictegravir, cabotegravir, dolutegravir, elvitegravir, and raltegravir are members of the latest class of antiretrovirals available to treat human immunodeficiency virus (HIV) infection, the integrase strand transfer inhibitors. Integrase strand transfer inhibitors are potent inhibitors of the HIV integrase enzyme with IC90/95 values in the low ogram per milliliter range and they retain antiviral activity against strains of HIV with acquired resistance to other classes of antiretrovirals. Each of the integrase strand transfer inhibitors have unique pharmacokinetic/pharmacodynamic properties, influencing their role in clinical use in specific subsets of patients. Cabotegravir, approved for use in Canada but not yet by the US Food and Drug Administration, is formulated in both oral and intramuscular formulations; the latter of which has shown efficacy as a long-acting extended-release formulation. Cabotegravir, raltegravir, and dolutegravir have minimal drug-drug interaction profiles, as their metabolism has minimal cytochrome P450 involvement. Conversely, elvitegravir metabolism occurs primarily via cytochrome P450 3A4 and requires pharmacokinetic boosting to achieve systemic exposures amenable to once-daily dosing. Bictegravir metabolism has similar contributions from both cytochrome P450 3A4 and uridine 5'-diphospho-glucuronosyltransferase 1A1. Bictegravir, dolutegravir, and raltegravir are recommended components of initial regimens for most people with HIV in the US adult and adolescent HIV treatment guidelines. This review summarizes and compares the pharmacokinetics and pharmacodynamics of the integrase strand transfer inhibitor agents, and describes specific pharmacokinetic considerations for persons with hepatic impairment, renal dysfunction, pregcy, and co-infections. Introduction: Oral pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) represent the cornerstones of HIV infection prevention and treatment. However, despite their high efficacy, the need to take daily oral pill(s) negatively impacts long-term patient adherence. In some cases, it can also be associated with drug-drug interactions and adverse gastrointestinal effects, as well as being a constant reminder to individuals of their HIV status. The availability of long-acting non-orally administered antiretroviral drugs could, therefore, be extremely useful. Cabotegravir (CAB) is a second-generation integrase strand transfer inhibitor, characterized by a relatively high genetic barrier and good antiretroviral potency, which is administrable as a long-acting injectable suspension (LAI CAB).Areas covered: The authors present and discuss the efficacy and available safety data of LAI CAB, either when co-administered with rilpivirine (RPV; LAI CAB + RPV) for the treatment of HIV infection, or when used as single agent for PrEP.Expert opinion: Cabotegravir has the potential to play a primary role in the treatment and prevention of HIV infection. The future availability of LAI CAB + RPV and LAI CAB may mark the beginning of an era of LAI ART and PrEP, respectively. Long-acting antiretroviral implants could help protect high-risk individuals from HIV infection. We describe the design and testing of a long-acting reservoir subcutaneous implant capable of releasing cabotegravir for several months. We compressed cabotegravir and excipients into cylindrical pellets and heat-sealed them in tubing composed of hydrophilic poly(ether-urethane) -. The implants have a 47 mm lumen length, 3.6 mm outer diameter, and 200 μm wall thickness. Four cabotegravir pellets were sealed in the membrane, with a total drug loading of 274 ± 3 mg. In vivo, the implants released 348 ± 107 μg/day (median value per implant, N = 41) of cabotegravir in rhesus macaques. Five implants generated an average cabotegravir plasma concentration of 373 ng/ml in rhesus macaques. The non-human primates tolerated the implant without gross pathology or microscopic signs of histopathology compared to placebo implants. Cabotegravir plasma levels in macaques dropped below detectable levels within two weeks after the removal of the implants. Author information: (1)Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. (2)Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. (3)Instituto de Pesquisa Clinica Evandro Chagas-Fiocruz, Rio de Janeiro, Brazil. (4)Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. (5)FHI 360, Durham, North Carolina, USA. (6)University of Colorado, Aurora, Colorado, USA. (7)Monogram Biosciences, South San Francisco, California, USA. (8)Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. (9)Prevention Science Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA. (10)Department of Medicine, University of California at Los Angeles, Los Angeles, California, USA. (11)ViiV Healthcare, Research Triangle Park, North Carolina, USA. (12)Gilead Sciences, Foster City, California, USA. (13)Hospital Velez Sarfield, Buenos Aires, Argentina. (14)St Jude Children's Research Hospital, Memphis, Tennessee, USA. (15)Desmond Tutu HIV Centre, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa. (16)Institute of HIV Research and Innovation, Bangkok, Thailand. (17)Center of Excellence in Transgender Health, Chulalongkorn University, Bangkok, Thailand. (18)Department of Medicine, University of North Carolina, North Carolina, USA. (19)Center for Clinical AIDS Research & Education, University of California, Los Angeles, Los Angeles, California, USA. Cabotegravir is a novel human immunodeficiency virus integrase enzyme inhibitor used for prevention and treatment of HIV infection. The combinational final dosage form, as extended release injection suspension in combination with rilpivirine and as cabotegravir tablets (for lead-in therapy), was recently approved in Canada, EU and in USA and is currently seeking approval also in other countries. The subject of this investigation was to study the degradation of cabotegravir under different stress conditions as per the International Council for Harmonization (ICH) guidelines. The drug substance was found to be stable in thermal, photolytic and basic stress conditions, but degraded under acidic and oxidative stress conditions. It was determined that four main degradation products of cabotegravir are formed in forced degradation studies. All four main degradation products were isolated using preparative chromatography and subjected to NMR and HRMS analysis in order to determine their structure. We proposed degradation pathways of cabotegravir under acidic stress conditions in solution based on the structure of isolated degradation products, cabotegravir degradation kinetic studies and degradation studies on two isolated key degradation products. Moreover, degradation pathway to predomit oxidation degradation product is proposed based on the adduct of cabotegravir and peroxide species, which was identified by LC-HRMS analysis. This is the first report to the best of our knowledge that describes characterized cabotegravir forced degradation impurities and provides insights into its degradation pathways. The retrovirus HIV-1 is the etiological agent of the decades-long AIDS pandemic. Although vaccination is the most common preexposure route to prevent acquisition of viral disease, scalable efficacious vaccination strategies have yet to be developed for HIV-1. By contrast, small molecule inhibitors of the HIV-1 enzymes reverse transcriptase, integrase, and protease have been developed that effectively block virus replication. Three different drug compounds are commonly prescribed for people living with HIV as once-daily oral tablets. Once-daily pills composed of two different reverse transcriptase inhibitors are moreover approved as preexposure prophylaxis (PrEP) treatment for virus naïve individuals who may partake in behaviors associated with increased risk of HIV-1 acquisition such as unprotected sex or injection drug use. Long-acting (LA) injectable HIV-1 enzyme inhibitors are at the same time being developed to sidestep adherence noncompliance issues that can arise from self-administered once-daily oral dosing regimens. Cabotegravir (CAB)-LA, which inhibits integrase strand transfer activity, has in recent clinical trials been shown to prevent HIV-1 acquisition more effectively than once-daily oral dosed reverse transcriptase inhibitors. In this Perspective, we examine bench to bedside aspects of CAB-LA treatment and development, starting from the biochemical basis of HIV-1 integration and pharmacological inhibition of integrase catalysis. We also review the results of recent clinical trials that evaluated CAB-LA, as well as the promises and challenges that surround its use for HIV/AIDS PrEP. Author information: (1)From the Center for Clinical AIDS Research and Education, David Geffen School of Medicine, University of California, Los Angeles (R.J.L., R.M.K.), the Lundquist Institute at Harbor-UCLA Medical Center, Torrance (E.D.), the San Francisco Department of Public Health, San Francisco (H.S.), and Gilead Sciences, Foster City (J.F.R.) - all in California; the Fred Hutchinson Cancer Research Center, Seattle (D.D., B.H., L. Cottle, M.L., Z.W.); the Louisiana State University Health Sciences Center, New Orleans (M.E.C.); Instituto Nacional de Infectologia Evandro Chagas-Fiocruz, Rio de Janeiro (L. Coelho, B.G.), University of São Paulo (E.G.K.), and Centro de Referência e Treinamento DST-AIDS-SP (J.V.M.), São Paulo, and Hospital Nossa Senhora da Conceição, Porto Alegre (B.S.) - all in Brazil; Via Libre (R.C.), Universidad Nacional Mayor de San Marcos (J.A.G.-C.), and Asociacion Civil Impacta Salud y Educacion (P.G., J.V.H.), Lima, and Asociacion Civil Selva Amazonica, Iquitos (J.C.H.) - all in Peru; the Research Institute for Health Sciences, Chiang Mai University, Chiang Mai (S.C.), and the Institute of HIV Research and Innovation, Bangkok (N.P.) - both in Thailand; the Division of HIV-AIDS Prevention, Centers for Disease Control and Prevention (E.F.D.), the School of Medicine (C.F.K., C.R.), and Rollins School of Public Health (C.R.), Emory University - both in Atlanta; the Perelman School of Medicine, University of Pennsylvania, Philadelphia (I.F.), and Pennsylvania State University, State College (S.D.F.) - both in Pennsylvania; St. Jude Children's Research Hospital, Memphis, TN (A.H.G.); University of North Carolina at Chapel Hill, Chapel Hill (H.V.T., J.J.E., M.S.C.), FHI 360, Durham (A. Asmelash, M.B., C.B., J.L., N.D.S., K.G.-F., A.J., M.M.), and ViiV Healthcare, Research Triangle (K.Y.S., W.S., D.M., A.R.) - all in North Carolina; Hospital General de Agudos José María Ramos Mejia (M.H.L.) and Fundación Huésped (O.S.) - both in Buenos Aires; the Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa (K.M.); University of Alabama at Birmingham, Birmingham (E.T.O., J.M.); Rutgers New Jersey Medical School, Newark (S. Swaminathan); Weill Cornell Medicine, New York (R.M.G.); Johns Hopkins University, Baltimore (P.R., P.S., E.P.-M., M.M., C.H., T.T.B., S.H.E., J.S.); University of Colorado Anschutz Medical Campus, Aurora (P.A.); Massachusetts General Hospital, Boston (C.P.); University of Miami, Coral Gables, FL (S. Safren); the Office of AIDS Research (T.H.H.) and Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD (K.S., A. Adeyeye). OBJECTIVE: We had previously shown that long-acting cabotegravir (CAB-LA) injections fully protected macaques from vaginal simian HIV (SHIV) infection. Here, we reassessed CAB-LA efficacy in the presence of depot medroxyprogesterone acetate and multiple sexually transmitted infections (STIs) that are known to increase HIV susceptibility in women. DESIGN: Two macaque models of increasing vaginal STI severity were used for efficacy assessment. METHODS: The first study (n = 11) used a double STI model that had repeated exposures to two vaginal STI, Chlamydia trachomatis and Trichomonas vaginalis. Six animals were CAB-LA treated and five were controls. The second study (n = 9) included a triple STI model with repeated exposures to C. trachomatis, T. vaginalis and syphilis, and the contraceptive, depot medroxyprogesterone acetate (DMPA). Six animals were CAB-LA treated and three were controls. All animals received up to 14 vaginal SHIV challenges. A survival analysis was performed to compare the number of SHIV challenges to infection in the drug-treated group compared with untreated controls over time. RESULTS: All six CAB-LA treated animals in both models, the double STI or the triple STI-DMPA model, remained protected after 14 SHIV vaginal challenges, while the untreated animals became SHIV-infected after a median of two challenges (log-rank P < 0.001) or one challenge (log-rank P = 0.002), respectively. Both models recapitulated human STI disease, with vaginal discharge, ulcers, and seroconversion. CONCLUSION: In these high and sustained susceptibility models spanning more than 3 months, CAB-LA maintained complete efficacy, demonstrating robustness of the CAB-LA dose used in clinical trials, and suggesting its insensitivity to multiple STIs and DMPA. PURPOSE OF REVIEW: Cabotegravir (CAB) and rilpivirine (RPV) is the first long-acting injectable antiretroviral therapy (ART) option approved for virologically suppressed adults with HIV-1. In addition, long-acting CAB is a promising agent for HIV preexposure prophylaxis (PrEP). This review focuses on phase 3 clinical trial results and implementation considerations for these long-acting ART and PrEP strategies. RECENT FINDINGS: Long-acting CAB and RPV administered every 4 weeks demonstrated noninferiority to oral ART through week 96 in both the ATLAS and FLAIR studies, whereas ATLAS-2M found similar efficacy through 96 weeks when the long-acting injectable ART was administered every 8 weeks instead of every 4 weeks. For prevention, two phase 3 trials were stopped early due to fewer incident HIV infections in participants receiving long-acting CAB every 8 weeks compared with daily oral tenofovir disoproxil fumarate-emtricitabine for PrEP. The long-acting therapies were well tolerated across all clinical trials. SUMMARY: Clinical trial results support the use of long-acting CAB for HIV PrEP and long-acting CAB and RPV as a switch strategy for adults with HIV-1 who are first virologically suppressed with oral ART. Implementation challenges persist, and data are urgently needed in populations who may benefit most from long-acting therapy, including adolescents, pregt individuals, and those with barriers to medication adherence.

What is the function of the protein SERT?

SERT is a Serotonin transporter.

Dynorphin (DYN) is an endogenous neurosecretory peptide which exerts its activity by binding to the family of G protein-coupled receptors, namely the kappa opioid receptor (KOR). Opioids are associated with pain, analgesia, and drug abuse, which play a central role in mood disorders with monoamine neurotransmitter interactions. Growing evidence demonstrates the cellular signaling cascades linked to KOR-mediated monoamine transporters regulation in cell models and native brain tissues. This chapter will review DYN/KOR role in mood and addiction in relevance to dopaminergic and serotonergic neurotransmissions. Also, we discuss the recent findings on KOR-mediated differential regulation of serotonin and dopamine transporters (SERT and DAT). These findings led to a better understanding of the role of DYN/KOR system in aminergic neurotransmission via its modulatory effect on both amine release and clearance. Detailed knowledge of these processes at the molecular level enables designing novel pharmacological reagents to target transporter motifs to treat mood and addiction and reduce unwanted side effects such as aversion, dysphoria, sedation, and psychomimesis. RATIONALE: MDMA or Ecstasy has made a resurgence in popularity and the majority of users consist of teenagers and adolescents. Therefore, it is important to determine whether MDMA causes long-term damage and what this damage entails. There is an ongoing debate about possible neurocognitive changes in 3,4-methylenedioxymethamphetamine (MDMA) users related to MDMA's neurotoxic potential. Multiple neuroimaging studies have shown that Ecstasy use leads to lower serotonin transporter (SERT) availability in multiple brain regions. This may express itself in a loss of cognitive functions like memory, attention and executive function. However, there is increasing evidence reporting that MDMA's induced serotonergic adaptations are reversible over time. The question we thus address is whether the recovery of SERT function predicts a recovery of cognitive function. OBJECTIVES: This review aims to investigate MDMA's long-term effects on SERT availability and cognitive functioning. METHODS: A literature search was performed in PubMed. Studies that investigated the effects of MDMA on both SERT availability and cognitive performance were eligible for inclusion. RESULTS: SERT availability positively correlated with time of abstinence, whereas memory performance did not show this correlation, but remained impaired in MDMA users. No significant correlation between SERT availability and memory function was found (r = 0.232, p = 0.581; r = 0.176, p = 0.677). CONCLUSIONS: The main findings of this review are that MDMA-use leads to an acute decrease in SERT availability and causes an impairment in cognitive functions, mostly memory. However, SERT availability recovers with sustained abstinence while memory function does not. This suggests that SERT availability is not a biomarker for MDMA-induced cognitive impairment and likely also not for MDMA-induced neurotoxicity. To explore mechanism of intestinal flora dysregulation promoting constipation, 60 specific pathogen-free (SPF) mice were used as research objects and were treated with constipation population fecal fluid gavage and distilled water gavage. Then, relationship between intestinal dysregulation and constipation in mice with biofilm-mediated intestinal flora was investigated in vitro. The results showed that recombit serotonin transporter (SERT) messenger ribonucleic acid (mRNA) level of the constipation population fecal fluid gavage group and the relative expression level of SERT mRNA were 1.61 ± 0.08 and 1.49 ± 0.06, which were higher markedly than those of distilled water group (P < 0.05). The level of 5-hydroxytryptamine (5-HT) in colonic tissue of the constipation population fecal fluid gavage group was 145.36 ± 14.12 ng/mL, and the expression level of 5-HT on the surface of epithelial cells of biofilm-positive colonic tissue was 20.11 ± 2.03, which were significantly lower than those of the distilled water group, with statistical significance (P < 0.05). Besides, the microbial sequencing of fecal flora indicated that The Akk and bacteroidetes ofconstipation population fecal fluid gavage group were higher hugely than those of distilled water group (P < 0.05).In conclusion, after the occurrence of constipation, the diversity of intestinal microflora decreased, and the probiotics reduced. Iintestinal microflora dysregulation would lead to increase of SERT expression level in defecation function and intestinal motility in mice, and the decrease of 5-HT, thereby changing the intestinal movement resulting in mucosal protective barrier damage,thereby causing changes in intestinal movement and the destruction of the intestinal mucosal protective barrier, which eventually resulted in constipation. The occurrence of constipation could be improved by regulating balance of intestinal flora, increasing the diversity of flora, and reducing the genus of opportunistic pathogens.

Are G-quadruplexes(G4) possible drug targets for glioblastoma?

The 2H2-6M(4)-oxazole telomestatin derivative (6OTD) targets Glioma stem cells through G4 stabilization and promotion of DNA damage responses. Therefore, G4s are promising therapeutic targets for glioblastoma.

Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. We have introduced a G-quadruplex (G4) ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy. The GROs that form parallel G4 structures, such as PU22, T40214 and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form non-parallel G4 structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the fluorescence resot energy transfer studies of fluorophore-labeled GROs show that the parallel G4 structures can be retained in CL1-0 cells, whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the non-parallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs. Interference with telomerase and telomere maintece is emerging as an attractive target for anticancer therapies. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA 3'-overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends, making these ligands good candidates for chemotherapeutic purposes. BRACO-19 is one of the most effective and specific ligand for telomeric G4. It is shown here that BRACO-19 suppresses proliferation and reduces telomerase activity in human glioblastoma cells, paralleled by the displacement of telomerase from nuclear to cytoplasm. Meanwhile, BRACO-19 triggers extensive DNA damage response at telomere, which may result from uncapping and disassembly of telomeric T-loop structure, characterized by the formation of anaphase bridge and telomere fusion, as well as the release of telomere-binding protein from telomere. The resulting dysfunctional telomere ultimately provokes p53 and p21-mediated cell cycle arrest, apoptosis and senescence. Notably, normal primary astrocytes do not respond to the treatment of BRACO-19, suggesting the agent's good selectivity for cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs for various tumors including maligt gliomas. G-quadruplex (G4) is a higher-order nucleic acid structure that is formed by guanine-rich sequences. G4 stabilization by small-molecule compounds called G4 ligands often causes cytotoxicity, although the potential medicinal impact of this effect has not been fully established. Here we demonstrate that a synthetic G4 ligand, Y2H2-6M(4)-oxazole telomestatin derivative (6OTD), limits the growth of intractable glioblastoma (grade IV glioma) and glioma stem cells (GSCs). Experiments involving a human cancer cell line panel and mouse xenografts revealed that 6OTD exhibits antitumor activity against glioblastoma. 6OTD inhibited the growth of GSCs more potently than it did the growth of differentiated non-stem glioma cells (NSGCs). 6OTD caused DNA damage, G1 cell cycle arrest, and apoptosis in GSCs but not in NSGCs. These DNA damage foci tended to colocalize with telomeres, which contain repetitive G4-forming sequences. Compared with temozolomide, a clinical DNA-alkylating agent against glioma, 6OTD required lower concentrations to exert anti-cancer effects and preferentially affected GSCs and telomeres. 6OTD suppressed the intracranial growth of GSC-derived tumors in a mouse xenograft model. These observations indicate that 6OTD targets GSCs through G4 stabilization and promotion of DNA damage responses. Therefore, G4s are promising therapeutic targets for glioblastoma. The G-quadruplex (G4) DNA, which has been developed as a potential anticancer target in drug screening and design, plays a crucial role in the oncogene transcription and translation. Tanshinone IIA derivatives with a planar heterocycle structure may function as G4 stabilizers. We present an innovative case of imidazole-based tanshinone IIA derivatives (1-8) especially compound 4 that improve the selectivity and the binding affinity with G4 DNA and enhance the target tumor inhibition. Cellular and in vivo experiments indicate that the tanshinone IIA derivative 4 inhibits the growth, metastasis, and angiogenesis of triple-negative breast cancer cells possibly through the stabilization of multiple G4 DNAs (e.g., c-myc, K-ras, and VEGF) to induce DNA damage. Further investigation of the intermolecular interaction and the molecular docking indicates that tanshinone IIA derivatives have better selective binding capability to various G4 DNAs than to double-stranded DNA. These findings provide guidance in modifying the molecular structures of tanshinone IIA derivatives and reveal their potential to function as specific G4 stabilizers.

Is RUNX1T1 associate with obesity?

Yes, the rs34269950 SNP of the RUNX1T1 gene was significantly associated with obesity risk and metabolic abnormalities. Specifically, compared to AA genotype, rs34269950 del/del genotype was associated with a 1.47 [95% confidence interval (CI): 1.01-2.14, P = 0.042] fold higher rate of obesity risk.

BACKGROUND: Runt-related transcription factor 1 (RUNX1T1) isoforms are involved in adipogenesis. RUNX1T1 is mediated by the fat mass and obesity-associated (FTO). However, the extent to which RUNX1T1 single-nucleotide polymorphisms (SNPs) are associated with obesity risk or metabolic abnormalities in a community population basis is unknown. METHODS: Samples were obtained from the Australian Crossroads study bio-bank. SNPs located in the coding region and 3'untranslated regions of RUNX1T1 with minor allele frequency ≥0.05 were analysed using Taqman genotyping assays. RESULTS: Eight candidate SNPs were genotyped successfully in 1440 participants. Of these SNPs only rs34269950 located in the 'RRACH' motif, the most common N6-methyladenosine (m6A) methylation modification site (recognized by FTO), was significantly associated with obesity risk and metabolic abnormalities. Specifically, compared to AA genotype, rs34269950 del/del genotype was associated with a 1.47 [95% confidence interval (CI): 1.01-2.14, P = 0.042] fold higher rate of obesity risk. Additionally, the del/del genotype was associated with a 60% increased risk of metabolic syndrome (MetS) [odds ratio (OR) = 1.60, 95% CI: 1.10-2.32, P = 0.015], in comparison to the AA genotype. Finally, rs34269950 del/del increased the risk of a larger waist circumference (OR = 1.65, 95% CI: 1.15-2.36, P = 0.007), but not other components of MetS. CONCLUSION: Our study demonstrates that RUNX1T1 rs34269950, located in a potential FTO recognition motif, is significantly associated with waist circumference. This provides novel evidence to suggest SNPs located in RRACH motif may be involved in RNA m6A modification and mechanistic pathways that influence abdominal obesity.

Is Adamts18 deficiency associated with cancer?

Yes. ADAMTS18 is a novel tumor suppressor and is critical to the pathology of human colorectal cancer. Adamts18 deficiency enhances tumorigenesis and intestinal inflammation through elevated Wnt/β-catenin and p38MAPK/ERK1/2 signaling and promotes colon cancer in this mouse model.

What causes Ocular Thelaziasis?

Ocular Thelaziasis is caused by Thelazia callipaeda.

Thelaziasis is an ocular infection of several mammals caused by nematodes of the genus Thelazia (Spirurida, Thelaziidae). The adults live in the lachrymal ducts and conjunctival sacs of infected animals, and transmission occurs by means of different species of muscids. T. rhodesi, T. skrjabini, and T. gulosa affect mainly cattle, inducing conjunctivitis, keratitis, and ocular discharge. This article describes the morphological features of adult T. rhodesi, T. skrjabini, and T. gulosa by scanning electron microscopy and highlights the most relevant differences for their identification. Human thelaziasis is a zoonotic eye disease caused by a nematode parasite called Thelazia. In India, seven human cases of Thelazia have been reported earlier. This is the first case report of an infant infected with Thelazia. During the month of July, 2012, the infant was presented with an eye problem to the eye clinic from a village of Dibrugarh. Five worms (three female and two male) were recovered from the left eye of the infant. Thelazia infection is rare in infant, and report of this case is suggestive of prevalence of infection in the area and warrants further investigation. Thelaziasis is an arthropod-born disease of the eye and adnexa caused by Thelazia callipaeda, a nematode parasite transmitted by drosophilid flies to carnivores and humans. Because of its distribution mainly confined to South Asian countries and Russia, it is commonly known as Oriental eye worm. It is often under-reported and not been given its due clinical importance. We report first case of human thelaziasis from Hassan District, Karnataka. Five creamy-white, translucent worms were removed from the conjunctival sac of a 74-year-old male patient. Based on morphological characters, the worms were identified as nematodes belonging to the genus Thelazia and speciation was confirmed by CDC, Atlanta as callipaeda. Rarity of the disease and its ability to cause both extra and intraocular manifestations leading to ocular morbidity is the reason for presenting this case. From the available data, this is the first case report from Karnataka, India. BACKGROUND: Thelazia callipaeda is the main causative organism in thelaziasis, commonly infecting orbital cavities and associated tissues of carnivores. Thelazia callipaeda infection is rarely reported in humans, especially in infants. CASE PRESENTATION: A 5-month-old male infant presented with 2 weeks of redness and increased secretions in the left eye. On examination, the left eye revealed the presence of one creamy thread-like mobile worm in the conjunctival sac. During surgical exploration, a total of 11 worms were extracted from the left eye. The worms were morphologically identified as seven female and four male T. callipaeda. Ocular symptoms resolved rapidly after the removal of the worms, with no recurrence after the 6-month follow-up. CONCLUSION: We present here detailed clinical and morphological information pertaining to T. callipaeda infection, which is considered to be a probably neglected parasitic disease of the eye. This case illustrates the importance of including thelaziasis into the differential diagnosis of ocular surface diseases, especially in infant patients. Thelaziasis is an ocular arthropod-borne, zoonotic disease of the eye infecting the conjunctival sac, lacrimal duct, and lacrimal gland caused by a nematode of the genus Thelazia. We report the first case of human ocular thelaziasis in Nepal in a 6-month-old child from a Rukum district, Nepal. The infant presented with conjunctivitis, and his visual acuity and dilated fundal examination were normal. A total of 6 worms were removed for identification. Collected nematodes were identified based on morphological keys as Thelazia callipaeda. The patient's symptoms improved after removal of the nematodes. An 80-year-old male reported to the clinic with complaints of diminished vision, foreign body sensation, and occasionally some black object moving in front of the right eye. Detailed ocular examination revealed three slender creamy white live worms in the conjunctival sac of the right eye. Total three worms were retrieved and sent to the pathology department for detailed examination. The worm was identified as gravid Thelazia callipaeda. As per the search results in PubMed and Cochrane search engine, this is the first report of human case of gravid Thelazia infestation with simultaneous existence of embryonated or ensheathed eggs and primary-stage larvae in the same worm.

What is the role of cytidine deaminase in healthy cells?

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination.

The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) cytidine deaminases classified into four families. These proteins function mainly in innate antiviral immunity and can also restrict endogenous retrotransposable element multiplication. The present study focuses on APOBEC3C (A3C), a member of the APOBEC3 subfamily. Some APOBEC3 proteins use their enzymatic activity on genomic DNA, inducing mutations and DNA damage, while other members facilitate DNA repair. Our results show that A3C is highly expressed in cells treated with DNA-damaging agents. Its expression is regulated by p53. Depletion of A3C slightly decreases proliferation and does not affect DNA repair via homologous recombination or nonhomologous end joining. The A3C interactomes obtained from control cells and cells exposed to the genotoxin etoposide indicated that A3C is a nucleolar protein. This was confirmed by the detection of either endogenous or ectopic A3C in nucleoli. Interestingly, we show that A3C is excluded from areas of DNA breaks in live cells. Our data also indicate that the C-terminal part of A3C is responsible for its nucleolar localization and exclusion from DNA damage sites. Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determit for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis. Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

Summarize the cause of autosomal dominant Spinocerebellar Ataxia type 3.

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3.

Spinocerebellar ataxia type 1 and type 3 (SCA1, SCA3) are autosomal domit neurodegenerative disorders caused by expanded CAG trinucleotide repeats in novel genes. In our collective of SCA1 and SCA3 families, we observed distortion of the Mendelian 1:1 segregation of the disease. The mutated alleles were preferentially transmitted by female carriers in SCA3, whereas a gender effect on clinical features such as age of onset was not obvious. The mechanism underlying segregation distortion remains to be established. Spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2) and Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) are three distinctive forms of autosomal domit spinocerebellar ataxia (SCA) caused by expansions of an unstable CAG repeat localized in the coding region of the causative genes. Another related disease, dentatorubropallidoluysian atrophy (DRPLA) is also caused by an unstable triplet repeat and can present as SCA in late onset patients. We investigated the frequency of the SCA1, SCA2, MJD/SCA3 and DRPLA mutations in 328 Brazilian patients with SCA, belonging to 90 unrelated families with various patterns of inheritance and originating in different geographic regions of Brazil. We found mutations in 35 families (39%), 32 of them with a clear autosomal domit inheritance. The frequency of the SCA1 mutation was 3% of all patients; and 6% in the domitly inherited SCAs. We identified the SCA2 mutation in 6% of all families and in 9% of the families with autosomal domit inheritance. The MJD/SCA3 mutation was detected in 30% of all patients; and in the 44% of the domitly inherited cases. We found no DRPLA mutation. In addition, we observed variability in the frequency of the different mutations according to geographic origin of the patients, which is probably related to the distinct colonization of different parts of Brazil. These results suggest that SCA may be occasionally caused by the SCA1 and SCA2 mutations in the Brazilian population, and that the MJD/SCA3 mutation is the most common cause of domitly inherited SCA in Brazil. Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3, SCA3), an autosomal domit neurological disorder, is caused by an abnormal expanded polyglutamine (polyQ) repeat in the ataxin-3 protein. The length of the expanded polyQ stretch correlates positively with the severity of the disease and inversely with the age at onset. To date, we cannot fully explain the mechanism underlying neurobiological abnormalities of this disease. Yet, accumulating reports have demonstrated the functions of ataxin-3 protein in the chaperone system, ubiquitin-proteasome system, and aggregation-autophagy, all of which suggest a role of ataxin-3 in the clearance of misfolded proteins. Notably, the SCA3 pathogenic form of ataxin-3 (ataxin-3(exp)) impairs the misfolded protein clearance via mechanisms that are either dependent or independent of its deubiquitinase (DUB) activity, resulting in the accumulation of misfolded proteins and the progressive loss of neurons in SCA3. Some drugs, which have been used as activators/inducers in the chaperone system, ubiquitin-proteasome system, and aggregation-autophagy, have been demonstrated to be efficacious in the relief of neurodegeneration diseases like Huntington's disease (HD), Parkinson's (PD), Alzheimer's (AD) as well as SCA3 in animal models and clinical trials, putting misfolded protein clearance on the list of potential therapeutic targets. Here, we undertake a comprehensive review of the progress in understanding the physiological functions of ataxin-3 in misfolded protein clearance and how the polyQ expansion impairs misfolded protein clearance. We then detail the preclinical studies targeting the elimination of misfolded proteins for SCA3 treatment. We close with future considerations for translating these pre-clinical results into therapies for SCA3 patients. Author information: (1)Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. (2)Zhengzhou University, Zhengzhou, He 450001, China. (3)School of life sciences, Zhengzhou University, Zhengzhou, He 450001, China. (4)Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310058, China; Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang 310058, China. (5)Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. Electronic address: [email protected]. (6)Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. Electronic address: [email protected]. BACKGROUND: Spinocerebellar ataxias (SCAs) are rare domitly inherited neurodegenerative disorders that lead to severe disability and premature death. OBJECTIVE: To better characterize the natural history of the most common SCAs, SCA1, SCA2, SCA3 and SCA6, we performed a meta-analysis of literature to determine disease progression, provide data for sample-sizes calculations for interventional trials and study the impact of geographical locations and study follow-up on disease progression. METHODS: A systematic literature search from MEDLINE and EMBASE databases for longitudinal natural history studies of SCA patients was conducted. Studies using the Scale for the Assessment and Rating Ataxia (SARA) as outcome measure were considered. Random-effect (RE) meta-analysis was applied to estimate pooled disease progression. RESULTS: Six studies with 1215 SCA patients enrolled between 2005 and 2016 were finally selected. Annual pooled SARA score increase was 1.83 (1.46-2.20) in patients with SCA1, 1.40 (1.19-1.61) in patients with SCA2, 1.41 (0.97-1.84) in patients with SCA3, and 0.81 (0.66-0.97) in patients with SCA6. For patients with SCA3, disease progression was faster in studies located in Asia and Europe than in the US. Two-arm interventional trials of 1-year duration to achieve 80% power and α level of 5% would require 92 patients per group with SCA1, 97 with SCA2, 115 with SCA3, and 430 with SCA6 to detect a 50% reduction in disease progression. CONCLUSION: This meta-analysis provides quantitative data on the progression of the most common spinocerebellar ataxias based on patient numbers that exceed those of previous studies and confirms that disease progression is faster in SCA1, intermediate in SCA2 and SCA3 and slower in SCA6, with similar rates of disease progression in SCA2 and SCA3 between different populations, suggesting a possibility of international collaborative studies. Nevertheless, individual-patient data meta-analysis is needed to better understand the risk factors that influence disease progression and improve patient stratification in interventional trials. Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a domit neurodegenerative disease caused by the expansion of a CAG repeat tract in ATXN3. Anticipation and worsening of clinical picture in subsequent generations were repeatedly reported, but there is no indication that SCA3/MJD frequency is changing. Thus, we performed a systematic review and meta-analysis on phenomena with potential effect on SCA3/MJD recurrency in populations: instability of CAG repeat transmissions, anticipation, fitness, and segregation of alleles. Transmission of the mutant allele was associated with an increase of 1.23 CAG repeats in the next generation, and the average change in age at onset showed an anticipation of 7.75 years per generation; but biased recruitments cannot be ruled out. Affected SCA3/MJD individuals had 45% more children than related controls. Transmissions from SCA3/MJD carriers showed that the expanded allele was segregated in 64% of their children. In contrast, transmissions from normal subjects showed that the minor allele was segregated in 54%. The present meta-analysis concluded that there is a segregation distortion favoring the expanded allele, among children of carriers. Therefore, further studies on transmissions and anticipation phenomena as well as more observations about fertility are required to clarify these selective forces over SCA3/MJD. Spinocerebellar Ataxia (SCA) is a heterogeneous adult-onset disorder with an autosomal domit inheritance pattern mainly caused by triplet repeat expansions. Clinical diagnosis of SCA is based on phenotypic features followed by confirmation through molecular diagnosis. To identify status of repeat range in Indian SCA cases and provide extended family screening, we enrolled 70 clinical SCA suspects. For molecular diagnosis, multiplex PCR (M-PCR) was used for common Indian SCA subtypes 1, 2, 3, 6, 7, 10, 12 and 17. TP-PCR was further used in SCA2, 7 and 10 to identify larger expansions. Eighteen out of 70 SCA suspects (25%) were found to be positive for various SCA subtypes- (5 SCA1 (28%), 6 SAC2 (34%), 2 SCA3 (12%), 3 SCA7 (16%) and one each for SCA6 (1%) and SCA17 (1%) subtypes). Genetic counselling and extended family screening were offered to all positive cases and yielded additional nine cases. We have established M-PCR and TP-PCR to detect the CAG repeat expansion in SCA suspects. This method can confirm SCA subtypes in a reliable, rapid and cost-effective way. Genetic characterization of SCA-related genes has great clinical relevance, as it could provide additional information and guidance to clinicians and family members regarding prognosis. Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive autosomal domit neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3. The accumulation of the mutant ataxin-3 proteins carrying expanded polyglutamine (polyQ) leads to selective degeneration of neurons. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies have been identified, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3. Induced pluripotent stem cells (iPSCs) can be used as the ideal cell model for the molecular pathogenesis of polyQ diseases. Abnormal CAG expansions mediated by CRISPR/Cas9 genome engineering technologies have shown promising potential for the treatment of polyQ diseases, including SCA3. In this study, SCA3-iPSCs can be corrected by the replacement of the abnormal CAG expansions (74 CAG) with normal repeats (17 CAG) using CRISPR/Cas9-mediated homologous recombination (HR) strategy. Besides, corrected SCA3-iPSCs retained pluripotent and normal karyotype, which can be differentiated into a neural stem cell (NSCs) and neuronal cells, and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the neuronal differentiation from normal control iPSCs (Ctrl-iPSCs), SCA3-iPSCs, and isogenic control SCA3-iPSCs. Furthermore, this study proved that the phenotypic abnormalities in SCA3 neurons, including aggregated IC2-polyQ protein, decreased mitochondrial membrane potential (MMP) and glutathione expressions, increased reactive oxygen species (ROS), intracellular Ca2+ concentrations, and lipid peroxidase malondialdehyde (MDA) levels, all were rescued in the corrected SCA3-NCs. For the first time, this study demonstrated the feasibility of CRISPR/Cas9-mediated HR strategy to precisely repair SCA3-iPSCs, and reverse the corresponding abnormal disease phenotypes. In addition, the importance of genetic control using CRISPR/Cas9-mediated iPSCs for disease modeling. Our work may contribute to providing a potential ideal model for molecular mechanism research and autologous stem cell therapy of SCA3 or other polyQ diseases, and offer a good gene therapy strategy for future treatment.

Through which pathway does the FTO-guided demethylation of GADD46 drive myogenesis?

FTO-mediated demethylation of GADD45B promotes myogenesis through the activation of p38 MAPK pathway.

N6-methyladenosine (m6A) modification plays a critical role in mammalian development. However, the role of m6A in the skeletal muscle development remains largely unknown. Here, we report a global m6A modification pattern of goat skeletal muscle at two key development stages and identified that the m6A modification regulated the expression of the growth arrest and DNA damage-inducible 45B (GADD45B) gene, which is involved in myogenic differentiation. We showed that GADD45B expression increased during myoblast differentiation, whereas the downregulation of GADD45B inhibits myogenic differentiation and mitochondrial biogenesis. Moreover, the expression of GADD45B regulates the expression of myogenic regulatory factors and peroxisome proliferator-activated receptor gamma coactivator 1 alpha by activating the p38 mitogen-activated protein kinase (MAPK) pathway. Conversely, the inactivation of p38 MAPK abolished the GADD45B-mediated myogenic differentiation. Furthermore, we found that the knockdown of fat mass and obesity-associated protein (FTO) increases GADD45B m6A modification and decreases the stability of GADD45B mRNA, which impairs myogenic differentiation. Our results indicate that the FTO-mediated m6A modification in GADD45B mRNA drives skeletal muscle differentiation by activating the p38 MAPK pathway, which provides a molecular mechanism for the regulation of myogenesis via RNA methylation.

What are the key characteristics of the syndrome caused by ANKRD17 loss-of-function variants?

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphia.

Is Ozanimod effective for Ulcerative Colitis?

Yes, Ozanimod is effective for Ulcerative Colitis.

The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here, we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of dextran sulfate sodium (DSS), after colitis induced by transfer of CD4+CD45RBhi cells, and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with inflammatory bowel disease (IBD) were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T-cell velocity and induced S1P1 degradation and retention of Naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function. BACKGROUND: Ozanimod (RPC1063) is an oral agonist of the sphingosine-1-phosphate receptor subtypes 1 and 5 that induces peripheral lymphocyte sequestration, potentially decreasing the number of activated lymphocytes circulating to the gastrointestinal tract. METHODS: We conducted a double-blind, placebo-controlled phase 2 trial of ozanimod in 197 adults with moderate-to-severe ulcerative colitis. Patients were randomly assigned, in a 1:1:1 ratio, to receive ozanimod at a dose of 0.5 mg or 1 mg or placebo daily for up to 32 weeks. The Mayo Clinic score was used to measure disease activity on a scale from 0 to 12, with higher scores indicating more severe disease; subscores range from 0 to 3, with higher scores indicating more severe disease. The primary outcome was clinical remission (Mayo Clinic score ≤2, with no subscore >1) at 8 weeks. RESULTS: The primary outcome occurred in 16% of the patients who received 1 mg of ozanimod and in 14% of those who received 0.5 mg of ozanimod, as compared with 6% of those who received placebo (P=0.048 and P=0.14, respectively, for the comparison of the two doses of ozanimod with placebo). Differences in the primary outcome between the group that received 0.5 mg of ozanimod and the placebo group were not significant; therefore, the hierarchical testing plan deemed the analyses of secondary outcomes exploratory. Clinical response (decrease in Mayo Clinic score of ≥3 points and ≥30% and decrease in rectal-bleeding subscore of ≥1 point or a subscore ≤1) at 8 weeks occurred in 57% of those receiving 1 mg of ozanimod and 54% of those receiving 0.5 mg, as compared with 37% of those receiving placebo. At week 32, the rate of clinical remission was 21% in the group that received 1 mg of ozanimod, 26% in the group that received 0.5 mg of ozanimod, and 6% in the group that received placebo; the rate of clinical response was 51%, 35%, and 20%, respectively. At week 8, absolute lymphocyte counts declined 49% from baseline in the group that received 1 mg of ozanimod and 32% from baseline in the group that received 0.5 mg. The most common adverse events overall were anemia and headache. CONCLUSIONS: In this preliminary trial, ozanimod at a daily dose of 1 mg resulted in a slightly higher rate of clinical remission of ulcerative colitis than placebo. The trial was not large enough or of sufficiently long duration to establish clinical efficacy or assess safety. (Funded by Receptos; TOUCHSTONE ClinicalTrials.gov number, NCT01647516.). Crohns disease (CD) and ulcerative colitis (UC) belong to chronic inflammatory bowel diseases, which are induced by autoimmune processes. While CD is characterized by over-activity of Th1, ILC1, and MAIT cells, UC is mediated by exaggerated activities of Th2 and ILC2 cells and cytokines they produce. Knowledge of the pathogenesis enabled a rational therapy based mostly on biologics and small molecules. TNF is the principal proinflammatory cytokine in both diseases. Anti-TNF monoclonal antibodies, mostly infliximab or adalimumab were therefore introduced to their treatment. Approximately 50-70 % of CD and more than 33 % of UC patients respond to primary treatment only, which resulted in the development of other biologics and small molecules. Out of them, monoclonal antibodies targeting adhesive molecules (vedolizumab, etrolizumab) and p40 chains shared by IL12 and IL23 (ustekinumab) have been already in clinical practice. There are also other small molecules in clinical trials: mongersen, tafacitinib, and ozanimod. Mongersen supports immunosuppressive activity of TGFβ; it has been tried for the treatment of CD. Tofacitinib inhibits activity of JAK kinases; it was shown to be effective in UC management. Ozanimod interferes with migrations of activated T cells to the site of inflammation and is a promising drug for the UC treatment.Key words: Crohns disease - mongersen - monoclonal antibodies - ozanimod - tofacitinib - ulcerative colitis. BACKGROUND AND AIMS: This analysis examined the long-term safety and efficacy of ozanimod in patients with moderately to severely active ulcerative colitis [UC] with ≥ 4 years of follow-up in the phase 2 TOUCHSTONE open-label extension [OLE]. METHODS: Patients receiving placebo or ozanimod HCl 0.5 mg or 1 mg during the double-blind period could enter the OLE [ozanimod HCl 1 mg daily]. Partial Mayo score [pMS] clinical response and remission were assessed through OLE week 200 and summarized descriptively using observed cases [OC] and non-responder imputation [NRI]. Endoscopy was required at OLE week 56 and the end of treatment. Parameters associated with endoscopy were summarized at weeks 56 and 104 [OC], and week 56 [NRI]. C-reactive protein and faecal calprotectin were assessed. Adverse events were monitored throughout the study. RESULTS: Of 197 patients receiving double-blind treatment, 170 entered the OLE. Discontinuation rates were 28% at year 1 and 15-18% annually through year 4. Partial Mayo measures indicated clinical response and remission rates at OLE week 200 of 93.3% and 82.7%, respectively, using OC and 41% and 37% with the more conservative NRI analysis. At weeks 56 and 104, respectively, histological remission rates were 46.3% and 38.5%, and endoscopic improvement rates were 46.4% and 46.5% [OC]. No new safety signals were identified during ≥ 4 years of follow-up. CONCLUSIONS: There was a high rate of continued study participation and long-term benefit with ozanimod HCl 1 mg daily based on clinical, histological and biomarker measures in patients with moderately to severely active UC in the TOUCHSTONE OLE. [NCT02531126]. OBJECTIVE: To review the pharmacological and clinical profile of ozanimod in the treatment of ulcerative colitis (UC). DATA SOURCES: A PubMed search was conducted from inception to July 2021 using the keywords ozanimod, ulcerative colitis, and sphingosine 1-phosphate receptor modulator. Information was also extracted from published abstracts and the package insert. STUDY SELECTION AND DATA EXTRACTION: Phase 2 and 3 studies and relevant literature on ozanimod pharmacological and clinical profiles were reviewed. DATA SYNTHESIS: Ozanimod approval was based on True North, a phase 3 trial evaluating ozanimod's efficacy and safety in the treatment of moderate to severe UC. Compared with placebo, ozanimod led to clinical remission in a significantly higher proportion of patients in both the induction and maintece phase. Additionally, for secondary end points of clinical response, endoscopic improvement, corticosteroid-free remission, and mucosal healing, ozanimod performed significantly better than placebo. Common adverse events included infections, headaches, hypertension, bradycardia, and liver enzyme elevations. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: Ozanimod is the first sphingosine 1-phosphate modulator to be approved for UC and is administered orally. Its efficacy profile is comparable with other UC medications. However, its safety profile is unique, requiring extensive assessments prior to initiation of and during treatment. Thus, it is unclear how ozanimod will be positioned in UC treatment. CONCLUSION: Ozanimod is another option in the growing arsenal of UC treatment. Although it offers a novel mechanism of action and is administered orally, there are important safety, dosing, and pharmacokinetic factors to consider prior to initiation and use.

What is the function of the protein PIEZO1?

Piezo1 is a key element of the mechanotransduction process and can transduce mechanical signals into biological signals by mediating Ca2+ influx, which in turn regulates cytoskeletal remodeling and stress alterations.

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the 'cap' region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the 'propeller' regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance. BACKGROUND: Mechanical ventilation can induce or aggravate lung injury, which is termed ventilator-induced lung injury (VILI). Piezo1 is a key element of the mechanotransduction process and can transduce mechanical signals into biological signals by mediating Ca2+ influx, which in turn regulates cytoskeletal remodeling and stress alterations. We hypothesized that it plays an important role in the occurrence of VILI, and investigated the underlying mechanisms. METHODS: High tidal volume mechanical ventilation and high magnitude cyclic stretch were performed on Sprague-Dawley rats, and A549 and human pulmonary microvascular endothelial cells, respectively, to establish VILI models. Immunohistochemical staining, flow cytometry, histological examination, enzyme-linked immunosorbent assay, western blotting, quantitative real-time polymerase chain reaction and survival curves were used to assess the effect of Piezo1 on induction of lung injury, as well as the signaling pathways involved. RESULTS: We observed that Piezo1 expression increased in the lungs after high tidal volume mechanical ventilation and in cyclic stretch-treated cells. Mechanistically, we observed the enhanced expression of RhoA/ROCK1 in both cyclic stretch and Yoda1-treated cells, while the deficiency or inhibition of Piezo1 dramatically antagonized RhoA/ROCK1 expression. Furthermore, blockade of RhoA/ROCK1 signaling using an inhibitor did not affect Piezo1 expression. GSMTx4 was used to inhibit Piezo1, which alleviated VILI-induced pathologic changes, water content and protein leakage in the lungs, and the induction of systemic inflammatory mediators, and improved the 7-day mortality rate in the model rats. CONCLUSIONS: These findings indicate that Piezo1 affects the development and progression of VILI through promotion of RhoA/ROCK1 signaling. BACKGROUND: To explore the role of the mechanosensitive ion channel Piezo1 in the proliferation and osteogenic differentiation of human dental follicle cells (hDFCs), and its mechanism, so as to provide the basis for the use of hDFCs to achieve bone regeneration. METHODS: hDFCs were obtained from fresh dental follicle tissues by enzymatic digestion, and cell phenotype and multipotential differentiation were identified. Identification of the expression of mechanosensitive ion channel Piezo1 was performed by immunofluorescence and immunohistochemistry. CCK-8 was used to determine the optimal concentration of the Piezo1 agonist, Yoda1. Then, according to the obtained results, Alizarin red staining, RT-PCR quantitative analysis and Western blot were used to further observe the osteogenic differentiation of hDFCs and its probable mechanism via Wnt/β-catenin signalling. The data were analysed by SPSS 22.0 software. RESULTS: The results of the concentration gradient experiments indicated that 0.5 µM Piezo1 agonist (Yoda1) enhanced the proliferation of hDFCs. Compared with the control group, a considerable number of calcium nodules showed that activating Piezo1 could promote the osteogenic differentiation of hDFCs. The relative mRNA and protein expression of Piezo1, ALP, RUNX2, OCN and BMP2 in the Piezo1 agonist group were higher than that of the control group. Furthermore, the expression of Wnt3a and β-catenin related to the classical osteogenic pathway were significantly up-regulated in the Piezo1 agonist group. CONCLUSION: Activating mechanosensitive ion channel Piezo1 with an appropriate concentration of Yoda1 has a positive effect on the proliferation and osteogenic differentiation of hDFCs. This mechanism of promoting osteogenic differentiation may be mediated by the Wnt/β-catenin pathway. 1.

Please list 3 biologic(monoclonal antibody) drugs used to treat migraine headaches.

Large molecule biologic antibody (mAb) approaches that are given subcutaneously to neutralize circulating CGRP peptide (fremanezumab, galcanezumab) or block CGRP receptors (erenumab) have shown consistent efficacy and tolerability in multicenter migraine prevention trials and are now approved for clinical use.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a validated target for the treatment of episodic migraine. Here we assess the safety, tolerability, and efficacy of TEV-48125, a monoclonal anti-CGRP antibody, in the preventive treatment of high-frequency episodic migraine. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, phase 2b study, we enrolled men and women (aged 18-65 years) from 62 sites in the USA who had migraine headaches 8-14 days per month. Using a randomisation list generated by a central computerised system and an interactive web response system, we randomly assigned patients (1:1:1; stratified by sex and use of concomitant preventive drugs) after a 28 day run-in period to three 28 day treatment cycles of subcutaneous 225 mg TEV-48125, 675 mg TEV-48125, or placebo. Investigators, patients, and the funder were blinded to treatment allocation. Patients reported headache information daily using an electronic diary. Primary endpoints were change from baseline in migraine days during the third treatment cycle (weeks 9-12) and safety and tolerability. The secondary endpoint was change relative to baseline in headache-days during weeks 9-12. Efficacy endpoints were analysed for the intention-to-treat population. Safety and tolerability were analysed using descriptive statistics. This trial is registered at ClinicalTrials.gov, number NCT02025556. FINDINGS: Between Jan 8, 2014, and Oct 15, 2014, we enrolled 297 participants: 104 were randomly assigned to receive placebo, 95 to receive 225 mg TEV-48125, and 96 to receive 675 mg TEV-48125. The least square mean (LSM) change in number of migraine-days from baseline to weeks 9-12 was -3.46 days (SD 5.40) in the placebo group, -6.27 days (5.38) in the 225 mg dose group, and -6.09 days (5.22) in the 675 mg dose group. The LSM difference in the reduction of migraine-days between the placebo and 225 mg dose groups was -2.81 days (95% CI -4.07 to -1.55; p<0.0001), whereas the difference between the placebo and 675 mg dose group was -2.64 days (-3.90 to -1.38; p<0.0001). LSM differences in the reduction of headache-days were -2.63 days (-3.91 to -1.34; p<0.0001) between the placebo group and 225 mg dose group and -2.58 days (-3.87 to 1.30; p <0.0001) between the placebo group and the 675 mg dose group. Adverse events occurred in 58 (56%) patients in the placebo group, 44 (46%) patients in the 225 mg dose group, and 57 (59%) patients in the 675 mg dose group; moderate or severe adverse events were reported for 29 (27%) patients, 24 (25%) patients, and 26 (27%) patients, respectively. INTERPRETATION: TEV-48125, at doses of 225 mg and 675 mg given once every 28 days for 12 weeks, was safe, well tolerated, and effective as a preventive treatment of high-frequency episodic migraine, thus supporting advancement of the clinical development programme to phase 3 clinical trials. FUNDING: Teva Pharmaceuticals. BACKGROUND: The calcitonin gene-related peptide (CGRP) pathway is a promising target for preventive therapies in patients with migraine. We assessed the safety and efficacy of AMG 334, a fully human monoclonal antibody against the CGRP receptor, for migraine prevention. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, phase 2 trial, patients aged 18-60 years with 4 to 14 migraine days per month were enrolled at 59 headache and clinical research centres in North America and Europe, and randomly assigned in a 3:2:2:2 ratio to monthly subcutaneous placebo, AMG 334 7 mg, AMG 334 21 mg, or AMG 334 70 mg using a sponsor-generated randomisation sequence centrally executed by an interactive voice response or interactive web response system. Study site personnel, patients, and the sponsor study personnel were masked to the treatment assignment. The primary endpoint was the change in monthly migraine days from baseline to the last 4 weeks of the 12-week double-blind treatment phase. The primary endpoint was calculated using the least squares mean at each timepoint from a generalised linear mixed-effect model for repeated measures. Safety endpoints were adverse events, clinical laboratory values, vital signs, and anti-AMG 334 antibodies. The study is registered with ClinicalTrials.gov, number NCT01952574. An open-label extension phase of up to 256 weeks is ongoing and will assess the long-term safety of AMG 334. FINDINGS: From Aug 6, 2013, to June 30, 2014, 483 patients were randomly assigned to placebo (n=160), AMG 334 7 mg (n=108), AMG 334 21 mg (n=108), or AMG 334 70 mg (n=107). The mean change in monthly migraine days at week 12 was -3·4 (SE 0·4) days with AMG 334 70 mg versus -2·3 (0·3) days with placebo (difference -1·1 days [95% CI -2·1 to -0·2], p=0·021). The mean reductions in monthly migraine days with the 7 mg (-2·2 [SE 0·4]) and the 21 mg (-2·4 [0·4]) doses were not significantly different from that with placebo. Adverse events were recorded in 82 (54%) patients who received placebo, 54 (50%) patients in the AMG 334 7 mg group, 54 (51%) patients in the AMG 334 21 mg group, and 57 (54%) patients in the AMG 334 70 mg group. The most frequently reported adverse events were nasopharyngitis, fatigue, and headache. Serious adverse events were reported for one patient in the AMG 334 7 mg group (ruptured ovarian cyst) and one patient in the AMG 334 70 mg group (migraine and vertigo); these events were judged to be unrelated to AMG 334 treatment. Nine (3%) of 317 patients had neutralising antibodies. No apparent association was recorded between patients with positive anti-AMG 334 antibodies and adverse events. No clinically significant vital signs, laboratory, or electrocardiogram findings were recorded. INTERPRETATION: These results suggest that AMG 334 70 mg might be a potential therapy for migraine prevention in patients with episodic migraine and support further investigation of AMG 334 in larger phase 3 trials. FUNDING: Amgen. PURPOSE OF REVIEW: The results of phase 2 randomized controlled trials for the prevention of episodic and chronic migraine demonstrating the efficacy and safety of four mAbs targeting the calcitonin gene-related peptide (CGRP) pathway [ALD403 (eptinezumab), AMG334 (erenumab), LY2951742 (galcanezumab) and TEV48125 (fremanezumab)] have been published recently, and phase 3 trials are in process. This development will change headache management fundamentally. We aim to summarize and compare the phase 2 data. RECENT FINDINGS: The change from baseline in the number of migraine days at the end of treatment in high-frequency episodic migraine was -1 (at weeks 5-8), -1.1 (at weeks 9-12), -1.2 (at weeks 9-12) and -2.6 (at weeks 9-12) days for ALD403, AMG344, LY2951742 and TEV48125 (225 mg), respectively. Number needed to treats for responders and odds ratio for any adverse event were 4.7, 6.2, 4.0 and 4.0 and 1.09, 0.96, 1.07 and 1.05, respectively. SUMMARY: All four CGRP antibodies display comparable efficacy that does not differ significantly from that of the currently available oral antimigraine drugs. However, their safety and tolerability profiles as well as low frequency of administration looks promising but remains to be verified in long-term and large-scale trials. Considerations related to pregcy, risk for cardiovascular effects and cost are subject for further evaluation. BACKGROUND: Fremanezumab, a humanized monoclonal antibody targeting calcitonin gene-related peptide (CGRP), is being investigated as a preventive treatment for migraine. We compared two fremanezumab dose regimens with placebo for the prevention of chronic migraine. METHODS: In this phase 3 trial, we randomly assigned patients with chronic migraine (defined as headache of any duration or severity on ≥15 days per month and migraine on ≥8 days per month) in a 1:1:1 ratio to receive fremanezumab quarterly (a single dose of 675 mg at baseline and placebo at weeks 4 and 8), fremanezumab monthly (675 mg at baseline and 225 mg at weeks 4 and 8), or matching placebo. Both fremanezumab and placebo were administered by means of subcutaneous injection. The primary end point was the mean change from baseline in the average number of headache days (defined as days in which headache pain lasted ≥4 consecutive hours and had a peak severity of at least a moderate level or days in which acute migraine-specific medication [triptans or ergots] was used to treat a headache of any severity or duration) per month during the 12 weeks after the first dose. RESULTS: Of 1130 patients enrolled, 376 were randomly assigned to fremanezumab quarterly, 379 to fremanezumab monthly, and 375 to placebo. The mean number of baseline headache days (as defined above) per month was 13.2, 12.8, and 13.3, respectively. The least-squares mean (±SE) reduction in the average number of headache days per month was 4.3±0.3 with fremanezumab quarterly, 4.6±0.3 with fremanezumab monthly, and 2.5±0.3 with placebo (P<0.001 for both comparisons with placebo). The percentage of patients with a reduction of at least 50% in the average number of headache days per month was 38% in the fremanezumab-quarterly group, 41% in the fremanezumab-monthly group, and 18% in the placebo group (P<0.001 for both comparisons with placebo). Abnormalities of hepatic function occurred in 5 patients in each fremanezumab group (1%) and 3 patients in the placebo group (<1%). CONCLUSIONS: Fremanezumab as a preventive treatment for chronic migraine resulted in a lower frequency of headache than placebo in this 12-week trial. Injection-site reactions to the drug were common. The long-term durability and safety of fremanezumab require further study. (Funded by Teva Pharmaceuticals; ClinicalTrials.gov number, NCT02621931 .). Treatment of migraine is on the cusp of a new era with the development of drugs that target the trigeminal sensory neuropeptide calcitonin gene-related peptide (CGRP) or its receptor. Several of these drugs are expected to receive approval for use in migraine headache in 2018 and 2019. CGRP-related therapies offer considerable improvements over existing drugs as they are the first to be designed specifically to act on the trigeminal pain system, they are more specific and they seem to have few or no adverse effects. CGRP receptor antagonists such as ubrogepant are effective for acute relief of migraine headache, whereas monoclonal antibodies against CGRP (eptinezumab, fremanezumab and galcanezumab) or the CGRP receptor (erenumab) effectively prevent migraine attacks. As these drugs come into clinical use, we provide an overview of knowledge that has led to successful development of these drugs. We describe the biology of CGRP signalling, summarize key clinical evidence for the role of CGRP in migraine headache, including the efficacy of CGRP-targeted treatment, and synthesize what is known about the role of CGRP in the trigeminovascular system. Finally, we consider how the latest findings provide new insight into the central role of the trigeminal ganglion in the pathophysiology of migraine. Objective - To evaluate 12-week changes from baseline of 2 disease-specific patient-reported outcome (PRO) measures in adults with migraine treated with galcanezumab, an investigational humanized antibody binding calcitonin gene-related peptide (CGRP), or placebo. Background - Preventing headache-related functional impairment is an important goal of migraine preventive treatment and a measurement target for PROs. Understanding which drugs have the potential to improve patient functioning in addition to preventing migraine headaches is vital to lessening patient burden. Design/Methods - This Phase 2b double-blind, randomized, placebo-controlled study enrolled adults with episodic migraine. Galcanezumab (120 mg subcutaneous injection; n = 60) or placebo (n = 127) was administered every 28 days for 12 weeks. Post hoc secondary analyses were conducted for those who completed 12 weeks of treatment on 2 PROs: The Migraine-Specific Quality of Life Questionnaire (MSQ) v2.1 and the Headache Impact Test™ (HIT-6). Results - Analysis of covariance revealed significant differences in least square mean changes from baseline between galcanezumab and placebo for all MSQ domains including total mean change placebo of 18.63, galcanezumab of 27.36 (95% CI 2.449, 15.008; P-value of .0067); Role Function-Restrictive mean change placebo of 22.40, galcanezumab of 31.92 (95% CI 2.636, 16.518; P-value of .0071); Role Function-Preventive mean change placebo of 13.43, galcanezumab of 19.76 (95% CI 0.476, 12.185; P-value of .0342); and Emotional Function mean change placebo of 16.88, galcanezumab of 26.61 (95% CI 2.789, 16.674; P-value of .0063). At baseline, mean number of migraine headache days (MHDs) did not correlate with MSQ total scores or HIT-6. At 12 weeks post-treatment, MHD correlated with MSQ and HIT-6 scores (all P < .0001). Change in MHD was associated with change in MSQ domains and change in HIT-6 scores (all P < .0001). Conclusions - In comparison with placebo, treatment with galcanezumab was associated with significant functional improvements as reflected by changes in MSQ scores. Change in MHD was associated with improvements in MSQ and reductions in HIT-6 scores, indicating the clinical importance of these changes in relation to PROs that measure function. BACKGROUND: A substantial proportion of patients with migraine does not respond to, or cannot tolerate, oral preventive treatments. Erenumab is a novel CGRP-receptor antibody with preventive efficacy in migraine. We assessed its efficacy and tolerability in patients with episodic migraine in whom previous treatment with two-to-four migraine preventives had been unsuccessful. METHODS: LIBERTY was a 12-week, double-blind, placebo-controlled randomised study at 59 sites in 16 countries. Eligible patients were aged 18-65 years and had a history of episodic migraine with or without aura for at least 12 months, had migraine for an average of 4-14 days per month during the 3 months before screening, and had been treated unsuccessfully (in terms of either efficacy or tolerability, or both) with between two and four preventive treatments. Eligible participants were randomly assigned (1:1) to receive either erenumab 140 mg (via two 70 mg injections) or placebo every 4 weeks subcutaneously for 12 weeks. Randomisation was by interactive response technology and was stratified by monthly frequency of migraine headache (4-7 vs 8-14 migraine days per month) during the baseline phase. Cenduit generated the randomisation list and assigned participants to groups. Participants, investigators, people doing various assessments, and the study sponsor were masked to treatment assignment. The primary endpoint was the proportion of patients achieving a 50% or greater reduction in the mean number of monthly migraine days during weeks 9-12. Efficacy was measured in the full analysis set, which included all randomly assigned patients who started their assigned treatment and completed at least one post-baseline monthly migraine day measurement. Safety and tolerability were assessed by recording adverse events and by physical examination, assessment of vital signs, clinical laboratory assessments, and electrocardiography. Safety was assessed in all randomly assigned patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT03096834. The trial is closed to new participants, but the open-label extension phase is ongoing. FINDINGS: Between March 20, 2017, and Oct 27, 2017, 246 participants were randomly assigned, 121 to the erenumab group and 125 to the placebo group. 95 of 246 (39%) participants had previously unsuccessfully tried two preventive drugs, 93 (38%) had tried three, and 56 (23%) had tried four. At week 12, 36 (30%) patients in the erenumab had a 50% or greater reduction from baseline in the mean number of monthly migraine days, compared with 17 (14%) in the placebo group (odds ratio 2·7 [95% CI 1·4-5·2]; p=0·002). The tolerability and safety profiles of erenumab and placebo were similar. The most frequent treatment-emergent adverse event was injection site pain, which occurred in seven (6%) participants in both groups. INTERPRETATION: Compared with placebo, erenumab was efficacious in patients with episodic migraine who previously did not respond to or tolerate between two and four previous migraine preventive treatments. Erenumab might be an option for patients with difficult-to-treat migraine who have high unmet needs and few treatment options. FUNDING: Novartis Pharma. BACKGROUND: Galcanezumab, a humanized monoclonal antibody that selectively binds to the calcitonin gene-related peptide, has demonstrated in previous Phase 2 and Phase 3 clinical studies (≤6-month of treatment) a reduction in the number of migraine headache days and improved patients' functioning. This study evaluated the safety and tolerability, as well as the effectiveness of galcanezumab for up to 12 months of treatment in patients with migraine. METHODS: Patients diagnosed with episodic or chronic migraine, 18 to 65 years old, that were not exposed previously to galcanezumab, were randomized to receive galcanezumab 120 mg or 240 mg, administered subcutaneously once monthly for a year. Safety and tolerability were evaluated by frequency of treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and adverse events (AEs) leading to study discontinuation. Laboratory values, vital signs, electrocardiograms, and suicidality were also analyzed. Additionally, overall change from baseline in the number of monthly migraine headache days, functioning, and disability were assessed. RESULTS: One hundred thirty five patients were randomized to each galcanezumab dose group. The majority of patients were female (> 80%) and on average were 42 years old with 10.6 migraine headache days per month at baseline. 77.8% of the patients completed the open-label treatment phase, 3.7% of patients experienced an SAE, and 4.8% discontinued due to AEs. TEAEs with a frequency ≥ 10% of patients in either dose group were injection site pain, nasopharyngitis, upper respiratory tract infection, injection site reaction, back pain, and sinusitis. Laboratory values, vital signs, or electrocardiograms did not show anyclinically meaningful differences between galcanezumab dosesOverall mean reduction in monthly migraine headache days over 12 months for the galcanezumab dose groups were 5.6 (120 mg) and 6.5 (240 mg). Level of functioning was improved and headache-related disability was reduced in both dose groups. CONCLUSION: Twelve months of treatment with self-administered injections of galcanezumab was safe and associated with a reduction in the number of monthly migraine headache days. Safety and tolerability of the 2 galcanezumab dosing regimens were comparable. TRIAL REGISTRATION: ClinicalTrials.gov as NCT02614287 , posted November 15, 2015. These data were previously presented as a poster at the International Headache Congress 2017: PO-01-184, Late-Breaking Abstracts of the 2017 International Headache Congress. (2017). Cephalalgia, 37(1_suppl), 319-374. Migraine affects the daily life of millions of people around the world. The most well-known disabling symptom associated with this illness is the intense headache. Nowadays, there are treatments that can diminish the level of pain. OnabotulinumtoxinA (BoNT-A) has become a very popular medication for treating migraine headaches in those cases in which other medication is not working, typically in chronic migraines. Currently, the positive response to Botox treatment is not clearly understood, yet understanding the mechanisms that determine the effectiveness of the treatment could help with the development of more effective treatments. To solve this problem, this paper sets up a realistic scenario of electronic medical records of migraineurs under BoNT-A treatment where some clinical features from real patients are labeled by doctors. Medical registers have been preprocessed. A label encoding method based on simulated annealing has been proposed. Two methodologies for predicting the results of the first and the second infiltration of the BoNT-A based treatment are contempled. Firstly, a strategy based on the medical HIT6 metric is described, which achieves an accuracy over 91%. Secondly, when this value is not available, several classifiers and clustering methods have been performed in order to predict the reduction and adverse effects, obtaining an accuracy of 85%. Some clinical features as Greater occipital nerves (GON), chronic migraine time evolution and others have been detected as relevant features when examining the prediction models. The GON and the retroocular component have also been described as important features according to doctors. Several lines of evidence pointed to an important role for CGRP in migraine. These included the anatomic colocalization of CGRP and its receptor in sensory fibers innervating pain-producing meningeal blood vessels, its release by trigeminal stimulation, the observation of elevated CGRP in the cranial circulation during migraine with normalization concomitant with headache relief by sumatriptan, and translational studies with intravenous (IV) CGRP that evoked migraine only in migraineurs. The development of small molecule CGRP receptor antagonists (CGRP-RAs) that showed clinical antimigraine efficacy acutely and prophylactically in randomized placebo-controlled clinical trials subsequently gave definitive pharmacological proof of the importance of CGRP in migraine. More recently, CGRP target engagement imaging studies using a CGRP receptor PET ligand [11 C]MK-4232 demonstrated that there was no brain CGRP receptor occupancy at clinically effective antimigraine doses of telcagepant, a prototypic CGRP-RA. Taken together, these data indicated that (1) the therapeutic site of action of the CGRP-RAs was peripheral not central; (2) that IV CGRP had most likely evoked migraine through an action at sites outside the blood-brain barrier; and (3) that migraine pain was therefore, at least in part, peripheral in origin. The evolution of CGRP migraine science gave impetus to the development of peripherally acting drugs that could modulate CGRP chronically to prevent frequent episodic and chronic migraine. Large molecule biologic antibody (mAb) approaches that are given subcutaneously to neutralize circulating CGRP peptide (fremanezumab, galcanezumab) or block CGRP receptors (erenumab) have shown consistent efficacy and tolerability in multicenter migraine prevention trials and are now approved for clinical use. Eptinezumab, a CGRP neutralizing antibody given IV, shows promise in late stage clinical development. Recently, orally administered next-generation small molecule CGRP-RAs have been shown to have safety and efficacy in acute treatment (ubrogepant and rimegepant) and prevention (atogepant) of migraine, giving additional CGRP-based therapeutic options for migraine patients. Author information: (1)Neuroscience Section, Department of Applied Clinical Sciences and Biotechnology, University of L'Aquila, via Vetoio, 67100, L'Aquila, Italy. [email protected]. (2)Department of Neurology, Danish Headache Center, Rigshospitalet Glostrup, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. (3)Department of Neurology, Charité Universitätsmedizin Berlin, Berlin, Germany. (4)Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands. (5)1st Department of Neurology, National and Kapodistrian University of Athens, Athens, Greece. (6)Department of Clinical and Molecular Medicine, Sapienza University, Rome, Italy. BACKGROUND: Chronic migraines affect approximately 2 percent of the U.S. population and cost an estimated $17 billion per year. OnabotulinumtoxinA (botulinum toxin type A) is a U.S. Food and Drug Administration-approved prophylactic medication for chronic migraine headaches and is best injected in a targeted fashion into specific trigger sites. The purpose of this study was to determine the cost-effectiveness of long-term, targeted botulinum toxin type A versus peripheral trigger site deactivation surgery for the treatment of migraine headaches. METHODS: A Markov model was constructed to examine long-term, targeted botulinum toxin type A versus peripheral trigger site deactivation surgery. Costs, utilities, and other model inputs were identified from the literature. One-way and probabilistic sensitivity analyses were performed. An incremental cost-effectiveness ratio under $50,000 per quality-adjusted life-year was considered cost-effective. RESULTS: The mean cost of peripheral trigger site deactivation surgery was $10,303, with an effectiveness of 7.06; whereas the mean cost of long-term, targeted botulinum toxin type A was $36,071, with an effectiveness of 6.34. Trigger-site deactivation surgery is more effective and less costly over the time horizon of the model. One-way sensitivity analysis revealed that surgery is the most cost-effective treatment in patients requiring treatment for greater than 6.75 years. CONCLUSIONS: Based on this model, peripheral trigger site deactivation surgery is the more cost-effective option for treating refractory migraine headaches requiring treatment beyond 6.75 years. The model reveals that peripheral trigger-site deactivation surgery is more effective and less costly than long-term, targeted botulinum toxin type A over the course of a patient's lifetime. Migraine is the third-most prevalent disease in the world and affects approximately 39 million individuals in the United States alone. Migraine occurs in nearly 1 in 7 individuals between 15 and 49 years of age and is 3 times more frequent in women than in men. The Food and Drug Administration (FDA) recently approved 3 new humanized monoclonal antibodies that target calcitonin gene-related peptide (CGRP): erenumab, fremanezumab, and galcanezumab. The agents either bind to the CGRP receptor (erenumab) or bind to the CGRP ligand (fremanezumab and galcanezumab) and block its binding to the receptor. All 3 products are indicated for preventative treatment of episodic or chronic migraine in adults. The available studies to date document that these agents reduce migraine attacks. The CGRP monoclonal antibodies offer patients new options once they have exhausted other treatments. Medical research continues to focus on developing specific treatment strategies, including biological products that are effective and have a good safety profile. Due to their novelty, an updated overall view is offered on some neurological diseases which benefit from monoclonal antibodies (mAbs), for better treatment in clinical decisions. An extensive literature review was performed using PubMed with the following search terms: 'monoclonal antibodies' and 'history of monoclonal antibodies' and 'monoclonal antibodies in neurology'. The following information was collected: the era before the discoveries of mAbs, the stage of implementation of biotechnologies for mAbs, and the clinical trials submitted at https://clinicaltrials.gov/ with patients suffering from neurological diseases treated with mAbs. Since 2004, mAbs have been used to treat several neurological diseases, yielding new therapeutic perspectives: natalizumab, alemtuzumab and ocrelizumab for multiple sclerosis, eculizumab for myasthenia gravis, erenumab and frenazumab for migraine, galcanezumab for migraine and cluster headache, eculizumab for neuromyelitis optica spectrum disorder. As in other cases, drug repurposing is applied to monoclonal antibodies, saving time and money. These innovative therapies are more effective and can treat previously untreatable diseases. As better understanding of the pathogenic mechanisms of neurological diseases is gained, additional mAbs are expected to be developed at a lower cost and with better safety profile compared with current treatment options. BACKGROUND: Galcanezumab is a monoclonal antibody (mAb) that binds calcitonin gene-related peptide (CGRP) and is indicated for the preventive treatment of migraine. Galcanezumab demonstrated early onset of effect in patients with migraine but it is unknown whether the same holds true for patients who have not benefited from multiple prior migraine preventives. METHODS: Patients with episodic or chronic migraine from a 3-month, randomized, double-blind, placebo-controlled, phase 3b study (CONQUER) who had 2 to 4 migraine preventive medication category failures in the past 10 years were randomized 1:1 to placebo (N = 230) or galcanezumab 120 mg/month (240 mg loading dose; N = 232). In this post-hoc analysis, change from baseline in number of monthly and weekly migraine headache days was assessed. Monthly onset of effect was the earliest month at which significant improvement with galcanezumab compared to placebo was achieved and maintained at all subsequent months. Weekly onset was the initial week at which statistical separation was achieved and maintained at all subsequent weeks during that month. Proportion of patients with migraine headache days in the first week of treatment, and patients achieving ≥50%, ≥75%, and 100% response by month and week were also assessed. RESULTS: Galcanezumab-treated patients had a significantly greater reduction in monthly migraine headache days starting at month 1, which remained significant for all subsequent months compared to placebo (all p ≤ 0.0001, month 1 mean change from baseline: placebo - 0.7; galcanezumab - 4.0). Weekly migraine headache days was significantly reduced in galcanezumab-treated patients starting at week 1 and continued for each subsequent week of month 1 compared to placebo (all p < 0.01, week 1 mean change from baseline: placebo - 0.2; galcanezumab - 1.1). A significantly smaller percentage of patients had a migraine headache on the first day after galcanezumab treatment compared to placebo (28.4% vs 39.2%) and at each subsequent day during week 1 (all p < 0.05). A greater proportion of galcanezumab-treated patients achieved ≥50%, ≥75%, and 100% response at months 1-3 (all p < 0.05) and at weeks 1-4 of month 1 compared to placebo (all p < 0.01). CONCLUSION: Galcanezumab showed early onset of effect beginning the day after treatment initiation in patients who had not previously benefited from migraine preventive treatments. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03559257 . Registered 18 June 2018. INTRODUCTION: Erenumab, a first-in-class monoclonal antibody targeting the calcitonin gene-related peptide pathway, was approved by the US Food and Drug Administration in 2018 for the prevention of migraine in adults. There is limited data available on its impact in real-world settings. The study aim was to characterize the real-world treatment profiles, clinical outcomes, and healthcare resource utilization of patients prescribed erenumab from select major US headache centers. METHODS: A retrospective chart review of patients with migraine treated with erenumab for at least 3 months across five major headache centers was conducted. Data was collected from patient charts between April 2019 and April 2020 and included patient and clinical characteristics, migraine medication use, and outpatient visits. The date of the first prescription fill of erenumab was defined as the index date. The baseline period comprised the 3 months prior to the index date and the study period comprised the at least 3 months on erenumab treatment. RESULTS: Data from a total of 1034 patients with chronic migraine with a mean of 9.3 months of erenumab treatment were analyzed. Patients were on average 48 years old, 86% were female, and 79% were white. Patients had a mean of 5 preventive treatment failures prior to erenumab initiation. Patients used a mean of 2 preventive treatments (excluding erenumab) and 2 acute treatments during baseline and study periods. Among patients with effectiveness data, 45% of patients had improvement in physician-reported migraine severity and 35% experienced at least 50% reduction in mean headache/migraine days per month. The average number of monthly outpatient visits was 0.43 and 0.30 before and after erenumab initiation, respectively. CONCLUSION: In this predomitly refractory chronic migraine population treated in select headache centers, patients had fewer headache/migraine days per month and outpatient visits after initiating erenumab. However, patients largely continued to be managed via a polypharmacy approach after erenumab initiation. BACKGROUND: Galcanezumab is a calcitonin gene-related peptide (CGRP) monoclonal antibody (mAb) indicated for the preventive treatment of migraine. While galcanezumab has demonstrated efficacy in patients who did not respond to prior preventive medications in general, its efficacy in patients who did not benefit from individual, commonly prescribed preventive treatments due to inadequate efficacy or safety/tolerability remains unknown. METHODS: CONQUER was a 3-month, randomized, double-blind, placebo-controlled, phase 3b study that enrolled patients with episodic or chronic migraine who had 2 to 4 migraine preventive medication category failures in the past 10 years. Patients were randomly assigned 1:1 to receive placebo (N = 230) or galcanezumab 120 mg/month (240 mg loading dose; N = 232). Post hoc analyses were conducted to determine the efficacy of galcanezumab in patients who had not benefited from six of the most commonly prescribed migraine preventive medications. The mean change from baseline in monthly migraine headache days and ≥ 50 % response rates were assessed over months 1-3. Improvement in Migraine-Specific Questionnaire Role Function-Restrictive (MSQ-RFR) scores were assessed at month 3. The endpoints were estimated via mixed model with repeated measures. RESULTS: The most common treatment failures due to inadequate efficacy or safety/tolerability, which at least 20 % of patients reported trying without benefit, included topiramate, amitriptyline, propranolol, valproate or divalproex, onabotulinum toxin A, and metoprolol. Patients who had not previously benefited from these treatments had a greater mean reduction in monthly migraine headache days across months 1-3 in the galcanezumab group compared to placebo (all p < 0.01). More patients treated with galcanezumab experienced a ≥ 50 % reduction from baseline in monthly migraine headache days across months 1-3 compared to placebo (all p < 0.05). Galcanezumab-treated patients had a greater improvement in mean MSQ-RFR scores at month 3 compared to placebo (all p < 0.01). CONCLUSIONS: In this population, galcanezumab was effective in reducing monthly migraine headache days, improving response rates, and enhancing quality of life in patients who had not previously benefited from topiramate, amitriptyline, propranolol, valproate or divalproex, onabotulinum toxin A, and/or metoprolol due to inadequate efficacy or safety/tolerability. TRIAL REGISTRATION: ClinicalTrials.gov NCT03559257 (CONQUER). OBJECTIVE: To evaluate the impact of fremanezumab on the severity and duration of remaining migraine attacks in patients with chronic migraine (CM) or episodic migraine (EM). BACKGROUND: Fremanezumab is a fully humanized monoclonal antibody (IgGΔa) that selectively targets calcitonin gene-related peptide and is efficacious in reducing migraine frequency. METHODS: This exploratory post hoc analysis included data from three randomized, double-blind, 12-week, phase 3 studies (HALO CM, HALO EM, and FOCUS). In all three studies, patients with CM or EM were randomized 1:1:1 to receive subcutaneous quarterly fremanezumab (month 1/2/3: 675 mg/placebo/placebo), monthly fremanezumab (month 1/2/3: 675 mg [CM], 225 mg [EM]/225 mg/225 mg), or matched monthly placebo. Changes from baseline were evaluated in the proportion of headache days of at least moderate severity, peak severity of headache days, mean monthly headache hours (of any severity and at least moderate severity), and mean headache hours per headache day of any severity. RESULTS: A total of 2843 patients were randomized with 2823 patients included in the efficacy analyses across all studies (HALO CM, N = 1121; HALO EM, N = 865; FOCUS, N = 837). At study baseline, mean (standard deviation [SD]) monthly number of headache days rated moderate or severe in the quarterly fremanezumab, monthly fremanezumab, and placebo groups, respectively, were 13.2 (5.5), 12.8 (5.8), and 13.3 (5.8) in HALO CM; 7.2 (3.1), 6.8 (2.9), and 6.9 (3.1) in HALO EM; and 12.4 (5.8), 12.7 (5.8), and 12.8 (5.9) in FOCUS. Patients experienced significant least-squares mean (LSM; 95% confidence interval) percent reductions from baseline in monthly number of headache days rated moderate or severe during the 12 weeks: HALO CM, quarterly fremanezumab, 34.5% (-39.8, -29.2) and monthly fremanezumab, 36.2% (-41.4, -31.0) vs. placebo, 19.6% (-20.0, -14.3); HALO EM, quarterly fremanezumab, 40.7% (-47.8, -33.5) and monthly fremanezumab, 43.4% (-50.4, -36.3) vs. placebo, 17.9% (-24.9, -11.0); and FOCUS, quarterly fremanezumab, 36.5% (-41.9, -31.1) and monthly fremanezumab, 38.6% (-44.0, -33.3) vs. placebo, 3.5% (-8.9, 1.8); all p < 0.0001. At study baseline, mean (SD) number of monthly headache hours rated moderate or severe in the quarterly fremanezumab, monthly fremanezumab, and placebo groups, respectively, were 66.4 (58.8), 68.0 (53.9), and 68.5 (57.0) in HALO CM; 33.3 (25.4), 31.7 (23.7), and 31.6 (23.2) in HALO EM; and 59.2 (54.7), 64.3 (65.2), and 65.9 (70.2) in FOCUS. Significant reductions were observed in LSM (standard error) number of monthly headache hours of at least moderate severity: HALO CM, quarterly fremanezumab, 24.4 (2.5) and monthly fremanezumab, 26.4 (2.3) vs. placebo, 14.1 (2.5); HALO EM, quarterly fremanezumab, 14.5 (1.4) and monthly fremanezumab, 15.5 (1.3) vs. placebo, 8.1 (1.3); and FOCUS, quarterly fremanezumab, 16.8 (3.0) and monthly fremanezumab, 18.3 (3.0) vs. placebo, 2.3 (3.0); all p < 0.001. CONCLUSION: These analyses demonstrated that quarterly or monthly treatment with fremanezumab significantly reduced headache severity and duration in patients with CM or EM, including in patients with documented inadequate response to two to four prior migraine preventive medication classes. BACKGROUND: Fremanezumab, a fully humanized monoclonal antibody (IgG2Δa) selectively targets the calcitonin gene-related peptide and has proven efficacy for the preventive treatment of migraine. In this study, we evaluated the long-term efficacy, safety, and tolerability of monthly and quarterly fremanezumab. METHODS: Episodic migraine and chronic migraine patients completing the 12-week double-blind period of the FOCUS trial entered the 12-week open-label extension and received 3 monthly doses of fremanezumab (225 mg). Changes from baseline in monthly migraine days, monthly headache days of at least moderate severity, days of acute headache medication use, days with photophobia/phonophobia, days with nausea or vomiting, disability scores, and proportion of patients achieving a ≥50% or  ≥75% reduction in monthly migraine days were evaluated. RESULTS: Of the 807 patients who completed the 12-week double-blind treatment period and entered the open-label extension, 772 patients completed the study. In the placebo, quarterly fremanezumab, and monthly fremanezumab dosing regimens, respectively, patients had fewer average monthly migraine days (mean [standard deviation] change from baseline: - 4.7 [5.4]; - 5.1 [4.7]; - 5.5 [5.0]), monthly headache days of at least moderate severity (- 4.5 [5.0]; - 4.8 [4.5]; - 5.2 [4.9]), days per month of acute headache medication use (- 4.3 [5.2]; - 4.9 [4.6]; - 4.8 [4.9]), days with photophobia/phonophobia (- 3.1 [5.3]; - 3.4 [5.3]; - 4.0 [5.2]), and days with nausea or vomiting (- 2.3 [4.6]; - 3.1 [4.5]; - 3.0 [4.4]). During the 12-week open-label extension, 38%, 45%, and 46% of patients, respectively, achieved a ≥50% reduction and 16%, 15%, and 20%, respectively, achieved a ≥75% reduction in monthly migraine days. Disability scores were substantially improved in all 3 treatment groups. There were low rates of adverse events leading to discontinuation (<1%). CONCLUSION: Fremanezumab demonstrated sustained efficacy up to 6 months and was well tolerated in patients with episodic migraine or chronic migraine and documented inadequate response to multiple migraine preventive medication classes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03308968 (FOCUS). Conflict of interest statement: SJT has received grants for research (no personal compensation) from Allergan/Abbvie, Amgen, Eli Lilly, Lundbeck, Neurolief, Novartis, Satsuma, and Zosano, has served as a consultant for Aeon, Allergan/Abbvie, AlphaSights, Amgen, Atheneum, Axsome Therapeutics, Becker Pharmaceutical Consulting, ClearView Healthcare Partners, CoolTech, CRG, Currax, DRG, Eli Lilly, ExpertConnect, FCB Health, GLG, Guidepoint Global, Health Science Communications, HMP Communications, Impel, InteractiveForums, Krog and Partners, Lundbeck, M3 Global Research, MJH Holdings, Neurolief, Novartis, Palion Medical, Pulmatrix, SAI MedPartners, Satsuma, Spherix Global Insights, Strategy Inc, System Analytic, Taylor and Francis, Teva, Theranica, UnityHA, Xoc, and Zosano, has received salary from Dartmouth‐Hitchcock Medical Center, American Headache Society, and the Thomas Jefferson University, and has received continuing medical education honoraria from the Annenberg Center for Health Sciences, American Academy of Neurology, American Headache Society, Catamount Medical Education, Diamond Headache Clinic, Forefront Collaborative, Haymarket Medical Education, Peerview, Medical Education Speakers Network, Migraine Association of Ireland, North American Center for CME, The Ohio State University, Physicians’ Education Resource, PlatformQ Education, Primed, Texas Neurological Society, WebMD/Medscape. SL has received advisory or consulting fees from Amgen, Alder, Biohaven, Impel, Eli Lilly, Lundbeck, and Teva, and receives compensation from Premera as a member of the Pharmacy & Therapeutics committee. MA has received personal fees from Alder, Allergan, Amgen, Alder, Eli Lilly, Novartis, and Teva; has participated in clinical trials as the principal investigator for Alder, Allergan, Amgen, ElectroCore, Eli Lilly, Novartis and Teva; has received a research grant form Novartis; has no ownership interest and does not own stocks of any pharmaceutical company; serves as associate editor of Cephalalgia, associate editor of The Journal of Headache and Pain, and associate editor of Headache, and is President of the International Headache Society. TS has received consulting fees from Alder, Allergan, Amgen, Aural Analytics, Avanir, Cipla, Dr. Reddy's, Eli Lilly, Ipsen Bioscience, Nocira, Novartis, Second Opinion, and Teva. JA has received consulting fees from Alder, Allergan, electroCore, Eli Lilly, Impel, Promius, and Teva; honoraria from Alder, Allergan, Amgen, Avanir, electroCore, Eli Lilly, Promius, and Teva; and serves as section editor for Current Pain and Headache Reports. JS, DEC, AW, and GPdSL are employees and stockholders of Amgen. JK is an employee and stockholder of Novartis. BACKGROUND: Calcitonin gene-related peptide (CGRP) (receptor) antibodies (erenumab, fremanezumab and galcanezumab) are increasingly used in prophylactic treatment of migraine. In the approval studies, severely affected patients with migraine and chronic daily headache without any headache free days were excluded. Thus, less is known about the effectiveness of CGRP antibody treatment in this cohort. METHODS: Clinical routine data of 32 patients with migraine and daily headache were analysed after three months of treatment with a CGRP antibody (16 erenumab, 7 galcanezumab, 9 fremanezumab), including changes of monthly headache days (MHD) monthly migraine days (MMD) and monthly acute medication intake (AMD) as well as migraine characteristics. Statistical analysis was performed with the Wilcoxon-Test. Migraine characteristics were analysed descriptively. RESULTS: The number of MHD was significantly reduced (mean reduction (standard error), p-value): (-4.2 (1.3), p = 0.009) as well as MMD (-4.3 (1.6), p = 0.033). Four patients (13 %) reached a 50 % reduction regarding MHD and 8 patients (25 %) regarding MMD, migraine duration and intensity improved under therapy. CONCLUSIONS: Despite the low responder rate, CGRP antibodies can be effective at least in a few cases of severely affected patients with drug resistant migraine and chronic daily headache. TRIAL REGISTRATION: Retrospective registered. BACKGROUND: Erenumab, the first-in-class fully human monoclonal antibody targeting the calcitonin gene-related peptide receptor, was shown to be efficacious and safe for the prophylactic treatment of migraine in adults in randomized clinical trials. Large-scale, real-world evidence in multi-centre settings is still needed to confirm these results. Erenumab patient profiles outside clinical trials and physicians' treatment patterns, as well as data from patients treated in Germany, a severely impacted population, are not published yet. METHODS: TELESCOPE was a multi-centre survey gathering real-world data from 45 German headache centres between July 2019 and December 2019. The project consisted of two parts. In the first part, treating physicians shared their experiences on current erenumab treatment with regard to patient profiles, treatment patterns and treatment responses. In the second part, a retrospective chart review was conducted of 542 migraine patients treated with erenumab for at least three months. Treatment responses focused on various aspects of patients' quality of life. RESULTS: The analysis of 542 patients' charts revealed that three-month treatment with erenumab significantly reduced monthly headaches, migraine and acute medication days. Furthermore, headache intensity and frequency were reduced in over 75 % and accompanying aura in 35 % of patients. The clinical global impression scale revealed a general improvement in 91 % of patients. According to the treating physicians' professional judgement, 83 % of patients responded to erenumab and 80 % were satisfied with the treatment. Physicians evaluated restricted quality of life, the number of monthly migraine days and previous, prophylactic treatments as the main components of the current patient profile for monoclonal antibody recipients. Based on the assessment of physicians, erenumab reduced migraine symptoms in 65 % and increased quality of life in more than 75 % of their patients. CONCLUSIONS: TELESCOPE confirms positive treatment responses with erenumab shown in clinical trials in a real-world multi-centre setting. The results show consistently positive experiences of physicians utilizing erenumab in clinical practice and underline that therapy with this monoclonal antibody is effective in migraine patients, particular in those, who have failed several prophylactic therapies.

What is the most common N6-methyladenosine (m6A) methylation modification site of RUNX1T1?

The RRACH motif is the most common N6-methyladenosine (m6A) methylation modification site of RUNX1T1.

BACKGROUND: Runt-related transcription factor 1 (RUNX1T1) isoforms are involved in adipogenesis. RUNX1T1 is mediated by the fat mass and obesity-associated (FTO). However, the extent to which RUNX1T1 single-nucleotide polymorphisms (SNPs) are associated with obesity risk or metabolic abnormalities in a community population basis is unknown. METHODS: Samples were obtained from the Australian Crossroads study bio-bank. SNPs located in the coding region and 3'untranslated regions of RUNX1T1 with minor allele frequency ≥0.05 were analysed using Taqman genotyping assays. RESULTS: Eight candidate SNPs were genotyped successfully in 1440 participants. Of these SNPs only rs34269950 located in the 'RRACH' motif, the most common N6-methyladenosine (m6A) methylation modification site (recognized by FTO), was significantly associated with obesity risk and metabolic abnormalities. Specifically, compared to AA genotype, rs34269950 del/del genotype was associated with a 1.47 [95% confidence interval (CI): 1.01-2.14, P = 0.042] fold higher rate of obesity risk. Additionally, the del/del genotype was associated with a 60% increased risk of metabolic syndrome (MetS) [odds ratio (OR) = 1.60, 95% CI: 1.10-2.32, P = 0.015], in comparison to the AA genotype. Finally, rs34269950 del/del increased the risk of a larger waist circumference (OR = 1.65, 95% CI: 1.15-2.36, P = 0.007), but not other components of MetS. CONCLUSION: Our study demonstrates that RUNX1T1 rs34269950, located in a potential FTO recognition motif, is significantly associated with waist circumference. This provides novel evidence to suggest SNPs located in RRACH motif may be involved in RNA m6A modification and mechanistic pathways that influence abdominal obesity.

Describe GREEKC

The COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC, CA15205, www.greekc.org) organized nine workshops in a four-year period, starting September 2016. The workshops brought together experts from all over the world working on various steps in the knowledge management process that focuses on understanding gene regulatory mechanisms.

Author information: (1)Systems Biology Group, Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway. Electronic address: [email protected]. (2)Faculty of Information & Communication Technology, University of Malta, Msida, Malta. (3)Departamento de Informática y Sistemas, Universidad de Murcia, IMIB-Arrixaca, CP 30100, Murcia, Spain. (4)Swiss Institute of Bioinformatics, Quartier Sorge, Bâtiment Amphipôle, 1015 Lausanne, Switzerland. (5)Systems Biology and Bioinformatics Laboratory (SysBioLab), Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal. (6)SIB Swiss Institute of Bioinformatics, 1 Rue Michel-Servet, 1204 Geneva, Switzerland. (7)Institute of Protein Research, Russian Academy of Sciences, 142290, Institutskaya 4, Pushchino, Russia. (8)Department of Biology, University of Rome Tor Vergata, Rome, Italy. (9)Department of Molecular Biology, Faculty of Science, Radboud University, PO box 9101, Nijmegen 6500HG, the Netherlands. (10)Functional Gene Annotation, Pre-clinical and Fundamental Science, Institute of Cardiovascular Science, University College London, 5 University Street, London WC1E 6JF, UK. (11)Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991, Gubkina 3, Moscow, Russia. (12)European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK. (13)Department DIBEST, University of Calabria, Rende, Italy. (14)Fondazione Human Technopole, Department of Biology, Via Cristina Belgioioso, 171, 20157 Milan, Italy. (15)Department of Biomedical Informatics, University of Utah, 421 Wakara Way #140, Salt Lake City, UT 84108, United States. (16)Institute of Medical Informatics, Statistics and Documentation, Medical University of Graz, Auenbruggerpl. 2, Graz, Austria. (17)Systems Biology Group, Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway. (18)European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK. (19)Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway.

Which receptors are targeted by Tirzepatide?

Tirzepatide is dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist that demonstrated substantially greater glucose control and weight loss (WL) compared with selective GLP-1RA dulaglutide.

OBJECTIVE: To determine the effect of tirzepatide, a dual agonist of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 receptors, on biomarkers of nonalcoholic steatohepatitis (NASH) and fibrosis in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: Patients with T2DM received either once weekly tirzepatide (1, 5, 10, or 15 mg), dulaglutide (1.5 mg), or placebo for 26 weeks. Changes from baseline in alanine aminotransferase (ALT), aspartate aminotransferase (AST), keratin-18 (K-18), procollagen III (Pro-C3), and adiponectin were analyzed in a modified intention-to-treat population. RESULTS: Significant (P < 0.05) reductions from baseline in ALT (all groups), AST (all groups except tirzepatide 10 mg), K-18 (tirzepatide 5, 10, 15 mg), and Pro-C3 (tirzepatide 15 mg) were observed at 26 weeks. Decreases with tirzepatide were significant compared with placebo for K-18 (10 mg) and Pro-C3 (15 mg) and with dulaglutide for ALT (10, 15 mg). Adiponectin significantly increased from baseline with tirzepatide compared with placebo (10, 15 mg). CONCLUSIONS: In post hoc analyses, higher tirzepatide doses significantly decreased NASH-related biomarkers and increased adiponectin in patients with T2DM. The effect of dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist (RA) tirzepatide on gastric emptying (GE) was compared to that of GLP-1RAs in non-clinical and clinical studies. GE was assessed following acute and chronic treatment with tirzepatide in diet-induced obese mice versus semaglutide or long-acting GIP analogue alone. Participants [with and without type 2 diabetes (T2DM)] from a phase 1, 4-week multiple dose study received tirzepatide, dulaglutide or placebo. GE was assessed by acetaminophen absorption. In mice, tirzepatide delayed GE to a similar degree to that achieved with semaglutide; however, these acute inhibitory effects were abolished after 2 weeks of treatment. GIP analogue alone had no effect on GE or on GLP-1's effect on GE. In participants with and without T2DM, once-weekly tirzepatide (≥5 and ≥4.5 mg, respectively) delayed GE after a single dose. This effect diminished after multiple doses of tirzepatide or dulaglutide in healthy participants. In participants with T2DM treated with an escalation schedule of tirzepatide 5/5/10/10 or 5/5/10/15 mg, a residual GE delay was still observed after multiple doses. These data suggest that tirzepatide's activity on GE is comparable to that of selective GLP-1RAs. INTRODUCTION: The glucagon-like peptide-1 (GLP-1) receptor agonists (RA) have increasingly gained prominence in the treatment of type 2 diabetes (T2D) based on their glycemic benefits and favorable body weight and cardiorenal effects. Despite this, continued development of therapeutics with superior efficacy is important to help address persistent challenges in the attainment of metabolic goals in many patients with T2D. AREAS COVERED: Tirzepatide is an unimolecular dual glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 RA in development for the treatment of T2D. This review summarizes key characteristics of tirzepatide and Phase 1 and Phase 2 clinical trial efficacy and safety results. Additionally, it provides an overview of the ongoing Phase 3 clinical trial program in T2D and briefly summarizes recently initiated studies in patients with obesity and nonalcoholic steatohepatitis. Information in this review comes primarily from published clinical trials, manufacturer's websites, and ClinicalTrials.gov. EXPERT OPINION: Based on data from Phase 2 trials, tirzepatide has the potential to be the most efficacious therapy in T2D with respect to both glucose and body weight control. Data from the ongoing Phase 3 clinical trial program should start to become available in late 2020 and will determine the future course of this promising therapeutic agent. CONTEXT: Novel dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist (RA) tirzepatide demonstrated substantially greater glucose control and weight loss (WL) compared with selective GLP-1RA dulaglutide. OBJECTIVE: Explore mechanisms of glucose control by tirzepatide. DESIGN: Post hoc analyses of fasting biomarkers and multiple linear regression analysis. SETTING: Forty-seven sites in 4 countries. PATIENTS OR OTHER PARTICIPANTS: Three hundred and sixteen subjects with type 2 diabetes. INTERVENTIONS: Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), placebo. MAIN OUTCOME MEASURES: Analyze biomarkers of beta-cell function and insulin resistance (IR) and evaluate WL contributions to IR improvements at 26 weeks. RESULTS: Homeostatic model assessment (HOMA) 2-B significantly increased with dulaglutide and tirzepatide 5, 10, and 15 mg compared with placebo (P ≤ .02). Proinsulin/insulin and proinsulin/C-peptide ratios significantly decreased with tirzepatide 10 and 15 mg compared with placebo and dulaglutide (P ≤ .007). Tirzepatide 10 and 15 mg significantly decreased fasting insulin (P ≤ .033) and tirzepatide 10 mg significantly decreased HOMA2-IR (P = .004) compared with placebo and dulaglutide. Markers of improved insulin sensitivity (IS) adiponectin, IGFBP-1, and IGFBP-2 significantly increased by 1 or more doses of tirzepatide (P < .05). To determine whether improvements in IR were directly attributable to WL, multiple linear regression analysis with potential confounding variables age, sex, metformin, triglycerides, and glycated hemoglobin A1c was conducted. WL significantly (P ≤ .028) explained only 13% and 21% of improvement in HOMA2-IR with tirzepatide 10 and 15 mg, respectively. CONCLUSIONS: Tirzepatide improved markers of IS and beta-cell function to a greater extent than dulaglutide. IS effects of tirzepatide were only partly attributable to WL, suggesting dual receptor agonism confers distinct mechanisms of glycemic control. Glucagon-like peptide 1 (GLP-1) based therapy is an established treatment option for the management of type 2 diabetes mellitus (T2DM) and is recommended early in the treatment algorithm owing to glycaemic efficacy, weight reduction and favourable cardiovascular outcomes. Glucose-dependent insulinotropic polypeptide (GIP), on the other hand, was thought to have no potential as a glucose-lowering therapy because of observations showing no insulinotropic effect from supraphysiological infusion in people with T2DM. However, emerging evidence has illustrated that co-infusion of GLP-1 and GIP has a synergetic effect, resulting in significantly increased insulin response and glucagonostatic response, compared with separate administration of each hormone. These observations have led to the development of a dual GIP/GLP-1 receptor agonist, known as a 'twincretin'. Tirzepatide is a novel dual GIP/GLP-1 receptor agonist formulated as a synthetic peptide containing 39 amino acids, based on the native GIP sequence. Pre-clinical trials and phase 1 and 2 clinical trials indicate that tirzepatide has potent glucose lowering and weight loss with adverse effects comparable to those of established GLP-1 receptor agonists. The long-term efficacy, safety and cardiovascular outcomes of tirzepatide will be investigated in the SURPASS phase 3 clinical trial programme. In this paper, we will review the pre-clinical and phase 1 and 2 trials for tirzepatide in the management of T2DM and give an overview of the SURPASS clinical trials. Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r-null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide. BACKGROUND: Tirzepatide is a novel dual glucose-dependent insulinotropic polypeptide and GLP-1 receptor agonist under development for the treatment of type 2 diabetes. We aimed to assess the efficacy and safety of tirzepatide versus titrated insulin degludec in people with type 2 diabetes inadequately controlled by metformin with or without SGLT2 inhibitors. METHODS: In this open-label, parallel-group, multicentre (122 sites), multinational (13 countries), phase 3 study, eligible participants (aged ≥18 years) had a baseline glycated haemoglobin (HbA1c) of 7·0-10·5%, body-mass index of at least 25 kg/m2, stable weight, and were insulin-naive and treated with metformin alone or in combination with an SGLT2 inhibitor for at least 3 months before screening. Participants were randomly assigned (1:1:1:1), using an interactive web-response system, to once-weekly subcutaneous injection of tirzepatide (5, 10, or 15 mg) or once-daily subcutaneous injection of titrated insulin degludec, and were stratified by country, HbA1c, and concomitant use of oral antihyperglycaemic medications. Tirzepatide was initially given at 2·5 mg and the dose was escalated by 2·5 mg every 4 weeks until the assigned dose was reached. Insulin degludec was initially given at 10 U per day and was titrated once weekly to a fasting self-monitored blood glucose of less than 5·0 mmol/L (<90 mg/dL), following a treat-to-target algorithm, for 52 weeks. The primary efficacy endpoint was non-inferiority of tirzepatide 10 mg or 15 mg, or both, versus insulin degludec in mean change from baseline in HbA1c at week 52. Key secondary efficacy endpoints were non-inferiority of tirzepatide 5 mg versus insulin degludec in mean change from baseline in HbA1c at week 52, superiority of all doses of tirzepatide versus insulin degludec in mean change from baseline in HbA1c and bodyweight, and the proportion of participants achieving HbA1c of less than 7·0% (<53 mmol/mol) at week 52. We used a boundary of 0·3% to establish non-inferiority in HbA1c difference between treatments. Efficacy and safety analyses were assessed in the modified intention-to-treat population (all participants who received at least one dose of study drug). This trial is registered with ClinicalTrials.gov, number NCT03882970, and is complete. FINDINGS: Between April 1 and Nov 15, 2019, we assessed 1947 participants for eligibility, 1444 of whom were randomly assigned to treatment. The modified intention-to-treat population was 1437 participants from the tirzepatide 5 mg (n=358), tirzepatide 10 mg (n=360), tirzepatide 15 mg (n=359), and insulin degludec (n=360) groups. From a mean baseline HbA1c of 8·17% (SD 0·91), the reductions in HbA1c at week 52 were 1·93% (SE 0·05) for tirzepatide 5 mg, 2·20% (0·05) for tirzepatide 10 mg, and 2·37% (0·05) for tirzepatide 15 mg, and 1·34% (0·05) for insulin degludec. The non-inferiority margin of 0·3% was met. The estimated treatment difference (ETD) versus insulin degludec ranged from -0·59% to -1·04% for tirzepatide (p<0·0001 for all tirzepatide doses). The proportion of participants achieving a HbA1c of less than 7·0% (<53 mmol/mol) at week 52 was greater (p<0·0001) in all three tirzepatide groups (82%-93%) versus insulin degludec (61%). At week 52, from a baseline of 94·3 kg (SD 20·1), all three tirzepatide doses decreased bodyweight (-7·5 kg to -12·9 kg), whereas insulin degludec increased bodyweight by 2·3 kg. The ETD versus insulin degludec ranged from -9·8 kg to -15·2 kg for tirzepatide (p<0·0001 for all tirzepatide doses). The most common adverse events in tirzepatide-treated participants were mild to moderate gastrointestinal events that decreased over time. A higher incidence of nausea (12-24%), diarrhoea (15-17%), decreased appetite (6-12%), and vomiting (6-10%) was reported in participants treated with tirzepatide than in those treated with insulin degludec (2%, 4%, 1%, and 1%, respectively). Hypoglycaemia (<54 mg/dL or severe) was reported in five (1%), four (1%), and eight (2%) participants on tirzepatide 5, 10, and 15 mg, respectively, versus 26 (7%) on insulin degludec. Treatment discontinuation due to an adverse event was more common in the tirzepatide groups than in the insulin degludec group. Five participants died during the study; none of the deaths were considered by the investigators to be related to the study treatment. INTERPRETATION: In patients with type 2 diabetes, tirzepatide (5, 10, and 15 mg) was superior to titrated insulin degludec, with greater reductions in HbA1c and bodyweight at week 52 and a lower risk of hypoglycaemia. Tirzepatide showed a similar safety profile to that of GLP-1 receptor agonists. FUNDING: Eli Lilly and Company. Publisher: Les agonistes des récepteurs du glucagon-like peptide-1 (GLP-1) sont les agents antidiabétiques qui, outre une baisse importante du taux d’hémoglobine glyquée, offrent la perte pondérale la plus marquée, en augmentant la satiété par un effet central, prédomit et périphérique. Le liraglutide et le sémaglutide sont développés pour le traitement de l’obésité, indépendamment de la présence d’un diabète de type 2. Trois approches sont possibles pour potentialiser la perte de poids: augmenter la posologie, compte tenu de l’existence d’une relation dose-réponse, ajouter un inhibiteur des sodium-glucose cotransporteurs 2 qui exerce un effet complémentaire grâce à une fuite calorique urinaire (glucosurie), ou encore combiner les effets de deux hormones incrétines (GLP-1 et Glucose-dependent Insulin Releasing Polypeptide), comme avec le puissant double agoniste tirzépatide en développement. There is a new generation of antiobesity drugs in development or just arriving on the scene. First, setmelanotide has been approved for three of the ultrarare genetic conditions that cause obesity-pro-opiomelanocortin deficiency, proprotein convertase subtilisin and kexin type 1 (an important enzyme in the melanocortin pathway) and leptin receptor deficiency. Setmelanotide marks the first in a personalized medicine approach to obesity. Second, semaglutide 2.4 mg once weekly has been submitted to regulators in the United States and the European Union for approval for patients with obesity (body mass index [BMI] ≥30 kg/m2) or overweight (BMI ≥27 kg/m2) and at least one weight related comorbidity. This drug has been studied in five phase 3 clinical trials, four discussed herein: semaglutide produces roughly twice as much weight loss as we have seen in older antiobesity medications. Semaglutide is already in use for treatment of diabetes and, as a glucagon-like peptide 1 (GLP-1) receptor analog, is part of a class of drugs used widely in diabetes. Tirzepatide, a glucose-insulin peptide and GLP-1 dual agonist is in phase 3 study for obesity management, and bimagrumab is a new agent in phase 2 with a unique mechanism of action; they are generating much interest. The purpose of this narrative review is lay the groundwork for a discussion of the clinical impact of these new medications on the clinical practice of obesity. Further, these developments shall be used to launch a speculation of what is likely to be their impact on the future of obesity pharmacotherapy. In a phase 2 trial of once-weekly tirzepatide (1, 5, 10, or 15 mg), dulaglutide (1.5 mg), or placebo, the dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist tirzepatide dose-dependently reduced HbA1c and body weight in patients with type 2 diabetes. In this post hoc analysis, inflammation, endothelial dysfunction, and cellular stress biomarkers were measured at baseline, 4, 12, and 26 weeks to evaluate the additional effects of tirzepatide on cardiovascular risk factors. At 26 weeks, tirzepatide 10 and 15 mg decreased YKL-40 (also known as chitinase-3 like-protein-1), intercellular adhesion molecule 1 (ICAM-1), leptin, and growth differentiation factor 15 levels versus baseline, and YKL-40 and leptin levels versus placebo and dulaglutide. Tirzepatide 15 mg also decreased ICAM-1 levels versus placebo and dulaglutide, and high-sensitivity C-reactive protein (hsCRP) levels versus baseline and placebo, but not dulaglutide. GlycA, interleukin 6, vascular cell adhesion molecule 1, and N-terminal-pro hormone B-type natriuretic peptide levels were not significantly changed in any group. YKL-40, hsCRP, and ICAM-1 levels rapidly decreased within 4 weeks of treatment with tirzepatide 10 and 15 mg, whereas the decrease in leptin levels was more gradual and did not plateau by 26 weeks. In this hypothesis-generating exploratory analysis, tirzepatide decreased several biomarkers that have been associated with cardiovascular risk. CONTEXT: Tirzepatide substantially reduced hemoglobin A1c (HbA1c) and body weight in subjects with type 2 diabetes (T2D) compared with the glucagon-like peptide 1 receptor agonist dulaglutide. Improved glycemic control was associated with lower circulating triglycerides and lipoprotein markers and improved markers of beta-cell function and insulin resistance (IR), effects only partially attributable to weight loss. OBJECTIVE: Assess plasma metabolome changes mediated by tirzepatide. DESIGN: Phase 2b trial participants were randomly assigned to receive weekly subcutaneous tirzepatide, dulaglutide, or placebo for 26 weeks. Post hoc exploratory metabolomics and lipidomics analyses were performed. SETTING: Post hoc analysis. PARTICIPANTS: 259 subjects with T2D. INTERVENTION(S): Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), or placebo. MAIN OUTCOME MEASURE(S): Changes in metabolite levels in response to tirzepatide were assessed against baseline levels, dulaglutide, and placebo using multiplicity correction. RESULTS: At 26 weeks, a higher dose tirzepatide modulated a cluster of metabolites and lipids associated with IR, obesity, and future T2D risk. Branched-chain amino acids, direct catabolic products glutamate, 3-hydroxyisobutyrate, branched-chain ketoacids, and indirect byproducts such as 2-hydroxybutyrate decreased compared to baseline and placebo. Changes were significantly larger with tirzepatide compared with dulaglutide and directly proportional to reductions of HbA1c, homeostatic model assessment 2-IR indices, and proinsulin levels. Proportional to metabolite changes, triglycerides and diglycerides were lowered significantly compared to baseline, dulaglutide, and placebo, with a bias toward shorter and highly saturated species. CONCLUSIONS: Tirzepatide reduces body weight and improves glycemic control and uniquely modulates metabolites associated with T2D risk and metabolic dysregulation in a direction consistent with improved metabolic health. Tirzepatide is a dual gastric inhibitory peptide/glucagon-like peptide 1 (GIP/GLP-1) receptor agonist formulated as a synthetic linear peptide, based on the native GIP sequence. It has a prolonged half-life of 5 days, which enables once-weekly dosing. Studies have hitherto demonstrated its superiority in achieving optimal glycaemic control and body weight management, as compared with various agents used in the treatment of type 2 diabetes mellitus (T2DM), including GLP-1 receptor agonists. Thus, it is expected to enrich our therapeutic armamentarium in T2DM. However, further experience, notably longer follow-up data and information on cardiovascular effects, is still needed. Tirzepatide is a novel once-a-week dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist, currently under trial to assess glycemic efficacy and safety in people with type 2 diabetes. A systematic review and meta-analysis were conducted to investigate the efficacy of tirzepatide on glycated hemoglobin (HbA1c, %), fasting serum glucose (mg/dL), and body weight (kg) in patients with uncontrolled type 2 diabetes (HbA1c > 7.0%). Mean changes for efficacy and proportions (safety) with corresponding 95% confidence intervals (CIs) were used to provide pooled estimates. A total of four randomized controlled trials, comprising 2783 patients of whom 69.4% (n = 1934) were treated with 5 mg (n = 646), 10 mg (n = 641), or 15 mg (n = 647) of tirzepatide, were compared to the placebo (n = 192) or the selective GLP-1 receptor agonist (n = 523). The pooled analysis showed that tirzepatide treatment resulted in a greater lowering of the HbA1c (-1.94%, 95% CI: -2.02 to -1.87), fasting serum glucose (-54.72 mg/dL, 95% CI: -62.05 to -47.39), and body weight (-8.47, 95% CI: -9.66 to -7.27). We also found that improvement in the HbA1c levels was still maintained at weeks 26 and 40 from the long-term trials. As for safety, only 3% experienced hypoglycemia, and 4% (95% CI: 2 to 6) experienced serious adverse events, while the discontinuation of therapy percentage was 7% (95% CI: 5 to 8). Tirzepatide significantly improved glycemic control and body weight and had an acceptable safety profile, indicating that it is an effective therapeutic option for glucose-lowering in patients with type 2 diabetes mellitus. Incretin hormones are peptides released in the intestine in response to the presence of nutrients in its lumen. The main incretins are glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 stimulates insulin secretion, inhibits glucagon secretion at pancreatic α cells and has also extrapancreatic influences as slowing of gastric emptying which increases the feeling of satiety. GIP is the main incretin hormone in healthy people, causative of most the incretin effects, but the insulin response after GIP secretion in type 2 diabetes mellitus (T2DM) is strongly reduced. Therefore, in the past GIP has been considered an unappealing therapeutic target for T2DM. This conception has been changing during recent years, since it has been reported that resistance to GIP can be reversed and its effectiveness restored by improving glycemic control. This fact paved the way for the development of a GIP receptor agonist-based therapy for T2DM, looking also for the possibility of finding a combined GLP-1/GIP receptor agonist. In this framework, the novel dual GIP and GLP-1 receptor agonist tirzepatide seems to be not just a new antidiabetic medication. Administered as a subcutaneous weekly injection, it is a manifold single pharmacological agent that has the ability to significantly lower glucose levels, as well as improve insulin sensitivity, reduce weight and amend dyslipidemia favorably modifying the lipid profile. Tirzepatide and additional dual GLP-1/GIP receptor agonists that could eventually be developed in the future seem to be a promising furthest advance for the management of several cardiometabolic settings. Obviously, it is too early to be overly hopeful since it is still necessary to determine the long-term effects of these compounds and properly verify the potential cardiovascular benefits. Anyway, we are currently facing a novel and very appealing therapeutic option.

Is esophageal adenocarcinoma associated with aberrant glycosylation?

Yes, Altered glycoprotein expression has been demonstrated in tissue from patients with Barrett's esophagus and esophageal cancer but the mechanisms regarding such changes are unknown.

Aberrant glycosylation has been implicated in various types of cancers and changes in glycosylation may be associated with signaling pathways during maligt transformation. Glycomic profiling of blood serum, in which cancer cell proteins or their fragments with altered glycosylation patterns are shed, could reveal the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease (N = 18), patients with high grade dysplasia (HGD, N = 11) and Barrett's esophagus (N = 5), and patients with esophageal adenocarcinoma (EAC, N = 50) in an attempt to delineate distinct differences in glycosylation between these groups. The relative intensities of 98 features were significantly different among the disease onsets; 26 of these correspond to known glycan structures. The changes in the relative intensities of three of the known glycan structures predicted esophageal adenocarcinoma with 94% sensitivity and better than 60% specificity as determined by receiver operating characteristic (ROC) analysis. We have demonstrated that comparative glycomic profiling of EAC reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate disease-free from HGD, disease-free from EAC, and HGD from EAC. The clinical utility of these glycan biomarkers requires further validation. It is generally accepted that esophageal adenocarcinoma arises from a Barrett's metaplastic lesion. Altered glycoprotein expression has been demonstrated in tissue from patients with Barrett's esophagus and esophageal cancer but the mechanisms regarding such changes are unknown. The bile acid deoxycholic acid (DCA) alters many cell signaling pathways and is implicated in esophageal cancer progression. We have demonstrated that DCA disrupts Golgi structure and affects protein secretion and glycosylation processes in cell lines derived from normal squamous epithelium (HET-1A) and Barrett's metaplastic epithelium (QH). Cell surface expression of glycans was identified using carbohydrate-specific probes (wheat germ agglutinate, conconavalin A, peanut agglutinin, lithocholic acid and Ulex europaeus agglutinin) that monitored N-glycosylation, O-glycosylation and core fucosylation in resting and DCA-treated cells. DCA altered intracellular localization and reduced cell surface expression of N-acetyl-D-glucosamine, α-methyl-mannopyranoside (Man/Glc) and fucose in both cell lines. Furthermore, DCA reduced the expression of epithelial growth factor receptor and E-cadherin in a manner analogous to treatment of cells with the N-glycan biosynthesis inhibitor tunicamycin. This is the first study to identify an altered Golgi structure and glycomic profile in response to DCA in esophageal epithelial cells, a process which could potentially contribute to metaplasia, dysplasia and cancer of the esophagus. Glycosylation is among the most common post translation modifications of proteins in humans. Decades of research have demonstrated that aberrant glycosylation can lead to maligt degeneration. Glycoproteomic studies in the past several years have identified techniques that can successfully characterize a glycan or glycan profile associated with a high-grade dysplastic or maligt state. This review summarizes the current glycomic and glycoproteomic literature with specific reference to esophageal cancer. Esophageal adenocarcinoma represents a highly morbid and mortal cancer with a defined progression from metaplasia (Barrett's esophagus) to dysplasia to neoplasia. This disease is highlighted because (1) differences in glycan profiles between the stages of disease progression have been described in the glycoproteomic literature; (2) a glycan biomarker that identifies a given stage may be used as a predictor of disease progression and thus may have significant influence over clinical management; and (3) the differences in glycan profiles between disease and disease-free states in esophageal cancer are more dramatic than in other cancers. This study aimed to investigate the association of IgG glycosylation and esophageal precancerosis for squamous cell carcinoma and determine its role in inflammation. Primary glycans selected by the least absolute shrinkage and selection operator (LASSO) algorithm were validated using univariate and multivariate logistics models plus restricted cubic spline functions. In total, 24 direct glycans and 27 derived traits were detected, among which four glycans and three derived traits were primarily selected. Then, GP5 (adjusted OR: 0.805), GP17 (adjusted OR: 1.305), G12n (adjusted OR: 1.271), Gal_1 (adjusted OR: 0.776) and Fuc (adjusted OR: 0.737) were validated and significantly associated with esophageal precancerosis. In addition, there was a consistent positive association in GP17 and G12n and a negative association in GP5, Gal_1, and Fuc by restricted cubic spline function. Compared with esophageal inflammation, GP17, G12n, and Fuc were still independently associated with precancerosis. In brief, the IgG glycosylation profile was independently associated with esophageal precancerosis beyond inflammation, which could be an early biomarker for esophageal cancer.Prevention Relevance: IgG glycosylation profile is associated with esophageal precancerosis and specific IgG glycans involves in the early stage of esophageal cancer, which is independent of inflammation.

What is the function of a chaperonin?

Molecular chaperones promote the correct folding of proteins in aggregation-prone cellular environments by stabilizing nascent polypeptide chains and providing appropriate folding conditions. They are involved in the development of pathological processes, including--atherosclerosis and coronary heart disease.

BACKGROUND: Mitochondria are known to be a major target during ischemic cardiac injury. Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which the heat shock or stress protein 70 is increased, there is a marked tolerance to ischemia/reperfusion injury. Two other heat shock proteins (HSP60 and HSP10) are known to form, within the mitochondria, a chaperonin complex that is important for mitochondrial protein folding and function. We were then interested in investigating whether increased expression of these two stress proteins is able to protect myogenic cells against ischemia/reperfusion injury. METHODS AND RESULTS: We generated recombit adenoviral vectors containing HSP60, HSP10, or a combination of the two genes. These adenoviral constructs overexpress significant amounts of these stress proteins in both rat neonatal cardiomyocytes and the myogenic H9 c2 cell line. Cells infected with an adenoviral construct overexpressing both HSP60 and HSP10 were found to be protected against simulated ischemia, whereas cells infected with adenoviral constructs overexpressing only HSP60 or HSP10 alone were not rendered tolerant to simulated ischemic injury. CONCLUSIONS: These results suggest that the simultaneous expression of these two proteins that form a chaperonin complex in the mitochondria plays an important role in the survival of myogenic cells after ischemia/reperfusion injury. Co-chaperonins from diverse organisms exhibit mobile loops which fold into a beta hairpin conformation upon binding to the chaperonin. GroES, Gp31, and human Hsp10 mobile loops exhibit a preference for the beta hairpin conformation in the free co-chaperonins, and the conformational dynamics of the human Hsp10 mobile loop appear to be restricted by nascent hairpin formation. Backbone conformational entropy must weigh against binding of co-chaperonins to chaperonins, and thus the conformational preferences of the loops may strongly influence chaperonin-binding affinity. Indeed, subtle mutations in the loops change GroEL-binding affinity and cause defects in chaperonin function, and these defects can be suppressed by mutations in GroEL which compensate for the changes in affinity. The fact that high-affinity co-chaperonin binding impairs chaperonin function has implications for the mechanism of chaperonin-assisted protein folding. Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia. Chaperonins are large oligomers made up of two superimposed rings, each enclosing a cavity used for the folding of other proteins. Among the chaperonins, the eukaryotic cytosolic chaperonin CCT is the most complex, not only with regard to its subunit composition but also with respect to its function, still not well understood. Unlike the more well studied eubacterial chaperonin GroEL, which binds any protein that presents stretches of hydrophobic residues, CCT recognises in its substrates specific binding determits and interacts with them through particular combinations of CCT subunits. Folding then occurs after the conformational changes induced in the chaperonin upon nucleotide binding have occurred, through a mechanism that, although still poorly defined, clearly differs from the one established for GroEL. Although CCT seems to be mainly involved in the folding of actin and tubulin, other substrates involved in various cellular roles are beginning to be characterised, including many WD40-repeat, 7-blade propeller proteins. Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like structure whose function is to deliver nonnative target proteins, principally actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated folding. Here we demonstrate that functional PFD can spontaneously assemble from its six constituent individual subunits (PFD1-PFD6), each expressed as a recombit protein. Using engineered forms of PFD assembled in vitro, we show that the tips of the PFD tentacles are required to form binary complexes with authentic target proteins. We show that PFD uses the distal ends of different but overlapping sets of subunits to form stable binary complexes with different target proteins, namely actin and alpha- and beta-tubulin. We also present data that suggest a model for the order of these six subunits within the hexamer. Our data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic chaperonin, has co-evolved specifically to facilitate the folding of its target proteins. Chaperonins assist in the acquisition of native protein structure in the cell by providing a shielded environment for a folding polypeptide chain, generated by the interior surface of their cylindrical structure. The folding chain is isolated from the highly crowded cytoplasm, but at the same time confined within the chaperonin folding cage. Both confinement and macromolecular crowding can affect folding kinetics and yields, the modus operandi of chaperonins and their interaction with their protegés. Recent experimental data, as well as computer simulations, provide increasing evidence that the particular physico-chemical conditions prevailing in the cellular interior have to be taken into account when trying to unravel the processes of cellular protein folding. The major heat shock protein, chaperonin 60, has been established to have intercellular signaling activity in addition to its established protein-folding function. Mycobacterium tuberculosis is one of a small proportion of bacteria to encode two chaperonin 60 proteins. We have demonstrated that chaperonin 60.1 from this bacterium is a very active stimulator of human monocytes. To determine structure/function relationships of chaperonin 60.1 we have cloned and expressed the apical, equatorial, and intermediate domains of this protein. We have found that the signaling activity of M. tuberculosis chaperonin 60.1 resides in the equatorial domain. This activity of the recombit equatorial domain was completely blocked by treating the protein with proteinase K, ruling out lipopolysaccharide contamination as the cause of the cell activation. Blockade of the activity of the equatorial domain by anti-CD14 monoclonal antibodies reveals that this domain activates monocytes by binding to CD14. Looking at the oligomeric state of the active proteins, using native gel electrophoresis and protein cross-linking we found that recombit M. tuberculosis chaperonin 60.1 fails to form the prototypic tetradecameric structure of chaperonin 60 proteins under the conditions tested and only forms dimers. It is therefore concluded that the monocyte-stimulating activity of M. tuberculosis Cpn60.1 resides in the monomeric subunit and within this subunit the biological activity is due to the equatorial domain. Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable "lid" structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determits for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding? The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division. Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome-wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well-conserved proteins with complex alpha/beta domains. In contrast, thermosome substrates comprise a group of faster-evolving proteins and contain a much wider range of different domain folds, including small all-alpha and all-beta modules, and a greater number of large multidomain proteins. Thus, the group II chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells. It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as oboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones. BACKGROUND: Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE) in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear. RESULTS: We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history. CONCLUSIONS: This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems. Chaperonins are universally conserved molecular machines that facilitate the proper -folding of nascent and partially folded polypeptides into their respective three-dimensional structures. These multimeric protein complexes utilize the energy derived from ATP hydrolysis to fuel a protein-folding mechanism that consists of multiple rounds of substrate binding, encapsulation, and eventual expulsion back into the cytosolic environment. In this portion of the chapter, the structure and function of group I and group II chaperonins are discussed. Furthermore, the general mechanism of chaperonin-mediated protein folding is addressed in addition to illustrating how viral phages such as Lambda, T4, and RB49 exploit the host machinery for the proper folding of viral gene products. Lastly, the phiEL chaperonin from phage EL is revealed to be the first virally encoded chaperonin and is proposed to function independently of the host chaperonin machinery. The molecular architecture of the phiEL chaperonin, coupled with its unique functional abilities, renders its characterization a challenge and further highlights its novelty as a potentially whole new class of chaperonins. Chaperonins are a class of molecular chaperones that assemble into a large double ring architecture with each ring constituting seven to nine subunits and enclosing a cavity for substrate encapsulation. The well-studied Escherichia coli chaperonin GroEL binds non-native substrates and encapsulates them in the cavity thereby sequestering the substrates from unfavorable conditions and allowing the substrates to fold. Using this mechanism, GroEL assists folding of about 10-15 % of cellular proteins. Surprisingly, about 30 % of the bacteria express multiple chaperonin genes. The presence of multiple chaperonins raises questions on whether they increase general chaperoning ability in the cell or have developed specific novel cellular roles. Although the latter view is widely supported, evidence for the former is beginning to appear. Some of these chaperonins can functionally replace GroEL in E. coli and are generally indispensable, while others are ineffective and likewise are dispensable. Additionally, moonlighting functions for several chaperonins have been demonstrated, indicating a functional diversity among the chaperonins. Furthermore, proteomic studies have identified diverse substrate pools for multiple chaperonins. We review the current perception on multiple chaperonins and their physiological and functional specificities. In review provides information about the function oft the body of chaperones and their role in the development of pathological processes, including--atherosclerosis and coronary heart disease. Marked comminications systems chaperones to the immune and endocrine systems, and inflammation. Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (>70 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells. Chaperonins are a subclass of molecular chaperones that assist cellular proteins to fold and assemble into their native shape. Much work has been done on Type I chaperonins, which has elucidated their elegant mechanism. Some debate remains about the details in these mechanisms, but nonetheless the roles of these in helping protein folding have been understood in great depth. In this review we discuss the known functions of atypical Type I chaperonins, highlighting evolutionary aspects that might lead chaperonins to perform alternate functions. Molecular chaperones promote the correct folding of proteins in aggregation-prone cellular environments by stabilizing nascent polypeptide chains and providing appropriate folding conditions. Prefoldins (PFDs) are molecular chaperones found in archaea and eukaryotes, generally characterized by a unique jellyfish-like hexameric structure consisting of a rigid beta-barrel backbone with protruding flexible coiled-coils. Unlike eukaryotic PFDs that mainly interact with cytoskeletal components, archaeal PFDs can stabilize a wide range of substrates; such versatility reflects PFD's role as a key element in archaeal chaperone systems, which often lack general nascent-chain binding chaperone components such as Hsp70. While archaeal PFDs mainly exist as hexameric complexes, their structural diversity ranges from tetramers to filamentous oligomers. PFDs bind and stabilize nonnative proteins using varying numbers of coiled-coils, and subsequently transfer the substrate to a group II chaperonin (CPN) for refolding. The distinct structure and specific function of archaeal PFDs have been exploited for a broad range of applications in biotechnology; furthermore, a filament-forming variant of PFD has been used to fabricate oscale architectures of defined shapes, demonstrating archaeal PFDs' potential applicability in otechnology. Molecular chaperones are key players in proteostasis, the balance between protein synthesis, folding, assembly and degradation. They are helped by a plethora of cofactors termed cochaperones, which direct chaperones towards any of these different, sometime opposite pathways. One of these is prefoldin (PFD), present in eukaryotes and in archaea, a heterohexamer whose best known role is the assistance to group II chaperonins (the Hsp60 chaperones found in archaea and the eukaryotic cytosolic) in the folding of proteins in the cytosol, in particular cytoskeletal proteins. However, over the last years it has become evident a more complex role for this cochaperone, as it can adopt different oligomeric structures, form complexes with other proteins and be involved in many other processes, both in the cytosol and in the nucleus, different from folding. This review intends to describe the structure and the many functions of this interesting macromolecular complex.

Can FTO promote pancreatic cancer development?

No, the m6A demethylase FTO suppresses pancreatic cancer tumorigenesis.

Pancreatic cancer is the deadliest maligcy of the digestive system and is the seventh most common cause of cancer-related deaths worldwide. The incidence and mortality of pancreatic cancer continue to increase, and its 5-year survival rate remains the lowest among all cancers. N6-methyladenine (m6A) is the most abundant reversible RNA modification in various eukaryotic messenger and long noncoding RNAs and plays crucial roles in the occurrence and development of cancers. However, the role of m6A in pancreatic cancer remains unclear. The present study aimed to explore the role of m6A and its regulators in pancreatic cancer and assess its underlying molecular mechanism associated with pancreatic cancer cell proliferation, invasion, and metastasis. Reduced expression of the m6A demethylase, fat mass and obesity-associated protein (FTO), was responsible for the high levels of m6A RNA modification in pancreatic cancer. Moreover, FTO demethylated the m6A modification of praja ring finger ubiquitin ligase 2 (PJA2), thereby reducing its mRNA decay, suppressing Wnt signaling, and ultimately restraining the proliferation, invasion, and metastasis of pancreatic cancer cells. Altogether, this study describes new, potential molecular therapeutic targets for pancreatic cancer that could pave the way to improve patient outcome.

Which disorder is caused by biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1)?

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB).

Author information: (1)Division of Molecular Medicine, Leeds Institute of Medical Research, University of Leeds, Leeds, UK. (2)School of Molecular and Cellular Biology, University of Leeds, Leeds, UK. (3)Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicines and Health, University of Manchester, Manchester, UK. (4)National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Centre, Tokyo, Japan. (5)Moorfields Eye Hospital, London, UK. (6)UCL Institute of Ophthalmology, London, UK. (7)Keio University School of Medicine, Tokyo, Japan. (8)Ghent University, Ghent, Belgium. (9)Division of Genetic Medicine, Centre Hospitalier Universitaire Vaudois (CHUV), University of Lausanne, Lausanne, Switzerland. (10)The Jikei University School of Medicine, Tokyo, Japan. (11)Mie University Graduate School of Medicine, Mie, Japan. (12)Department of Genetics, Faculty of Science, Hazara University Mansehra, Dhodial, Pakistan. (13)Clinical Research Center, Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland. (14)School of Biomedical Sciences, University of Leeds, Leeds, UK. (15)Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. (16)St Thomas's Hospital, London, UK. (17)Department of Ophthalmology, Great Ormond Street Hospital, London, UK. (18)Department of Genetics and Genome Biology, University of Leicester, Leicester, UK. (19)Department of Ophthalmology, University Hospital Basel, Basel, Switzerland. (20)Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France. (21)Manchester Centre for Genomic Medicine, Saint Mary's Hospital, Manchester University NHS Foundation Trust, Manchester, UK. (22)Leeds Teaching Hospitals NHS Trust, St James' University Hospital, Leeds, UK. (23)School of Physics and Astronomy, University of Leeds, Leeds, UK.

Which disease is treated with Tebentafusp?

Tebentafusp is used for treatment of Metastatic Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular maligcy in adults. UMs are usually initiated by a mutation in GNAQ or GNA11, unlike cutaneous melanomas, which usually harbour a BRAF or NRAS mutation. The annual incidence in Europe and the USA is ~6 per million population per year. Risk factors include fair skin, light-coloured eyes, congenital ocular melanocytosis, ocular melanocytoma and the BAP1-tumour predisposition syndrome. Ocular treatment aims at preserving the eye and useful vision and, if possible, preventing metastases. Enucleation has largely been superseded by various forms of radiotherapy, phototherapy and local tumour resection, often administered in combination. Ocular outcomes are best with small tumours not extending close to the optic disc and/or fovea. Almost 50% of patients develop metastatic disease, which usually involves the liver, and is usually fatal within 1 year. Although UM metastases are less responsive than cutaneous melanoma to chemotherapy or immune checkpoint inhibitors, encouraging results have been reported with partial hepatectomy for solitary metastases, with percutaneous hepatic perfusion with melphalan or with tebentafusp. Better insight into tumour immunology and metabolism may lead to new treatments. PURPOSE OF REVIEW: Uveal melanoma is a distinct subtype of melanoma characterized by a unique biology and divergent response to immune therapies. In this review, we will discuss our current understanding of the pathophysiology of uveal melanoma, systemic treatment options for advanced disease, and potential future therapeutic directions. RECENT FINDINGS: Although treatment with single-agent checkpoint blockade has been generally disappointing, the results of combined checkpoint blockade are modestly more promising. Several alternative systemic therapeutic approaches have been or are currently being investigated, including two agents that have been taken into registration-intent clinical trials: tebentafusp, a T cell redirecting agent, and IDE196, an oral protein kinase C inhibitor. Treatment of advanced uveal melanoma remains challenging, however, encouraging results from novel agents offer hope for improvement in the near future. PURPOSE: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp. PATIENTS AND METHODS: Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment. RESULTS: Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway-related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival. CONCLUSIONS: These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp. A bispecific fusion protein designed to redirect T cells toward a melanoma-associated antigen helped prolong survival among patients with an aggressive form of eye cancer. If approved, tebentafusp could become the standard of care for metastatic uveal melanoma-but only for those patients with a particular HLA allele. Author information: (1)From Mount Vernon Cancer Centre, Northwood (P.N.), the Clatterbridge Cancer Centre NHS Foundation Trust, Wirral (J.J.S.), the University of Liverpool, Liverpool (J.J.S.), and Immunocore, Abingdon (S.E.A., C.H., H.G.) - all in the United Kingdom; the Department of Dermatology and the National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg (J.C.H.), the Department of Dermatology and Allergy, University Hospital, Ludwig Maximilian University of Munich, Munich (M.S.), the Department of Hematology and Oncology, Charité-Comprehensive Cancer Center (S.O.), Berlin, and the Department of Dermatology and the Center for Integrated Oncology, University Hospital Cologne, Cologne (C.M.) - all in Germany; Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland (P.R.); Institut Roi Albert II des Cliniques Universitaires Saint-Luc and Université Catholique de Louvain, Brussels (J.-F.B.); Princess Margaret Cancer Centre, Toronto (M.O.B.); Massachusetts General Hospital Cancer Center, Boston (R.J.S.); the Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland (R.D.); Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh (J.M.K.); Sidney Kimmel Cancer Center, Thomas Jefferson University Hospital, Philadelphia (M.O.); Kinghorn Cancer Centre, Saint Vincent's Hospital, Darlinghurst, NSW, Australia (A.M.J.); Memorial Sloan Kettering Cancer Center (A.N.S.) and Irving Medical Center, Columbia University (R.D.C.) - both in New York; Institut d'Investigació Biomèdica de Bellvitge-Centro de Investigación Biomédica en Red de Oncología, Institut Català d'Oncologia, Barcelona (J.M.P.); University of Iowa Hospitals and Clinics, Iowa City (M.M.); Duke University, Durham, NC (A.K.S.S.); Earle A. Chiles Research Institute, Providence Cancer Institute, Portland, OR (B.C.); N.N. Blokhin Cancer Research Center, Moscow (L.D.); Centre Antoine Lacassagne, Nice (L.G.) and Institut Curie, Paris Sciences and Letters Research University, Paris (S.P.-N.) - both in France; Winship Cancer Institute, Emory University, Atlanta (M.Y.); and the Angeles Clinic and Research Institute, a Cedars-Sinai Affiliate, Los Angeles (O.H.). Uveal melanoma is a rare neoplasm with poor prognosis in the metastatic setting. Unlike cutaneous melanoma, treatment with kinase inhibitors or immune checkpoint inhibitors is not effective. Glycoprotein 100 (Gp-100) is a protein highly expressed in melanocytes and melanoma that has recently been effectively targeted by tebentafusp, a first-in-class bispecific protein of the immune-mobilizing monoclonal T cell receptors against cancer (ImmTACs) family. Tebentafusp targets tumor cells that express a peptide of Gp-100 presented by HLA*A0201, creating an immune synapse that kills targeted tumor cells. Recently, a randomized phase III trial reported an overall survival benefit for tebentafusp in patients with untreated metastatic uveal melanoma. The aim of this comprehensive review is to summarize evidence of Gp-100 as a therapeutic target in melanoma, and the preclinical and clinical development of tebentafusp as a novel therapeutic strategy for patients with uveal melanoma.

Is Lysozyme abundant in human tears?

Yes, lysozyme is the most prevalent protein in tear fluid.

We studied the behaviour of three lipid tear products, commercialised by the same brand, as Langmuir films at the air/liquid interface to simulate the ocular environment. No significant differences were observed in the surface behaviour of two of them disclosing the same composition, but commercialised for different applications. The interaction of several subphases, namely sodium chloride, glucose, albumin and lysozyme present in the natural tear, with the lipid films was assessed at room temperature and the temperature of human tear using surface pressure-area isotherms and elastic modulus plots. There is a notable influence of sodium chloride and the proteins albumin and lysozyme on the surface pressure-area isotherm of the lipid Langmuir films. Albumin shifted this isotherm to lower areas while an opposite shift was caused by lysozyme. These studies could be useful for the formulation of new lipid-containing artificial tears, and for increasing the confidence of the customers in commercial eye care formulations. Lysozyme (Lyz) is a naturally occurring enzyme that operates against Gram-positive bacteria and leads to cell death. This antimicrobial enzyme forms the part of the innate defense system of nearly all animals and exists in their somatic discharges such as milk, tears, saliva and urine. Increased Lyz level in serum is an important indication of several severe diseases and so, precise diagnosis of Lyz is an urgent need in biosensing assays. Up to know, various traditional and modern techniques have been introduced for Lyz determination. Although the traditional methods suffer from some significant limitations such as time-consuming, arduous, biochemical screening, bacterial colony isolation, selective enrichment and requiring sophisticated instrumentation or isotope labeling, some new modern approaches like aptamer-based biosensors (aptasensors) and quantum dot (QD) omaterials are the main goal in Lyz detection. Electrochemical and optical sensors have been highlighted because of their adaptability and capability to decrease the drawbacks of common methods. Using an aptamer-based biosensor, sensor selectivity is enhanced due to the specific recognition of the analyte. Thereby, in this review article, the recent advances and achievements in electrochemical and optical aptasensing detection of Lyz based on different QD omaterials and detection methods have been discussed in detail. PURPOSE: To evaluate the concentration of tear lysozyme in individuals with Sjogren´s syndrome, meibomian gland dysfunction, and non-dry-eye disease. METHODS: Ninety subjects were recruited for this study, including 30 with Sjogren´s syndrome, 30 with meibomian gland dysfunction, and 30 with non-dry-eye disease. All subjects were referred to participate in the study based on a "dry eye" investigation. They underwent a complete ocular surface ophthalmic examination encompassing ocular surface disease index, biomicroscopy, tear break-up time, Schirmer test type I, conjunctival vital staining with fluorescein and lissamine green, tear lysozyme concentration, and impression cytology. RESULTS: Clinical tests yielded the following results: ocular surface disease index Sjogren´s syndrome: 64.5 ± 22.6 meibomian gland dysfunction: 43.5 ± 21.4, non-dry-eye disease: 6.7 ± 4.3 (p=0.02 between groups); Schirmer I test (mm/5 min): Sjogren´s syndrome: 4.95 ± 2.25, meibomian gland dysfunction: 13.28 ± 1.53, non-dry-eye disease 13.70 ± 1.39 (p<0.01 Sjogren´s syndrome vs. non-dry-eye disease and p<0.01 meibomian gland dysfunction vs. non-dry-eye disease); tear break-up time (seconds): Sjogren´s syndrome: 3.97 ± 1.47, meibomian gland dysfunction: 3.95 ± 0.86, non-dry-eye disease: 7.25 ± 1.90 (p<0.01 Sjogren´s syndrome vs. non-dry-eye disease and p<0.01 meibomian gland dysfunction vs. non-dry-eye disease); Lissamine green score: Sjogren´s syndrome-dry-eye: 6.18 ± 2.14, meibomian gland dysfunction-dry-eye: 5.27 ± 1.27, non-dry-eye disease: 1.52 ± 0.97 (p<0.01 Sjogren´s syndrome vs. non-dry-eye disease and p<0.01 meibomian gland dysfunction vs. non-dry-eye disease); impression cytology score: Sjogren´s syndrome: 1.88 ± 0.92, meibomian gland dysfunction: 1.67 ± 0.56, non-dry-eye: 0.45 ± 0.44 (p<0.01 Sjogren´s syndrome vs. non-dry-eye disease and p<0.01 meibomian gland dysfunction vs. non-dry-eye disease) and; tear lysozyme concentration (µg/mL): Sjogren´s syndrome: 751.25 ± 244.73, meibomian gland dysfunction: 1423.67 ± 182.75, non-dry-eye disease: 1409.90 ± 188.21 (p<0.01 Sjogren´s syndrome vs. non-dry-eye disease and p<0.01 Sjogren´s syndrome vs. meibomian gland dysfunction). CONCLUSION: The concentration of lysozyme in the tears was lower in Sjögren's syndrome patients than in meibomian gland dysfunction and non-dry-eye disease groups. Hence, the lacrimal lysozyme could be considered as a simple, non-invasive, and economical biomarker to differentiate between Sjögren's syndrome dry eye disease and meibomian gland dysfunction dry eye disease.

What is Neuromedin U (NmU)

Neuromedin U (NmU) is a highly conserved neuropeptide and has multiple physiological and pathophysiological roles detected, ranging from smooth muscle contraction, feeding, energy balance to tumorigenesis, stress responses, and inflammation.

Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding. Neuromedin U (NMU) is a brain-gut peptide whose peripheral activities are well-understood but whose central actions have yet to be clarified. The recent identification of two NMU receptors in rat brain has provided a springboard for further investigation into its role in the central nervous system. Intracerebroventricular administration of NMU to free-feeding rats decreased food intake and body weight. Conversely, NMU increased gross locomotor activity, body temperature, and heat production. NMU, a potent endogenous anorectic peptide, serves as a catabolic signaling molecule in the brain. Further investigation of the biochemical and physiological functions of NMU will help our better understanding of the mechanisms of energy homeostasis. Neuromedin U (NMU) is a hypothalamic peptide that has been recently found to reduce food intake, but few is known about its other functions in the central nervous system. We here studied behavioral activities induced by an intracerebroventricular (ICV) administration of NMU in rats and mice. NMU increased gross locomotor activity, face washing behavior, and grooming. NMU-induced stress response was significantly abolished by pretreatment with an antagonist of corticotropin-releasing hormone (CRH), alpha-helical CRH (9-41) (alpha-hCRH), or anti-CRH IgG. NMU did not induce locomotor activity in CRH knockout mice. NMU that interacts anatomically and/or functionally with the CRH system is a novel physiological regulator of stress response. Neuromedin U (NMU) is a brain-gut peptide, which peripherally stimulates smooth muscle, increases of blood pressure, alters ion transport in the gut, controls local blood flow, and regulates adrenocortical function. Although intracerebroventricular (i.c.v.) administration of NMU is known to decrease food intake and body weight, little is known about its effect on other physiological functions. We examined the effects of i.c.v. administration of NMU on mean arterial pressure (MAP), heart rate (HR), and plasma norepinephrine in conscious rats. Neuromedin U (0.05 and 0.5 nmol) provoked an increase in MAP (93.8 +/- 0.5 to 123.5 +/- 1.7 and 94.7 +/- 0.8 to 132.7 +/- 3.0 mm Hg, respectively) and HR (334.9 +/- 6.0 to 494.1 +/- 6.9 and 346.3 +/- 3.3 to 475.1 +/- 8.9 beats/min, respectively). In contrast, plasma norepinephrine increased only with a high dose of neuromedin U. Intravenously administered NMU (0.5 nmol) elicited a small and short lasting increase in MAP, compared to that by i.c.v. NMU. These results indicate that central neuromedin U regulates sympathetic nervous system activity and affects cardiovascular function. The neuropeptide neuromedin U (NMU) has been shown to have significant effects on cardiovascular, gastrointestinal and CNS functions. The peptide was first isolated from the porcine spinal cord and later shown to be present in spinal cords of other species. Little is known about the distribution of neuromedin U receptors (NMURs) in the spinal cord and the spinal action of the peptide. Here we report on the expression of NMURs and a potential role in nociception in the rat spinal cord using a combination of behavioral and electrophysiological studies. Receptor autoradiography showed that NMU-23 binding was restricted to the superficial layers of spinal cord, a region known to be involved in the control of nociception. In situ hybridization analysis indicated the mRNA of NMUR2 was located in the same region (laminae I and IIo) as NMU-23 binding, while the mRNA for NMU receptor 1 was observed in a subpopulation of small diameter neurons of dorsal root ganglia. Intrathecal (i.t.) administration of neuromedin U-23 (0.4-4.0 nmol/10 microl) dose-dependently decreased both the mechanical threshold to von Frey hair stimulation and the withdrawal latency to a noxious thermal stimulus. Mechanical allodynia was observed between 10 and 120 min, peaking at 30 min and heat hyperalgesia was observed 10-30 min after i.t. administration of NMU-23. A similar mechanical allodynia was also observed following i.t. administration of NMU-8 (0.4-4 nmol/10 microl). A significant enhancement of the excitability of flexor reflex was induced by intrathecal administration of NMU-23 (4 nmol/10 microl). Evoked responses to touch and pinch stimuli were increased by 439+/-94% and 188+/-36% (P<0.01, n=6) respectively. The behavioral and electrophysiological data demonstrate, for the first time, a pro-nociceptive action of NMU. The restricted distribution of NMU receptors to a region of the spinal cord involved in nociception suggests that this peptide receptor system may play a role in nociception. OBJECTIVES: Neuromedin U (NmU) is a neuropeptide with anorexigenic activity. Two receptor subtypes (NmUR1 and NmUR2) confer the effects of NmU on target cells. We have recently demonstrated that NmU reduces insulin secretion from isolated pancreatic islets. Aim of our current study is to investigate the role of somatostatin at mediating the effects of NmU on insulin secretion. METHODS: Expression of NmU in the pancreas was detected by immunohistochemistry. Insulin and somatostatin secretion from in situ perfused rat pancreas and isolated pancreatic islets was measured by radioimmunoassay. The paracrine effects of somatostatin within pancreatic islets were blocked by cyclosomatostatin, a somatostatin receptor antagonist. RESULTS: Receptor subtype NmUR1, but not NmUR2, was expressed in the endocrine pancreas, predomitly in the periphery. Neuromedin U reduced insulin secretion from in situ perfused rat pancreas and stimulated somatostatin secretion from isolated pancreatic islets. Neuromedin U stimulated somatostatin secretion at both physiological and supraphysiological glucose concentrations. Cyclosomatostatin increased insulin secretion and reduced NmU-induced inhibition of insulin secretion. CONCLUSIONS: Neuromedin U reduces insulin and increases somatostatin secretion. Blockade of somatostatin action abolishes the inhibition of insulin secretion by NmU. The results of the study suggest that somatostatin mediates the inhibitory action of NmU on insulin secretion. Neuromedin U (NMU) and neuromedin S (NMS) are structurally related neuropeptides that have been reported to modulate energy homeostasis. Pharmacological data have shown that NMU and NMS inhibit food intake when administered centrally and that NMU increases energy expenditure. Additionally, NMU-deficient mice develop obesity, whereas transgenic mice overexpressing NMU are lean and hypophagic. Two high-affinity NMU/NMS receptors, NMUR1 and NMUR2, have been identified. NMUR1 is predomitly expressed in the periphery, whereas NMUR2 is predomitly expressed in the brain, suggesting that the effects of centrally administered NMU and NMS are mediated by NMUR2. To evaluate the role of NMUR2 in the regulation of energy homeostasis, we characterized NMUR2-deficient (Nmur2(-/-)) mice. Nmur2(-/-) mice exhibited a modest resistance to diet-induced obesity that was at least in part due to reduced food intake. Acute central administration of NMU and NMS reduced food intake in wild-type but not in Nmur2(-/-) mice. The effects on activity and core temperature induced by centrally administered NMU were also absent in Nmur2(-/-) mice. Moreover, chronic central administration of NMU and NMS evoked significant reductions in body weight and sustained reductions in food intake in mice. In contrast, Nmur2(-/-) mice were largely resistant to these effects. Collectively, these data demonstrate that the anorectic and weight-reducing actions of centrally administered NMU and NMS are mediated predomitly by NMUR2, suggesting that NMUR2-selective agonists may be useful for the treatment of obesity. BACKGROUND: Neuromedin U (NmU) belongs to the neuromedin family, comprising a series of neuropeptides involved in the gut-brain axis and including neuromedins B and C (bombesin-like), K (neurokinin B), L (neurokinin A or neurotensin), N, S, and U. CONTENT: Although initially isolated from porcine spinal cord on the basis of their ability to induce uterine smooth muscle contraction, these peptides have now been found to be expressed in several different tissues and have been ascribed numerous functions, from appetite regulation and energy balance control to muscle contraction and tumor progression. NmU has been detected in several species to date, particularly in mammals (pig, rat, rabbit, dog, guinea pig, human), but also in amphibian, avian, and fish species. The NmU sequence is highly conserved across different species, indicating that this peptide is ancient and plays an important biological role. Here, we summarize the main structural and functional characteristics of NmU and describe its many roles, highlighting the jack-of-all-trades nature of this neuropeptide. SUMMARY: NmU involvement in key processes has outlined the possibility that this neuropeptide could be a novel target for the treatment of obesity and cancer, among other disorders. Although the potential for NmU as a therapeutic target is obvious, the multiple functions of this molecule should be taken into account when designing an approach to targeting NmU and/or its receptors. Neuromedin U (NMU), a neuropeptide isolated from porcine spinal cord and named because of its activity as a rat uterus smooth muscle contraction inducer, is emerging as a new player in the tumorigenesis and/or metastasis of many types of cancers. Expressed in a variety of tissues, NMU has been shown to possess many important activities in the central nervous system as well as on the periphery. Along with the main structural and functional features of NMU and its currently known receptors, we summarized a growing number of recently published data from different tissues and cells that associate NMU activity with cancer development and progression. We ask if, based on current reports, NMU can be included as a marker of these processes and/or considered as a therapeutic target. Neuromedin U (NMU) is a highly conserved neuropeptide that has been implicated in the stress response. To better understand how it influences various aspects of the stress response, we studied the effects of intracerebroventricular NMU-8 administration on stress-related behavior and activity of the hypothalamus-pituitary-adrenal (HPA) axis in male C57BL/6J mice. We investigated these NMU-8 effects when mice remained in their home cage and when they were challenged by exposure to forced swim stress. NMU-8 administration resulted in increased grooming behavior in mice that remained in their home cage and in a significant increase in c-Fos immunoreactivity in the paraventricular hypothalamus (PVH) and arcuate nucleus (ARC). Surprisingly, NMU-8 administration significantly decreased plasma corticosterone concentrations. Furthermore, NMU-8 administration increased immobility in the forced swim test in both naïve mice and mice that were previously exposed to swim stress. The effect of NMU-8 on c-Fos immunoreactivity in the PVH was dependent on previous exposure to swim stress given that we observed no significant changes in mice exposed for the first time to swim stress. In contrast, in the ARC we observed a significant increase in c-Fos immunoreactivity regardless of previous stress exposure. Interestingly, NMU-8 administration also significantly decreased plasma corticosterone concentrations in mice that were exposed to single forced swim stress, while this effect was no longer observed when mice were exposed to forced swim stress for a second time. Taken together, our data indicate that NMU-8 regulates stress responsiveness and suggests that its effects depend on previous stress exposure. Asthma is a major inflammatory airway disease with high incidence and mortality rates. The Global Initiative for Asthma released a report called 'The Global Burden of Asthma' in 2004. However, the specific pathogenesis of asthma remains unclear. An increasing number of studies have demonstrated that neuromedin U (NMU) plays a pleiotropic role in the pathogenesis of asthma. NMU is a highly structurally conserved neuropeptide that was first purified from porcine spinal cord and named for its contractile effect on the rat uterus. NMU amplifies type 2 innate lymphoid cell (ILC2)-driven allergic lung inflammation. The NMU receptors (NMURs), designated as NMUR1 and NMUR2, belong to the G protein-coupled receptor family. NMUR1 has also been found in immune cells, including ILC2s, mast cells and eosinophils. In view of the important roles of NMU in the pathogenesis of asthma, the present review evaluates the potential mechanisms underlying the impact of NMU on asthma and its association with asthma therapy. BACKGROUND: Neuromedin U (NMU) is a neuropeptide belonging to the neuromedin family. Recently, significant associations between NMU and several cancers have been reported. However, no studies have examined the association between NMU and hepatocellular carcinoma (HCC). The purpose of this study was to examine the role of NMU in HCC. METHODS: An enzyme-linked immunosorbent assay was used to measure the level of NMU protein in the sera of patients with hepatic hemangioma and HCC. NMU and cytokine mRNA expression was assessed in HCC samples via RT-qPCR. A tissue microarray consisting of 228 HCC peri- and intra-tumor tissues was used to detect NMU expression via immunohistochemical analysis. The association between NMU expression and overall survival (OS) and disease-free survival (DFS) was analyzed by Kaplan-Meier curves, the log-rank test, and Cox proportional hazard model. RESULTS: The level of NMU protein was increased in the sera of HCC patients (p = 0.006). NMU was expressed in intercellular space, rather than in hepatocytes or HCC cells. The prognosis of HCC patients with high NMU expression in peri-tumor tissue was significantly poorer than that of patients with low NMU expression (OS: p = 0.002, DFS: p = 0.033). Peri-tumor NMU expression was also a significant independent prognostic factor for OS (hazard ratio: 1.541, 95% confidence interval: 1.092-2.175, p = 0.014). The level of NMU expression was positively associated with M2 macrophage percentage and the levels of type-2 inflammatory cytokines in HCC tissue. CONCLUSIONS: NMU may serve as a novel prognostic biomarker for HCC patients, although further validation is needed in the future. The activation of M2 macrophages and a type-2 inflammatory response may involve in the role of NMU in patients with HCC. Neuromedin U (NMU) is a bioactive neuropeptide, highly distributed in the gastrointestinal tract and the central nervous system. NMU has various physiological functions related to feeding behavior, energy metabolism, stress responses, circadian rhythmicity and inflammation. Recently, several reports indicate that the central NMU system plays an important role in the reward systems in the brain. However, the underlying molecular mechanisms are not yet fully defined. In this study, we found that some of cocaine-induced c-Fos immunoreactive cells were co-localized with NMU in the nucleus accumbens (NAc), caudate putamen (CPu), and basolateral amygdala (BLA), which are key brain regions associated with the brain reward system, in wild type mice. Whereas, a treatment with cocaine did not influence the kinetics of NMU or NMU receptors mRNA expression in these brain regions, and NMU-knockout mice did not show any higher preference for cocaine compared with their control mice. These results indicate that cocaine has some effect on NMU expressing neurons related to the brain reward system, and this suggests NMU system may have a role on the brain reward systems activated by cocaine.

Is FTY720 FDA approved?

Yes, FTY720 was approved by the US Food and Drug Administration (FDA) in 2010.

FTY720 (Fingolimod) is a known sphingosine-1-phosphate (S1P) receptor agonist that exerts strong anti-inflammatory effects and was approved as the first oral drug for the treatment of multiple sclerosis by the US Food and Drug Administration (FDA) in 2010. FTY720 is mainly associated with unique functional "antagonist" and "agonist" mechanisms. The functional antagonistic mechanism is mediated by the transient down-regulation and degradation of S1P receptors on lymphocytes, which prevents lymphocytes from entering the blood stream from the lymph node. This subsequently results in the development of lymphopenia and reduces lymphocytic inflammation. Functional agonistic mechanisms are executed through S1P receptors expressed on the surface of various cells including neurons, astrocytes, microglia, and blood vessel endothelial cells. These functions might play important roles in regulating anti-apoptotic systems, modulating brain immune and phagocytic activities, preserving the Blood-Brain-Barrier (BBB), and the proliferation of neural precursor cells. Recently, FTY720 have shown receptor-independent effects, including intracellular target bindings and epigenetic modulations. Many researchers have recognized the positive effects of FTY720 and launched basic and clinical experiments to test the use of this agent against stroke. Although the mechanism of FTY720 has not been fully elucidated, its efficacy against cerebral stroke is becoming clear, not only in animal models, but also in ischemic stroke patients through clinical trials. In this article, we review the data obtained from laboratory findings and preliminary clinical trials using FTY720 for stroke treatment.

Describe SPar-K

SPar-K is a method to search for archetypical chromatin architectures by partitioning a set of genomic regions characterized by chromatin signal profiles around ChIP-seq peaks and other kinds of functional sites. This method efficiently deals with problems of data heterogeneity, limited misalignment of anchor points and unknown orientation of asymmetric patterns.

SUMMARY: We present SPar-K (Signal Partitioning with K-means), a method to search for archetypical chromatin architectures by partitioning a set of genomic regions characterized by chromatin signal profiles around ChIP-seq peaks and other kinds of functional sites. This method efficiently deals with problems of data heterogeneity, limited misalignment of anchor points and unknown orientation of asymmetric patterns. AVAILABILITY AND IMPLEMENTATION: SPar-K is a C++ program available on GitHub https://github.com/romaingroux/SPar-K and Docker Hub https://hub.docker.com/r/rgroux/spar-k. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

What is the mechanism of action of donanemab?

Donanemab is a new monoclonal antibody that uniquely targets Aβ(p3-42), a pyroglutamate form of Amyloid-β (Aβ) exclusively found in plaques.

BACKGROUND: A hallmark of Alzheimer's disease is the accumulation of amyloid-β (Aβ) peptide. Doemab, an antibody that targets a modified form of deposited Aβ, is being investigated for the treatment of early Alzheimer's disease. METHODS: We conducted a phase 2 trial of doemab in patients with early symptomatic Alzheimer's disease who had tau and amyloid deposition on positron-emission tomography (PET). Patients were randomly assigned in a 1:1 ratio to receive doemab (700 mg for the first three doses and 1400 mg thereafter) or placebo intravenously every 4 weeks for up to 72 weeks. The primary outcome was the change from baseline in the score on the Integrated Alzheimer's Disease Rating Scale (iADRS; range, 0 to 144, with lower scores indicating greater cognitive and functional impairment) at 76 weeks. Secondary outcomes included the change in scores on the Clinical Dementia Rating Scale-Sum of Boxes (CDR-SB), the 13-item cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-Cog13), the Alzheimer's Disease Cooperative Study-Instrumental Activities of Daily Living Inventory (ADCS-iADL), and the Mini-Mental State Examination (MMSE), as well as the change in the amyloid and tau burden on PET. RESULTS: A total of 257 patients were enrolled; 131 were assigned to receive doemab and 126 to receive placebo. The baseline iADRS score was 106 in both groups. The change from baseline in the iADRS score at 76 weeks was -6.86 with doemab and -10.06 with placebo (difference, 3.20; 95% confidence interval, 0.12 to 6.27; P = 0.04). The results for most secondary outcomes showed no substantial difference. At 76 weeks, the reductions in the amyloid plaque level and the global tau load were 85.06 centiloids and 0.01 greater, respectively, with doemab than with placebo. Amyloid-related cerebral edema or effusions (mostly asymptomatic) occurred with doemab. CONCLUSIONS: In patients with early Alzheimer's disease, doemab resulted in a better composite score for cognition and for the ability to perform activities of daily living than placebo at 76 weeks, although results for secondary outcomes were mixed. Longer and larger trials are necessary to study the efficacy and safety of doemab in Alzheimer's disease. (Funded by Eli Lilly; TRAILBLAZER-ALZ ClinicalTrials.gov number, NCT03367403.). INTRODUCTION: Alzheimer's disease is the leading cause of disability and poor health, takes a huge emotional and ficial burden on family caregivers, and is costly. Doemab (LY3002813) is a new monoclonal antibody that uniquely targets Aβ(p3-42), a pyroglutamate form of Amyloid-β (Aβ) exclusively found in plaques. AREAS COVERED: The phase 2 trial of doemab in participants with early symptomatic Alzheimer's disease, TRAILBLAZER-ALZ. Doemab reduced cerebral plaque but not tau load and only marginally improved the primary outcome of cognition and activities of daily living (p = 0.04) without altering individual measures of these. EXPERT OPINION: In TRAILBLAZER-ALZ, anticholinesterase use was given at the beginning but not the end of the trial, and thus, it is not known whether changes in this or other medicines were involved in the outcome with doemab. Tau load (measured with flortuacipir PET) may be a biomarker of cognition but was not altered by doemab. As there is no clear evidence that removing cerebral amyloid plaques with Aβ antibodies, such as doemab, improves cognition and the activities of daily living in Alzheimer's disease, clinical trials with these agents should be abandoned, and more time and money should spend on further investigating the underlying cause of Alzheimer's disease. The discussion of whether amyloid plaque Aβ is a valid drug target to fight Alzheimer's disease (AD) has been a matter of scientific dispute for decades. This question can only be settled by successful clinical trials and the approval of disease-modifying drugs. However, many clinical trials with antibodies against different regions of the amyloid Aβ peptide have been discontinued, as they did not meet the clinical endpoints required. Recently, passive immunization of AD patients with Doemab, an antibody directed against the N-terminus of pyroglutamate Aβ, showed beneficial effects in a phase II trial, supporting the concept that N-truncated Aβ is a relevant target for AD therapy. There is long-standing evidence that N-truncated Aβ variants are the main variants found in amyloid plaques besides full-length Aβ1-42, t, therefore their role in triggering AD pathology and as targets for drug development are of interest. While the contribution of pyroglutamate Aβ3-42 to AD pathology has been well studied in the past, the potential role of Aβ4-42 has been largely neglected. The present review will therefore focus on Aβ4-42 as a possible drug target based on human and mouse pathology, in vitro and in vivo toxicity, and anti-Aβ4-X therapeutic effects in preclinical models. The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of β-amyloid (AβΝ3pE) in the brains of AD patients. In this work, we identified potent QC inhibitors with subomolar IC50 values that were up to 290-fold higher than that of PQ912, which is currently being tested in Phase II clinical trials. Among the tested compounds, the cyclopentylmethyl derivative (214) exhibited the most potent in vitro activity (IC50 = 0.1 nM), while benzimidazole (227) showed the most promising in vivo efficacy, selectivity and druggable profile. 227 significantly reduced the concentration of pyroform Aβ and total Aβ in the brain of an AD animal model and improved the alternation behavior of mice during Y-maze tests. The crystal structure of human QC (hQC) in complex with 214 indicated tight binding at the active site, supporting that the specific inhibition of QC results in potent in vitro and in vivo activity. Considering the recent clinical success of doemab, which targets AβΝ3pE, small molecule-based QC inhibitors may also provide potential therapeutic options for early-stage AD treatment. Compelling evidence suggests that pyroglutamate-modified Aβ (pGlu3-Aβ; AβN3pG) peptides play a pivotal role in the development and progression of Alzheimer's disease (AD). Approaches targeting pGlu3-Aβ by glutaminyl cyclase (QC) inhibition (Varoglutamstat) or monoclonal antibodies (Doemab) are currently in clinical development. Here, we aimed at an assessment of combination therapy of Varoglutamstat (PQ912) and a pGlu3-Aβ-specific antibody (m6) in transgenic mice. Whereas the single treatments at subtherapeutic doses show moderate (16-41%) but statistically insignificant reduction of Aβ42 and pGlu-Aβ42 in mice brain, the combination of both treatments resulted in significant reductions of Aβ by 45-65%. Evaluation of these data using the Bliss independence model revealed a combination index of ≈1, which is indicative for an additive effect of the compounds. The data are interpreted in terms of different pathways, in which the two drugs act. While PQ912 prevents the formation of pGlu3-Aβ in different compartments, the antibody is able to clear existing pGlu3-Aβ deposits. The results suggest that combination of the small molecule Varoglutamstat and a pE3Aβ-directed monoclonal antibody may allow a reduction of the individual compound doses while maintaining the therapeutic effect. One of the central aims in Alzheimer's disease (AD) research is the identification of clinically relevant drug targets. A plethora of potential molecular targets work very well in preclinical model systems both in vitro and in vivo in AD mouse models. However, the lack of translation into clinical settings in the AD field is a challenging endeavor. Although it is long known that N-terminally truncated and pyroglutamate-modified Abeta (AβpE3) peptides are abundantly present in the brain of AD patients, form stable and soluble low-molecular weight oligomers, and induce neurodegeneration in AD mouse models, their potential as drug target has not been generally accepted in the past. This situation has dramatically changed with the report that passive immunization with doemab, an AβpE3-specific antibody, cleared aymloid plaques and stabilized cognitive deficits in a group of patients with mild AD in a phase II trial. This review summarizes the current knowledge on the molecular mechanisms of generation of AβpE, its biochemical properties, and the intervention points as a drug target in AD.

Is cytokeratin a tumor marker?

Yes, cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) is a tumor marker.

PURPOSE: To explore the concentration of squamous cell carcinoma antigen (SCCA), cytokeratin fragment antigen 21-1 (CYFRA21-1) in patients with laryngeal squamous cell carcinoma (LSCC) and its correlation with tumorigenesis and progression. METHODS: A total of 78 patients with LSCC admitted to our hospital from February 2010 to January 2016 were enrolled as the research group (RG), and another 41 healthy volunteers from the same period were selected as the control group (CG). The serum concentrations of SCCA and CYFRA21-1 in patients with LSCC were detected by ELISA, whose diagnostic value in LSCC were further analyzed by ROC curve. The prognosis and survival curves of patients with LSCC were observed according to the median value of serum SCCA and CYFRA21-1 concentrations. RESULTS: The concentration of CYFRA21-1 and SCCA in the RG was significantly higher than that in the CG (p < 0.050). The SCCA and CYFRA21-1 identified a significant difference in smoking, lymphatic metastasis, TNM staging, and differentiation degree (p < 0.050). The survival rate of the SCCA low-concentration group was significantly better than that of the high-concentration group, p < 0.050. The survival rate of the CYFRA21-1 low-concentration group was markedly better than that of the high-concentration group, p < 0.050. CONCLUSIONS: SCCA and CYFRA21-1 are highly concentrated in LSCC patients, which have good diagnostic efficacy for LSCC. In addition, they play some certain role in the occurrence and development of LSCC, and are expected to be markers for early diagnosis and prognosis of this disease. PURPOSE: We aimed to develop a novel method for orthotopic colon cancer model, using tissue adhesive in place of conventional surgical method. MATERIALS AND METHODS: RFP HCT 116 cell line were used to establish the colon cancer model. Fresh tumor tissue harvested from a subcutaneous injection was grafted into twenty nude mice, divided into group A (suture method) and group B (tissue adhesive method). For the group A, we fixed the tissue on the serosa layer of proximal colon by 8-0 surgical suture. For the group B, tissue adhesive (10 μL) was used to fix the tumor. The mortality, tumor implantation success, tumor metastasis, primary tumor size, and operation time were compared between the two groups. Dissected tumor tissue was analyzed for the histology and immunohistochemistry. Also, we performed tumor marker analysis. RESULTS: We observed 30% increase in graft success and 20% decrease in mortality, by using tissue adhesive method, respectively. The median colon tumor size was significantly increased by 4 mm and operation time was shortened by 6.5 minutes. The H&E showed similar tumor structure between the two groups. The immunohistochemistry staining for cancer antigen 19-9, carcinoembryonic antigen, cytokeratin 20, and Ki-67 showed comparable intensities in both groups. Real-time quantitative reverse transcription analysis showed eight out of nine tumor markers are unchanged in the tissue adhesive group. Western blot indicated the tissue adhesive group expressed less p-JNK (apototic marker) and more p-MEK/p-p38 (proliferation marker) levels. CONCLUSION: We concluded the tissue adhesive method is a quick and safe way to generate orthotopic, colon cancer model. OBJECTIVE: The main aims of this study were to explore the relationships between serum tumor markers and connective tissue disease-related interstitial lung disease (CTD-ILD) and to evaluate the clinical value of tumor markers for investigating interstitial lung disease (ILD) in patients with connective tissue disease (CTD). METHODS: The study included 235 patients with CTD (90 CTD without ILDs, 145 CTD-ILD). Clinical information and the levels of inflammatory and tumor markers, including carbohydrate antigen (CA) 19-9, CA125, carcinoembryonic antigen (CEA), CA153, and cytokeratin 19 fragments (CYFRA21-1), were obtained in all the patients. RESULTS: A significant difference between CTD with or without ILD and higher levels of tumor markers was observed in the CTD-ILD group, including CA19-9 (p<0.001), CEA (p<0.001), CA153 (p<0.001), and CYFRA21-1 (p<0.001). There was no significant difference in serum tumor marker levels in the various types of CTD (rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, inflammatory myositis, systemic sclerosis, and mixed connective tissue disease). The levels of CA153 [odds ratio (OR)=1.159] and CYFRA21-1 (OR=2.269) were clearly related to the risk of CTD-ILD. The diagnostic value of CA153 [area under receiver operating characteristic curve (AUC)=0.736] and CYFRA21-1 (AUC=0.718) was confirmed for ILDs in CTD patients, at cut-off values of 9.45 U/mL and 2.13 ng/mL, respectively. CONCLUSION: There is a positive correlation between serum tumor marker levels and CTD-ILD. Higher levels of CA153 and CYFRA21-1 suggest an increased risk of developing ILD and may therefore be useful as biomarkers for detecting CTD-ILD in the clinical setting.

What are TAMs in cancer therapy?

TAMs are Tumor Associated Macrophages and are important in Cancer therapy.

Macrophages are one of the principal host cell populations in solid tumors. They are capable, due to their plasticity, of acquiring phenotypes that either combat (M1 type) or promote (M2 type) neoplastic growth. These cells, known as tumor-associated macrophages (TAMs), play complex but pivotal roles in the outcome of photodynamic therapy (PDT) of maligt lesions. Among the various parenchymal and stromal cell populations found in tumors, TAMs have been shown to have the greatest capacity for the uptake of systemically administered photosensitizers. Both the tumor-localizing property of photosensitizers and their tumor-localized fluorescence could be partly attributed to the activity of TAMs. Since resident TAMs with accumulated high photosensitizer content will sustain high degrees of PDT damage, this population (predomitly M2 in most tumors) is selectively destroyed, and during the ensuing inflammatory reaction is replaced with newly invading macrophages of M1 phenotype. These macrophages are sentinels responding to DAMP signals from PDT-treated tumor cells and in turn are mobilized to generate a variety of inflammatory/immune mediators and opsonins. They have a critical role in contributing to the therapeutic effect of PDT by mediating disposal of killed cancer cells and by processing/presenting tumor antigens to T lymphocytes. However, TAMs accumulating in the later post-PDT phase can acquire the M2 (healing) phenotype, and could have a role in tumor recurrence by releasing factors that promote angiogenesis and the survival/proliferation of remaining cancer cells. Various therapeutic strategies modulating TAM activity in the PDT response have potential for clinical use for improving PDT-mediated tumor control. Cancer therapy has developed around the concept of killing, or stopping the growth of, the cancer cells. Molecularly targeted therapy is the modern expression of this paradigm. Increasingly, however, the realization that the cancer has co-opted the normal cells of the stroma for its own survival has led to the concept that the tumor microenvironment (TME) could be targeted for effective therapy. In this review, we outline the importance of tumor-associated macrophages (TAM), a major component of the TME, in the response of tumors to cancer therapy. We discuss the normal role of macrophages in wound healing, the major phenotypes of TAMs, and their role in blunting the efficacy of cancer treatment by radiation and anticancer drugs, both by promoting tumor angiogenesis and by suppressing antitumor immunity. Finally, we review the many preclinical studies that have shown that the response of tumors to irradiation and anticancer drugs can be improved, sometimes markedly so, by depleting TAMs from tumors or by suppressing their polarization from an M1 to an M2 phenotype. The data clearly support the validity of clinical testing of combining targeting TAMs with conventional therapy. Clin Cancer Res; 23(13); 3241-50. ©2017 AACR. Eliciting effective antitumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF-1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or cosecreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset cosecreting TNF-α, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, and on TNF-α and IFN-γ. Together, this study demonstrates the potential for targeting TAMs to convert a "cold" into an "inflamed" tumor microenvironment capable of eliciting protective T cell responses. Purpose: Cancer stem-like cells (CSC) contribute to the progression and androgen deprivation therapy (ADT) resistance of prostate cancer. As CSCs depend on their specific niche, including tumor-associated macrophages (TAM), elucidating the network between CSCs and TAMs may help to effectively inhibit the progression and ADT resistance of prostate cancer.Experimental Design: The underlying intracellular mechanism that sustains the stem-like characteristics of CSCs in prostate cancer was assessed via RNA sequencing, co-immunoprecipitation, chromatin immunoprecipitation, and other assays. A coculture system and cytokine antibody arrays were used to examine the interaction network between CSCs and TAMs. In addition, an orthotopic prostate cancer model was established to evaluate the in vivo effects of the combined targeting of CSCs and their interaction with TAMs on ADT resistance.Results: Autophagy-related gene 7 (ATG7) facilitated the transcription of OCT4 via β-catenin, which binds to the OCT4 promoter, promoting CSC characteristics in prostate cancer, including self-renewal, tumor initiation, and drug resistance. In addition, CSCs remodeled their specific niche by educating monocytes/macrophages toward TAMs, and the CSC-educated TAMs reciprocally promoted the stem-like properties of CSCs, progression and ADT resistance of prostate cancer via IL6/STAT3. Furthermore, the combined targeting of CSCs and their interaction with TAMs by inhibiting ATG7/OCT4 and IL6 receptor effectively ameliorated ADT resistance in an orthotopic prostate cancer model.Conclusions: Targeting CSCs and their niche may prove to be a more powerful strategy than targeting CSCs alone, providing a rational approach to ameliorating ADT resistance in prostate cancer. Clin Cancer Res; 24(18); 4612-26. ©2018 AACR. Tumorigenesis is not only determined by the intrinsic properties of cancer cells but also by their interactions with components of the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME. During initial stages of tumor development, macrophages can either directly promote antitumor responses by killing tumor cells or indirectly recruit and activate other immune cells. As genetic changes occur within the tumor or T helper 2 (TH 2) cells begin to dominate the TME, TAMs begin to exhibit an immunosuppressive protumor phenotype that promotes tumor progression, metastasis, and resistance to therapy. Thus, targeting TAMs has emerged as a strategy for cancer therapy. To date, TAM targeting strategies have focused on macrophage depletion and inhibition of their recruitment into the TME. However, these strategies have shown limited therapeutic efficacy, although trials are still underway with combination therapies. The fact that macrophages have the potential for antitumor activity has moved the TAM targeting field toward the development of TAM-reprogramming strategies to support this antitumor immune response. Here, we discuss the various roles of TAMs in cancer therapy and their immunosuppressive properties, as well as implications for emerging checkpoint inhibitor-based immunotherapies. We review state-of-the-art TAM-targeting strategies, focusing on current ones at the preclinical and clinical trial stages that aim to reprogram TAMs as an oncological therapy. Tumor-associated macrophages (TAMs) represent the most abundant innate immune cells in tumors. TAMs, exhibiting anti-inflammatory phenotype, are key players in cancer progression, metastasis and resistance to therapy. A high TAM infiltration is generally associated with poor prognosis, but macrophages are highly plastic cells that can adopt either proinflammatory/antitumor or anti-inflammatory/protumor features in response to tumor microenvironment stimuli. In the context of cancer therapy, many anticancer therapeutics, apart from their direct effect on tumor cells, display different effects on TAM activation status and density. In this review, we aim to evaluate the indirect effects of anticancer therapies in the modulation of TAM phenotypes and pro/antitumor activity. In the last decade, it has been well-established that tumor-infiltrating myeloid cells fuel not only the process of carcinogenesis through cancer-related inflammation mechanisms, but also tumor progression, invasion, and metastasis. In particular, tumor-associated macrophages (TAMs) are the most abundant leucocyte subset in many cancers and play a major role in the creation of a protective niche for tumor cells. Their ability to generate an immune-suppressive environment is crucial to escape the immune system and to allow the tumor to proliferate and metastasize to distant sites. Conventional therapies, including chemotherapy and radiotherapy, are often not able to limit cancer growth due to the presence of pro-tumoral TAMs; these are also responsible for the failure of novel immunotherapies based on immune-checkpoint inhibition. Several novel therapeutic strategies have been implemented to deplete TAMs; however, more recent approaches aim to use TAMs themselves as weapons to fight cancer. Exploiting their functional plasticity, the reprogramming of TAMs aims to convert immunosuppressive and pro-tumoral macrophages into immunostimulatory and anti-tumor cytotoxic effector cells. This shift eventually leads to the reconstitution of a reactive immune landscape able to destroy the tumor. In this review, we summarize the current knowledge on strategies able to reprogram TAMs with single as well as combination therapies. OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal maligcy and lacks effective treatment. We aimed to understand molecular mechanisms of the intertwined interactions between tumour stromal components in metastasis and to provide a new paradigm for PDAC therapy. DESIGN: Two unselected cohorts of 154 and 20 patients with PDAC were subjected to correlation between interleukin (IL)-33 and CXCL3 levels and survivals. Unbiased expression profiling, and genetic and pharmacological gain-of-function and loss-of-function approaches were employed to identify molecular signalling in tumour-associated macrophages (TAMs) and myofibroblastic cancer-associated fibroblasts (myoCAFs). The role of the IL-33-ST2-CXCL3-CXCR2 axis in PDAC metastasis was evaluated in three clinically relevant mouse PDAC models. RESULTS: IL-33 was specifically elevated in human PDACs and positively correlated with tumour inflammation in human patients with PDAC. CXCL3 was highly upregulated in IL-33-stimulated macrophages that were the primary source of CXCL3. CXCL3 was correlated with poor survival in human patients with PDAC. Mechanistically, activation of the IL-33-ST2-MYC pathway attributed to high CXCL3 production. The highest level of CXCL3 was found in PDAC relative to other cancer types and its receptor CXCR2 was almost exclusively expressed in CAFs. Activation of CXCR2 by CXCL3 induced a CAF-to-myoCAF transition and α-smooth muscle actin (α-SMA) was uniquely upregulated by the CXCL3-CXCR2 signalling. Type III collagen was identified as the CXCL3-CXCR2-targeted adhesive molecule responsible for myoCAF-driven PDAC metastasis. CONCLUSIONS: Our work provides novel mechanistic insights into understanding PDAC metastasis by the TAM-CAF interaction and targeting each of these signalling components would provide an attractive and new paradigm for treating pancreatic cancer. Tumor-associated macrophages (TAMs) of M2 phenotype have mediated the immunosuppression in a tumor microenvironment, facilitating the escape of tumor cells from immunosurveillance. Reprograming the immunosuppressive M2 TAMs to immunostimulatory M1 phenotype can activate the antitumor immune responses for cancer immunotherapy. Herein, hollow iron oxide (Fe3O4) oparticles (NPs) were employed to reprogram M2 TAMs toward M1 TAMs, aiming to release proinflammatory cytokines and recruit T cells to kill tumor cells. After loaded with l-arginine (l-Arg) and sealed with poly(acrylic acid) (PAA), hollow Fe3O4 NPs were fabricated into LPFe3O4 NPs, which could release l-Arg based on pH-responsive PAA and produce nitric oxide (NO) with the help of nitric oxide synthase (iNOS) overexpressed by M1 TAMs, as a result of additional tumor elimination for gas therapy. In vitro and in vivo studies demonstrate that LPFe3O4 NPs could effectively reprogram M2 to M1 macrophages, activating T cells, releasing TNF-α, and producing high levels of NO, leading to synergistic tumor therapy. Endocrine therapy is the standard treatment for estrogen receptor (ER)-positive breast cancer, but tumors eventually develop resistance. However, endocrine therapy resistance mechanisms mediated through interactions between breast cancer cells and tumor-associated macrophages (TAMs) are still unclear. Here, we characterized sodium/glucose cotransporter 1 (SGLT1) overexpression drives the highly glycolytic phenotype of tamoxifen-resistant breast cancer cells where enhanced lactic acid secretion promotes M2-like TAM polarization via the hypoxia-inducible factor-1α/signal transducer and activator of transcription-3 pathway. In turn, M2-like TAMs activate breast cancer cells through EGFR/PI3K/Akt signaling, providing feedback to upregulate SGLT1 and promote tamoxifen resistance and accelerate tumor growth in vitro and in vivo. Higher expression of SGLT1 and CD163+ TAMs was associated with endocrine-resistant ER-positive breast cancers. Our study identifies a novel vicious cycle of metabolic reprogramming, M2-like TAM polarization, and endocrine therapy resistance, which involves SGLT1, proposing SGLT1 as a therapeutic target to overcome endocrine therapy resistance in breast cancer. Inflammation is a fundamental physiological response orchestrated by innate immune cells to restore tissue homeostasis. Specialized pro-resolving mediators (SPMs) are involved in active resolution of inflammation but when inflammation is incomplete, chronic inflammation creates a favorable environment that fuels carcinogenesis and cancer progression. Conventional cancer therapy also strengthens cancer-related inflammation by inducing massive tumor cell death that activate surrounding immune-infiltrating cells such as tumor-associated macrophages (TAMs). Macrophages are key actors of both inflammation and its active resolution due to their plastic phenotype. In line with this high plasticity, macrophages can be hijacked by cancer cells to support tumor progression and immune escape, or therapy resistance. Impaired resolution of cancer-associated inflammation supported by TAMs may thus reinforces tumor progression. From this perspective, recent evidence suggests that stimulating macrophage's pro-resolving functions using SPMs can promote inflammation resolution in cancer and improve anticancer treatments. Thus, TAMs' re-education toward an antitumor phenotype by using SPMs opens a new line of attack in cancer treatment. Here, we review SPMs' anticancer capacities with special attention regarding their effects on TAMs. We further discuss how this new therapeutic approach could be envisioned in cancer therapy. Melanoma is one of the most common maligt tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35-40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4+Fop3+ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8+ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.

What is the relationship between RUNX1T1 and FTO?

FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1.

The fat mass and obesity-associated (FTO) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression (FTO-4) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout (FTO-KO) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity.

Which java utility has been developed for class hidden markov models?

JUCHMME is an open-source software package designed to fit arbitrary custom Hidden Markov Models (HMMs) with a discrete alphabet of symbols, and is used for biological sequence analysis and class hidden markov models in Java EE 8 and Java EE 9.

SUMMARY: JUCHMME is an open-source software package designed to fit arbitrary custom Hidden Markov Models (HMMs) with a discrete alphabet of symbols. We incorporate a large collection of standard algorithms for HMMs as well as a number of extensions and evaluate the software on various biological problems. Importantly, the JUCHMME toolkit includes several additional features that allow for easy building and evaluation of custom HMMs, which could be a useful resource for the research community. AVAILABILITY AND IMPLEMENTATION: http://www.compgen.org/tools/juchmme, https://github.com/pbagos/juchmme. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

What is the mechanism of action of Etesevimab?

Etesevimab is a neutralizing antibody indicated for treatment of coronavirus disease 2019 (COVID-19) in patients with early mild or moderate disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread rapidly worldwide. This study is the first to report the tolerability, safety, pharmacokinetics (PK), and immunogenicity of a recombit human anti-SARS-CoV-2 monoclonal antibody, etesevimab (CB6, JS016, LY3832479, or LY-CoV016), in healthy adults. This paper describes a randomized, double-blind, placebo-controlled, phase 1 study. A total of 40 participants were enrolled to receive a single intravenous dose of either etesevimab or placebo in one of four sequential ascending intravenous dose cohorts. All 40 participants completed the study. Seventeen (42.5%) participants experienced 22 treatment emergent adverse events (TEAEs) that were drug-related, and the rates of these TEAEs among different dose cohorts were numerically comparable. No difference was observed between the combined etesevimab group and the placebo group. The exposure after etesevimab infusion increased in an approximately proportional manner as the dose increased from 2.5 to 50 mg/kg. The elimination half-life (t1/2) value did not differ among different dose cohorts and was estimated to be around 4 weeks. Etesevimab was well tolerated after administration of a single dose at a range of 2.5 mg/kg to 50 mg/kg in healthy Chinese adults. The PK profiles of etesevimab in healthy volunteers showed typical monoclonal antibody distribution and elimination characteristics. (This study has been registered at ClinicalTrials.gov under identifier NCT04441918.). Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation. The severity of coronavirus disease 2019 (COVID-19) ranges from mild to death, with high morbidity and mortality rates reported amongst a vulnerable subset of patients termed high risk. While vaccines remain the primary option for COVID-19 prevention, neutralizing monoclonal antibodies (mAbs), such as bamlanivimab and etesevimab, have been shown to benefit certain subpopulations after exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unlike vaccine-derived immunity that develops over time, administration of neutralizing mAbs is an immediate and passive immunotherapy, with the potential to reduce disease progression, emergency room visits, hospitalizations, and death. Bamlanivimab alone and together with etesevimab hold emergency use authorizations in several countries globally, with countries increasingly transitioning to the use of bamlanivimab and etesevimab together and other authorized mAbs on the basis of their evolving variant landscape, regulatory authorizations, and access to drugs. The current guidelines for the administration of bamlanivimab alone or together with etesevimab are informed by an iterative process of testing and development. Herein the rationale for these guidelines is provided by sharing the learnings that have been gathered throughout the development process of these mAbs. In addition, this review addresses the most common clinical questions received from health care professionals (HCPs) and patients regarding indicated population, dose, use with other medications and vaccines, duration of protection, and variants in clinical practice. As prevalence of SARS-CoV-2 variants can differ by country and state, prescribing HCPs should consider the prevalence of bamlanivimab and etesevimab resistant variants in their area, where data are available, regarding potential efficacy impact when considering treatment options.Trial Registration: ClinicalTrials.gov identifier: NCT04427501; NCT04411628; NCT04497987; NCT04634409. Patients with the monogenic immune dysregulatory syndrome autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene, uniformly carry neutralizing autoantibodies directed against type-I interferons (IFNs) and many develop autoimmune pneumonitis, both of which place them at high risk for life-threatening COVID-19 pneumonia. Bamlanivimab and etesevimab are monoclonal antibodies (mAbs) that target the SARS-CoV-2 spike protein and block entry of SARS-CoV-2 in host cells. The use of bamlanivimab and etesevimab early during infection was associated with reduced COVID-19-associated hospitalization and death in patients at high risk for progressing to severe disease, which led the US Food and Drug Administration to issue an emergency use authorization for their administration in non-hypoxemic, non-hospitalized high-risk patients. However, the safety and efficacy of these mAbs has not been evaluated in APECED patients. We enrolled two siblings with APECED on an IRB-approved protocol (NCT01386437) and admitted them prophylactically at the NIH Clinical Center for evaluation of mild-to-moderate COVID-19. We assessed the safety and clinical effects of early treatment with bamlanivimab and etesevimab. The administration of bamlanivimab and etesevimab was well tolerated and was associated with amelioration of COVID-19 symptoms and prevention of invasive ventilatory support, admission to the intensive care, and death in both patients without affecting the production of antibodies to the nucleocapsid protein of SARS-CoV-2. If given early in the course of COVID-19 infection, bamlanivimab and etesevimab may be beneficial in APECED and other high-risk patients with neutralizing autoantibodies directed against type-I IFNs. This review takes into consideration the principal vaccines developed against the SARS-CoV-2 in this unprecedented period of Covid-19 pandemic. We evaluated the mechanism of action of each vaccine as well as the efficacy, the safety and the storage temperature. In addition, the problem of the dose units, the vaccinal strategy, the activity of alternative compounds such as the monoclonal antibodies and especially the issue of the virus variants were also described in detail. Four vaccines are currently used in Italy: Pfizer-BioNTech mRNA BNT162b2 (Comirnaty) (USA), Moderna mRNA 1273 (USA), Astra-Zeneca ChAdOx1-S (recombit) viral vector adenovirus belonging to Oxford (UK) and Pomezia (Italy), Janssen (two recombit viral vector adenoviruses) belonging to Johnson & Johnson (USA). The efficacy of Pfizer and Moderna for preventing disease or severe disease results 95-87.5% and 94.5-100%, respectively. The efficacy of Astra-Zeneca and Janssen is about 70% and 65%, respectively; in the case of Janssen, it depends on the geographical area ranging from 72% to 57%. The problem of the administrated doses (one dose, two doses from the same vaccine or from different vaccines, half dose) is also discussed. The vaccination strategy based on the age group remains the simplest, most transparent and fair criterion. This strategy is also based on accelerating the administration of the vaccines, so that as many subjects as possible can be vaccinated quickly for achieving the "herd immunity". The monoclonal antibodies appeared to be a valid solution for the treatment of Covid-19 disease. Two antibodies (bamlanivimab and etesevimab) have just been approved by the FDA. They could also be used for the infection by virus variants which represent a big problem due to their higher transmissibility and virulence and to their lower response to the vaccines. Bamlanivimab and etesevimab are neutralizing antibodies indicated for treatment of coronavirus disease 2019 (COVID-19) in patients with early mild or moderate disease. We present the use of pharmacokinetic/pharmacodynamic (PK/PD) modeling that characterizes the timecourse of viral load obtained from 2,970 patients from 2 phase II clinical trials. The model was used for identification of optimal doses that would result in at least 90% of patients achieving serum drug concentrations that result in 90% of maximum drug effect (IC90) for at least 28 days. The serum IC90 (95% confidence interval) was estimated to be 4.2 (3.2-4.3) µg/mL for bamlanivimab and 12.6 (9.7-12.8) µg/mL for etesevimab. Observed clinical trial data confirmed PK and PK/PD model predictions that doses of 700 mg bamlanivimab and 1,400 mg etesevimab would result in maximum reduction in viral load, with no additional effect seen at higher doses. No dose adjustment is recommended as age, sex, race, baseline viral load, and hepatic impairment did not have a significant impact on the PK of the antibodies. Earlier drug administration resulted in greater reductions in viral load, demonstrating the importance of receiving treatment as soon as possible. Relative to placebo, typical reduction in viral load over a 7-day period was estimated to be 80 or 93% (drug administered 4 days or 1 day after the onset of symptoms, respectively), P < 0.0001. PK/PD modeling and simulation was pivotal throughout the drug development and emergency use authorization process. OBJECTIVES: To evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness. PATIENTS AND METHODS: We evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients). RESULTS: The NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients. CONCLUSION: Two-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced. INTRODUCTION: Treatments for subjects with Covid-19 are required. One approach is neutralizing monoclonal antibodies. Bamlanivimab and etesevimab are monoclonal antibodies to SARS-CoV-2. AREAS COVERED: This evaluation is of the phase 3 BLAZE-1 clinical trial, which was of bamlanivimab plus etesevimab in adult ambulatory participants with a risk factor for, and mild to moderate, Covid-19 illness. The primary outcome was Covid 19 related hospitalization of ≥ 24 hours or death from any cause by day 29, and this occurred in 2.1% subjects in the bamlanivimab/etesevimab group, compared to 7.0% in the placebo group. EXPERT OPINION: In the pandemic, the attempts by the FDA to shorten approval processes for medicines and by journals to make information available in a timely manner are admirable. However, these shortened processes made negotiating the details of BLAZE-1 and producing accurate and critical appraisals difficult. It seems to me that if there are any benefits of bamlanivimab alone in Covid-19, they are not clear-cut. Bamlanivimab has limited effects against the beta and gamma variants and is not effective against the delta variant. Thus, the benefits of bamlanivimab/etesevimab in the phase 3 of the BLAZE-1 may be solely due to etesevimab, and this needs to be tested.

List known pseudokinases.

TRIB1 TRIB2 TRIB3 MLKL ULK4 HER3 CASK

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determits involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response. Despite advancements in management of acute myocardial infarction, this disease remains one of the leading causes of death. Timely reestablishment of epicardial coronary blood flow is the cornerstone of therapy; however, substantial amount of damage can occur as a consequence of cardiac ischaemia/reperfusion (I/R) injury. It has been previously proposed that the pathway leading to major cell death, apoptosis, is responsible for cardiac I/R injury. Nevertheless, there is compelling evidence to suggest that necroptosis, a programmed necrosis, contributes remarkably to both myocardial injury and microcirculatory dysfunction following cardiac I/R injury. Receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like pseudokinase (MLKL) are shown as the major mediators of necroptosis. In addition to the traditional perception that RIPK1/RIPK3/MLKL-dependent plasma membrane rupture is fundamental to this process, several RIPK3-related pathways such as endoplasmic reticulum stress and mitochondrial fragmentation have also been implicated in cardiac I/R injury. In this review, reports from both in vitro and in vivo studies regarding the roles of necroptosis and RIPK3-regulated necrosis in cardiac I/R injury have been collectively summarized and discussed. Furthermore, reports on potential interventions targeting these processes to attenuate cardiac I/R insults to the heart have been presented in this review. Future investigations adding to the knowledge obtained from these previous studies are needed in the pursuit of discovering the most effective pharmacological agent to improve cardiac I/R outcomes. Hypereosinophilia (HE) has been defined as persistent eosinophilia >1.5 × 109/L; it is broadly divided into primary HE (clonal or neoplastic; HEN), secondary/reactive HE (HER), or HE of undetermined significance (HEUS) when no cause is identified. The use of myeloid next-generation sequencing (NGS) panels has led to the detection of several mutations in patients previously diagnosed with HEUS, reassigning some patients to the category of HEN, specifically the World Health Organization category of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Here, we describe a novel somatic JAK1 pseudokinase domain mutation (R629_S632delinsSA) in a patient with HE that had initially been characterized as a variant of uncertain significance. We performed functional studies that demonstrated that this mutation results in growth factor independence of Ba/F3 cells in vitro and activation of the JAK-STAT pathway. These effects were abrogated by the JAK1/JAK2 inhibitor ruxolitinib. R629_S632delinsSA is the first known somatic mutation in JAK1 linked to a clonal eosinophilic neoplasm, and highlights the importance of the JAK-STAT pathway in eosinophil survival. BACKGROUND: Protein kinases are among the largest druggable family of signaling proteins, involved in various human diseases, including cancers and neurodegenerative disorders. Despite their clinical relevance, nearly 30% of the 545 human protein kinases remain highly understudied. Comparative genomics is a powerful approach for predicting and investigating the functions of understudied kinases. However, an incomplete knowledge of kinase orthologs across fully sequenced kinomes severely limits the application of comparative genomics approaches for illuminating understudied kinases. Here, we introduce KinOrtho, a query- and graph-based orthology inference method that combines full-length and domain-based approaches to map one-to-one kinase orthologs across 17 thousand species. RESULTS: Using multiple metrics, we show that KinOrtho performed better than existing methods in identifying kinase orthologs across evolutionarily divergent species and eliminated potential false positives by flagging sequences without a proper kinase domain for further evaluation. We demonstrate the advantage of using domain-based approaches for identifying domain fusion events, highlighting a case between an understudied serine/threonine kinase TAOK1 and a metabolic kinase PIK3C2A with high co-expression in human cells. We also identify evolutionary fission events involving the understudied OBSCN kinase domains, further highlighting the value of domain-based orthology inference approaches. Using KinOrtho-defined orthologs, Gene Ontology annotations, and machine learning, we propose putative biological functions of several understudied kinases, including the role of TP53RK in cell cycle checkpoint(s), the involvement of TSSK3 and TSSK6 in acrosomal vesicle localization, and potential functions for the ULK4 pseudokinase in neuronal development. CONCLUSIONS: In sum, KinOrtho presents a novel query-based tool to identify one-to-one orthologous relationships across thousands of proteomes that can be applied to any protein family of interest. We exploit KinOrtho here to identify kinase orthologs and show that its well-curated kinome ortholog set can serve as a valuable resource for illuminating understudied kinases, and the KinOrtho framework can be extended to any protein-family of interest. CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non-small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3-DXd is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3-DXd across a series of EGFR inhibitor-resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3-DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3-DXd. The combination of osimertinib and HER3-DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients. SIGNIFICANCE: EGFR inhibition leads to increased HER3 membrane expression and promotes HER3-DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3-DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18.

Where is the body would the Peyer's patches be found

Peyer's patches (PPs) play a major role in intestinal mucosal immunity and are located in the gut.

In the rabbit intestine Peyer's patches can easily be distinguished. They are found in the jejunum and ileum, but not in the duodenum. The highest number observed is seven. In most animals the first, i.e. proximally situated patch has a rather well-defined place in the beginning of the jejunum; similarly, the last, i.e. distally situated one mostly has its place in the end of the ileum. The demarcation of the three classical sections of the small intestine is discussed from a comparative viewpoint with special criticism as to what is called ileum in man. Unlike the Peyer's patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyer's patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyer's patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyer's patch is of a special type that sheds carbonic anhydrase-rich, 50-ometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyer's patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyer's patches with respect to their lymphocyte population and the ileal Peyer's patch with respect to their follicle-associated epithelium. During the course of their recirculation through the body, blood-borne lymphocytes specifically adhere to high endothelial venules (HEV) within secondary lymphoid organs such as peripheral lymph nodes (PN) and gut-associated Peyer's patches (PP). This adherence event, which initiates the extravasation of the lymphocyte, is highly specific in terms of the class of lymphocyte and the anatomic location of the HEV. We review evidence that the lymphocyte adhesive molecule ('homing receptor') involved in attachment to PN HEV is a carbohydrate-binding receptor (lectin-like) with specificity for mannose-6-phosphate (M6P)-like ligands. We describe the use of a novel cytochemical probe for the detection and characterization of cell surface carbohydrate-binding receptors. Using a M6P-based probe, we show that the carbohydrate-binding receptor on lymphocytes is closely-related or identical to the MEL-14 antigen, a putative homing receptor identified by a monoclonal antibody. Evidence is presented that the lymphocyte attachment sites on both PN and PP HEV are inactivated by mild periodate oxidation and hence are probably carbohydrate in nature. Yet, the sites are biochemically distinguishable in that one class (PN) requires sialidase-sensitive structures whereas the other (PP) does not. We raise the possibility that diversity in the carbohydrate-based recognition determits on HEV may underlie the adhesive specificities in this system. The distribution and number of Peyer's patches (5) in two species of marsupial mice, Antechinus swainsonii and Antechinus stuartii was found to be the same even though the length of the intestine in the latter species was half that of the former. Both species lack a caecum and appendix. The position of the Peyer's patches is unusual in that the first three Peyer's patches are on the right side of the small intestine whereas the penultimate and ultimate Peyer's patches are large, contain many lymphoid follicles and are in an anti-mesenteric position in the small intestine and sometimes in the large intestine (ultimate Peyer's patch). The number of Peyer's patches in eutherian mice of similar size, and intestinal length is greater (13) although the number of Peyer's patch lymphoid follicles per centimeter intestine is less (1.8) than in A. swainsonii (4) and A. stuartii (4.4). Marsupial mice have most of their lymphoid follicles confined to a few large Peyer's patches, whereas eutherian mice have fewer lymphoid follicles per unit intestinal length, more Peyer's patches (with fewer lymphoid follicles) evenly distributed along the intestine and more single lymphoid follicles interspersed between them. Adaptive immune protection of mucous membranes is provided mainly by secretory IgA (SIgA) antibodies. This first-line defence is accomplished through an ingenious cooperation between the mucosal B-cell system and the epithelial glycoprotein called secretory component (SC). This is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA), which is the major humoral mediator substance of the whole immune system. Transmembrane SC belongs to the Ig supergene family and functions as a general pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B-cells responsible for local pIg production are initially stimulated in lymphoepithelial structures, particularly the Peyer's patches in the distal small intestine, from which they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory site. There is currently great interest in exploiting this integrated or "common' mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce therapeutic peripheral tolerance to ameliorate T-cell-mediated autoimmune diseases. Much remains to be learnt about antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses, and how normal homeostasis is maintained in the intestinal mucosa. Considerable information has accumulated about various types of immune deviation that may lead to local or extraintestinal hypersensitivity reactions against luminal antigen, but the crucial mechanisms remain obscure. BACKGROUND AND AIMS: Peyer's patches play a major role in intestinal immunity, are portals of entry for significant pathogens, and may be important in Crohn's disease. Whereas their microscopic anatomy and immune function are well described, surprisingly little is known of their macroscopic anatomy and distribution. Our aim was to assess their number, area, and distribution in the normal distal ileum, with particular reference to patient age. METHODS: Distal ilea (200 cm) obtained at autopsy from 55 adults without intestinal disease were opened along the mesenteric border, fixed in acetic acid, and transilluminated. Peyer's patches were counted, and the length, breadth, and distance from the ileocecal valve were recorded. RESULTS: Patches were most numerous in the terminal 10-15 cm where they formed a lymphoid ring. More proximal patches were oval, antimesenteric, and irregularly spaced. By area, 46% of patch tissue occurred in the terminal 25 cm. The mean number of patches ranged from 29.4 +/- 5.4 in the youngest group studied, to 19.0 +/- 3.0 in the oldest. Total patch area was greatest in the group aged 21-30 (47.4 +/- 1.0 cm2). CONCLUSION: Peyer's patches are concentrated in the distal 25 cm of ileum but extend proximally for 200 cm. The variation in their size, shape, and distribution in different individuals is greater than often appreciated and may influence the presentation of diseases centered on these structures. In small rumits, the development of B cells differs from that in mice or in man. The anti-body repertoire is expanded in the Peyer's patches of the terminal ileum where each B-cell follicle is found by a few cells. To investigate the amount of founder clones in bovine ileal follicles, we have used sex mismatched cattle twins. These animals are chimeric due to placental anastomoses. Y-chromosome targeted in situ hybridization was used to trace donor-derived cells of the male genotype in a female recipient (called a freemartin). A strong clustering of lymphoid cells originating from either twin was seen in the ileal Peyer's patches (IPPs). Furthermore, the follicles displayed a low amount of immunoglobulin heavy chain gene configurations in comparison with the splenic or jejunal follicles. These findings strongly suggest that as in sheep, the B-cell follicles in cattle IPPs develop oligoclonally. Pro-inflammatory mediators hold important functions in human body in response to infection, trauma and vascular disease. However, their action is down regulated by the release of anti-inflammatory cytokines, thus restoring a balance which reflects the immune status of a given individual. Recent studies have stressed out the importance of circulating levels of cytokines for forensic purposes even if there is a lack of studies regarding the role of post-mortem mucosa-associated lymphoid tissue. In this respect, Peyer's patches (PP), represent one of the most important immunological site of the body and the major component of the gut -associated lymphoid tissue. The aim of this study was to evaluate post-mortem PP immune response in 40 serial autopsy cases of people who died from natural and traumatic death. The study examined spontaneous release of the following cytokines by fresh isolated PP cells: interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, IL-10, IL-6, IL-1 beta, and IL-8. Results will show that higher levels of TNF-alpha, IL-6, IL-1 beta, and IL-8 are statistically correlated with the traumatic death group. From a forensic point of view these data demonstrate that fundamental lymphoid organs, such as PP, may have a potential in diagnosing the cause of death. Peyer's patch have been extensively studied as a major inductive site for mucosal immunity within the small intestine. The intestinal mucosa contains numerous dendritic cells, which induce either protective immunity to infectious agents or tolerance to innocuous antigens, including food and commensal bacteria. Although during the past few years, several subsets of human mucosal dendritic cells have been described, a precise characterization of the different mouse mucosal dendritic cells subpopulations remains to be achieved with regard to their phenotype and localization in Peyer's patch. In this report, we have investigated by immunofluorescence on cryosection and by flow cytometry, the phenotype and the localization of dendritic cells into Peyer's patch of C57Bl/6 mouse intestine using dendritic cells markers. Positive and double staining for CD11c and BDCA-2, pDC/IPC, DC-LAMP, DC-SIGN, TLR8 and Langerin have been observed revealing new mouse intestinal DC subsets. This study provides new insight in the understanding of mucosal immune responses induced by natural processes as infections but also new perspectives for the evaluation of oral vaccines. Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a "carrier of Salmonella," TLR11 works as a "blocker of Salmonella" to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection. BACKGROUND: Oral immunization has numerous advantages over parenteral administrations. In addition to ease administration, more effective pathogen elimination on the mucosa before spreading into the blood circulation, constitutes the main benefit. This is particularly true for pathogens that enter the body through the oral route. On the other hand, it is the most challenging administration route for peptides, proteins and recombit antigens due to gastrointestinal (GI) tract, numerous barriers including the harsh environment and the inherent weak immunogenicity. In addition to the adjuvant properties, polymeric particles arise as the most promising strategy to overcome poor antigen bioavailability/ stability upon oral administration. The Peyer's patches have been considered an important structure of the gut associate lymphoid tissue (GALT) for the initiation of the immune response towards particulate oral antigens. OBJECTIVE: The transport mechanism of both, o and microparticles across intestinal mucosa, particularly throughout Peyer's patches, is discussed in this review. CONCLUSION: We provide a short and concise update (last decade) focused on the importance of particle physicochemical properties, M-cell ligands and size-dependent transport and intracellular fate concerning Peyer's patches targeted oral vaccination. In mammals, macrophages (MF) are present in virtually all tissues where they serve many different functions linked primarily to the maintece of homeostasis, innate defense against pathogens, tissue repair and metabolism. Although some of these functions appear common to all tissues, others are specific to the homing tissue. Thus, MF become adapted to perform particular functions in a given tissue. Accordingly, MF express common markers but also sets of tissue-specific markers linked to dedicated functions. One of the largest pool of MF in the body lines up the wall of the gut. Located in the small intestine, Peyer's patches (PP) are primary antigen sampling and mucosal immune response inductive sites. Surprisingly, although markers of intestinal MF, such as F4/80, have been identified more than 30 years ago, MF of PP escaped any kind of phenotypic description and remained "unknown" for decades. In absence of MF identification, the characterization of the PP mononuclear phagocyte system (MPS) functions has been impaired. However, taking into account that PP are privileged sites of entry for pathogens, it is important to understand how the latter are handled by and/or escape the PP MPS, especially MF, which role in killing invaders is well known. This review focuses on recent advances on the PP MPS, which have allowed, through new criteria of PP phagocyte subset identification, the characterization of PP MF origin, diversity, specificity, location and functions. The early replication of certain prion strains within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally acquired prions utilize specialized gut epithelial cells known as M cells to enter Peyer's patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer's patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer's patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in Peyer's patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility.IMPORTANCE The accumulation of orally acquired prions within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within Peyer's patches. Some gastrointestinal pathogens utilize molecules, such as the cellular prion protein PrPC, expressed on gut epithelial cells to enter Peyer's patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer's patches was dependent upon PrPC expression in the gut epithelium. We show that orally acquired prions can establish infection in Peyer's patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility. Sex differences in immunity are well described in the literature and thought to be mainly driven by sex hormones and sex-linked immune response genes. The gastrointestinal tract (GIT) is one of the largest immune organs in the body and contains multiple immune cells in the GIT-associated lymphoid tissue, Peyer's patches and elsewhere, which together have profound effects on local and systemic inflammation. The GIT is colonised with microbial communities composed of bacteria, fungi and viruses, collectively known as the GIT microbiota. The GIT microbiota drives multiple interactions locally with immune cells that regulate the homeostatic environment and systemically in diverse tissues. It is becoming evident that the microbiota differs between the sexes, both in animal models and in humans, and these sex differences often lead to sex-dependent changes in local GIT inflammation, systemic immunity and susceptibility to a range of inflammatory diseases. The sexually dimorphic microbiome has been termed the 'microgenderome'. Herein, we review the evidence for the microgenderome and contemplate the role it plays in driving sex differences in immunity and disease susceptibility. We further consider the impact that biological sex might play in the response to treatments aimed at manipulating the GIT microbiota, such as prebiotics, live biotherapeutics, (probiotics, synbiotics and bacteriotherapies) and faecal microbial transplant. These alternative therapies hold potential in the treatment of both psychological (e.g., anxiety, depression) and physiological (e.g., irritable bowel disease) disorders differentially affecting males and females. BACKGROUND AND AIM: Peyer's patches (PPs) play a major role in intestinal mucosal immunity; however, their role in ulcerative colitis (UC) is not well investigated. We evaluated endoscopic features of PPs on narrow-band imaging with magnifying endoscopy (NBI-ME) and investigated their association with clinical factors. METHODS: We prospectively recruited 105 patients with UC, 18 with Crohn's disease, 16 with disease control, and 33 healthy control subjects at three institutions from 2014 to 2017. NBI-ME images of the villi of PPs were evaluated according to the Villi Index, and patients were divided into the Villi Index low (L) and high (H) types. The 1-year sustained clinical remission rate was evaluated between L-type and H-type PPs in patients with UC. RESULTS: The proportions of patients with H-type PPs were significantly higher among UC, Crohn's disease, and disease control patients than among healthy control patients (P = 0.0125, 0.018, 0.0007). In UC, age, gender, endoscopic score, and extent of disease involvement were not significantly different between L-type and H-type PPs, whereas the sustained clinical remission rate was significantly higher in L-type PPs than in H-type PPs (88% [57/65] vs 65% [17/26], P = 0.019). Multivariate analysis revealed that the L type of PPs was a significant factor for sustained clinical remission (odds ratio 3.8, 95% confidence interval 1.1-12.9, P = 0.033). CONCLUSIONS: Patients with UC showed endoscopic alterations in PPs on NBI-ME, and highly altered appearance of PPs can be associated with a high risk of clinical relapse in patients with UC. The small intestine hosts specialized lymphoid structures, the Peyer's patches, that face the gut lumen and are overlaid with unique epithelial cells, called microfold (M) cells. M cells are considered to constitute an important route for antigen uptake in the mucosal immune system. Here, we used intravital microscopy to define immune cell populations, which are in close contact with M cells and potentially sample antigen. We present live evidence that DCs enter M cell pockets and highlight the abundance of mononuclear phagocytes in these structures. Taking advantage of the respective reporter animals, we focused on classical DCs that express Zbtb46 and analyzed how these cells interact with M cells in steady state and sample antigen for T cell activation in the Peyer's patches following challenge. Peyer's patches are gut-associated lymphoid tissue located throughout the intestinal wall. Peyer's patches consist of highly organized ovoid-shaped follicles, classified as non-encapsulated lymphatic tissues, populated with B cells, T cells, macrophages, and dendritic cells and function as an organism's intestinal surveillance. Limited work compares the gene profiles of Peyer's patches derived from different intestinal regions. In the current study, we first performed whole transcriptome analysis using RNAseq to compare duodenal and ileal Peyer's patches obtained from the small intestine of Long Evans rats. Of the 12,300 genes that were highly expressed, 18.5% were significantly different between the duodenum and ileum. Using samples obtained from additional subjects (n = 10), we validated the novel gene expression patterns in Peyer's patches obtained from the three regions of the small intestine. Rats had a significantly reduced number of Peyer's patches in the duodenum in comparison to either the jejunum or ileum. Regional differences in structural, metabolic, and immune-related genes were validated. Genes such as alcohol dehydrogenase 1, gap junction protein beta 2, and serine peptidase inhibitor clade b, member 1a were significantly reduced in the ileum in comparison to other regions. On the other hand, genes such as complement C3d receptor type, lymphocyte cytosolic protein 1, and lysozyme C2 precursor were significantly lower in the duodenum. In summary, the gene expression pattern of Peyer's patches is influenced by intestinal location and may contribute to its role in that segment.

Is tofacitinib a JAK inhibitor?

Yes, tofacitinib is a small JAK inhibitor.

Several cytokines signalling via Janus Kinase (JAK) proteins have been implicated in the pathogenesis of immune-mediated inflammatory diseases, including psoriasis and rheumatoid arthritis (RA). Tofacitinib, a small JAK inhibitor, is approved for the treatment of RA and has demonstrated good efficacy in psoriasis phase III clinical trials. In this work, we analysed the in vitro effects of tofacitinib on the functions of human dendritic cells (DCs) and macrophages. When assessing the effects of tofacitinib on monocyte-derived DCs, we observed reduced differentiation of monocytes into immature DCs, as evidenced by a decreased transcription of CD209 and CD80. Phenotype assessment in the presence of tofacitinib suggested a switch towards a M1-like macrophage phenotype, as evidenced by the expression of M1 markers such as iNOS, as well as cytokines typically expressed by M1 cells, including IL-12 and IL-23. Of note, Arginase1 and CD200R, typically expressed by M2 cells, were absent on tofacitinib-treated DCs. Furthermore, tofacitinib affected the response of differentiated DCs to maturation stimuli such as LPS and IFNγ, resulting in a partial up-regulation of IL-23 and down-regulation of IL-12, as assessed by qPCR. When investigating macrophage development, we found that tofacitinib inhibited the ability of monocytes to differentiate and polarize into regulatory M2 macrophages, while rather enhancing the ability to develop into inflammatory M1-like macrophages, as evidenced by decreased expression of the M2 marker CD200R and enhanced production of IL-12 and IL-23. In conclusion, tofacitinib impacts the differentiation of human DCs and macrophages, it particularly favours generation of M1-like pro-inflammatory macrophages.

Describe the GenomeAsia 100K Project

The GenomeAsia 100K Project (GenomeAsia100K Project) aims to identify and catalogue genetic variation, population structure, disease associations and founder effects in populations across Asia and worldwide. It includes a whole-genome sequencing reference dataset from 1,739 individuals of 219 population groups and 64 countries across Asia, as well as a population-wide association dataset.

The underrepresentation of non-Europeans in human genetic studies so far has limited the diversity of individuals in genomic datasets and led to reduced medical relevance for a large proportion of the world's population. Population-specific reference genome datasets as well as genome-wide association studies in diverse populations are needed to address this issue. Here we describe the pilot phase of the GenomeAsia 100K Project. This includes a whole-genome sequencing reference dataset from 1,739 individuals of 219 population groups and 64 countries across Asia. We catalogue genetic variation, population structure, disease associations and founder effects. We also explore the use of this dataset in imputation, to facilitate genetic studies in populations across Asia and worldwide.

Which disease is treated with Risdiplam?

Risdiplam is approved for spinal muscular atrophy.

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA. AIMS: Risdiplam (RG7916, RO7034067) is an orally administered, centrally and peripherally distributed, survival of motor neuron 2 (SMN2) mRNA splicing modifier for the treatment of spinal muscular atrophy (SMA). The objectives of this entry-into-human study were to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of risdiplam, and the effect of the strong CYP3A inhibitor itraconazole on the PK of risdiplam in healthy male volunteers. METHODS: Part 1 had a randomized, double-blind, adaptive design with 25 subjects receiving single ascending oral doses of risdiplam (ranging from 0.6-18.0 mg, n = 18) or placebo (n = 7). A Bayesian framework was applied to estimate risdiplam's effect on SMN2 mRNA. The effect of multiple doses of itraconazole on the PK of risdiplam was also assessed using a two-period cross-over design (n = 8). RESULTS: Risdiplam in the fasted or fed state was well tolerated. Risdiplam exhibited linear PK over the dose range with a multi-phasic decline with a mean terminal half-life of 40-69 h. Food had no relevant effect, and itraconazole had only a minor effect on plasma PK indicating a low fraction of risdiplam metabolized by CYP3A. The highest tested dose of 18.0 mg risdiplam led to approximately 41% (95% confidence interval 27-55%) of the estimated maximum increase in SMN2 mRNA. CONCLUSIONS: Risdiplam was well tolerated and proof of mechanism was demonstrated by the intended shift in SMN2 splicing towards full-length SMN2 mRNA. Based on these data, Phase 2/3 studies of risdiplam in patients with SMA are now ongoing. Risdiplam (Evrysdi™) is an orally administered, survival motor neuron 2 (SMN2)-directed RNA splicing modifier being developed by Roche, PTC Therapeutics Inc and the SMA Foundation for the treatment of the spinal muscular atrophy. The small molecule is designed to treat spinal muscular atrophy caused by mutations in chromosome 5q leading to SMN protein deficiency. The drug boosts the ability of an alternative gene SMN2 to produce full-length and functional SMN protein. In August 2020, Evrysdi™ (risdiplam) received its first approval in the USA for the treatment of spinal muscular atrophy in patients 2 months of age and older. Risdiplam is in pre-registration for this indication in numerous countries worldwide, including the European Union, Brazil, Chile, China, Indonesia, Russia, South Korea and Taiwan. This article summarizes the milestones in the development of risdiplam leading to this first approval for spinal muscular atrophy. The phenotype of spinal muscular atrophy (SMA) has been changing with the recent availability of three FDA-approved treatments: intrathecal nusinersen, intravenous onasemnogene abeparvovec-xioi, and enteral risdiplam. The degree of improvement in muscle strength and respiratory health varies with SMA genotype, severity of baseline neuromuscular and pulmonary impairment, medication used, and timing of the first dose. A spectrum of pulmonary outcomes has been reported with these novel medications when used early and in conjunction with proactive multidisciplinary management of comorbidities. In this review, we summarize the reported impact of these novel therapies on pulmonary well-being and the improving trajectory of pulmonary morbidity, compared to the natural history of SMA. The importance of ongoing clinical monitoring albeit the improved phenotype is reiterated. We also discuss the limitations of the current SMA-therapy trials and offer suggestions for future clinical-outcome studies and long-term monitoring. OBJECTIVE: Evaluation of ophthalmologic safety with focus on retinal safety in patients with spinal muscular atrophy (SMA) treated with risdiplam (EVRYSDI®), a survival of motor neuron 2 splicing modifier associated with retinal toxicity in monkeys. Risdiplam was approved recently for the treatment of patients with SMA, aged ≥ 2 months in the United States, and is currently under Health Authority review in the EU. METHODS: Subjects included patients with SMA aged 2 months-60 years enrolled in the FIREFISH, SUNFISH, and JEWELFISH clinical trials for risdiplam. Ophthalmologic assessments, including functional assessments (age-appropriate visual acuity and visual field) and imaging (spectral domain optical coherence tomography [SD-OCT], fundus photography, and fundus autofluorescence [FAF]), were conducted at baseline and every 2-6 months depending on study and assessment. SD-OCT, FAF, fundus photography, and threshold perimetry were evaluated by an independent, masked reading center. Adverse events (AEs) were reported throughout the study. RESULTS: A total of 245 patients receiving risdiplam were assessed. Comprehensive, high-quality, ophthalmologic monitoring assessing retinal structure and visual function showed no retinal structural or functional changes. In the youngest patients, SD-OCT findings of normal retinal maturation were observed. AEs involving eye disorders were not suggestive of risdiplam-induced toxicity and resolved with ongoing treatment. INTERPRETATION: Extensive ophthalmologic monitoring conducted in studies in patients with SMA confirmed that risdiplam does not induce ophthalmologic toxicity in pediatric or adult patients with SMA at the therapeutic dose. These results suggest that safety ophthalmologic monitoring is not needed in patients receiving risdiplam, as also reflected in the United States Prescribing Information for risdiplam. Recent advances in our understanding of RNA biology have uncovered crucial roles for RNA in multiple disease states, ranging from viral and bacterial infections to cancer and neurological disorders. As a result, multiple laboratories have become interested in developing drug-like small molecules to target RNA. However, this development comes with multiple unique challenges. For example, RNA is inherently dynamic and has limited chemical diversity. In addition, promiscuous RNA-binding ligands are often identified during screening campaigns. This Tutorial Review overviews important considerations and advancements for generating RNA-targeted small molecules, ranging from fundamental chemistry to promising small molecule examples with demonstrated clinical efficacy. Specifically, we begin by exploring RNA functional classes, structural hierarchy, and dynamics. We then discuss fundamental RNA recognition principles along with methods for small molecule screening and RNA structure determination. Finally, we review unique challenges and emerging solutions from both the RNA and small molecule perspectives for generating RNA-targeted ligands before highlighting a selection of the "Greatest Hits" to date. These molecules target RNA in a variety of diseases, including cancer, neurodegeneration, and viral infection, in cellular and animal model systems. Additionally, we explore the recently FDA-approved small molecule regulator of RNA splicing, risdiplam, for treatment of spinal muscular atrophy. Together, this Tutorial Review showcases the fundamental role of chemical and molecular recognition principles in enhancing our understanding of RNA biology and contributing to the rapidly growing number of RNA-targeted probes and therapeutics. In particular, we hope this widely accessible review will serve as inspiration for aspiring small molecule and/or RNA researchers. BACKGROUND: Type 1 spinal muscular atrophy is a rare, progressive neuromuscular disease that is caused by low levels of functional survival of motor neuron (SMN) protein. Risdiplam is an orally administered, small molecule that modifies SMN2 pre-messenger RNA splicing and increases levels of functional SMN protein. METHODS: We report the results of part 1 of a two-part, phase 2-3, open-label study of risdiplam in infants 1 to 7 months of age who had type 1 spinal muscular atrophy, which is characterized by the infant not attaining the ability to sit without support. Primary outcomes were safety, pharmacokinetics, pharmacodynamics (including the blood SMN protein concentration), and the selection of the risdiplam dose for part 2 of the study. Exploratory outcomes included the ability to sit without support for at least 5 seconds. RESULTS: A total of 21 infants were enrolled. Four infants were in a low-dose cohort and were treated with a final dose at month 12 of 0.08 mg of risdiplam per kilogram of body weight per day, and 17 were in a high-dose cohort and were treated with a final dose at month 12 of 0.2 mg per kilogram per day. The baseline median SMN protein concentrations in blood were 1.31 ng per milliliter in the low-dose cohort and 2.54 ng per milliliter in the high-dose cohort; at 12 months, the median values increased to 3.05 ng per milliliter and 5.66 ng per milliliter, respectively, which represented a median of 3.0 times and 1.9 times the baseline values in the low-dose and high-dose cohorts, respectively. Serious adverse events included pneumonia, respiratory tract infection, and acute respiratory failure. At the time of this publication, 4 infants had died of respiratory complications. Seven infants in the high-dose cohort and no infants in the low-dose cohort were able to sit without support for at least 5 seconds. The higher dose of risdiplam (0.2 mg per kilogram per day) was selected for part 2 of the study. CONCLUSIONS: In infants with type 1 spinal muscular atrophy, treatment with oral risdiplam led to an increased expression of functional SMN protein in the blood. (Funded by F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02913482.). Not too long ago, the concept of selectively targeting mRNA with small molecules was perceived as a formidable scientific challenge. The discovery of small molecule splicing modifiers and the development of risdiplam for the treatment of spinal muscular atrophy (SMA) have firmly established proof of concept for this exciting new platform and transformed a scientific curiosity into a viable technology to target disease. Today, several approaches to target mRNA with small molecules, supported by biophysical and screening methods, are in place to deliver new drugs with high therapeutic relevance. Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode. INTRODUCTION/AIMS: There are currently three medications approved for spinal muscular atrophy (SMA), but the use of these medications in combination has not been well described. METHODS: This is a retrospective report of four cases of SMA treated with dual onasemnogene and risdiplam therapy at our institution. RESULTS: Following onasemnogene therapy, all four patients experienced a perceived plateau of therapeutic benefit, at which time daily risdiplam was started. Transient fatigue and weakness was seen in two patients following risdiplam initiation, but this resolved within 1 mo. One patient was hospitalized with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and post-viral pneumonia, weeks following risdiplam initiation. No other adverse effects related to onasemnogene and risdiplam combination therapy were identified and all patients experienced objective and subjective improvement. DISCUSSION: Combination therapy with onasemnogene and risdiplam in patients with SMA appears to be well-tolerated. Further large prospective trials are needed to determine whether dual therapy is more efficacious than monotherapy, and to identify rare adverse events that may occur with the use of combination therapy. Risdiplam (Evrysdi) improves motor neuron function in patients with spinal muscular atrophy (SMA) and has been approved for the treatment of patients ≥2 months old. Risdiplam exhibits time-dependent inhibition of cytochrome P450 (CYP) 3A in vitro. While many pediatric patients receive risdiplam, a drug-drug interaction (DDI) study in pediatric patients with SMA was not feasible. Therefore, a novel physiologically-based pharmacokinetic (PBPK) model-based strategy was proposed to extrapolate DDI risk from healthy adults to children with SMA in an iterative manner. A clinical DDI study was performed in healthy adults at relevant risdiplam exposures observed in children. Risdiplam caused an 1.11-fold increase in the ratio of midazolam area under the curve with and without risdiplam (AUCR)), suggesting an 18-fold lower in vivo CYP3A inactivation constant compared with the in vitro value. A pediatric PBPK model for risdiplam was validated with independent data and combined with a validated midazolam pediatric PBPK model to extrapolate DDI from adults to pediatric patients with SMA. The impact of selected intestinal and hepatic CYP3A ontogenies on the DDI susceptibility in children relative to adults was investigated. The PBPK analysis suggests that primary CYP3A inhibition by risdiplam occurs in the intestine rather than the liver. The PBPK-predicted risdiplam CYP3A inhibition risk in pediatric patients with SMA aged 2 months-18 years was negligible (midazolam AUCR of 1.09-1.18) and included in the US prescribing information of risdiplam. Comprehensive evaluation of the sensitivity of predicted CYP3A DDI on selected intestinal and hepatic CYP3A ontogeny functions, together with PBPK model-based strategy proposed here, aim to guide and facilitate DDI extrapolations in pediatric populations. Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient. Spinal muscular atrophy (SMA) is one of the leading causes of death in infants related to the degeneration of neurons. Currently, there are no curative treatment options for SMA, and many options available may not be feasible. This review presents the background, clinical studies, and indications for the use of Risdiplam in treating SMA. SMA causes a decrease in the production of survival motor neuron proteins (SMN) and current treatments target to increase the expression of SMN. Risdiplam is the first and only oral medication to be approved to treat SMA. As an SMN2 splicing modifier, it has provided stronger systemic therapies than previous intrathecal and gene replacement therapies. There have been many efforts to treat SMA with multidisciplinary approaches. These include intrathecal injections to gene replacement therapies. However, these have been faced with limitations such as reaching a good therapeutic dose in systemic tissues, route of administration, and price. Risdiplam is currently the only orally administered drug approved by the FDA for the treatment of SMA. It not only provides a good therapeutic window to systemic tissues but allows for a non-invasive approach in infants. Further investigation and comparison on the safety profile of Risdiplam due to its broader systemic effect should be considered with other available therapies. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous deletions or mutations in survival motor neuron gene 1 (SMN1). Currently, the primary therapeutic strategy for SMA is to increase the level of SMN via correcting SMN2 splicing (nusinersen and risdiplam). However, some patients with SMA do not respond to such treatments, thereby warranting a need to develop new therapeutic strategies. We have previously reported that SMN2 expression is epigenetically regulated by DNA methylation levels of the SMN2 promoter region. In the present study, we determined that methyl-CpG-binding protein 2 (MeCP2) may bind to this critical promoter region (nt-167 to 43). Antisense oligonucleotides (ASO-P1 and ASO-P2) were designed to target the key methylation sites in the SMN2 promoter region, which enhanced the overall transcription and functional protein expression levels in the SMA cell lines. These results were similar to those observed in nusinersen-treated SMA cells. Moreover, a combined treatment of ASO-P1 and ASO-NUS in SMA cell lines further increases fl-SMN2 transcript and SMN protein levels. The delivery of ASO-P1 to the central nervous system of severe SMA mice corrected the molecular, pathological, and functional phenotypes of this disease and increased survival rates. Our findings suggest that the key methylation regions in the SMN2 promoter region may be a novel therapeutic target for SMA.

What is protein palmitoylation?

Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16‑carbon fatty acid palmitate, is the most common acylation, and it enhances the membrane stability of ion channels. This post-translational modification (PTM) determines a functional mechanism of ion channel life cycle from maturation and membrane trafficking to localization.

Rho GTPases play central roles in a wide variety of cellular processes, including cytoskeletal dynamics, cell adhesion and cell polarity. RhoU and RhoV are Rho GTPases that have some atypical properties compared with classical Rho family members, such as the presence of N- and C-terminal extension regions, unusual GDP/GTP cycling and post-translational modification by palmitoylation but not prenylation. Their activity and localization is regulated by the N-terminal and C-terminal regions, and so far no GEFs or GAPs have been identified for them. Similar to Rac and Cdc42, they interact with PAK serine/threonine kinases, and in the case of PAK4, this interaction leads to RhoU protein stabilization. In cells, RhoU and RhoV alter cell shape and cell adhesion, which probably underlies some of the phenotypes reported for these proteins in vivo, for example in heart development and epithelial morphogenesis. However, the molecular basis for these functions of RhoU and RhoV remains to be characterized. Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle. Protein S-palmitoylation is an important post-translational modification (PTM) in blood stages of the malaria parasite, Plasmodium falciparum. S-palmitoylation refers to reversible covalent modification of cysteine residues of proteins by saturated fatty acids. In vivo, palmitoylation is regulated by concerted activities of DHHC palmitoyl acyl transferases (DHHC PATs) and acyl protein thioesterases (APTs), which are enzymes responsible for protein palmitoylation and depalmitoylation, respectively. Here, we investigate the role of protein palmitoylation in red blood cell (RBC) invasion by P. falciparum merozoites. We demonstrate for the first time that free merozoites require PAT activity for microneme secretion in response to exposure to the physiologically relevant low [K+] environment, characteristic of blood plasma. We have adapted copper catalyzed alkyne azide chemistry (CuAAC) to image palmitoylation in merozoites and found that exposure to low [K+] activates PAT activity in merozoites. Moreover, using acyl biotin exchange chemistry (ABE) and confocal imaging, we demonstrate that a calcium dependent protein kinase, PfCDPK1, an essential regulator of key invasion processes such as motility and microneme secretion, undergoes dynamic palmitoylation and localizes to the merozoite membrane. Treatment of merozoites with the PAT inhibitor, 2-bromopalmitate (2-BP), effectively inhibits microneme secretion and RBC invasion by the parasite, thus opening the possibility of targeting P. falciparum PATs for antimalarial drug discovery to inhibit blood stage growth of malaria parasites.

Αre plants from the genus Strychnos the original source of curare?

Species of plants from the genus Strychnos are the source of curare.

The history to about 1850 of the muscle-relaxant poison curare is discussed, especially the developments leading to the botanical identification of the plants that yield the alkaloidal active principles: Loganiaceae (Strychnos species) and Menispermaceae (Abuta, Chondrodendron, and Curarea species). One of the earliest encounters with the poison appears to have been during the exploration of the Lake Maracaibo region in Colombia by Alonso Pérez de Tolosa in 1548. It is pointed out (yet again) that Sir Walter Ralegh did not bring back the poison to Europe in 1595 and that it was Keymis who first came across the word ourari when exploring the lower reaches of the Orinoco in 1596. Gumilla, La Condamine, Ulloa, Veigl, and others gave much additional information about the poison during the 18th century. Scientific studies began in the latter part of the century when Schreber listed the botanical identities of four of the plant components entering into the curare prepared by the Akawai Indians of Surinam. As far as is known, none of these people actually saw curare being made. Thereafter, progress was rapid. Humboldt and Bonpland were the first trained scientists to witness the preparation of the poison, at the very beginning of the 19th century. Subsequent exploration by Martius and Spix, Poeppig, Youd, the Schomburgk brothers, De Castelnau and Deville, Spruce, and others, up to the middle of the century, extended and deepened botanical and ethnological knowledge of curare. Study of its physiology started at about that time with the classical experiments of Rudolf von Koelliker and Claude Bernard. The ethnobotanical uses of South American species of Strychnos L. (Loganiaceae) are reviewed, with the exception of their major rôle in the preparation of curare, which will be dealt with in detail elsewhere. Medicinal uses are less common than is the case with the African and Asian species of the genus. About 140 samples, mostly of leaves, belonging to 53 species, have been screened for alkaloids. As with species from other parts of the world, the stem bark and root bark tend to be a richer source than leaves. Nor-harman is present in extracts from S. barnhartiana leaves. Pyridino-indolo-quinolizidinone (angustine-type) bases are also found in several species. The occurrence and pharmacology of the (non-curarizing) alkaloids known to be present in South American Strychnos species is reviewed.

When was galcanezumab approved by FDA?

Galcanezumab was approved by the FDA in September 2018.

Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (CLR + RAMP1) offer considerable improvements over existing drugs in migraine prophylaxis and are the first designed to act on the trigeminal pain system. Erenumab is approved by the FDA and EMA and has reached the market since May 2018. Two antibodies, fremanezumab and galcanezumab, directed towards the CGRP ligand, were approved by the FDA in September 2018. To view this Bench to Bedside, open or download the PDF.

Are variants in FHF2 (also known as FGF13) associated with encephalopathy?

Yes. FHF2 (also known as FGF13) variants are a cause of infantile-onset developmental and epileptic encephalopathy.

Author information: (1)Institute of Medical Genetics, University Hospital of Wales, Cardiff CF14 4XW, UK; Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK. Electronic address: [email protected]. (2)Department of Biological Sciences, Hunter College of City University, 695 Park Avenue, New York, NY 10065, USA; Program in Biology, Graduate Center of City University, 365 Fifth Avenue, New York, NY 10016, USA. (3)Neurology and Molecular Neuroscience Research, Institute of Life Science, Swansea University Medical School, Swansea University, Swansea SA2 8PP, UK. (4)Neurology and Molecular Neuroscience Research, Institute of Life Science, Swansea University Medical School, Swansea University, Swansea SA2 8PP, UK; Neurology department, Morriston Hospital, Swansea Bay University Hospital Health Board, Swansea SA6 6NL, UK. (5)Paediatric Neurology, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK. (6)Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK. (7)Institute of Medical Genetics, University Hospital of Wales, Cardiff CF14 4XW, UK. (8)Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust and Institute of Human Development, University of Manchester, Manchester M13 9WL, UK. (9)Department of Paediatric Neurology, Royal Manchester Children's Hospital, Oxford Road, Manchester M13 9WL, UK. (10)Department of Neurology, Salford Royal Hospital NHS Foundation Trust, Stott Lane, Salford M6 8HD, UK. (11)Department of Radiology, Alder Hey Children's NHS Foundation Trust, Eaton Road, Liverpool L12 2AP, UK. (12)West Midlands Regional Genetics Service, Clinical Genetics Unit, Birmingham Women's Hospital, Birmingham B15 2TG, UK. (13)Department of Paediatrics, McMaster University, 1200 Main St. W., Hamilton, ON L8N 3Z5, Canada. (14)Department of Pediatrics, Peking University First Hospital, Xicheng District, Beijing 100034, China. (15)Cipher Gene Ltd, Beijing, China. (16)Genomics England, London EC1M 6BQ, UK. (17)Neurology and Molecular Neuroscience Research, Institute of Life Science, Swansea University Medical School, Swansea University, Swansea SA2 8PP, UK; Faculty of Medicine and Health, Camperdown, University of Sydney, NSW 2006, Australia. (18)Neurology and Molecular Neuroscience Research, Institute of Life Science, Swansea University Medical School, Swansea University, Swansea SA2 8PP, UK; Kids Neuroscience Centre, Kids Research, Children Hospital at Westmead, Sydney, NSW 2145, Australia; Brain and Mind Centre, Faculty of Medicine and Health, University of Sydney, NSW 2050, Australia.

Tofersen has been developed for treatment of which disease?

Tofersen is an antisense oligonucleotide that mediates the degradation of superoxide dismutase 1 (SOD1) messenger RNA to reduce SOD1 protein synthesis is being studied for the treatment of amyotrophic lateral sclerosis due to SOD1 mutations.

BACKGROUND: Tofersen is an antisense oligonucleotide that mediates the degradation of superoxide dismutase 1 (SOD1) messenger RNA to reduce SOD1 protein synthesis. Intrathecal administration of tofersen is being studied for the treatment of amyotrophic lateral sclerosis (ALS) due to SOD1 mutations. METHODS: We conducted a phase 1-2 ascending-dose trial evaluating tofersen in adults with ALS due to SOD1 mutations. In each dose cohort (20, 40, 60, or 100 mg), participants were randomly assigned in a 3:1 ratio to receive five doses of tofersen or placebo, administered intrathecally for 12 weeks. The primary outcomes were safety and pharmacokinetics. The secondary outcome was the change from baseline in the cerebrospinal fluid (CSF) SOD1 concentration at day 85. Clinical function and vital capacity were measured. RESULTS: A total of 50 participants underwent randomization and were included in the analyses; 48 participants received all five planned doses. Lumbar puncture-related adverse events were observed in most participants. Elevations in CSF white-cell count and protein were reported as adverse events in 4 and 5 participants, respectively, who received tofersen. Among participants who received tofersen, one died from pulmonary embolus on day 137, and one from respiratory failure on day 152; one participant in the placebo group died from respiratory failure on day 52. The difference at day 85 in the change from baseline in the CSF SOD1 concentration between the tofersen groups and the placebo group was 2 percentage points (95% confidence interval [CI], -18 to 27) for the 20-mg dose, -25 percentage points (95% CI, -40 to -5) for the 40-mg dose, -19 percentage points (95% CI, -35 to 2) for the 60-mg dose, and -33 percentage points (95% CI, -47 to -16) for the 100-mg dose. CONCLUSIONS: In adults with ALS due to SOD1 mutations, CSF SOD1 concentrations decreased at the highest concentration of tofersen administered intrathecally over a period of 12 weeks. CSF pleocytosis occurred in some participants receiving tofersen. Lumbar puncture-related adverse events were observed in most participants. (Funded by Biogen; ClinicalTrials.gov number, NCT02623699; EudraCT number, 2015-004098-33.).

What is the cause of lactose intolerance?

Lactose intolerance is a common condition caused by lactase deficiency and may result in symptoms of lactose malabsorption (bloating, flatulence, abdominal discomfort, and change in bowel habits). Four clinical subtypes of lactose intolerance may be distinguished, namely lactase deficiency in premature infants, congenital lactase deficiency, adult-type hypolactasia and secondary lactase intolerance.

Lactose intolerance is a common condition caused by lactase deficiency and may result in symptoms of lactose malabsorption (bloating, flatulence, abdominal discomfort, and change in bowel habits). As current data is limited, the aim of our study was to assess the efficacy of probiotics with a β-galactosidase activity on symptoms of lactose malabsorption and on the lactose hydrogen breath test (LHBT). The study group comprised eight symptomatic female patients with a positive LHBT. Patients were treated for 6 months with a probiotic formula with β-galactosidase activity (Bio-25, Ambrosia-SupHerb, Israel). All patients completed a demographic questionnaire as well as a diary for the assessment of symptom severity and frequency at entry, every 8 weeks, and at the end of the treatment period. Measurements of hydrogen (H2) levels (parts per million, ppm) at each of these time points were also performed. End points were a decrease of 50% in symptom severity or frequency, and the normalization (decrease below cutoff point of 20 ppm) of the breath test. Mean age and mean body mass index (BMI) were 36.4 ± 18.6 years and 23.2 kg/m2, respectively. Compared to baseline scores, the frequency of most symptoms, and the severity of bloating and flatulence, improved after treatment. Normalization of LHBT was obtained in only two patients (25%). In this pilot study, Bio-25, a unique formulation of probiotics with β-galactosidase activity, demonstrated symptom resolution in most patients with lactose malabsorption. A larger randomized trial is warranted to confirm these preliminary findings. INTRODUCTION: Adult lactose intolerance (ALI) significantly alters calcium intake and absorption, and thus may promote osteoporosis. ALI is a recessive condition with a geographical north-south gradient characterised by decreased levels of intestinal lactase. PCR-based genotyping of lactase (LCT) gene polymorphisms is a safe and easy way to diagnose ALI and may complement diagnostic procedures to identify individuals at risk for reduced calcium intake and subsequently osteoporosis due to lactose malabsorption. Therefore, we investigated the frequency of ALI and its influence on calcium intake, markers of bone metabolism and bone mineral density (BMD) in a cohort of Turkish immigrants living in Germany. MATERIALS AND METHODS: We investigated single nucleotide polymorphisms of the LCT gene, calcium intake, markers of bone metabolism and BMD in 183 Turkish immigrants. RESULTS: ALI was diagnosed in 154 out of 183 (81%) probands. ALI was significantly associated with self-reported lactose intolerance (p < 0.001) and dislike for dairy products (p < 0.01). Osteopenia was diagnosed in 59 out of 183 (32%) and osteoporosis in 15 out of 183 (8%) probands. Probands with reduced BMD had ALI in 86%. All probands had a decreased calcium intake [mg/week]. There was no significant association between ALI, calcium intake, markers of bone metabolism or BMD. CONCLUSION: Turkish immigrants mostly have ALI and overall show a reduced calcium intake per week. However, ALI did not significantly influence calcium intake, markers of bone metabolism or BMD in this cohort. Therefore, ALI in Turkish immigrants does not seem to be a risk factor for osteoporosis. Dairy products have been a key component of dietary guidance in the United States for more than 100 years. In light of major advances in the understanding of dietary intake and metabolism, the aim of this review was to examine whether dairy should remain a single commodity in federal guidance. Considerations include recognizing that a substantial proportion of the world's adult population (65%-70%) exhibits lactase nonpersistence, a reduced ability to metabolize lactose to glucose and galactose. Shifts in the US population, including a greater proportion of African Americans and Asians, are of key importance because several studies have shown a markedly higher prevalence of lactase nonpersistence and, consequently, a lower dairy intake among these groups. While cow's milk alternatives are available, families who use them will pay up to an additional $1400 per year compared with those who are able to consume dairy products. Dietary guidance also contains downstream effects for government assistance, such as the US Department of Agriculture's National School Lunch Program and School Breakfast Program. For reasons like these, Canada has recently removed dairy as a separate food group in national dietary guidance. The results of the present review suggest that consideration of this modification when developing population-level guidelines in the United States is warranted. Lactose is a reducing sugar consisting of galactose and glucose, linked by a β (1→4) glycosidic bond, considered as an antioxidant due to its α-hydroxycarbonyl group. Lactose is widely ingested through the milk and other unfermented dairy products and is considered to be one of the primary foods. On the other hand, lactose is also considered as one of the most widely used excipients for the development of pharmaceutical formulations. In this sense, lactose has been related to numerous drug-excipient or drug-food pharmacokinetic interactions. Intolerance, maldigestion and malabsorption of carbohydrates are common disorders in clinical practice, with lactose-intolerance being the most frequently diagnosed, afflicting 10% of the world's population. Four clinical subtypes of lactose intolerance may be distinguished, namely lactase deficiency in premature infants, congenital lactase deficiency, adult-type hypolactasia and secondary lactase intolerance. An overview of the main uses of lactose in human nutrition and in the pharmaceutical industry and the problems derived from this circumstance are described in this review. Lactose intolerance (LI) is characterized by the presence of primarily gastrointestinal clinical signs resulting from colonic fermentation of lactose, the absorption of which is impaired due to a deficiency in the lactase enzyme. These clinical signs can be modified by several factors, including lactose dose, residual lactase expression, concurrent ingestion of other dietary components, gut-transit time, and enteric microbiome composition. In many of individuals with lactose malabsorption, clinical signs may be absent after consumption of normal amounts of milk or, in particular, dairy products (yogurt and cheese), which contain lactose partially digested by live bacteria. The intestinal microbiota can be modulated by biotic supplementation, which may alleviate the signs and symptoms of LI. This systematic review summarizes the available evidence on the influence of prebiotics and probiotics on lactase deficiency and LI. The literature search was conducted using the MEDLINE (via PUBMED) and SCOPUS databases following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and included randomized controlled trials. For each study selected, the risk of bias was assessed following the Cochrane Collaboration methodology. Our findings showed varying degrees of efficacy but an overall positive relationship between probiotics and LI in relation to specific strains and concentrations. Limitations regarding the wide heterogeneity between the studies included in this review should be taken into account. Only one study examined the benefits of prebiotic supplementation and LI. So further clinical trials are needed in order to gather more evidence.

What is Etizolam?

Etizolam is a benzodiazepine analogue that is approved for use in Japan, Italy and India as an anxiolytic drug with a pharmacologic profile similar to that of the classic benzodiazepines. Neurochemical research suggests that etizolam may have selectivity for the subpopulation of Y-aminobutyric acid type A receptors associated with anxiety (ie, alpha1, beta2, gamma2).

A double-blind study was carried out in 30 female patients with generalized anxiety disorders associated with depressive symptoms to compare the effectiveness and tolerability of etizolam and alprazolam. Patients were allocated at random to receive one or other drug at a dosage of 0.5 mg twice daily for 5 weeks. Assessments were made on entry and after 3 and 5 weeks of treatment using the Hamilton rating scales for anxiety and for depression. The results showed that both drugs had marked anxiolytic and antidepressive activity, there being significant reductions after treatment in mean total rating scores compared to baseline. Although there was no statistically significant difference between the two drugs, there was a trend for etizolam to be more effective in relieving anxiety somatization symptoms. Apart from moderate daytime drowsiness in a few patients, both drugs were considered to be extremely well tolerated. Etizolam, an anti-anxiety agent which is an antagonist of platelet-activating factor receptors, was administered to patients with chronic subdural hematoma (CSH) after hematoma removal to assess the effectiveness for preventing recurrence compared with control patients not given the drug after surgery. The remaining volumes of subdural hematomas on brain computed tomography were measured approximately 1 month after removal. Volume in the etizolam group (15 patients) was significantly smaller than in the control group (24 patients). Hematoma recurrence was not detected in the etizolam group 3 months after surgery, but occurred in the control group. The difference was significant. Etizolam administration may be useful for the prevention of recurrence of CSH. BACKGROUND: Etizolam is an anxiolytic drug with a pharmacologic profile similar to that of the classic benzodiazepines. Neurochemical research suggests that etizolam may have selectivity for the subpopulation of Y-aminobutyric acid type A receptors associated with anxiety (ie, alpha1, beta2, gamma2). This property, plus its characterization as a ligand with fewer of the adverse events typical of full agonists (impaired cognitive function, tolerance, and dependence), led to its selection for this study. OBJECTIVES: The primary aim of this study was to test for the noninferiority of etizolam 0.5 mg BID versus placebo in affecting cognitive function in patients with mild to moderate anxiety disorder of recent onset (<1 month). Anxiety measures and tolerability were also assessed. METHODS: Patients between the ages of 18 and 65 years were eligible for enrollment. This double-blind, placebo-controlled study was performed in 5 centers in Italy using a 2-treatment, 3-period crossover design. Patients were randomized to 3-week sequences of either etizolam-placebo-placebo or placebo-etizolam-etizolam. They were evaluated at 4 scheduled visits (screening and days 7, 14, and 21). Cognitive function was assessed using scores from the Wechsler Adult Intelligence Scale (WAIS) Digit Span test (total forward and backward scores and the time required to perform the test). Anxiety was measured using the Hamilton Anxiety Rating Scale (HAM-A) and the State-Trait Anxiety Inventory (STAI) for screening and to monitor adequacy of therapy. Blood pressure, heart rate, weight, and adverse events were also recorded. RESULTS: A total of 77 white patients were enrolled (mean age, 33.3 years [range, 22-60 years]; 62.3% female; mean weight, 65.2 kg). With a power of 0.80, the difference between the effects of etizolam and placebo on WAIS Digit Span performance was not significant for total score (0.102 [90% CI, -0.130 to 0.335]) or time required for completion (0.029 second [90% CI, -0.574 to 0.632]). Anxiety, as measured using the HAM-A and STAI instruments, did not differ significantly between groups. No significant differences were found between etizolam 0.5 mg BID and placebo for cardiovascular events, weight changes, or adverse events. Mild or moderate somnolence was reported by 7 of 77 patients (9.1% [3 patients while receiving etizolam and 4 patients while receiving etizolam and placebo]). CONCLUSIONS: No significant differences between etizolam 0.5 mg BID and placebo were found for cognitive function or anxiety measures in these patients with anxiety. Etizolam was well tolerated. The prevalence of benzodiazepine consumption in Japan is one of the highest worldwide. Etizolam is the most abused drug of the benzodiazepine class. The treatment of benzodiazepine dependence is difficult. We report a case of successful dosage reduction in a 24-year-old female patient with etizolam dependence. She was diagnosed with etizolam dependence at the age of 22 years old. We proposed a benzodiazepine dependence treatment that involved replacing etizolam with a long-acting benzodiazepine class drug in conjunction with a long-term weaning plan. However, the patient refused the treatment and insisted that reducing the number of tablets would increase her anxiety. After providing a detailed explanation and receiving consent from the patient, a treatment regimen consisting of fine granules of etizolam mixed with lactose granules was begun with the aim of reducing the percentage of etizolam at a rate of 0.3 mg/week. The treatment of etizolam dependence in this patient was successful. This treatment strategy may be an effective option for patients who are difficult to treat with conventional methods, or who have anxiety regarding the reduction of the amount of the drug itself. Tizanidine hydrochloride, α2-receptor stimulant, is a central muscle relaxant. Etizolam is a benzodiazepine-based anti-anxiety agent. Both drugs are widely used for the treatment of a variety of muscle pain and frequently used together in Japan. We experienced a case of complicating prolonged myocardial dysfunction in a 56-year-old woman. Six hours after overdose of 48 mg tizanidine and 24 mg etizolam, she showed sinus bradycardia and peripheral vascular resistance decreasing shock. At that time new ST-T depressions were recognized in electrocardiography (ECG); however, structural heart diseases were interpreted as negative by other examinations. Intravenous norepinephrine infusion was useful to maintain the hemodynamic stability. ECG reversed to normal findings on day 14; however, the cardiac nuclear medicine studies on day 30 showed severe fatty metabolic disorder and sympathetic denervation. Non-sustained ventricular tachycardia was detected. Complete recovery of the myocardium damage required one year. For one mechanism, it was suggested that over-stimulation of α2-receptor by tizanidine inhibited the norepinephrine secretion and reuptake at pre-synaptic surface of adipose cell and cardiac sympathetic nerve. We want to suggest that the cardiologist should consider the risk of fatal arrhythmia and long-term myocardium toxicity as the poisoning of the central muscle relaxant and benzodiazepine agent. <Learning objective: We experienced a case of complicating prolonged myocardial dysfunction in a 56-year-old woman. Six hours after overdose of 48 mg tizanidine and 24 mg etizolam, she showed sinus bradycardia and peripheral vascular resistance decreasing shock. The cardiac nuclear medicine studies on day 30 showed severe fatty metabolic disorder and sympathetic denervation. Non-sustained ventricular tachycardia was detected. Complete recovery of the myocardium damage required one year.>. Etizolam is a drug from the thienotriazoldiazepine class, widely prescribed as anxiolytic due to its apparently secure toxicological profile. Nevertheless, some recent cases of etizolam dependence, intoxications and fatalities associated to its abuse have been reported in the international literature. For this reason, the drug listed as new psychoactive substance (NPS) by the World Health Organization (WHO) since 2015. Euphoric effect at high dosage is the first cause of its recreational use that has determined a wider distribution in the illicit market. An experimental study was performed to obtain evidence that etizolam at low therapeutic dosages is a drug with negligible influence on the psychomotor performances involved in driving. The psychomotor performance was assessed by performing different tests, such as critical tracking task (CTT), critical flicker fusion (CFF), choice reaction time (CRT), visual vigilance task (VVT), response competition test (RCT) in a group of 16 healthy volunteers after a single administration of etizolam at two different dosages (0.25 mg or 1.00 mg) in comparison to placebo. The test results showed that etizolam at 0.25 mg and 1.00 mg had no significant effect on vigilance, short term memory, psychomotor coordination or speed in decision making. Differently, abuse of etizolam to obtain the euphoric effects at presumably high dosages or in combination with other psychoactive substances could be fatal. The negligible side effects on mental and behavioral function demonstrated by this study, could represent an incitement to abuse, which can be strongly discouraged with correct information on differences between its correct use and its misuse. Etizolam is a novel psychoactive substance and novel benzodiazepine of the thienotriazolodiazepine class, which has recently seen an increasing trend in use worldwide. We report a case series of 10 decedents with etizolam and opioids in their systems. Death investigation, expanded toxicology and medical investigation information were included for contextualization of etizolam in death. Etizolam was detected and confirmed within peripheral and cardiac blood, urine, vitreous humor and, in one case, gastric fluid, by liquid chromatography-tandem mass spectrometry and liquid chromatography-quadrupole time of flight mass spectrometry methodologies. Death investigation indicated nonmedical use of most drugs. Medical investigation commonly noted pulmonary edema, cardiomegaly and cerebral swelling. The majority of the decedents appeared to be unaware of the presence of etizolam and succumbed to the mixed drug toxicity of their routine depressant and narcotic analgesic drug of abuse in combination with etizolam. Etizolam use continues to be observed and poses as a potentially lethal contribution to multiple drug toxicity, especially in the age of the opioid crisis. Assessment of analytes like etizolam requires up-to-date methodologies and vigilance in testing to better characterize the toxicology and interpret the contribution to death. Etizolam is a benzodiazepine analogue that is approved for use in Japan, Italy and India but has recently appeared as a nonapproved product on the illicit drug market in Europe and North America. Etizolam was identified in a crystalline material seized at a Kentucky racetrack, raising concerns that this drug may have been used in racing. The aim of this study was to characterize the metabolism and excretion of etizolam in horses to generate information on its disposition and to incorporate the correct urinary and serum target analytes into anti-doping screening procedures. Etizolam was administered both intravenous and orally at a dose of 0.1 mg/kg of body weight to three horses using a two-way crossover design. Pre-administration and post-administration serum and urine samples were collected and experiments conducted to identify potential metabolites in these samples. Additionally, in vitro metabolism studies using horse liver S9 were undertaken to complement the in vivo metabolism studies. Numerous metabolites were id1entified in both serum and urine in additional to parent drug, with α-hydroxy-etizolam producing the most abundant analytical signal (in terms of signal intensity and duration of detection) of the identified metabolites in both matrices. Therefore, α-hydroxy-etizolam is considered to be the most appropriate analyte for detection for anti-doping purposes. Analytical methods were developed and validated and then applied to post-administration samples to generate concentrations of etizolam and its major metabolites in serum and urine, resulting in excretion profiles that can be used to guide approaches to detecting the use of the drug. Etizolam is a thienodiazepine that although licensed for clinical usage in Japan, India and South Korea is commonly abused and detected in post-mortem cases around the world. To date, there are limited data in the literature to allow for the interpretation of blood concentrations of etizolam in post-mortem cases. A liquid chromatography with tandem mass spectrometry method was used to quantitate etizolam concentrations in 28 post-mortem cases where etizolam was detected. The median concentration of etizolam in femoral blood was 8.5 ng/mL (range 1.0-172.0 ng/mL; n = 24); in antemortem plasma, the etizolam concentration range was 4-44 ng/mL (n = 4). The mean age of the individuals abusing etizolam was 38.5 ± 8.4 years (median 39 years), with the majority being male (86%). In all of the cases, multiple drugs were detected, with the most common being pregabalin (61%) followed by morphine/heroin (54%), diazepam (54%) and benzoylecgonine (21%), illustrating the increasing problem of poly-substance use in drug abusers. The cause of death in the cases in which etizolam was detected was multi-drug toxicity in 87.5% of the cases, with 12.5% unrelated to drug use (hangings and blunt-force trauma). These data will further help forensic practitioners with the interpretation of post-mortem etizolam concentrations. Introduction: The use of novel designer drugs has increased worldwide over the years. Etizolam is a designer benzodiazepine (BZD) that has raised concern because of its growing non-medical use, liability to tolerance and dependence, and related harms. Studies exploring the abuse liability and cognitive effects of etizolam outside the therapeutic doses are lacking. Aims: To explore the abuse liability of etizolam and the characteristics of patients affected by etizolam high-dose dependence in a nationwide tertiary referral addiction unit. To document the cognitive changes to etizolam high-dose use. Design and Methods: Sociodemographic and clinical data on subjects with etizolam high-dose use were retrospectively collected from a database of 1,293 patients consecutively admitted to the Addiction Medicine Unit, Verona University Hospital, Italy for detoxification from high-dose BZDs or Z-drugs dependence. Thorough neuropsychological testing explored the cognitive side effects of high-dose etizolam use. Results: We found eleven etizolam high-dose users, of which eight used etizolam only, and three used etizolam with other BZDs/zolpidem. All the patients were prescribed etizolam for medical reasons, i.e., anxiety and/or insomnia. Neuropsychological evaluation showed deficits of working memory, visuospatial memory and executive function in a 27-year-old woman who used etizolam 15 mg daily. Discussion: Our findings suggest that abuse and dependence liability of etizolam should be considered a public health and social problem. They offer preliminary evidence on the cognitive side effects of etizolam high-dose use. Conclusions: This report offers new information on the potential harms of etizolam in patients who are prescribed this drug for medical reasons.

Is AZD9668 a VEGF mRNA drug?

AZD9668 is a reversible and selective inhibitor of neutrophil elastase.

BACKGROUND: Neutrophil elastase (NE) is implicated in chronic obstructive pulmonary disease (COPD). AZD9668 is a reversible and selective inhibitor of NE, well tolerated at doses of 60 mg bid during Phase I/IIa development. METHODS: This 12-week, randomised, double-blind, placebo-controlled, Phase IIb, trial (NCT01023516), investigated the efficacy and safety of AZD9668 (60 mg bid) versus placebo in patients with symptomatic COPD and a history of exacerbation receiving maintece budesonide/formoterol. Primary outcome variable: forced expiratory volume in one second (FEV1). Secondary endpoints included: post-bronchodilator FEV1, pre- and post-bronchodilator forced vital capacity, FEV6, forced expiratory flow between 25% and 75% of vital capacity and inspiratory capacity; peak expiratory flow and FEV1 measured at home; EXAcerbations of Chronic pulmonary disease Tool and Breathlessness, Cough and Sputum Scores; St George's respiratory questionnaire for COPD (SGRQ-C) scores; exacerbations; and safety assessments. RESULTS: Six hundred and fifteen patients were randomised: placebo (302), AZD9668 60 mg bid (313). AZD9668 showed no effect on lung function: change in mean pre-bronchodilator FEV1 versus placebo was 0.01L (95% confidence interval: -0.03, 0.05; p=0.533). AZD9668 did not significantly improve respiratory signs and symptoms, SGRQ-C score or time to first exacerbation. Adverse events were similar for AZD9668 and placebo. CONCLUSIONS: Three months' treatment with AZD9668 did not improve lung function, respiratory signs and symptoms or SGRQ-C score when added to budesonide/formoterol maintece therapy in patients with COPD. In the absence of definitive biomarkers of short-term disease progression, further research is needed to determine the optimal duration of studies to evaluate NE inhibitors as disease-modifying agents.

What is caused by de novo VPS4A mutations?

De novo VPS4A mutations cause multisystem disease with abnormal neurodevelopment. VPS4A normal function is required for multiple human developmental and cellular processes.

Author information: (1)Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK; Department of Medical Genetics, University of Cambridge, Cambridge CB2 0QQ, UK. (2)Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome 00161, Italy. (3)Department of Haematology, NHS Blood and Transplant Centre, University of Cambridge, Cambridge CB2 0XY, UK; NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK. (4)Department of Medical Genetics, University of Cambridge, Cambridge CB2 0QQ, UK; European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. (5)Microscopy Area, Core Facilities, Istituto Superiore di Sanità, Rome 00161, Italy. (6)Department of Medical Genetics, University of Cambridge, Cambridge CB2 0QQ, UK. (7)Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK; Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK. (8)Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome 00161, Italy; Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome 00146, Italy. (9)Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome 00146, Italy. (10)Genomics England, London, UK. (11)Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome 00168, Italy. (12)Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome 00168, Italy; Università Cattolica del Sacro Cuore, Rome 00168, Italy. (13)Unit of Mechanisms, Biomarkers and Models, Department of Environment and Health, Istituto Superiore di Sanità, Rome 00161, Italy. (14)Department of Clinical Genetics, Great Ormond Street Hospital, London WC1N 3JH, UK. (15)Department of Paediatric Neurology, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, UK. (16)Department of Clinical Genetics, Liverpool Women's Hospital, Liverpool L8 7SS, UK. (17)Department of Medical Genetics, Guys' and St Thomas' NHS Foundation Trust, London SE1 9RT, UK. (18)Clinical Genetics, Birmingham Women's and Children's NHS Foundation Trust, Birmingham B15 2TG, UK. (19)Phoenix Children's Hospital, Phoenix, AZ 76109, USA. (20)Cook Children's Medical Centre, Fort Worth, TX 76104, USA. (21)Colchester Hospital, East Suffolk and North Essex NHS Foundation Trust, Essex CO4 5JL, UK. (22)Ophthalmology Department, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, UK. (23)Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genova 16163, Italy; Area of Neuroscience, SISSA, Trieste 34136, Italy. (24)Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome 00146, Italy. Electronic address: [email protected]. (25)Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK; Department of Medical Genetics, University of Cambridge, Cambridge CB2 0QQ, UK. Electronic address: [email protected].

What is the use of pegcetacoplan?

Pegcetacoplan is promising for paroxysmal nocturnal haemoglobinuria and Geographic Atrophy.

PURPOSE: Geographic atrophy (GA), a late stage of age-related macular degeneration (AMD), is a major cause of blindness. Even while central visual acuity remains relatively well preserved, GA often causes considerable compromise of visual function and quality of life. No treatment currently exists. We evaluated the safety and efficacy of pegcetacoplan, a complement C3 inhibitor, for treatment of GA. DESIGN: Prospective, multicenter, randomized, sham-controlled phase 2 study. PARTICIPANTS: Two hundred forty-six patients with GA. METHODS: Patients with GA were assigned randomly in a 2:2:1:1 ratio to receive intravitreal injections of 15 mg pegcetacoplan monthly or every other month (EOM) or sham intravitreal injections monthly or EOM for 12 months with follow-up at months 15 and 18. Area and growth of GA were measured using fundus autofluorescence imaging. MAIN OUTCOME MEASURES: The primary efficacy end point was mean change in square root GA lesion area from baseline to month 12. Secondary outcome measures included mean change from baseline in GA lesion area without the square root transformation, distance of GA lesion from the fovea, best-corrected visual acuity (BCVA), low-lumice BCVA, and low-lumice visual acuity deficit. The primary safety end point was the number and severity of treatment-emergent adverse events. RESULTS: In patients receiving pegcetacoplan monthly or EOM, the GA growth rate was reduced by 29% (95% confidence interval [CI], 9-49; P = 0.008) and 20% (95% CI, 0-40; P = 0.067) compared with the sham treatment group. Post hoc analysis showed that the effect was greater in the second 6 months of treatment, with observed reductions of 45% (P = 0.0004) and 33% (P = 0.009) for pegcetacoplan monthly and EOM, respectively. Two cases of culture-positive endophthalmitis and 1 case of culture-negative endophthalmitis occurred in the pegcetacoplan monthly group. New-onset investigator-determined exudative AMD was reported more frequently in pegcetacoplan-treated eyes (18/86 eyes [20.9%] and 7/79 eyes [8.9%] in monthly and EOM groups, respectively) than in sham-treated eyes (1/81 eyes [1.2%]). CONCLUSIONS: Local C3 inhibition with pegcetacoplan resulted in statistically significant reductions in the growth of GA compared with sham treatment. Phase 3 studies will define the efficacy and safety profile further. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, life-threatening hematologic disease characterized by chronic complement-mediated hemolysis and thrombosis. Despite treatment with eculizumab, a C5 inhibitor, 72% of individuals remain anemic. Pegcetacoplan (APL-2), a PEGylated C3 inhibitor, has the potential to provide more complete hemolysis control in patients with PNH. This open-label, phase Ib study was designed to assess the safety, tolerability, and pharmacokinetics of pegcetacoplan in subjects with PNH who remained anemic during treatment with eculizumab. Pharmacodynamic endpoints were also assessed as an exploratory objective of this study. Data are presented for six subjects in cohort 4 who received treatment for up to 2 years. In total, 427 treatment-emergent adverse events (TEAEs) were reported, 68 of which were possibly related to the study drug. Eight serious TEAEs occurred in two subjects; three of these events were considered possibly related to the study drug. Pegcetacoplan pharmacokinetic concentrations accumulated with repeated dosing, and steady state was reached at approximately 6-8 weeks. Lactate dehydrogenase levels were well controlled by eculizumab at baseline. Pegcetacoplan increased hemoglobin levels and decreased both reticulocyte count and total bilirubin in all six subjects. Improvements were observed in Functional Assessment of Chronic Illness Therapy Fatigue scores. Two subjects discontinued for reasons unrelated to pegcetacoplan. All four subjects who completed the study transitioned to pegcetacoplan monotherapy following eculizumab discontinuation and avoided transfusions. In this small study, pegcetacoplan therapy was generally well-tolerated, and resulted in an improved hematological response by achieving broad hemolysis control, enabling eculizumab discontinuation. OBJECTIVES: To evaluate clinical characteristics of eyes in which investigator-determined new-onset exudative age-related macular degeneration (eAMD) developed during the FILLY trial. DESIGN: Post hoc analysis of the phase 2 study of intravitreal pegcetacoplan in geographic atrophy (GA). SUBJECTS: Patients with GA secondary to age-related macular degeneration (AMD), n = 246. INTERVENTION: Either 15 mg intravitreal pegcetacoplan or sham given monthly or every other month for 12 months followed by a 6-month off-treatment period. MAIN OUTCOME MEASURES: Time of new eAMD onset in the study eye, history of eAMD in the fellow eye, presence of double-layer sign (DLS) on structural OCT in the study eye, changes in retinal anatomic features by structural OCT and fluorescein angiography (FA), and changes in visual acuity. RESULTS: Exudation was reported in 26 study eyes across treatment groups over 18 months. Mean time to eAMD diagnosis was 256 days (range, 31-555 days). Overall, a higher proportion of patients with a baseline history of eAMD in the fellow eye (P = 0.016) and a DLS in the study eye (P = 0.0001) demonstrated eAMD. Among study eyes in which eAMD developed, 18 of 26 (69%) had history of fellow-eye eAMD and 19 of 26 (73.1%) had DLS at baseline, compared with 76 of 217 study eyes (35%; P = 0.0007) and 70 of 215 study eyes (32.5%; P < 0.0001), respectively, in which eAMD did not develop. All 21 patients with structural OCT imaging at the time of eAMD diagnosis demonstrated subretinal fluid, intraretinal cysts, or both consistent with exudation. Among 17 patients who underwent FA at eAMD diagnosis, 10 showed detectable macular neovascularization (MNV), all occult lesions. Development of eAMD did not have an appreciable impact on visual acuity, and all patients responded to anti-vascular endothelial growth factor (VEGF) therapy. CONCLUSIONS: Intravitreal pegcetacoplan slowed the rate of GA growth and was associated with an unexpected dose-dependent increased incidence of eAMD with no temporal clustering of onset. Exudative AMD seemed to be associated with baseline eAMD in the contralateral eye and a DLS, suggestive of nonexudative MNV, in the study eye. The safety profile of pegcetacoplan was acceptable to proceed to phase 3 studies without adjustments to enrollment criteria. BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease characterized by chronic complement-mediated hemolysis. C5 inhibition controls intravascular hemolysis in untreated PNH but cannot address extravascular hemolysis. Pegcetacoplan, a pegylated peptide targeting proximal complement protein C3, potentially inhibits both intravascular and extravascular hemolysis. METHODS: We conducted a phase 3 open-label, controlled trial to assess the efficacy and safety of pegcetacoplan as compared with eculizumab in adults with PNH and hemoglobin levels lower than 10.5 g per deciliter despite eculizumab therapy. After a 4-week run-in phase in which all patients received pegcetacoplan plus eculizumab, we randomly assigned patients to subcutaneous pegcetacoplan monotherapy (41 patients) or intravenous eculizumab (39 patients). The primary end point was the mean change in hemoglobin level from baseline to week 16. Additional clinical and hematologic markers of hemolysis and safety were assessed. RESULTS: Pegcetacoplan was superior to eculizumab with respect to the change in hemoglobin level from baseline to week 16, with an adjusted (least squares) mean difference of 3.84 g per deciliter (P<0.001). A total of 35 patients (85%) receiving pegcetacoplan as compared with 6 patients (15%) receiving eculizumab no longer required transfusions. Noninferiority of pegcetacoplan to eculizumab was shown for the change in absolute reticulocyte count but not for the change in lactate dehydrogenase level. Functional Assessment of Chronic Illness Therapy-Fatigue scores improved from baseline in the pegcetacoplan group. The most common adverse events that occurred during treatment in the pegcetacoplan and eculizumab groups were injection site reactions (37% vs. 3%), diarrhea (22% vs. 3%), breakthrough hemolysis (10% vs. 23%), headache (7% vs. 23%), and fatigue (5% vs. 15%). There were no cases of meningitis in either group. CONCLUSIONS: Pegcetacoplan was superior to eculizumab in improving hemoglobin and clinical and hematologic outcomes in patients with PNH by providing broad hemolysis control, including control of intravascular and extravascular hemolysis. (Funded by Apellis Pharmaceuticals; PEGASUS ClinicalTrials.gov, NCT03500549.). The FDA approval of pegcetacoplan (Empaveli), a PEGylated compstatin-based C3 therapeutic, as a new treatment for paroxysmal nocturnal hemoglobinuria (PNH) marks a milestone in the history of complement drug discovery. Almost 15 years after the approval of the first complement-specific drug for PNH, the anti-C5 antibody eculizumab, a novel class of complement inhibitors with a distinct mechanism of action finally enters the clinic. This landmark decision broadens the spectrum of available complement therapeutics, offering patients with unmet clinical needs or insufficient responses to anti-C5 therapy an alternative treatment option with a broad activity profile. Here we present a brief historical account of this newly approved complement drug, consolidating its approval within the long research record of the compstatin family of peptidic C3 inhibitors. Pegcetacoplan (Empaveli™) is a PEGylated pentadecapeptide developed by Apellis Pharmaceuticals for the treatment of complement-mediated diseases. It binds to complement component 3 (C3) and its activation fragment C3b, controlling the cleavage of C3 and the generation of the downstream effectors of complement activation and thus both C3b-mediated extravascular haemolysis and terminal complement-mediated intravascular haemolysis. Pegcetacoplan is the first C3-targeted paroxysmal nocturnal haemoglobinuria (PNH) therapy to be approved (in May 2021) in the USA, where it is indicated for the treatment of adults with PNH, including those switching from C5 inhibitor therapy with eculizumab and ravulizumab. A regulatory assessment of pegcetacoplan for the treatment of PNH is currently underway in the EU and Australia. Pegcetacoplan is also being investigated as a therapeutic option in other complement-mediated diseases, including age-related macular degeneration, C3 glomerulopathy and autoimmune haemolytic anaemia. The recommended dosage regimen of pegcetacoplan is 1080 mg twice weekly, administered as a subcutaneous infusion via an infusion pump with a ≥ 20 mL reservoir. This article summarizes the milestones in the development of pegcetacoplan leading to this first approval for the treatment of adults with PNH. Complement is an elaborate system of innate immunity. Genetic variants and autoantibodies leading to excessive complement activation are implicated in a variety of human diseases. Among them, the hematologic disease paroxysmal nocturnal hemoglobinuria (PNH) remains the prototypic model of complement activation and inhibition. Eculizumab, the first-in-class complement inhibitor, was approved for PNH in 2007. Addressing some of the unmet needs, a long-acting C5 inhibitor, ravulizumab, and a C3 inhibitor, pegcetacoplan, have also now been approved for PNH. Novel agents, such as factor B and factor D inhibitors, are under study, with very promising results. In this era of several approved targeted complement therapeutics, selection of the proper drug must be based on a personalized approach. Beyond PNH, complement inhibition has also shown efficacy and safety in cold agglutinin disease, primarily with the C1s inhibitor of the classical complement pathway sutimlimab, as well as with pegcetacoplan. Furthermore, C5 inhibition with eculizumab and ravulizumab, as well as inhibition of the lectin pathway with narsoplimab, is being investigated in transplantation-associated thrombotic microangiopathy. With this revolution of next-generation complement therapeutics, additional hematologic entities, such as delayed hemolytic transfusion reaction or immune thrombocytopenia, might also benefit from complement inhibitors. Therefore, this review aims to describe state-of-the-art knowledge of targeting complement in hematologic diseases, focusing on (1) complement biology for the clinician, (2) complement activation and therapeutic inhibition in prototypic complement-mediated hematologic diseases, (3) hematologic entities under investigation for complement inhibition, and (4) other complement-related disorders of potential interest to hematologists.

What is the activity of a Oligosaccharyltransferases ?

oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid odiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life. Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation. Lipid-linked oligosaccharides (LLOs) play an important role in the N-glycosylation pathway as the donor substrate of oligosaccharyltransferases (OSTs), which are responsible for the en bloc transfer of glycan chains onto a nascent polypeptide. The lipid component of LLO in both eukarya and archaea consists of a dolichol, and an undecaprenol in prokarya, whereas the number of isoprene units may change between species. Given the potential relevance of LLOs and their related enzymes to diverse biotechnological applications, obtaining reliable LLO models from distinct domains of life could support further studies on complex formation and their processing by OSTs, as well as protein engineering on such systems. In this work, molecular modeling techniques, such as quantum mechanics calculations, molecular dynamics simulations, and metadynamics were employed to study eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol), bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol), and archaeal (Glc1-Man1-Gal1-Man1-Glc1-Gal1-Glc1-P-Dolichol) LLOs in membrane bilayers. Microsecond molecular dynamics simulations and metadynamics calculations of LLOs revealed that glycan chains are more prone to interact with the membrane lipid head groups, while the PP linkages are positioned at the lipid phosphate head groups level. The dynamics of isoprenoid chains embedded within the bilayer are described, and membrane dynamics and related properties are also investigated. Overall, there are similarities regarding the structure and dynamics of the eukaryotic, the bacterial, and the archaeal LLOs in bilayers, which can support the comprehension of their association with OSTs. These data may support future studies on the transferring mechanism of the oligosaccharide chain to an acceptor protein. Bioconjugate vaccines, consisting of polysaccharides attached to carrier proteins, are enzymatically generated using prokaryotic glycosylation systems in a process termed bioconjugation. Key to bioconjugation are a group of enzymes known as oligosaccharyltransferases (OTases) that transfer polysaccharides to engineered carrier proteins containing conserved amino acid sequences known as sequons. The most recently discovered OTase, PglS, has been shown to have the broadest substrate scope, transferring many different types of bacterial glycans including those with glucose at the reducing end. However, PglS is currently the least understood in terms of the sequon it recognizes. PglS is a pilin-specific O-linking OTase that naturally glycosylates a single protein, ComP. In addition to ComP, we previously demonstrated that an engineered carrier protein containing a large fragment of ComP is also glycosylated by PglS. Here we sought to identify the minimal ComP sequon sufficient for PglS glycosylation. We tested >100 different ComP fragments individually fused to Pseudomonas aeruginosa exotoxin A (EPA), leading to the identification of an 11-amino acid sequence sufficient for robust glycosylation by PglS. We also demonstrate that the placement of the ComP sequon on the carrier protein is critical for stability and subsequent glycosylation. Moreover, we identify novel sites on the surface of EPA that are amenable to ComP sequon insertion and find that Cross-Reactive Material 197 fused to a ComP fragment is also glycosylated. These results represent a significant expansion of the glycoengineering toolbox as well as our understanding of bacterial O-linking sequons. Glycosylation is increasingly recognised as a common protein modification within bacterial proteomes. While great strides have been made in identifying species that contain glycosylation systems, our understanding of the proteins and sites targeted by these systems is far more limited. Within this work we explore the conservation of glycoproteins and glycosylation sites across the pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach, we generate high-confidence glycoproteomes in two widely utilized B. cenocepacia strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively at Serine residues and that glycoproteins/glycosylation sites are highly conserved across B. cenocepacia isolates. This preference for glycosylation at Serine residues is observed across at least 9 Burkholderia glycoproteomes, supporting that Serine is the domit residue targeted by PglL-mediated glycosylation across the Burkholderia genus. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharyltransferases that target conserved and shared protein substrates.

On what chromosome would the MKKS gene for McKusick-Kaufman(AKA Kaufman-McKusick) syndrome be found?

The MKKS gene is mapped to chromosome 20

McKusick-Kaufman syndrome (MKKS, MIM 236700) is a human developmental anomaly syndrome comprising hydrometrocolpos (HMC), postaxial polydactyly (PAP) and congenital heart disease (CHD). MKKS has been mapped in the Old Order Amish population to 20p12, between D20S162 and D20S894 (ref. 3). Here we describe the identification of a gene mutated in MKKS. We analysed the approximately 450-kb candidate region by sample sequencing, which revealed the presence of several known genes and EST clusters. We evaluated candidate transcripts by northern-blot analysis of adult and fetal tissues. We selected one transcript with widespread expression, MKKS, for analysis in a patient from the Amish pedigree and a sporadic, non-Amish case. The Old Order Amish patient was found to be homozygous for an allele that had two missense substitutions and the non-Amish patient was a compound heterozygote for a frameshift mutation predicting premature protein truncation and a distinct missense mutation. The MKKS predicted protein shows amino acid similarity to the chaperonin family of proteins, suggesting a role for protein processing in limb, cardiac and reproductive system development. We believe that this is the first description of a human disorder caused by mutations affecting a putative chaperonin molecule. We report a 19-year-old, non-Amish Caucasian female patient with primary amenorrhea caused by complete lack of Müllerian fusion with vaginal agenesis or Müllerian aplasia (MA), postaxial polydactyly (PAP), and tetralogy of Fallot. The genital tract anomaly of MA with and without renal or skeletal anomalies comprises Mayer-Rokitansky-Kuster-Hauser syndrome, which has not been reported with tetralogy of Fallot. The phenotypic triad of anomalies most closely resembled McKusick-Kaufman syndrome (MKS; OMIM 236700), a rare multiple congenital anomaly syndrome comprised of hydrometrocolpos (HMC), PAP, and congenital heart malformation that is inherited in an autosomal recessive pattern. While upper reproductive tract anomalies have not been reported with MKS, they have been reported with Bardet-Biedl syndrome (BBS), a syndrome that significantly overlaps with MKS. Both MKS and BBS can be caused by mutations in the MKKS or BBS6 gene on chromosome 20p12 and BBS is also associated with mutations in other genes (BBS1, BBS2, BBS4, and BBS7). To address this heterogenity, we sequenced the causative genes in MKS and BBS but no mutations in these five genes were identified. Fluorescence in situ hybridization (FISH) excluded large deletions of chromosome 20p12 and microsatellite marker studies confirmed biparental inheritance for all of the known BBS loci. The dual midline fusion defects of tetralogy of Fallot and MA suggests that either this patient has a unique syndrome with a distinct genetic etiology or that she has a genetically heterogeneous or variant form of MKS. McKusick-Kaufman syndrome (MKS, OMIM #236700) is a rare syndrome inherited in an autosomal recessive pattern with a phenotypic triad comprising hydrometrocolpos (HMC), postaxial polydactyly (PAP), and congenital cardiac disease (CHD). The syndrome is caused by mutations in the MKKS gene mapped onto chromosome 20p12 between D20S162 and D20S894 markers. Mutations in the same gene causes Bardet-Biedl-6 syndrome (BBS-6, OMIM #209900) inherited in an autosomal recessive pattern. BBS-6 comprises retinitis pigmentosa, polydactyly, obesity, mental retardation, renal and genital anomalies. HMC, CHD, and PAP defects can also occur in BBS-6, and there is a significant clinical overlap between MKS and BBS-6 in childhood. We describe a new borderline case of MKS and BBS syndrome and suggest insights for understanding correlation between MKKS gene mutations and clinical phenotype. Here, we report the results of molecular analysis of MKKS in a female proband born in an Italian nonconsanguineous healthy family that presents HMC and PAP. The mutational screening revealed the presence of two different heterozygous missense variants (p.242A>S in exon 3, p.339 I>V in exon 4) in the MKKS gene, and a nucleotide variation in 5'UTR region in exon 2 (-417 A>C).

Which maternal CYP2D6 related 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.

Describe the web application VICTOR

VICTOR is a free, dependency-free visual analytics web application that allows the visual comparison of the results of various clustering algorithms. It can handle multiple cluster set results simultaneously and compare them using ten different metrics. Clustering results can be filtered and compared with the use of data tables or interactive heatmaps, bar plots, correlation networks, sankey and circos plots.

Clustering is the process of grouping different data objects based on similar properties. Clustering has applications in various case studies from several fields such as graph theory, image analysis, pattern recognition, statistics and others. Nowadays, there are numerous algorithms and tools able to generate clustering results. However, different algorithms or parameterizations may produce quite dissimilar cluster sets. In this way, the user is often forced to manually filter and compare these results in order to decide which of them generate the ideal clusters. To automate this process, in this study, we present VICTOR, the first fully interactive and dependency-free visual analytics web application which allows the visual comparison of the results of various clustering algorithms. VICTOR can handle multiple cluster set results simultaneously and compare them using ten different metrics. Clustering results can be filtered and compared to each other with the use of data tables or interactive heatmaps, bar plots, correlation networks, sankey and circos plots. We demonstrate VICTOR's functionality using three examples. In the first case, we compare five different network clustering algorithms on a Yeast protein-protein interaction dataset whereas in the second example, we test four different parameters of the MCL clustering algorithm on the same dataset. Finally, as a third example, we compare four different meta-analyses with hierarchically clustered differentially expressed genes found to be involved in myocardial infarction. VICTOR is available at http://victor.pavlopouloslab.info or http://bib.fleming.gr:3838/VICTOR.

What is GLS-5700?

GLS-5700 is a synthetic, consensus DNA vaccine encoding the ZIKV premembrane and envelope proteins that was tested for Zika virus disease.

1. BACKGROUND: Zika virus (ZIKV) infection has been associated with prolonged viral excretion in human semen and causes testicular atrophy and infertility in 10-week-old immunodeficient mice. METHODS: Male IFNAR-/- mice, knockout for type I interferon receptor, were immunized with GLS-5700, a deoxyribonucleic acid-based vaccine, before a subcutaneous ZIKV challenge with 6 × 105 plaque-forming units at 13 weeks of age. On day 28 postinfection, testes and epididymides were collected in some mice for histological and functional analyses, whereas others were mated with naive female wild-type C57BL/6J. RESULTS: Although all mice challenged with ZIKV developed viremia, most of them were asymptomatic, showed no weight loss, and survived infection. On day 28 postinfection, none of the unvaccinated, infected mice (9 of 9) exhibited abnormal spermatozoa counts or motility. However, 33% (3 of 9) and 36% (4 of 11) of mated males from this group were infertile, from 2 independent studies. Contrarily, males from the noninfected and the vaccinated, infected groups were all fertile. On days 75 and 207 postinfection, partial recovery of fertility was observed in 66% (2 of 3) of the previously infertile males. CONCLUSIONS: This study reports the effects of ZIKV infection on male fertility in a sublethal, immunodeficient mouse model and the efficacy of GLS-5700 vaccination in preventing male infertility. BACKGROUND: Although Zika virus (ZIKV) infection is typically self-limiting, other associated complications such as congenital birth defects and the Guillain-Barré syndrome are well described. There are no approved vaccines against ZIKV infection. METHODS: In this phase 1, open-label clinical trial, we evaluated the safety and immunogenicity of a synthetic, consensus DNA vaccine (GLS-5700) encoding the ZIKV premembrane and envelope proteins in two groups of 20 participants each. The participants received either 1 mg or 2 mg of vaccine intradermally, with each injection followed by electroporation (the use of a pulsed electric field to introduce the DNA sequence into cells) at baseline, 4 weeks, and 12 weeks. RESULTS: The median age of the participants was 38 years, and 60% were women; 78% were White and 22% Black; in addition, 30% were Hispanic. At the interim analysis at 14 weeks (i.e., after the third dose of vaccine), no serious adverse events were reported. Local reactions at the vaccination site (e.g., injection-site pain, redness, swelling, and itching) occurred in approximately 50% of the participants. After the third dose of vaccine, binding antibodies (as measured on enzyme-linked immunosorbent assay) were detected in all the participants, with geometric mean titers of 1642 and 2871 in recipients of 1 mg and 2 mg of vaccine, respectively. Neutralizing antibodies developed in 62% of the samples on Vero-cell assay. On neuronal-cell assay, there was 90% inhibition of ZIKV infection in 70% of the serum samples and 50% inhibition in 95% of the samples. The intraperitoneal injection of postvaccination serum protected 103 of 112 IFNAR knockout mice (bred with deletion of genes encoding interferon-α and interferon-β receptors) (92%) that were challenged with a lethal dose of ZIKV-PR209 strain; none of the mice receiving baseline serum survived the challenge. Survival was independent of the neutralization titer. CONCLUSIONS: In this phase 1, open-label clinical trial, a DNA vaccine elicited anti-ZIKV immune responses. Further studies are needed to better evaluate the safety and efficacy of the vaccine. (Funded by GeneOne Life Science and others; ZIKA-001 ClinicalTrials.gov number, NCT02809443.).

Is histone variant H3.3K27M associated with gliomas?

Yes, Diffuse intrinsic pontine gliomas (DIPG) are the most aggressive brain tumors in children with 5-year survival rates of only 2%. About 85% of all DIPG are characterized by a lysine-to-methionine substitution in histone 3, which leads to global H3K27 hypomethylation accompanied by H3K27 hyperacetylation.

Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered H3K27me3 activity including within super-enhancers, which exhibited perturbed transcriptional function. This was largely independent of H3.3 DNA binding. The K27M and G34R mutations induced several of the same pathways suggesting key shared oncogenic mechanisms including activation of neurogenesis and NOTCH pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH pathway gene knockdown and drug inhibition, reducing their viability in culture. Reciprocal editing of cells generally produced reciprocal effects on tumorgenicity in xenograft assays. Overall, our findings define common and distinct K27M and G34R oncogenic mechanisms, including potentially targetable pathways. Diffuse intrinsic pontine gliomas (DIPG) are the most aggressive brain tumors in children with 5-year survival rates of only 2%. About 85% of all DIPG are characterized by a lysine-to-methionine substitution in histone 3, which leads to global H3K27 hypomethylation accompanied by H3K27 hyperacetylation. Hyperacetylation in DIPG favors the action of the Bromodomain and Extra-Terminal (BET) protein BRD4, and leads to the reprogramming of the enhancer landscape contributing to the activation of DIPG super enhancer-driven oncogenes. The activity of the acetyltransferase CREB-binding protein (CBP) is enhanced by BRD4 and associated with acetylation of nucleosomes at super enhancers (SE). In addition, CBP contributes to transcriptional activation through its function as a scaffold and protein bridge. Monotherapy with either a CBP (ICG-001) or BET inhibitor (JQ1) led to the reduction of tumor-related characteristics. Interestingly, combined treatment induced strong cytotoxic effects in H3.3K27M-mutated DIPG cell lines. RNA sequencing and chromatin immunoprecipitation revealed that these effects were caused by the inactivation of DIPG SE-controlled tumor-related genes. However, single treatment with ICG-001 or JQ1, respectively, led to activation of a subgroup of detrimental super enhancers. Combinatorial treatment reversed the inadvertent activation of these super enhancers and rescued the effect of ICG-001 and JQ1 single treatment on enhancer-driven oncogenes in H3K27M-mutated DIPG, but not in H3 wild-type pedHGG cells. In conclusion, combinatorial treatment with CBP and BET inhibitors is highly efficient in H3K27M-mutant DIPG due to reversal of inadvertent activation of detrimental SE programs in comparison with monotherapy.

What is another name for keratomileusis?

Report the outcomes of laser in situ keratomileusis (LASIK) for high myopia correction after long-term follow-up

BACKGROUND: LASIK (Laser in situ keratomileusis) is used in refractive surgery especially for correction of higher degrees of myopia. Preservation of Bowman's layer as well as less postoperative pain and the slight to absent subepithelial haze are regarded as advantages compared to photorefractive keratectomy (PRK). However, numerous serious complications have been described in the literature. PATIENTS AND METHODS: LASIK treatment was performed elsewhere in two patients to treat myopia or myopic astigmatism between -6 and -9 diopters (D). An astigmatism of -6 D was corrected with the LASIK method in another patient with keratoconus. Progressive corneal ectasia of up to seven diopters occurred in all four eyes within a few months. CONCLUSION: Corneal ectasia can occur after LASIK even in low degrees of myopia of less than ten diopters. Recently, -12 D has been specified as the upper limit for this technique. It is especially important to rule out an early keratoconus or a forme fruste of keratoconus preoperatively since keratectasia with particularly rapid progression can occur in such cases: we would like to designate this as "maligt keratoconus". PURPOSE: Ectasia after laser in situ keratomileusis (LASIK) is a rare but serious complication. Prevention includes proper patient selection with detection of those at particular risk. Causes of ectasia include predisposition, excessive ablation with less than 250 microm of residual stromal bed, thicker than normal flap, irregular corneal thickness, and different ablation rates. METHODS: We evaluated corneal curvature patterns and their relationship to corneal topography and pachymetry maps. RESULTS: Corneal topography (axial, tangential, and altimetric) and pachymetry map characteristics of normally astigmatic corneas, keratoconus, false-positive and false-negative cases, as well as contact lens-induced warpage are discussed. CONCLUSIONS: Preoperative pachymetry maps for LASIK surgery allow accurate case selection through detection of borderline cases, and provide important documentation of preoperative status, as well as useful information for improving surgical strategy. Another important parameter is the asphericity index. BACKGROUND: Laser epithelial keratomileusis (LASEK) is a new keratorefractive procedure for the correction of myopia and myopic astigmatism, which may combine advantages and eliminate disadvantages of photorefractive keratectomy (e.g. pain, corneal haze) and laser in situ keratomileusis (e.g. flap and interface complications, dry eye, keratectasia). We present the results of 108 consecutively LASEK-treated eyes with a follow-up period of 12 months. PATIENTS AND METHODS: LASEK was performed on 108 consecutive eyes with myopia or myopic astigmatism using a keracor 117 excimer laser. The mean preoperative refraction was -4.12+/-1.30 diopters (D) spherical equivalent range: -1.75 to -6.0 D and maximal cylinder was 3.25 D. Results of the 12 months visit are available for 101 eyes (93.5%). RESULTS: No serious complications were observed. After 12 months, SE was within +/-1.0 D of emmetropia in 96% and within +/-0.5 D in 86% of the eyes; 6 eyes had to be retreated. None of the eyes showed haze worse than grade 1 or lost more than one line of best-corrected visual acuity. Uncorrected visual acuity (UCVA) was > or =20/20 in 80% and > or =20/40 in 98%. CONCLUSIONS: Laser epithelial keratomileusis (LASEK) seems to be safe and effective in treatment of myopia and myopic astigmatism of up to -6.0 D. Preliminary results compare favourably with those after photorefractive keratectomy and laser in situ keratomileusis. Haze formation after LASEK seems to be low. Coverage of the stromal wound with a vital epithelial flap could positively influence postoperative wound healing reactions. Photorefractive keratectomy, laser epithelial keratomileusis (LASEK) and Epi-LASIK are all variants of a similar type refractive surgery involving laser on the surface of the cornea and differ mainly in management of the epithelium. Although laser in situ keratomileusis (LASIK) is currently the most popular form of refractive surgery, LASEK is the procedure of choice in some patients. We highlight potential complications of LASEK and how these may be managed. Following laser refractive surgery, corneal thickness is reduced, which has implications for intraocular pressure measurement and glaucoma screening and management. This is particularly important following surface laser procedures where no evidence of previous surgery may be visible. In the event that cataract surgery is required at a later date, correct calculation of the appropriate intraocular lens power can be difficult and it thus important that patients are given their preoperative keratometry readings and refraction. Compared with LASIK patients, those who undergo LASEK are considered to be at lower risk of corneal ectasia. Improved understanding of wound healing post LASEK and better postoperative pain management are ongoing challenges. Keratomileusis, brainchild of Jose I. Barraquer Moner, was conceived and developed as the first stromal sculpting method to correct refractive error in 1948. The word "keratomileusis" literally means "sculpting" of the "cornea." Barraquer's first procedures involved freezing a disc of anterior corneal tissue before removing stromal tissue with a lathe. Over the years, the procedure continued to develop, first through the Barraquer-Krumeich-Swinger non-freeze technique where tissue was removed from the underside of the disc by a second pass of the microkeratome. In-situ keratomileusis was later developed by passing the microkeratome a second time directly on the stromal bed. The procedure became known as automated lamellar keratoplasty with the invention of an automated microkeratome and was further refined by replacing the disc without sutures and later by stopping the microkeratome before the end of the pass to create a hinged flap, as first demonstrated in 1989. The history of the excimer laser dates back to 1900 and the quantum theory, eventually leading to the discovery that 193-nm ultraviolet excimer laser pulses could photoablate tissue without thermal damage. Ultrastructural and wound healing studies confirmed that large area ablation could be performed in the central cornea. This was described as photorefractive keratectomy in 1986 and the first sighted eyes were treated in 1988. An excimer laser was first used to sculpt from the stromal bed under a hinged flap created manually using a trephine and scalpel in 1988. The incorporation of a microkeratome in 1990 finally led to laser in situ keratomileusis-LASIK-as we know it today. A 33-year-old man who underwent uneventful laser in situ keratomileusis (LASIK) developed pressure-induced stromal edema resulting in an interface haze in both eyes and a pocket of fluid under the flap of the right eye 10 days after surgery, while receiving topical fluorometholone. Intraocular pressure by applanation tonometry was 16 mm Hg in his right eye (erroneous result due to the fluid in the interface) and 34 mm Hg in his left eye. After discontinuation of steroids and addition of ocular hypotensive medication, interface fluid collection disappeared in his right eye. Visual acuity improved and haze diminished in both eyes. This case illustrates that in the same patient a post-LASIK edema induced syndrome may be present with or without fluid in the interface, suggesting that both clinical pictures could be manifestations of a broad spectrum of the same condition. We suggest a new name for this non-inflammatory disorder: post-LASIK edema-induced keratopathy (PLEK). Refractive surgery includes all procedures which are primarily targeted at changing the refractive power of the eye. Currently laser-assisted in situ keratomileusis (LASIK) is indicated to correct myopia of up to - 8 D, hyperopia up to + 3 D and astigmatism up to 5 D. Photorefractive keratectomy (PRK) and laser epithelial keratomileusis (LASEK) are primarily recommended for myopia up to - 6 D and for greater refractive errors, phakic intraocular lenses (IOL) are the first choice (myopia greater than - 6 D and hyperopia greater than + 3 D). If presbyopia is present in addition to the high refractive error, refractive lens exchange is another alternative. OBJECTIVE: Report the outcomes of laser in situ keratomileusis (LASIK) for high myopia correction after long-term follow-up. METHODS: Retrospective, consecutive, clinical study including 70 eyes that underwent LASIK using a 500 Hertz (Hz) repetition rate excimer laser and femtosecond technology for flap creation. Visual, refractive, corneal aberrations, and correlation among the variables were evaluated during 3 years. In addition, 34 eyes were followed during 5 years. RESULTS: Significant improvement of 17 logMAR lines was observed in uncorrected vision at 5 years (p < 0.01). This was consistent with spherical equivalent reduction that came from mean preoperative -7.79 diopters (D) to 5 years postoperative -0.24 D (p < 0.01). Significant induction of primary spherical aberration and coma aberration was also found (p < 0.01) at 3 months with levels of 0.61 µm and 0.47 µm, respectively, with no further changes at 5 years (p > 0.05). Pearson correlation showed that the amount of high-order aberrations at 3 months postoperativeley was significantly correlated with the changes in the keratometry throughout the 5 years (delta K) (R2 0.242 p = 0.05). Finally, preoperative corneal asphericity showed an inverse correlation with the delta K (R2 -308 p = 0.01). CONCLUSIONS: Results from this study suggests that LASIK for high myopia correction using 500 Hz repetition rate excimer laser provides stable visual, refractive and aberrometric results after 5 years of follow-up. A more prolate cornea and the amount of higher-order aberrations induced after LASIK are factors that negatively impact in the long-term stability of the procedure. PURPOSE: Laser in situ keratomileusis (LASIK) and small-incision lenticule extraction (SMILE) are popular refractive surgeries. The objective refractive outcomes of LASIK and SMILE have been studied extensively; both procedures have comparable safety, efficacy, and predictability. However, owing to various psychosocial factors, refractive patients may report dissatisfaction despite good postoperative vision. Hence the importance of studies on subjective patient-reported outcomes. This review discusses the role of psychometric-technique-based validated questionnaires when evaluating subjective outcomes. It also summarizes the literature on patient-reported outcomes for LASIK and SMILE. DESIGN: A literature search was performed on PubMed database to identify studies that have assessed patient-reported outcomes for LASIK and SMILE. RESULTS: Several studies have looked into patient-reported outcome measures for LASIK, but the number of equivalent studies for SMILE is limited. Questionnaires (validated and non-validated) are used to evaluate patient-reported outcomes. Validated questionnaires are designed based on psychometric techniques, such as Classic Test Theory, Item Response Theory, and Rasch analysis. The Quality of Life Impact of Refractive Correction (QIRC) questionnaire, a validated questionnaire administered to both LASIK and SMILE patients, suggests that both groups have comparable vision-related quality of life in the first few months postoperatively; but SMILE might confer a slight advantage in the later postoperative period (postoperative month 6). CONCLUSIONS: Future LASIK-SMILE comparative studies utilizing standardized validated questionnaires for patient-reported outcome measures with longer follow-up durations would be a welcome contribution to this important aspect of refractive surgery. PURPOSE: To provide an overview of the currently available retreatment methods after myopic small-incision lenticule extraction (SMILE). DESIGN: Systematic literature review. METHODS: The PubMed library was searched for articles containing the terms "small-incision lenticule extraction" and "enhancement" or "retreatment". The last search was performed on May 1, 2019. RESULTS: In contrast to laser in-situ keratomileusis (LASIK), which can be retreated by a flap relift, repeat SMILE retreatment is currently not approved and only seldomly performed. As substitutes, surface ablation, cap-to-flap conversion using the CIRCLE program in the VisuMax platform, and thin-flap LASIK have been recently established. While all options offer safety and efficacy comparable to LASIK retreatments, each has its patient-specific advantages and disadvantages. While surface ablation preserves the flap-free approach of the primary procedure, the aspect of pain and a slow visual recovery might render it less attractive as compared with CIRCLE and thin-flap LASIK which offer quick recovery, however at the price of flap creation. Besides, each retreatment method generates specific tissue responses and has a different impact on corneal biomechanics, which is strongly dependent on the previous SMILE parameters, especially the cap thickness. CONCLUSIONS: Refractive enhancement after SMILE is currently mostly performed by surface ablation, CIRCLE cap-to-flap conversion or thin-flap LASIK, which all offer safety and efficacy comparable to LASIK retreatments. In this review, a detailed overview over each method, its technical aspects, and specific advantages and disadvantages is given.

In which motif of the RUNX1T1 protein is the rs34269950 SNP located?

The rs34269950 SNP of RUNX1T1 is located in the 'RRACH' motif.

BACKGROUND: Runt-related transcription factor 1 (RUNX1T1) isoforms are involved in adipogenesis. RUNX1T1 is mediated by the fat mass and obesity-associated (FTO). However, the extent to which RUNX1T1 single-nucleotide polymorphisms (SNPs) are associated with obesity risk or metabolic abnormalities in a community population basis is unknown. METHODS: Samples were obtained from the Australian Crossroads study bio-bank. SNPs located in the coding region and 3'untranslated regions of RUNX1T1 with minor allele frequency ≥0.05 were analysed using Taqman genotyping assays. RESULTS: Eight candidate SNPs were genotyped successfully in 1440 participants. Of these SNPs only rs34269950 located in the 'RRACH' motif, the most common N6-methyladenosine (m6A) methylation modification site (recognized by FTO), was significantly associated with obesity risk and metabolic abnormalities. Specifically, compared to AA genotype, rs34269950 del/del genotype was associated with a 1.47 [95% confidence interval (CI): 1.01-2.14, P = 0.042] fold higher rate of obesity risk. Additionally, the del/del genotype was associated with a 60% increased risk of metabolic syndrome (MetS) [odds ratio (OR) = 1.60, 95% CI: 1.10-2.32, P = 0.015], in comparison to the AA genotype. Finally, rs34269950 del/del increased the risk of a larger waist circumference (OR = 1.65, 95% CI: 1.15-2.36, P = 0.007), but not other components of MetS. CONCLUSION: Our study demonstrates that RUNX1T1 rs34269950, located in a potential FTO recognition motif, is significantly associated with waist circumference. This provides novel evidence to suggest SNPs located in RRACH motif may be involved in RNA m6A modification and mechanistic pathways that influence abdominal obesity.

What is caused by SCUBE3 loss of function?

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Author information: (1)Department of Physiology, School of Medicine, Taipei Medical University, 110301 Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, 115201 Taipei, Taiwan. (2)Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy. (3)Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany; Department of Otolaryngology - Head and Neck Surgery, Eberhard Karls University, 72076 Tübingen, Germany. (4)NIHR Oxford Biomedical Research Centre, Wellcome Centre for Human Genetics, University of Oxford, OX3 7BN Oxford, UK. (5)Genetics and Molecular Cell Sciences Research Centre, St George's University of London, Cranmer Terrace, SW17 0RE London, UK. (6)Centogene AG, 18055 Rostock, Germany. (7)Department of Clinical Genetics, Leiden University Medical Center, 2300 RC Leiden, the Netherlands. (8)Department of Paediatric Endocrinology, Emma Children's Hospital, Amsterdam University Medical Center, 1105 AZ Amsterdam, the Netherlands. (9)Department of Cell and Molecular Biology & Microbiology, University of Isfahan, 8174673441 Isfahan, Iran. (10)Confocal Microscopy Core Facility, Research Laboratories, IRCCS Ospedale Pediatrico Bambino Gesù, 00146 Rome, Italy. (11)Department of Genetics, King Faisal Specialist Hospital and Research Center, 11211 Riyadh, Saudi Arabia; Qatar Biomedical Research Institute, Hamad Bin Khalifa University, 34110 Doha, Qatar. (12)Pediatric Endocrinology Unit, Ruth Rappaport Children's Hospital, Rambam Healthcare Campus, 352540 Haifa, Israel; Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, 352540 Haifa, Israel. (13)Pediatric Endocrinology Unit, Ruth Rappaport Children's Hospital, Rambam Healthcare Campus, 352540 Haifa, Israel. (14)Edmond and Lily Safra Children's Hospital, Sheba Medical Center, 52621 Tel-Hashomer, Israel; The Sackler Faculty of Medicine, Tel-Aviv University, 6997801 Tel-Aviv, Israel. (15)Department of Pediatric Endocrinology, Gaziantep Cengiz Gökcek Maternity & Children's Hospital, 27010 Gaziantep, Turkey. (16)Department of Pediatrics, Leiden University Medical Center, 2333ZA Leiden, the Netherlands. (17)Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany; Institute of Bioinformatics, Julius Maximilians University, 97070 Würzburg, Germany. (18)Unidade de Endocrinologia Genética, Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo, 01246903 Sao Paulo, Brazil. (19)Unidade de Genética do Instituto da Criança, Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo, 05403000 Sao Paulo, Brazil. (20)Sheba Cancer Research Center, Sheba Medical Center, 52621 Tel-Hashomer, Israel; Wohl Institute for Translational Medicine, Sheba Medical Center, 52621 Tel-Hashomer, Israel. (21)Department of Clinical Genetics, Guy's and St Thomas' NHS Foundation Trust, SE1 9RT London, UK; Birmingham Women's and Children's NHS Foundation Trust, University of Birmingham, B4 6NH Birmingham, UK. (22)Department of Paediatrics, Sheikh Khalifa Medical City, 51900 Abu Dhabi, United Arab Emirates. (23)Johns Hopkins Aramco Healthcare, 34465 Dhahran, Saudi Arabia. (24)Department of Clinical Genetics, Guy's and St Thomas' NHS Foundation Trust, SE1 9RT London, UK. (25)Edmond and Lily Safra Children's Hospital, Sheba Medical Center, 52621 Tel-Hashomer, Israel; The Sackler Faculty of Medicine, Tel-Aviv University, 6997801 Tel-Aviv, Israel; Wohl Institute for Translational Medicine, Sheba Medical Center, 52621 Tel-Hashomer, Israel. (26)Department of Medical Genetics, Kasturba Medical College, Manipal Academy of Higher Education, Manipal 576104, India. (27)Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany. (28)Department of Genetics, King Faisal Specialist Hospital and Research Center, 11211 Riyadh, Saudi Arabia. (29)Institute of Biomedical Sciences, Academia Sinica, 115201 Taipei, Taiwan; Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 110301 Taipei, Taiwan; Institute of Pharmacology, School of Medicine, National Yang-Ming University, 112304, Taipei, Taiwan. Electronic address: [email protected]. (30)Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy. Electronic address: [email protected].

Which drugs are included in the CAPOX chemotherapy regimen for colorectal cancer?

CAPOX chemotherapy regimen for colorectal cancer includes capecitabine plus oxaliplatin.

BACKGROUND: Cetuximab is an IgG1 monoclonal antibody targeting the epidermal growth factor receptor and is able to reverse the resistance to irinotecan in patients with metastatic colorectal cancer (mCRC). This phase II trial evaluates the safety and efficacy of cetuximab combined with capecitabine and oxaliplatin (CAPOX) in the treatment of patients with mCRC progressing under oxaliplatin-based chemotherapy. PATIENTS AND TREATMENT: Forty patients with mCRC were treated with cetuximab (loading dose 400 mg/m(2) and then 250 mg/m(2) i.v. weekly) in combination with CAPOX (d(1): L-OHP 85 mg/m(2) and d(1-7) capecitabine 2000 mg/m(2) every 2 weeks). Thirty-one (77.5%) and nine (22.5%) patients had oxaliplatin-refractory and -resistant disease, respectively; in addition, 32 (80%) patients had also progressed on prior irinotecan-based chemotherapy. RESULTS: One hundred and thirty-four cycles were administered (median of four cycles per patient). Main toxic effects included grade 3-4 neutropenia (12.5%), grade 3/4 diarrhea (7.5%), grade 3 fatigue (2.5%), and grade 2-3 neurotoxicity (22.5%). One (2.5%) complete and seven (17.5%) partial responses were achieved [overall objective response rate (ORR): 20%; 95% confidence interval (CI): 9% to 32%)], whereas 11 (27.5%) patients had stable disease [disease control rate (DCR): 47.5%; 95% CI: 30.2% to 64.5%]. The ORR and DCR were 18.7% and 46.8%, respectively, in patients with oxaliplatin-refractory disease. The median time to tumor progression was 3 months, the median survival 10.7 months and the probability of 1-year survival rate 53.4%. CONCLUSIONS: The combination of cetuximab plus CAPOX is safe and has a promising activity in patients with mCRC refractory or resistant to oxaliplatin. Colorectal cancer is the second most common site of cancer for both men and women in New Zealand (NZ). Survival, especially with metastatic disease, has improved considerably over the last decade with the introduction of new chemotherapeutic agents. A questionnaire-based survey was conducted to document variations in chemotherapy prescription patterns throughout NZ. Out of 25 medical oncologists, responses were obtained from 22 (88%). The patient with stage III colon cancer was offered either 5-fluorouracil/leucovorin, most commonly on the weekly bolus schedule, or capecitabine monotherapy. Chemotherapy was also offered by the majority (65%) of respondents to the patient with 'high-risk' stage II colon cancer. Several chemotherapy combinations are available in NZ in the metastatic setting, with the most popular being oxaliplatin/capecitabine combination (CAPOX) (35%) or irinotecan/5-FU combination (FOLFIRI) (23%). None of the respondents would commence chemotherapy solely on the basis of a rising carcinoembryonic antigen (CEA). Two-thirds of respondents would recommend chemotherapy for the patient with resectable liver metastases, either before or after surgery. Our survey indicates that chemotherapy prescriptions for patients with colon cancer in NZ, though not uniform, are mostly in line with international recommendations. Colorectal cancer (CRC) is a worldwide public health problem, with nearly 800,000 new cases diagnosed each year, resulting in approximately 500,000 deaths. When advanced metastatic disease is diagnosed, CRC is associated with a poor prognosis, and 5-year survival rates are in the range of 5%-8%. Chemotherapy has been the mainstay approach for patients with advanced CRC. For nearly 40 years, the main drug used for this disease was the fluoropyrimidine 5-fluorouracil (5-FU). Significant advances have been made in chemotherapy treatment options for patients with metastatic disease, such that improvements in 2-year survival are now being reported with median survival rates of 21 months to 24 months. Over the past 10 years, 3 new cytotoxic chemotherapy agents have been approved by the FDA for metastatic CRC. These compounds include the topoisomerase I inhibitor irinotecan, the third-generation platinum analogue oxaliplatin, and the oral fluoropyrimidine capecitabine. Since 2004, 3 novel biologic agents have been approved by the FDA, and they include the anti-epidermal growth factor receptor antibodies cetuximab and panitumumab and the anti-vascular endothelial growth factor bevacizumab. The oral fluoropyrimidine capecitabine has been effectively and safely combined with irinotecan (CAPIRI) and/or oxaliplatin (CAPOX). Three randomized phase III studies have now shown that CAPOX is equivalent to FOLFOX (5-FU/leucovorin/oxaliplatin)-based regimens. Significant interest has centered around combining capecitabine-based cytotoxic regimens with the biologic agents, and specifically, bevacizumab and cetuximab. This review will update the current status of these capecitabine-based combination regimens. BACKGROUND: To evaluate the efficacy and tolerance of capecitabine (CAP) given every other week and biweekly oxaliplatin (OX; modified CAPOX regimen) in patients with advanced colorectal cancer previously treated with irinote- can-based frontline chemotherapy. METHODS: Sixty-seven patients were enrolled; the median age was 62 years and 62 (92.5%) had a performance status (ECOG) of 0-1. OX and CAP were administered at the dose of 100 mg/m(2) on day 1 and 2,000 mg/m(2) on days 1-7, respectively, every 2 weeks. RESULTS: A total of 429 treatment cycles were administered. Grade 3/4 neutropenia and thrombocytopenia were observed in 4 (6%) and 2 (3%) patients, respectively. Febrile neutropenia complicated 1 treatment cycle. The main nonhematologic toxicities were grade 2/3 peripheral sensory neurotoxicity (10% of patients) and grade 3/4 diarrhea (7%). In an intention-to-treat analysis, 3 (4.5%) complete and 13 (19.4%) partial responses (overall response rate 24%) were observed. Seventeen (24.5%) patients had stable and 27 (40.3%) progressive disease. The median time to tumor progression and overall survival were 5 months and 11.3 months, respectively. CONCLUSIONS: The results indicate that the modified CAPOX regimen is safe and effective as salvage treatment in patients with advanced colorectal cancer who were previously treated with irinotecan-based frontline therapy. Colon cancer with DNA mismatch repair (MMR) defects reveals distinct clinical and pathologic features, including a better prognosis but reduced response to 5-fluorouracil (5-FU)-based chemotherapy. A current standard treatment for recurrent or metastatic colon cancer uses capecitabine plus oxaliplatin (CAPOX), or continuous-infusion fluorouracil plus oxaliplatin (FOLFOX). This study investigated the effect of MMR status on the treatment outcomes for CAPOX and FOLFOX as first-line combination chemotherapy in recurrent or metastatic colon cancer. We analyzed 171 patients who had been treated with CAPOX or FOLFOX as first-line combination chemotherapy in recurrent or metastatic colon adenocarcinoma between February 2004 and July 2008. Tumor expression of the MMR proteins, MLH1 and MSH2, was detected by immunohistochemistry (IHC) in surgically resected tumor specimens. The microsatellite instability (MSI) was analyzed by polymerase chain reaction (PCR) amplification, using fluorescent dye-labeled primers specific to microsatellite loci. Tumors with MMR defect were defined as those demonstrating a loss of MMR protein expression (MMR-D) and/or a microsatellite instability-high (MSI-H) genotype. In all, 75 patients (44%) received FOLFOX, and 96 patients (56%) received CAPOX as first-line combination chemotherapy. The incidence of colon cancer with MMR defect was 10/171 (6%). Colon cancers with MMR defect (MSI-H and/or MMR-D) are more commonly located in proximal to the splenic flexure (p=0.03). The MMR status did not significantly influence the overall response (p=0.95) to first-line CAPOX or FOLFOX treatment in patients with recurrent or metastatic colon cancer. According to the MMR status, there was no significant difference for PFS (p=0.50) and OS (p=0.47) in patients with recurrent or metastatic colon cancer treated with first-line CAPOX or FOLFOX. In colon cancers with MMR defect, there was no significant difference for PFS (p=0.48) and OS (p=0.56) between CAPOX and FOLFOX as first-line combination chemotherapy. However, in MMR intact, there was significant difference for OS between CAPOX and FOLFOX (p=0.04). OS was significantly better in patients treated with CAPOX when compared to patients with FOLFOX. The MMR status does not predict the effect of oxaliplatin-based combination chemotherapy as 1st line in recurrent or metastatic colon cancers. CAPOX in the first-line treatment of recurrent or metastatic colon cancer with MMR intacts showed a superior OS compared with FOLFOX unlike colon cancer with MMR defects. PURPOSE: Capecitabine has shown similar efficacy to 5-fluorouracil (5-FU); a regimen containing 2 weeks of capecitabine/oxaliplatin (CapOx) has demonstrated noninferiority to infusional 5-FU/oxaliplatin/leucovorin (FOLFOX) for the treatment of metastatic colorectal cancer (mCRC). This phase II study explores the efficacy and safety of a 2-day course of oxaliplatin/capecitabine (2DOC), with oxaliplatin given on day 1 and capecitabine given orally every 8 hours in high doses over 6 doses, mimicking FOLFOX6. PATIENTS AND METHODS: This phase II study was conducted by the University of Wisconsin Carbone Cancer Center. Eligible patients with mCRC received oxaliplatin 100 mg/m2 intravenously (I.V.) over 2 hours followed by leucovorin 20 mg/m2 I.V. bolus and 5-FU 400 mg/m2 I.V. bolus on day 1 and day 15. Capecitabine was administered at 1500 mg/m2 orally every 8 hours over 6 doses starting on day 1 and day 15. RESULTS: A total of 45 patients were enrolled; 44 were evaluated for response. Seventeen patients (39%) had objective responses. Median time to progression was 6.8 months, and median overall survival (OS) was 17.5 months. The most common side effects were grade 1/2 neuropathy, fatigue, and nausea. Severe hand-foot syndrome (HFS) was rare. CONCLUSION: The overall response rate with the 2DOC regimen is similar to published CapOx regimens, and time to progression and OS are similar. The incidence of HFS, diarrhea, and mucositis were lower compared with published results of 2-week schedules of capecitabine. The 2DOC regimen merits further study as a more convenient regimen than infusional 5-FU with less HFS when compared with a 2-week administration of capecitabine. PURPOSE: The AIO KRK-0104 randomized phase II trial investigated the efficacy and safety of cetuximab combined with capecitabine and irinotecan (CAPIRI) or capecitabine and oxaliplatin (CAPOX) in the first-line treatment of metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: A total of 185 patients with mCRC were randomly assigned to cetuximab (400 mg/m(2) day 1, followed by 250 mg/m(2) weekly) plus CAPIRI (irinotecan 200 mg/m(2), day 1; capecitabine 800 mg/m(2) twice daily days 1 through 14, every 3 weeks; or cetuximab plus CAPOX (oxaliplatin 130 mg/m(2) day 1; capecitabine 1,000 mg/m(2) twice daily day 1 through 14, every 3 weeks). The primary study end point was objective response rate (ORR). RESULTS: In the intention-to-treat patient population (n = 177), ORR was 46% (95% CI, 35 to 57) for CAPIRI plus cetuximab versus 48% (95% CI, 37 to 59) for CAPOX plus cetuximab. Analysis of the KRAS gene mutation status was performed in 81.4% of the intention to treat population. Patients with KRAS wild-type in the CAPIRI plus cetuximab arm showed an ORR of 50.0%, a PFS of 6.2 months and an OS of 21.1 months. In the CAPOX plus cetuximab arm, an ORR of 44.9%, a PFS of 7.1 months and an OS of 23.5 months were observed. While ORR and PFS were comparable in KRAS wild-type and mutant subgroups, a trend toward longer survival was associated with KRAS wild-type. Both regimens had manageable toxicity profiles and were safe. CONCLUSION: This randomized trial demonstrates that the addition of cetuximab to CAPIRI or CAPOX is effective and safe in first-line treatment of mCRC. In the analyzed regimens, ORR and PFS did not differ according to KRAS gene mutation status. BACKGROUND: Despite lack of a true comparative study, the folfox (5-fluorouracil-leucovorin-oxaliplatin) and capox (capecitabine-oxaliplatin) regimens are believed to be similar in their efficacy and tolerability in the treatment of stage iii colorectal cancer. However, that belief has been disputed, because real-life data suggest that the capox regimen is more toxic, leading to more frequent reductions in the delivered dose intensity-thus raising questions about the effect of dose intensity on clinical outcomes. METHODS: A retrospective data review for two Canadian institutions, the Segal Cancer Centre and the Tom Baker Cancer Centre, considered patients diagnosed with stage iii colorectal cancer during 2006-2013. Primary endpoints were dose intensity and toxicity, with a secondary endpoint of disease-free survival. RESULTS: The study enrolled 180 eligible patients (80 at the Segal Cancer Centre, 100 at the Tom Baker Cancer Centre). Of those 180 patients, 75 received capox, and 105 received mfolfox6. In the capox group, a significant dose reduction was identified for capecitabine compared with 5-fluorouracil in mfolfox6 group (p = 0.0014). Similarly, a significant dose reduction was observed for oxaliplatin in mfolfox6 compared with oxaliplatin in capox (p = 0.0001). Compared with the patients receiving capox, those receiving mfolfox6 were twice as likely to experience a treatment delay of more than 1 cycle-length (p = 0.03855). Toxicity was more frequent in patients receiving mfolfox6 (nausea: 30% vs. 18%; diarrhea: 47% vs. 24%; peripheral sensory neuropathy: 32% vs. 3%). At a median follow-up of 40 months, preliminary data showed no difference in disease-free survival (p = 0.598). Pooled data from both institutions were also separately analyzed, and no significant differences were found. CONCLUSIONS: Our results support the use of capox despite a lack of head-to-head randomized trial data. PURPOSE: Oxaliplatin, an important chemotherapeutic agent in colorectal cancer, causes chemotherapy-induced peripheral neuropathy (CIPN), for which prophylactic or therapeutic interventions are lacking. We aimed to investigate changes in upper extremities, activities of daily living (ADL), and health-related quality of life (HRQoL) parameters after the first chemotherapy cycle. METHODS: Thirty-eight colorectal cancer patients scheduled to receive the leucovorin, 5'-fluorouracil, oxaliplatin (FOLFOX) therapy or the capecitabine, oxaliplatin (CAPOX) therapy, participated. Patients underwent objective assessment of sensory function, muscular strength, and manual dexterity and answered the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC QLQ-C30) and the Disabilities of the Arm, Shoulder, and Hand-Disability/Symptom (DASH-DS) questionnaires for subjective assessment. The CIPN was assessed at baseline and prior to the second drug cycle. RESULTS: Light touch sensation in both hands worsened significantly after the first drug cycle, though no significant changes were observed in muscular strength and manual dexterity. The QLQ-C30 analysis showed that Physical Functioning, Role Functioning, Nausea and Vomiting, and Dyspnea were significantly worse, whereas Emotional Functioning was improved. The DASH-DS analysis revealed significant worsening of dysfunction and subjective symptoms. CONCLUSIONS: Our results suggest that light touch sensation may worsen even in the absence of multiple chemotherapy cycles. Even if arm and hand function (muscular strength and manual dexterity) is apparently intact, patients may experience dysfunction and decreased HRQoL. For preserving or improving patients' ADL and HRQoL, it is imperative to provide support at chemotherapy initiation. BACKGROUND: Folate level was proposed to be a predictor for fluoropyrimidine-related toxicity. We conducted a prospective study to determine the association between serum and red-cell folate and capecitabine-related toxicity in patients with colorectal cancers. MATERIALS AND METHODS: Eligibility criteria included diagnosis of colorectal cancers; eligible patients who were scheduled to undergo capecitabine monotherapy or capecitabine-oxaliplatin (CAPOX) for adjuvant or palliative purposes. Exclusion criteria included concomitant radiotherapy or chemotherapy other than capecitabine or CAPOX and creatinine clearance <30 mL/min. Fasting serum and red-cell folate were measured prior to chemotherapy. Capecitabine was administered at 2,500 mg/m2 per day (monotherapy) or 2,000 mg/m2 per day (CAPOX) for 14 days every 3 weeks. The toxicity of the first four cycles was documented by clinical investigators who were blinded to folate levels. RESULTS: A total of 144 patients were recruited, of whom 126 were eligible; 40 patients had capecitabine alone, and 86 patients received CAPOX. The rates of grade 2 and grade 3 toxicity were 63.5% and 14.3%, respectively. Nausea and vomiting were the most common grade ≥2 adverse event (47.7%), followed by hand-foot syndrome (25.4%), diarrhea (23.1%), and neutropenia (22.3%). Combination with oxaliplatin (odds ratio [OR], 2.77; p = .043) and serum folate (OR, 10.33; p = .002) were independent predictors of grade ≥2 toxicity. Red-cell folate was not predictive of toxicity. For every 10 nmol/L increment in serum folate, the risk of grade ≥2 toxicity increased by 9%. CONCLUSION: Serum folate level, but not red-cell folate, was associated with higher rate of grade ≥2 toxicity during capecitabine-based treatment. Excessive folate intake may be avoided before and during capecitabine-based chemotherapy. IMPLICATIONS FOR PRACTICE: This is the first prospective study to evaluate the association between serum folate level and capecitabine-related toxicity in patients with colon cancers. It shows that higher serum folate level is associated with increased risks of moderate to severe toxicity during capecitabine-based treatment. Excessive folate intake should be avoided before and during capecitabine-based chemotherapy. BACKGROUND Colorectal cancer (CRC) is considered to be a worldwide health problem because of its increasing incidence and prevalence. Surgery offers an opportunity for cure, but the postoperative recurrence rate is still high despite the advancement of chemotherapy. This study aimed to assess the efficacy and safety of prolonged capecitabine chemotherapy following CAPOX chemotherapy for stage III  CRC after radical surgery. MATERIAL AND METHODS This study included 212 patients with stage III CRC undergoing open radical surgery from July 2010 to June 2015. Among those patients, 104 patients received prolonged capecitabine chemotherapy (prolonged group) following 8 cycles of CAPOX regimen chemotherapy, while the other 108 patients (control group) received no prolonged chemotherapy. The prolonged chemotherapy consisted of capecitabine (1000 mg/m² per day for 2 weeks) and was repeated every 3 weeks for 8 cycles at most. Long-term survival and toxicities were retrospectively compared. RESULTS Patient characteristics did not differ between the 2 groups. For all patients, no significant difference was found in the 3-year disease-free survival (DFS) (P=0.7775) or 3-year overall survival (OS) rates between the 2 groups (P=0.5787). The prolonged group had significantly higher frequency of hand-foot syndrome (P=0.0267) and paresthesia (P=0.0164). In further subgroup analyses, no benefit for 3-year DFS or 3-year OS of prolonged capecitabine chemotherapy was found in colon cancer or rectal cancer. CONCLUSIONS Prolonged capecitabine chemotherapy following CAPOX regimen chemotherapy failed to improve the survival of patients with stage III CRC after radical surgery. IMPORTANCE: The addition of oxaliplatin to the standard 6-month fluorouracil-based adjuvant chemotherapy in stage II colorectal cancer has been reported to reduce the risk of relapse although it does not increase survival. The Three or Six Colon Adjuvant (TOSCA) trial compared 3 months with 6 months of adjuvant fluoropyrimidine and oxaliplatin-based chemotherapy in patients with stage III colon cancer. The utility remains unknown. OBJECTIVE: To assess the noninferiority and toxic effects of 3 vs 6 months of FOLFOX (fluorouracil, leucovorin, and oxaliplatin) or CAPOX (capecitabine plus oxaliplatin) adjunct chemotherapy among patients with high-risk stage II resected colorectal cancer enrolled in the TOSCA trial. DESIGN, SETTING, AND PARTICIPANTS: The TOSCA study was a noninferiority phase 3 randomized clinical trial conducted from June 2007 to March 2013 in 130 Italian centers. Included patients had resected colorectal cancer located 12 cm from the anal verge by endoscopy or above the peritoneal reflection at surgery. In this preplanned study assessing the per-protocol population, 5-year relapse-free survival was evaluated in 1254 patients with high-risk stage II resected colorectal cancer who had received adjuvant FOLFOX (fluorouracil, leucovorin, and oxaliplatin) or CAPOX (capecitabine plus oxaliplatin). INTERVENTIONS: Patients were originally randomized (1:1) in the TOSCA trial to receive 3 months (experimental group) or 6 months (control) of standard doses of FOLFOX or CAPOX at the discretion of the treating physician. MAIN OUTCOME AND MEASURES: A hazard ratio of at least 1.2 between the 3-month and 6-month chemotherapy groups was set to reject the null hypothesis of noninferiority. RESULTS: Overall, 1254 patients (mean [SD] age, 62.4 [9.8] years; 565 women [45.1%]) with clinical high-risk stage II resected colorectal cancer were analyzed at a median follow-up of 62 months (interquartile range, 53-71) months. Of them, 301 patients (24.0%) had pT4N0M0 tumors, and the remaining 953 patients (76.0%) had high-risk pT3N0M0 tumors; 776 patients (61.9%) received FOLFOX and 478 (38.1%) received CAPOX. The 5-year relapse-free survival was 82.2% for the 3-month arm and 88.2% for the 6-month arm, with an estimated hazard ratio of 1.41 (95% CI, 1.05-1.89; P = .86 for noninferiority). For CAPOX, the 5-year relapse-free survival was similar in the 2 arms (difference, 0.76% favoring the 6-month arm; 95% CI, -6.28% to 7.80%), whereas for FOLFOX, the difference was pronounced: 8.56% in favor of the longer-duration arm (95% CI, 3.45%-13.67%). Nevertheless, the test for an interaction between duration and regimen was not statistically significant. Neurotoxicity was approximately 5 times lower in the shorter duration arm than in the longer duration arm. CONCLUSIONS AND RELEVANCE: In the 3-month arm, the treatment was significantly less toxic than in the 6-month arm. Noninferiority was not shown for 5-year relapse-free survival. However, a possible regimen effect was observed, suggesting that either 3 months of CAPOX or 6 months of FOLFOX therapy can be used whenever an oxaliplatin doublet is indicated for treatment of patients with stage II colorectal cancer. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT0064660. A 74-year-old man underwent distal gastrectomy for gastric cancer(CY1, fStage Ⅳ). About 18 months after surgery, abdominal CT scans revealed multiple lymph node metastases along the portal vein. Systemic chemotherapy was administered comprising a capecitabine/oxaliplatin(CAPOX)regimen. After 4 courses of chemotherapy, an adverse reaction of Grade 2 diarrhea and peripheral neuropathy occurred, although regression of the lymph node metastasis was confirmed. Ramucirumab was administered as the second-line regimen, but CT imaging revealed lymph node progression after several courses. Although irinotecan(CPT-11)was selected as the third-line chemotherapy, the lymph node progression remained uncontrolled. Nivolumab was selected as the fourth-line chemotherapy. After 23 courses, nivolumab immunotherapy induced a partial response to the lymph node metastasis. Nivolumab immunotherapy continues to be administered until now, 5 years after the operation. We experienced a case of lymph node metastasis from gastric cancer successfully treated with nivolumab chemotherapy. WHAT IS KNOWN AND OBJECTIVE: High interindividual response variability was reported with capecitabine and oxaliplatin (CAPOX) regimen in colorectal cancer (CRC). The single nucleotide polymorphisms (SNPs) of the genes related to drug efflux transport (ABCB1) and DNA repair (ERCC) could result in altered tumour response. Hence, this study was designed to assess the influence of ABCB1, ERCC-1 and ERCC-2 gene polymorphisms on tumour response to CAPOX treatment in CRC patients of South Indian origin. PATIENTS AND METHODS: A total of 145 newly diagnosed CRC patients were included in the final analysis. Response to CAPOX treatment in the adjuvant setting was assessed in terms of disease-free survival rate (DFSR) and overall survival rate (OSR) at 3 years, whereas in the palliative setting, the response was assessed as progression-free survival rate (PFSR) and OSR at 3 years. Five millilitres of the venous blood sample was collected from each patient for genomic DNA extraction by the manual phenol-chloroform method. Genotyping and allelic discrimination analysis were done using real-time PCR (RT-PCR). RESULTS AND DISCUSSION: With ABCB1 gene polymorphism rs1045642 (A > G), patients with AG/GG genotype showed better DFSR [P value = .02, OR = 2 (CI: 1.5-3)] and PFSR [P value = .02, OR = 1.6 (CI: 1.1-2.5)] when compared to AA genotype in the adjuvant and palliative settings, respectively. Similarly with rs1128503 (A > G) polymorphism, patients with AG/GG genotype were found to have better DFSR [P value = .02, OR = 1.9 (CI: 1.3-3)] and PFSR [P value = .01, OR = 2 (CI: 1.1-3.7)] when compared to AA genotype. However, we did not find any association between CAPOX response and ABCB1 gene polymorphisms in a binary logistic regression when non-genetic predictors were considered for analysis. We did not find any association with ERCC1 (rs11615 A > G) and ERCC2 (rs13181 T > G) gene polymorphisms with respect to CAPOX response in either of the treatment settings. WHAT IS NEW AND CONCLUSION: The response to CAPOX treatment was found to be influenced by the ABCB1 gene variants (rs1128503 and rs1045642), thereby strengthening their predictive role. No association was found between ERCC1 (rs11615 A > G), ERCC2 (rs13181 T > G) gene polymorphisms and tumour response to CAPOX treatment in CRC patients of South Indian origin. Colorectal cancer is one of the most common maligcies diagnosed in Canada. Currently, adjuvant colorectal cancer treatment primarily includes chemotherapeutic regimens such as FOLFOX6 (5-fluorouracil, leucovorin, oxaliplatin) or CAPOX (capecitabine, oxaliplatin), as well as alternative regimens such as TOMOX (raltitrexed, oxaliplatin). However, the prevalence of drug shortages in today's society may make these preferred regimens inaccessible. The purpose of this case report is to highlight the tolerability of an alternative adjuvant regimen (pemetrexed plus oxaliplatin) that has undergone both phase I and II clinical trials for the treatment of colorectal cancer. The patient presented in this case report is a 57-year-old female diagnosed with Stage III colon cancer. This patient received seven cycles of pemetrexed plus oxaliplatin and experienced several adverse events, with the majority of them being mild in nature including fatigue and cold dysesthesia. However, the patient also experienced progressive neuropathy which required a dose reduction and subsequent discontinuation of oxaliplatin. Overall, pemetrexed and oxaliplatin's tolerability seems comparable to other regimens used to treat colorectal cancer and could potentially be an option to consider in the future for alternative treatment of colorectal cancer pending further trials. OBJECTIVE: To assess the effectiveness and safety of treatment consisting of extending chemotherapy (ECT) with capecitabine following capecitabine plus oxaliplatin (CAPOX) chemotherapy for stage 3 gastric carcinoma (GC) after D2 gastrectomy. PATIENTS AND METHODS: In this retrospective study, we included 214 patients with stage 3 GC who underwent D2 gastrectomy between January 2012 and April 2014. The CAPOX regimen chemotherapy was administrated to all of the patients as adjuvant therapy. The CAPOX regimen consisted of capecitabine (1000 mg/m2, in 2 divided doses for 14 d) and oxaliplatin (130 mg/m2 given on Day 1), repeated every 21 d for 8 cycles. Following CAPOX chemotherapy, 102 of these patients received extending chemotherapy (the ECT group) with capecitabine, whereas 112 patients (the control group) received no ECT. The ECT consisted of capecitabine (1000 mg/m2, in 2 divided doses for 14 d), repeated every 21 d for 8 cycles at most. The chemotherapy was discontinued if unacceptable toxicity or disease progression occurred or upon the request of the patient. All cases were followed up, and overall survival (OS), recurrence-free survival (RFS), and toxicities were compared. RESULTS: The ECT group exhibited a distinctly higher 5-year OS (p=0.0468) and RFS (p=0.0483) than those of the control group. The incidence of hand-foot syndrome was markedly greater in the ECT group (p=0.0043). No toxicity-related death occurred. CONCLUSIONS: Extending chemotherapy with capecitabine following the CAPOX regimen chemotherapy provides significant survival benefit for stage 3 GC after D2 gastrectomy. Conflict of interest statement: Conflict of interest statement G.N. reports receiving honoraria from Chugai, Janssen, Yakult Honsha, Taiho, Eli Lilly, Miyarinsan and Takeda and research funding to his institution from Eli Lilly, outside the submitted work. H.T. reports receiving honoraria from Bayer, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eli Lilly, MBL, Merck Bio Pharma, Mitsubishi Tanabe Pharma, MSD, Nippon Kayaku, Novartis, Sanofi, Taiho, Takeda and Yakult Honsha and research funding to his institution from Daiichi Sankyo, Isofol, MSD, Novartis, Ono, Pfizer, Sumitomo Dainippon Pharma, Sysmex and Takeda, outside the submitted work. K.U. reports receiving honoraria from Eli Lilly and Chugai, outside the submitted work. K.M. reports receiving honoraria from Chugai, outside the submitted work. M.A. reports receiving grant from Kyowa Kirin, outside the submitted work. Y.A. reports receiving grants and personal fees from Chugai, Kyowa Hakko Kirin, Nippon Kayaku, Yakult Honsha, Mochida, Ono, Taiho and Daiichi Sankyo; personal fees from Eli Lilly, Novartis, Bayer, Bristol-Myers Squibb, Sawai, Tsumura and Shionogi and grants from Eisai, outside the submitted work. K.M. reports receiving grants from Daiichi Sankyo, Parexel International, Shionogi Pharma, Sumitomo Dainippon, MSD, Pfizer, Mediscience Planning and Solasia Pharma; grants and personal fees from Ono and Sanofi and personal fees from Takeda, Taiho, Bristol-Myers Squibb, Bayer, Eli Lilly and Chugai, outside the submitted work. Y.K. reports receiving honoraria from Taiho, Ono, Daiichi Sankyo, Eli Lilly, Chugai, Yakult Honsha, MSD, Nippon Kayaku, Sawai, Tsumura and Miyarinsan and research funding to his institution from Taiho, Chugai, Eli Lilly, Sanofi, Nippon Kayaku, Ono, Otsuka, Takeda, MSD and Pfizer, outside the submitted work. LESSONS LEARNED: Three-month adjuvant capecitabine plus oxaliplatin in combination (CAPOX) appeared to reduce recurrence, with mild toxicity in postcurative resection of colorectal cancer liver metastases (CLM). Recurrence in patients who underwent the 3-month adjuvant CAPOX after resection of CLM was most commonly at extrahepatic sites. BACKGROUND: The role of neoadjuvant and adjuvant chemotherapy in the management of initially resectable colorectal cancer liver metastases (CLM) is still unclear. We evaluated the feasibility of 3-month adjuvant treatment with capecitabine plus oxaliplatin in combination (CAPOX) for postcurative resection of CLM. METHODS: Patients received one cycle of capecitabine followed by four cycles of CAPOX as adjuvant chemotherapy after curative resection of CLM. Oral capecitabine was given as 1,000 mg/m2 twice daily for 2 weeks in a 3-week cycle, and CAPOX consisted of oral capecitabine plus oxaliplatin 130 mg/m2 on day 1 in a 3-week cycle. Primary endpoint was the completion rate of adjuvant chemotherapy. Secondary endpoints included recurrence-free survival (RFS), overall survival (OS), dose intensity, and safety. RESULTS: Twenty-eight patients were enrolled. Median age was 69.5 years, 54% of patients had synchronous metastases, and 29% were bilobar. Mean number of lesions resected was two, and mean size of the largest lesion was 31 mm. Among patients, 20 (71.4%; 95% confidence interval, 53.6%-89.3%) completed the protocol treatment and met its primary endpoint. The most common grade 3 or higher toxicity was neutropenia (29%). Five-year recurrence-free survival and overall survival were 65.2% and 87.2%, respectively. CONCLUSION: Three-month adjuvant treatment with CAPOX is tolerable and might be a promising strategy for postcurative resection of CLM. BACKGROUND: At present, colorectal cancer is routinely treated with adjuvant radiotherapy and chemotherapy postoperatively. The adverse effects (AEs) of chemotherapy usually interrupt the treatment of chemotherapy. Traditional Chinese medicine (TCM) has demonstrated great potential in improving patients' clinical symptoms, regulating the immune function, improving the life quality, and reducing the AEs of chemotherapy. AIM: To observe the clinical efficacy of Yiqi Jianpi anti-cancer prescription combined with chemotherapy in patients with colorectal cancer after operation. METHODS: Data from patients diagnosed with colorectal cancer between January 2019 and February 2021 were collected from Liaoning Cancer Hospital and Institute and the Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine. Patients receiving the chemotherapy regimen of capecitabine plus oxaliplatin (CAPOX) after radical resection of colorectal cancer were prospectively collected and randomly divided into an experimental group and a control group. The experimental group was given Yiqi Jianpi anti-cancer prescription combined with the CAPOX regimen, while the control group was given the CAPOX regimen alone. After six cycles of chemotherapy, the scores of TCM symptoms, Karnofsky performance scale (KPS) score, levels of T-cell subsets, and AEs after chemotherapy of the two groups were compared. RESULTS: A total of 70 patients were randomly divided into either an experimental group (n = 35, no dropout) or a control group (n = 33, with 2 dropouts). Compared with the control group, the experimental group improved significantly (P < 0.05) in scores of TCM symptoms, KPS score, levels of T-cell subsets, and AEs of chemotherapy. CONCLUSION: Yiqi Jianpi anti-cancer prescription can effectively improve spleen deficiency, regulate the immune function, and alleviate the AEs of chemotherapy, so as to improve the life quality of patients with good therapeutic effects and application prospect in clinical practice.

Is FKBP52 encoding a chaperone ?

Yes, FKBP52 is a co-chaperone.

The present study aimed to examine the expression of FK‑506 binding protein 52 (FKBP52) in the ovary tissues of rats with polycystic ovarian syndrome (PCOS) and its action on mediating androgen receptor (AR) through the mitogen‑activated protein kinase (MAPK)/extracellular signal‑regulated kinase (ERK) pathway. PCOS model rats were established by dehydroepiandrosterone injection. Enzyme‑linked immunosorbent assay (ELISA) measured serum sex hormones. Hematoxylin and eosin (H&E) staining was used to examine histological changes of the ovarian tissues. The expression levels of FKBP52 were detected by immunohistochemical (IHC) staining, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis and western blotting (WB). In addition, RT‑qPCR analysis was used to detect the mRNA expression of AR, and WB was used to detect the protein expression levels of AR, ERK1/2 and phosphorylated (p‑)ERK1/2. In granulosa cell (GC) experiments, primary GCs were extracted and cultured. FKBP4 is the FKBP52‑encoding gene, therefore, adenovirus vectors Ad‑Oe‑FKBP4‑EGFP and Ad‑siRNA‑FKBP4‑EGFP were constructed to examine the association among the above factors using the RT‑qPCR and WB methods. In the animal experiment, the vaginal smear, H&E staining and ELISA results showed that the PCOS model was successfully established. The IHC staining revealed that the expression of FKBP52 in the GCs of the PCOS model group was higher than the remaining groups (P<0.01). The mRNA and expression levels of FKBP52 and AR in the PCOS model rats were significantly increased, when compared with levels in the other rats (P<0.05). The expression level of p‑ERK1/2 was also higher (P<0.05). In the GC experiment, following overexpression of the FKBP4 gene, the mRNA and expression levels of FKBP52 and AR were increased (P<0.05). The expression level of p‑ERK1/2 was also increased (P<0.05). Following FKBP4 gene silencing, the mRNA and expression levels of FKBP52 and AR were decreased (P<0.05). The expression level of ERK1/2 was also decreased (P<0.05). However, the expression level of p‑ERK1/2 was increased (P<0.05). In conclusion, the upregulation of co‑chaperone FKBP52 may mediate the activation of AR through the MAPK/ERK pathway. BACKGROUND: Stress exposure as well as psychiatric disorders are often associated with abnormalities in brain structure or connectivity. The co-chaperone FK506-binding protein 51 (FKBP51) is a regulator of the stress system and is associated with a risk to develop stress-related mental illnesses. PURPOSE: To assess the effect of a general FKBP51 knockout on brain structure and connectivity in male mice. STUDY TYPE: Animal study. ANIMAL MODEL: Two cohorts of FKBP51 knockout (51KO) and wildtype (WT) mice. The first cohort was comprised of n = 18 WT and n = 17 51KOs; second cohort n = 10 WT and n = 9 51KOs. FIELD STRENGTH/SEQUENCE: 9.4T/3D gradient echo (VBM), DTI-EPI (DTI). ASSESSMENT: Voxel-based morphometry (VBM) and diffusion tensor imaging (DTI). For VBM, all procedures were executed in SPM12. DTI: FMRIB Software Library (FSL) Tract Based Statistics (TBSS) were integrated within DTI-TK, allowing the creation of a mean FA skeleton. A voxelwise statistical analysis was applied between WT and 51KO mice. STATISTICAL TEST: Volumetric differences were collected at a threshold of P < 0.005, and only clusters surviving a familywise error correction on the cluster level (pFWE, cluster <0.05) were further considered. VBM data were analyzed using a two-sample t-test. The Threshold Free Cluster Enhancement (TFCE) method was used to derive uncorrected-P statistical results at a P-level of 0.01. RESULTS: The structural analysis revealed two clusters of significantly larger volumes in the hypothalamus, periaqueductal gray, and dorsal raphe region of WT animals. DTI measurements, however, demonstrated statistically higher fractional anisotropy (FA) values for 51KO animals in locations including the anterior commissure, fornix, and posterior commissure/superior colliculus commissure region. DATA CONCLUSION: This study used in vivo structural MRI and DTI to demonstrate that a lack of FKBP51 leads to alterations in brain architecture and connectivity in male mice. These findings are of particular translational relevance for our understanding of the neuroanatomy underlying the interaction of FKBP5 genetic status, stress susceptibility, and psychiatric disorders. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 1.

Isotocin is an homolog of what hormone?

Isotocin is a homolog of oxytocin.

A wide range of physiological and behavioral processes, such as social, sexual, and maternal behaviors, learning and memory, and osmotic homeostasis are influenced by the neurohypophysial peptides oxytocin and vasopressin. Disruptions of these hormone systems have been linked to several neurobehavioral disorders, including autism, Prader-Willi syndrome, affective disorders, and obsessive-compulsive disorder. Studies in zebrafish promise to reveal the complex network of regulatory genes and signaling pathways that direct the development of oxytocin- and vasopressin-like neurons, and provide insight into factors involved in brain disorders associated with disruption of these systems. Isotocin, which is homologous to oxytocin, is expressed early, in a simple pattern in the developing zebrafish brain. Single-minded 1 (sim1), a member of the bHLH-PAS family of transcriptional regulatory genes, is required for terminal differentiation of mammalian oxytocin cells and is a master regulator of neurogenesis in Drosophila. Here we show that sim1 is expressed in the zebrafish forebrain and is required for isotocin cell development. The expression pattern of sim1 mRNA in the embryonic forebrain is dynamic and complex, and overlaps with isotocin expression in the preoptic area. We provide evidence that the role of sim1 in zebrafish neuroendocrine cell development is evolutionarily conserved with that of mammals. BACKGROUND: The mammalian neurohypophysial hormones, vasopressin and oxytocin are involved in osmoregulation and uterine smooth muscle contraction respectively. All jawed vertebrates contain at least one homolog each of vasopressin and oxytocin whereas jawless vertebrates contain a single neurohypophysial hormone called vasotocin. The vasopressin homolog in non-mammalian vertebrates is vasotocin; and the oxytocin homolog is mesotocin in non-eutherian tetrapods, mesotocin and [Phe2]mesotocin in lungfishes, and isotocin in ray-finned fishes. The genes encoding vasopressin and oxytocin genes are closely linked in the human and rodent genomes in a tail-to-tail orientation. In contrast, their pufferfish homologs (vasotocin and isotocin) are located on the same strand of DNA with isotocin gene located upstream of vasotocin gene separated by five genes, suggesting that this locus has experienced rearrangements in either mammalian or ray-finned fish lineage, or in both lineages. The coelacanths occupy a unique phylogenetic position close to the divergence of the mammalian and ray-finned fish lineages. RESULTS: We have sequenced a coelacanth (Latimeria menadoensis) BAC clone encompassing the neurohypophysial hormone genes and investigated the evolutionary history of the vertebrate neurohypophysial hormone gene locus within a comparative genomics framework. The coelacanth contains vasotocin and mesotocin genes like non-mammalian tetrapods. The coelacanth genes are present on the same strand of DNA with no intervening genes, with the vasotocin gene located upstream of the mesotocin gene. Nucleotide sequences of the second exons of the two genes are under purifying selection implying a regulatory function. We have also analyzed the neurohypophysial hormone gene locus in the genomes of opossum, chicken and Xenopus tropicalis. The opossum contains two tandem copies of vasopressin and mesotocin genes. The vasotocin and mesotocin genes in chicken and Xenopus, and the vasopressin and mesotocin genes in opossum are linked tail-to-head similar to their orthologs in coelacanth and unlike their homologs in human and rodents. CONCLUSION: Our results indicate that the neurohypophysial hormone gene locus has experienced independent rearrangements in both placental mammals and teleost fishes. The coelacanth genome appears to be more stable than mammalian and teleost fish genomes. As such, it serves as a valuable outgroup for studying the evolution of mammalian and teleost fish genomes. The present study using zebrafish as a model explores the role of isotocin, a homolog of oxytocin, in controlling ion regulatory mechanisms. Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos. Whole-body Cl(-), Ca(2+), and Na(+) contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants. In contrast, overexpression of isotocin caused an increase in ionocyte numbers. Isotocin morpholino caused significant suppression of foxi3a mRNA expression, while isotocin cRNA stimulated foxi3a mRNA expressions at the tail-bud stage of zebrafish embryos. The density of P63 (an epidermal stem cell marker)-positive cells was downregulated by isotocin morpholinos and was upregulated by isotocin cRNA. Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes. The nonapeptides arginine vasopressin (AVP; including its non-mammalian homolog arginine vasotocin, AVT) and oxytocin (OT; including its non-mammalian homologs mesotocin, MT, and isotocin, IT) regulate social behavior, including aggression and reproduction, via receptors conserved across vertebrates. In monogamous prairie voles, the vasopressin and oxytocin pathways are crucially important for pair-bond formation, specifically by influencing affiliative behavior toward the mate and aggression toward non-mates. Monogamous social systems are found in diverse taxa. We hypothesized that the AVT/IT pathways are associated with mating behavior in monogamous teleost fishes. We used the monogamous convict cichlid, Amatitlania nigrofasciata, to test this idea. In the first experiment, we treated males with a general nonapeptide receptor antagonist during pair-bond formation. Control males were treated with vehicle. On the first day of treatment we observed a significant reduction in both affiliative behavior toward the potential mate and aggression toward neighbors. However, the antagonist did not prevent the pair-bond from forming and the behavioral effects disappeared on subsequent treatment days. In the second experiment, we administered on three consecutive days the AVP/OT receptor antagonist to males that were in an established pair-bond. In established pairs, male affiliation towards the mate and aggressive behavior towards territorial neighbors were not affected by the antagonist. Our results indicate that the basic social behaviors typically mediated by the AVP/OT pathways may provide the building blocks necessary for monogamous mating behavior. Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish. Pro-social effects of oxytocin and its homologues are well-documented in birds and mammals. However, in fishes, the effect of isotocin, the homologue of oxytocin, on social behaviour is less clear. Studies in fishes have generally shown no effect of isotocin on social behaviours or even an anti-social effect. In our study, we measured association preference for conspecifics in 92 adult guppies (46 females and 46 males), half of which were injected with isotocin and the other half with an isotocin antagonist. We found that individuals injected with isotocin spent 29% more time associating with conspecifics than individuals injected with an isotocin antagonist. The effect of isotocin on association time did not differ between males and females. Our study provides some of the first evidence of a pro-social effects of isotocin in a fish and suggests that in fishes, isotocin may have a homologous role to oxytocin, at least in promoting shoaling behaviour.

Fingolimod is a selective antagonist for which molecule?

Fingolimod is a selective S1P1 functional antagonist by induction of irreversible S1P1 internalization and degradation.

Does αCGRP have amyloidogenic properties?

Yes. αCGRP, a 37-residue-long peptide hormone, is a novel amyloidogenic member of the CGRP family

Author information: (1)Section of Cell Biology and Biophysics, Department of Biology, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 157 01, Greece. Electronic address: [email protected]. (2)Section of Cell Biology and Biophysics, Department of Biology, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 157 01, Greece. Electronic address: [email protected]. (3)Section of Cell Biology and Biophysics, Department of Biology, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 157 01, Greece. Electronic address: [email protected]. (4)Section of Cell Biology and Biophysics, Department of Biology, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 157 01, Greece. Electronic address: [email protected]. (5)Department of Pharmacy, University of Patras, Patras 26504, Greece. Electronic address: [email protected]. (6)Section of Cell Biology and Biophysics, Department of Biology, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 157 01, Greece. Electronic address: [email protected]. (7)Section of Cell Biology and Biophysics, Department of Biology, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 157 01, Greece. Electronic address: [email protected].