The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

Functional genomic diversity is correlated with neutral genomic diversity in populations of an endangered rattlesnake.

Theory predicts that genetic erosion in small, isolated populations of endangered species can be assessed using estimates of neutral genetic variation, yet this widely used approach has recently been questioned in the genomics era. Here, we leverage a chromosome-level genome assembly of an endangered rattlesnake (Sistrurus catenatus) combined with whole genome resequencing data (N = 110 individuals) to evaluate the relationship between levels of genome-wide neutral and functional diversity over historical and future timescales. As predicted, we found positive correlations between genome-wide estimates of neutral genetic diversity (π) and inferred levels of adaptive variation and an estimate of inbreeding mutation load, and a negative relationship between neutral diversity and an estimate of drift mutation load. However, these correlations were half as strong for projected future levels of neutral diversity based on contemporary effective population sizes. Broadly, our results confirm that estimates of neutral genetic diversity provide an accurate measure of genetic erosion in populations of a threatened vertebrate. They also provide nuance to the neutral-functional diversity controversy by suggesting that while these correlations exist, anthropogenetic impacts may have weakened these associations in the recent past and into the future.

HERV1-env Induces Unfolded Protein Response Activation in Autoimmune Liver Disease: A Potential Mechanism for Regulatory T Cell Dysfunction.

Regulatory T cells (Tregs) are not terminally differentiated but can acquire effector properties. Here we report an increased expression of human endogenous retrovirus 1 (HERV1-env) proteins in Tregs of patients with de novo autoimmune hepatitis and autoimmune hepatitis, which induces endoplasmic reticulum (ER) stress. HERV1-env-triggered ER stress activates all three branches (IRE1, ATF6, and PERK) of the unfolded protein response (UPR). Our coimmunoprecipitation studies show an interaction between HERV1-env proteins and the ATF6 branch of the UPR. The activated form of ATF6α activates the expression of RORC and STAT3 by binding to promoter sequences and induces IL-17A production. Silencing of HERV1-env results in recovery of Treg suppressive function. These findings identify ER stress and UPR activation as key factors driving Treg plasticity (species: human).

Treatment response and clinical event-free survival in autoimmune hepatitis: A Canadian multicentre cohort study.

BACKGROUND & AIMS: Treatment outcomes for people living with autoimmune hepatitis (AIH) are limited by a lack of specific therapies, as well as limited well-validated prognostic tools and clinical trial endpoints. We sought to identify predictors of outcome for people living with AIH. METHODS: We evaluated the clinical course of people with AIH across 11 Canadian centres. Biochemical changes were analysed using linear mixed-effect and logistic regression. Clinical outcome was dynamically modelled using time-varying Cox proportional hazard modelling and landmark analysis. RESULTS: In 691 patients (median age 49 years, 75.4% female), with a median follow-up of 6 years (25th-75th percentile, 2.5-11), 118 clinical events occurred. Alanine aminotransferase (ALT) normalisation occurred in 63.8% of the cohort by 12 months. Older age at diagnosis (odd ratio [OR] 1.19, 95% CI 1.06-1.35) and female sex (OR 1.94, 95% CI 1.18-3.19) were associated with ALT normalisation at 6 months, whilst baseline cirrhosis status was associated with reduced chance of normalisation at 12 months (OR 0.52, 95% CI 0.33-0.82). Baseline total bilirubin, aminotransferases, and IgG values, as well as initial prednisone dose, did not predict average ALT reduction. At baseline, older age (hazard ratio [HR] 1.25, 95% CI 1.12-1.40), cirrhosis at diagnosis (HR 3.67, 95% CI 2.48-5.43), and elevated baseline total bilirubin (HR 1.36, 95% CI 1.17-1.58) increased the risk of clinical events. Prolonged elevations in ALT (HR 1.07, 95% CI 1.00-1.13) and aspartate aminotransferase (HR 1.13, 95% CI 1.06-1.21), but not IgG (HR 1.01, 95% CI 0.95-1.07), were associated with higher risk of clinical events. Higher ALT at 6 months was associated with worse clinical event-free survival. CONCLUSION: In people living with AIH, sustained elevated aminotransferase values, but not IgG, are associated with poorer long-term outcomes. Biochemical response and long-term survival are not associated with starting prednisone dose. IMPACT AND IMPLICATIONS: Using clinical data from multiple Canadian liver clinics treating autoimmune hepatitis (AIH), we evaluate treatment response and clinical outcomes. For the first time, we apply mixed-effect and time-varying survival statistical methods to rigorously examine treatment response and the impact of fluctuating liver biochemistry on clinical event-free survival. Key to the study impact, our data is 'real-world', represents a diverse population across Canada, and uses continuous measurements over follow-up. Our results challenge the role of IgG as a marker of treatment response and if normalisation of IgG should remain an important part of the definition of biochemical remission. Our analysis further highlights that baseline markers of disease severity may not prognosticate early treatment response. Additionally, the initial prednisone dose may be less relevant for achieving aminotransferase normalisation. This is important for patients and treating clinicians given the relevance and importance of side effects.

Unfermented ß-fructan Fibers Fuel Inflammation in Select Inflammatory Bowel Disease Patients.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBD) are affected by dietary factors, including nondigestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial, but not all fibers are alike, and some patients with IBD report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in patients with IBD, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation. METHODS: Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5 g/L), or fermentation supernatants (24-hour anaerobic fermentation) and immune responses (cytokine secretion [enzyme-linked immunosorbent assay/meso scale discovery] and expression [quantitative polymerase chain reaction]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software. RESULTS: Unfermented dietary ß-fructan fibers induced proinflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining ß-fructan supplementation. The proinflammatory response to intact ß-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of ß-fructans by human gut whole microbiota cultures reduced the proinflammatory response, but only when microbes were collected from patients without IBD or patients with inactive IBD. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance. CONCLUSION: Although fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities. The study is publicly accessible at the U.S. National Institutes of Health database (clinicaltrials.gov identification number NCT02865707).

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 µM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Anti-vascular endothelial growth factor biosimilars for neovascular age-related macular degeneration.

RATIONALE: Neovascular age-related macular degeneration (AMD) is a progressive eye disease characterized by choroidal neovascularization (CNV) and is a leading cause of vision loss and disability worldwide. Although intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy is an effective treatment option that helps to prevent vision loss or to improve visual acuity in people with neovascular AMD, treatment imposes a significant financial burden on patients and healthcare systems. A biosimilar is a biological product that has been developed to be nearly identical to a previously approved biological product. The use of biosimilars may help reduce costs and so may increase patient access to effective biologic medicines with similar levels of safety to the drugs on which they are based. OBJECTIVES: To assess the benefits and harms of anti-VEGF biosimilar agents compared with their corresponding anti-VEGF agents (i.e. the reference products) that have obtained regulatory approval for intravitreal injections in people with neovascular AMD. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, two other databases, and two trials registries together with reference checking and contact with study authors to identify studies that are included in the review. The latest search date was 2 June 2023. ELIGIBILITY CRITERIA: We included randomized controlled trials (RCTs) that compared approved anti-VEGF biosimilars with their reference products for treating the eyes of adult participants (≥ 50 years) who had an active primary or recurrent choroidal neovascularization lesion secondary to neovascular AMD. OUTCOMES: Our outcomes were: best-corrected visual acuity (BCVA), central subfield thickness (CST), vision-related quality of life, serious ocular and non-ocular adverse events (AE), treatment-emergent adverse events (TEAEs), anti-drug antibodies (ADAs), and serum concentrations of biosimilars and reference drugs. RISK OF BIAS: We assessed the risk of bias (RoB) for seven outcomes reported in a summary of findings table by using the Cochrane RoB 2 tool. SYNTHESIS METHODS: We synthesized results for each outcome using meta-analysis, where possible, by calculating risk ratios (RR) and mean differences (MD) with 95% confidence intervals (CI) for dichotomous outcomes and continuous outcomes, respectively. Where this was not possible due to the nature of the data, we summarized the results narratively. We used GRADE to assess the certainty of evidence for prespecified outcomes. INCLUDED STUDIES: We included nine parallel-group multi-center RCTs that enrolled a total of 3814 participants (3814 participating eyes), with sample sizes that ranged from 160 to 705 participants per study. The mean age of the participants in these studies ranged from 67 to 76 years, and the proportion of women ranged from 26.5% to 58.7%. Ranibizumab (Lucentis) was the reference product in seven studies, and aflibercept (Eyelea) was the reference product in two others. All the included studies had been supported by industry. The follow-up periods ranged from 12 to 52 weeks (median 48 weeks). Five studies (56%) were conducted in multi-country settings across Europe, North America and Asia, two studies in India, and one each in Japan and the Republic of Korea. We judged all the included studies to have met high methodological standards. SYNTHESIS OF RESULTS: With regard to efficacy, our meta-analyses demonstrated that anti-VEGF biosimilars for neovascular AMD resulted in little to no difference compared with the reference products for BCVA change at 8 to 12 weeks (MD -0.55 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, 95% CI -1.17 to 0.07; 8 studies, 3603 participants; high-certainty evidence) and the proportion of participants who lost fewer than 15 letters in BCVA at 24 to 48 weeks (RR 0.99, 95% CI 0.98 to 1.01; 7 studies, 2658 participants; moderate-certainty evidence). Almost all participants (96.6% in the biosimilar group and 97.0% in the reference product group) lost fewer than 15 letters in BCVA. The evidence from two studies suggested that there was no evidence of difference between biosimilars and reference products in vision-related quality of life measured by the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ-25) summary scores at 24 to 48 weeks (MD 0.82, 95% CI -0.70 to 2.35; 2 studies, 894 participants; moderate-certainty evidence). With regard to the safety profile, meta-analyses also revealed little to no difference between anti-VEGF biosimilars and the reference products for the proportion of participants who experienced serious ocular AEs (RR 1.24, 95% CI 0.68 to 2.26; 7 studies, 3292 participants; moderate-certainty evidence), and for TEAEs leading to investigational product discontinuation or death (RR 0.96, 95% CI 0.63 to 1.46; 8 studies, 3497 participants; moderate-certainty evidence). Overall, 1.4% of participants in the biosimilar group and 1.2% in the reference product group experienced serious ocular adverse events. The most frequently documented serious ocular AEs were retinal hemorrhage and endophthalmitis. Although the evidence is of low certainty due to imprecision, meta-analysis suggested that anti-VEGF biosimilars led to no difference compared with the reference products for cumulative incidence of ADAs (RR 0.84, 95% CI 0.58 to 1.22; 8 studies, 3066 participants; low-certainty evidence) or mean maximum serum concentrations (MD 0.42 ng/mL, 95% CI -0.22 to 1.05; subgroup of 3 studies, 100 participants; low-certainty evidence). We judged the overall risk of bias to be low for all studies. AUTHORS' CONCLUSIONS: In our review, low to high certainty evidence suggests that there is little to no difference, to date, between the anti-VEGF biosimilars approved for treating neovascular AMD and their reference products in terms of benefits and harms. While anti-VEGF biosimilars may be a viable alternative to reference products, current evidence for their use is based on a limited number of studies - particularly for comparison with aflibercept - with sparse long-term safety data, and infrequent assessment of quality of life outcomes. Our effect estimates and conclusions may be modified once findings have been reported from studies that are currently ongoing, and studies of biosimilar agents that are currently in development. FUNDING: Cochrane Eyes and Vision US Project is supported by grant UG1EY020522, National Eye Institute, National Institutes of Health. Takeshi Hasegawa and Hisashi Noma were supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant numbers: 22H03554, 19K03092, 24K06239). REGISTRATION: Protocol available via doi.org/10.1002/14651858.CD015804.

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

Phylogenetically diverse diets favor more complex venoms in North American pitvipers.

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants.

BACKGROUND: The red junglefowl, the wild outgroup of domestic chickens, has historically served as a reference for genomic studies of domestic chickens. These studies have provided insight into the etiology of traits of commercial importance. However, the use of a single reference genome does not capture diversity present among modern breeds, many of which have accumulated molecular changes due to drift and selection. While reference-based resequencing is well-suited to cataloging simple variants such as single-nucleotide changes and short insertions and deletions, it is mostly inadequate to discover more complex structural variation in the genome. METHODS: We present a pangenome for the domestic chicken consisting of thirty assemblies of chickens from different breeds and research lines. RESULTS: We demonstrate how this pangenome can be used to catalog structural variants present in modern breeds and untangle complex nested variation. We show that alignment of short reads from 100 diverse wild and domestic chickens to this pangenome reduces reference bias by 38%, which affects downstream genotyping results. This approach also allows for the accurate genotyping of a large and complex pair of structural variants at the K feathering locus using short reads, which would not be possible using a linear reference. CONCLUSIONS: We expect that this new paradigm of genomic reference will allow better pinpointing of exact mutations responsible for specific phenotypes, which will in turn be necessary for breeding chickens that meet new sustainability criteria and are resilient to quickly evolving pathogen threats.

Wide Voltage Swing Potentiostat with Dynamic Analog Ground to Expand Electrochemical Potential Windows in Integrated Microsystems.

Electrochemical measurements are vital to a wide range of applications such as air quality monitoring, biological testing, food industry, and more. Integrated circuits have been used to implement miniaturized and low-power electrochemical potentiostats that are suitable for wearable devices. However, employing modern integrated circuit technologies with low supply voltage precludes the utilization of electrochemical reactions that require a higher potential window. In this paper, we present a novel circuit architecture that utilizes dynamic voltage at the working electrode of an electrochemical cell to effectively enhance the supported voltage range compared to traditional designs, increasing the cell voltage range by 46% and 88% for positive and negative cell voltages, respectively. In return, this facilitates a wider range of bias voltages in an electrochemical cell, and, therefore, opens integrated microsystems to a broader class of electrochemical reactions. The circuit was implemented in 180 nm technology and consumes 2.047 mW of power. It supports a bias potential range of 1.1 V to -2.12 V and cell potential range of 2.41 V to -3.11 V that is nearly double the range in conventional designs.

Venom Gene Sequence Diversity and Expression Jointly Shape Diet Adaptation in Pitvipers.

Understanding the joint roles of protein sequence variation and differential expression during adaptive evolution is a fundamental, yet largely unrealized goal of evolutionary biology. Here, we use phylogenetic path analysis to analyze a comprehensive venom-gland transcriptome dataset spanning three genera of pitvipers to identify the functional genetic basis of a key adaptation (venom complexity) linked to diet breadth (DB). The analysis of gene-family-specific patterns reveals that, for genes encoding two of the most important venom proteins (snake venom metalloproteases and snake venom serine proteases), there are direct, positive relationships between sequence diversity (SD), expression diversity (ED), and increased DB. Further analysis of gene-family diversification for these proteins showed no constraint on how individual lineages achieved toxin gene SD in terms of the patterns of paralog diversification. In contrast, another major venom protein family (PLA2s) showed no relationship between venom molecular diversity and DB. Additional analyses suggest that other molecular mechanisms-such as higher absolute levels of expression-are responsible for diet adaptation involving these venom proteins. Broadly, our findings argue that functional diversity generated through sequence and expression variations jointly determine adaptation in the key components of pitviper venoms, which mediate complex molecular interactions between the snakes and their prey.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

Greater Transplant-Free Survival in Patients Receiving Obeticholic Acid for Primary Biliary Cholangitis in a Clinical Trial Setting Compared to Real-World External Controls.

BACKGROUND & AIMS: The Primary Biliary Cholangitis (PBC) Obeticholic Acid (OCA) International Study of Efficacy (POISE) randomized, double-blind, placebo-controlled trial demonstrated that OCA reduced biomarkers associated with adverse clinical outcomes (ie, alkaline phosphatase, bilirubin, aspartate aminotransferase, and alanine aminotransferase) in patients with PBC. The objective of this study was to evaluate time to first occurrence of liver transplantation or death in patients with OCA in the POISE trial and open-label extension vs comparable non-OCA-treated external controls. METHODS: Propensity scores were generated for external control patients meeting POISE eligibility criteria from 2 registry studies (Global PBC and UK-PBC) using an index date selected randomly between the first and last date (inclusive) on which eligibility criteria were met. Cox proportional hazards models weighted by inverse probability of treatment assessed time to death or liver transplantation. Additional analyses (Global PBC only) added hepatic decompensation to the composite end point and assessed efficacy in patients with or without cirrhosis. RESULTS: During the 6-year follow-up, there were 5 deaths or liver transplantations in 209 subjects in the POISE cohort (2.4%), 135 of 1381 patients in the Global PBC control (10.0%), and 281 of 2135 patients in the UK-PBC control (13.2%). The hazard ratios (HRs) for the primary outcome were 0.29 (95% CI, 0.10-0.83) for POISE vs Global PBC and 0.30 (95% CI, 0.12-0.75) for POISE vs UK-PBC. In the Global PBC study, HR was 0.20 (95% CI, 0.03-1.22) for patients with cirrhosis and 0.31 (95% CI, 0.09-1.04) for those without cirrhosis; HR was 0.42 (95% CI, 0.21-0.85) including hepatic decompensation. CONCLUSIONS: Patients treated with OCA in a trial setting had significantly greater transplant-free survival than comparable external control patients.

Ursodeoxycholic Acid Treatment-Induced GLOBE Score Changes Are Associated With Liver Transplantation-Free Survival in Patients With Primary Biliary Cholangitis.

INTRODUCTION: Treatment of primary biliary cholangitis (PBC) can improve the GLOBE score. We aimed to assess the association between changes in the GLOBE score (ΔGLOBE) and liver transplantation (LT)-free survival in patients with PBC who were treated with ursodeoxycholic acid (UDCA). METHODS: Among UDCA-treated patients within the Global PBC cohort, the association between ΔGLOBE (ΔGLOBE 0-1 : during the first year of UDCA, ΔGLOBE 1-2 : during the second year) and the risk of LT or death was assessed through Cox regression analyses. RESULTS: Overall, 3,775 UDCA-treated patients were included; 3,424 (90.7%) were female, the median age was 54.0 (interquartile range [IQR] 45.9-62.4) years, and the median baseline GLOBE score was 0.25 (IQR -0.47 to 0.96). During a median follow-up of 7.2 (IQR 3.7-11.5) years, 730 patients reached the combined end point of LT or death. The median ΔGLOBE 0-1 was -0.27 (IQR -0.56 to 0.02). Cox regression analyses, adjusted for pretreatment GLOBE score and ΔGLOBE 0-12 , showed that ΔGLOBE was associated with LT or death (adjusted hazard ratio 2.28, 95% confidence interval 1.81-2.87, P < 0.001). The interaction between baseline GLOBE score and ΔGLOBE 0-1 was not statistically significant ( P = 0.296). The ΔGLOBE 1-2 was associated with LT or death (adjusted hazard ratio 2.19, 95% confidence interval 1.67-2.86, P < 0.001), independently from the baseline GLOBE score and the change in GLOBE score during the first year of UDCA. DISCUSSION: UDCA-induced changes in the GLOBE score were significantly associated with LT-free survival in patients with PBC. While the relative risk reduction of LT or death was stable, the absolute risk reduction was heavily dependent on the baseline prognosis of the patient.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

MOTIVATION: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. RESULTS: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.

Ethnicity, disease severity, and survival in Canadian patients with primary biliary cholangitis.

BACKGROUND AND AIMS: We investigated associations between ethnicity, survival, and disease severity in a diverse Canadian cohort of patients with primary biliary cholangitis (PBC). APPROACH AND RESULTS: Patients with PBC were included from the Canadian Network for Autoimmune Liver Disease. Ethnicity was defined using a modified list adopted from Statistics Canada, and ethnicities with small samples were grouped. Clinical events were defined as liver decompensation, HCC, liver transplantation, or death. Clinical event-free and liver transplantation-free survival were analyzed using Cox regression. Trajectories of serum liver function tests were assessed over time using mixed-effects regression. Health-related quality of life was assessed using the Short Form 36, the PBC-40 questionnaire, and the 5-D Itch scale and analyzed using mixed-effects regression. The cohort included 1538 patients with PBC from six sites and was comprised of 82% White, 4.7% Indigenous, 5.5% East Asian, 2.6% South Asian, and 5.1% miscellaneous ethnicities. Indigenous patients were the only ethnic group with impaired liver transplant-free and event-free survival compared to White patients (HR, 3.66; 95% CI, 2.23-6.01; HR, 3.09; 95% CI, 1.94-4.92). Indigenous patients were more likely to have a clinical event before diagnosis (10%) than all other ethnic groups despite similar age at diagnosis. Indigenous patients presented with higher alkaline phosphatase, total bilirubin, and GLOBE scores than White patients; and these relative elevations persisted during follow-up. CONCLUSIONS: Indigenous Canadians with PBC present with advanced disease and have worse long-term outcomes compared to White patients.

Optimizing therapy in primary biliary cholangitis: Alkaline phosphatase at six months identifies one-year non-responders and predicts survival.

BACKGROUND AND AIMS: Patients with primary biliary cholangitis (PBC) and insufficient response to ursodeoxycholic acid (UDCA), currently assessed after 1 year, are candidates for second-line therapy. The aims of this study are to assess biochemical response pattern and determine the utility of alkaline phosphatase (ALP) at six months as a predictor of insufficient response. METHODS: UDCA-treated patients in the GLOBAL PBC database with available liver biochemistries at one year were included. POISE criteria were used to assess response to treatment, defined as ALP <1.67 × upper limit of normal (ULN) and normal total bilirubin at one year. Various thresholds of ALP at six months were evaluated to predict insufficient response based on negative predictive value (NPV) and that with nearest to 90% NPV was selected. RESULTS: For the study, 1362 patients were included, 1232 (90.5%) female, mean age of 54 years. The POISE criteria were met by 56.4% (n = 768) of patients at one year. The median ALP (IQR) of those who met POISE criteria compared to those who did not was 1.05 × ULN (0.82-1.33) vs. 2.37 × ULN (1.72-3.69) at six months (p < .001). Of 235 patients with serum ALP >1.9 × ULN at six months, 89% did not achieve POISE criteria (NPV) after one year of UDCA. Of those with insufficient response by POISE criteria at one year, 210 (67%) had an ALP >1.9 × ULN at six months and thus would have been identified early. CONCLUSIONS: We can identify patients for second-line therapy at six months using an ALP threshold of 1.9 × ULN, given that approximately 90% of these patients are non-responders according to POISE criteria.

Evidence of a Novel Mitochondrial Signature in Systemic Sclerosis Patients with Chronic Fatigue Syndrome.

Symptoms of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are common in rheumatic diseases, but no studies report the frequency of these in early systemic sclerosis. There are no known biomarkers that can distinguish between patients with ME/CFS, although mitochondrial abnormalities are often demonstrated. We sought to assess the prevalence of ME/CFS in limited cutaneous SSc (lcSSc) patients early in their disease (<5 years from the onset of non-Raynaud's symptoms) and to determine if alterations in mitochondrial electron transport chain (ETC) transcripts and mitochondrial DNA (mtDNA) integrity could be used to distinguish between fatigued and non-fatigued patients. All SSc patients met ACR/EULAR classification criteria. ME/CFS-related symptoms were assessed through validated questionnaires, and the expression of ETC transcripts and mtDNA integrity were quantified via qPCR. SSc patients with ME/CFS could be distinguished from non-fatigued patients through ETC gene analysis; specifically, reduced expression of ND4 and CyB and increased expression of Cox7C. ND4 and CyB expression correlated with indicators of disease severity. Further prospective and functional studies are needed to determine if this altered signature can be further utilized to better identify ME/CFS in SSc patients, and whether ME/CFS in early SSc disease could predict more severe disease outcomes.

Genomic Adaptations to Salinity Resist Gene Flow in the Evolution of Floridian Watersnakes.

The migration-selection balance often governs the evolution of lineages, and speciation with gene flow is now considered common across the tree of life. Ecological speciation is a process that can facilitate divergence despite gene flow due to strong selective pressures caused by ecological differences; however, the exact traits under selection are often unknown. The transition from freshwater to saltwater habitats provides strong selection targeting traits with osmoregulatory function. Several lineages of North American watersnakes (Nerodia spp.) are known to occur in saltwater habitat and represent a useful system for studying speciation by providing an opportunity to investigate gene flow and evaluate how species boundaries are maintained or degraded. We use double digest restriction-site associated DNA sequencing to characterize the migration-selection balance and test for evidence of ecological divergence within the Nerodia fasciata-clarkii complex in Florida. We find evidence of high intraspecific gene flow with a pattern of isolation-by-distance underlying subspecific lineages. However, we identify genetic structure indicative of reduced gene flow between inland and coastal lineages suggesting divergence due to isolation-by-environment. This pattern is consistent with observed environmental differences where the amount of admixture decreases with increased salinity. Furthermore, we identify significantly enriched terms related to osmoregulatory function among a set of candidate loci, including several genes that have been previously implicated in adaptation to salinity stress. Collectively, our results demonstrate that ecological differences, likely driven by salinity, cause strong divergent selection which promotes divergence in the N. fasciata-clarkii complex despite significant gene flow.