Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite.

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.

The evolution of mendelian randomization for investigating drug effects.

Dipender Gill and Stephen Burgess discuss the accompanying study by James Yarmolinsky and colleagues investigating the associations between genetically-proxied inhibition of antihypertensive drug targets and risk of common cancer subtypes using Mendelian randomization.

Influence of Ribavirin on Mumps Virus Population Diversity.

Frequent mumps outbreaks in vaccinated populations and the occurrence of neurological complications (e.g., aseptic meningitis or encephalitis) in patients with mumps indicate the need for the development of more efficient vaccines as well as specific antiviral therapies. RNA viruses are genetically highly heterogeneous populations that exist on the edge of an error threshold, such that additional increases in mutational burden can lead to extinction of the virus population. Deliberate modulation of their natural mutation rate is being exploited as an antiviral strategy and a possibility for rational vaccine design. The aim of this study was to examine the ability of ribavirin, a broad-spectrum antiviral agent, to introduce mutations in the mumps virus (MuV) genome and to investigate if resistance develops during long-term in vitro exposure to ribavirin. An increase in MuV population heterogeneity in the presence of ribavirin has been observed after one passage in cell culture, as well as a bias toward C-to-U and G-to-A transitions, which have previously been defined as ribavirin-related. At higher ribavirin concentration, MuV loses its infectivity during serial passaging and does not recover. At low ribavirin concentration, serial passaging leads to a more significant increase in population diversity and a stronger bias towards ribavirin-related transitions, independently of viral strain or cell culture. In these conditions, the virus retains its initial growth capacity, without development of resistance at a whole-virus population level.

Novel Approaches to Prevent Epileptogenesis After Traumatic Brain Injury.

Traumatic brain injury (TBI) is defined as an alteration in brain function or other evidence of brain pathology caused by an external force. When epilepsy develops following TBI, it is known as post-traumatic epilepsy (PTE). PTE occurs in a subset of patients suffering from different types and severities of TBI, occurs more commonly following severe injury, and greatly impacts the quality of life for patients recovering from TBI. Similar to other types of epilepsy, PTE is often refractory to drug treatment with standard anti-seizure drugs. No therapeutic approaches have proven successful in the clinic to prevent the development of PTE. Therefore, novel treatment strategies are needed to stop the development of PTE and improve the quality of life for patients after TBI. Interestingly, TBI represents an excellent clinical opportunity for intervention to prevent epileptogenesis as typically the time of initiation of epileptogenesis (i.e., TBI) is known, the population of at-risk patients is large, and animal models for preclinical studies of mechanisms and treatment targets are available. If properly identified and treated, there is a true opportunity to prevent epileptogenesis after TBI and stop seizures from ever happening. With that goal in mind, here we review previous attempts to prevent PTE both in animal studies and in humans, we examine how biomarkers could enable better-targeted therapeutics, and we discuss how genetic variation may predispose individuals to PTE. Finally, we highlight exciting new advances in the field that suggest that there may be novel approaches to prevent PTE that should be considered for further clinical development.

Genomic methylation variations predict the susceptibility of six chemotherapy related adverse effects and cancer development for Chinese colorectal cancer patients.

Colorectal cancer (CRC) remains a major concern with high morbidity and mortality worldwide. Despite the positive influence of chemotherapy on the decline in CRC mortality, the negative influence of chemotherapy-related adverse effects (CRAEs) caused by capecitabine (Cap) remains a challenging problem. DNA methylation alteration plays a pivotal role in gene expression regulation. Here, we aimed to screen reliable and novel biomarkers for CRC diagnosis and CRAE prediction using the advanced Illumina Infinium MethylationEPIC (850 K) BeadChip. Paired tumor and normal tissues from 21 Chinese CRC patients who received Cap-based adjuvant chemotherapy were analyzed. CRC-related methylation was characterized by hypermethylated promoter islands and hypomethylated intragenic openseas; CRAE-related methylation was characterized by hyper- (or hypo-) methylated intragenic (or intergenic) regions. Based on three types of methylation profiles (differentially methylated probes, differentially methylated regions, and gene-function-differentially methylated regions), pathway enrichment analyses revealed that CRC-related genes were significantly enriched in the neuronal system, metabolism of RNA, and extracellular matrix organization; CRAE-related genes were abundantly enriched in pathways controlling regeneration functions and immune response. Finally, based on genes within the mostly related pathways and LASSO logistic regression selection, the integrated-methylation-marker systems developed here demonstrated high discriminative accuracy in both CRC diagnosis (AUROC = 1) and CRAE prediction (AUROC = 0.817-1). In conclusion, we conducted a comprehensive DNA methylation analysis of CRC patients with chemotherapy, which provided new insights into the formation of CRC and CRAEs. Most importantly, our findings identified potentially CRAE-related metabolic pathways and markers, providing a valuable reference for personalized medicine promising better safety. Trail registration:ClinicalTrials.gov,NCT03030508, Registered 25 January 2017,https://www.clinicaltrials.gov/ct2/show/NCT03030508?term=NCT03030508&draw=2&rank=1.

Pharmacogenetic Predictors of Cannabidiol Response and Tolerability in Treatment-Resistant Epilepsy.

In patients with treatment-resistant epilepsy (TRE), cannabidiol (CBD) produces variable improvement in seizure control. Patients in the University of Alabama at Birmingham CBD Expanded Access Program (EAP) were enrolled in the genomic study and genotyped using the Affymetrix Drug Metabolizing Enzymes and Transporters plus array. Associations between variants and CBD response (≥50% seizure reduction) and tolerability (diarrhea, sedation, and abnormal liver function) was evaluated under dominant and recessive models. Expression quantitative trait loci (eQTL) influencing potential CBD targets was evaluated in the UK Brain Expression Consortium data set (Braineac), and genetic co-expression examined. Of 169 EAP patients, 112 (54.5% pediatric and 50.0% female) were included in the genetic analyses. Patients with AOX1 rs6729738 CC (aldehyde oxidase; odds ratio (OR) 6.69, 95% confidence interval (CI) 2.19-20.41, P = 0.001) or ABP1 rs12539 (diamine oxidase; OR 3.96, 95% CI 1.62-9.73, P = 0.002) were more likely to respond. Conversely, patients with SLC15A1 rs1339067 TT had lower odds of response (OR 0.06, 95% CI 0.01-0.56, P = 0.001). ABCC5 rs3749442 was associated with lower likelihood of response and abnormal liver function tests, and higher likelihood of sedation. The eQTL revealed that rs1339067 decreased GPR18 expression (endocannabinoid receptor) in white matter (P = 5.6 × 10-3 ), and rs3749442 decreased hippocampal HTR3E expression (serotonin 5-HT3E ; P = 8.5 × 10-5 ). Furthermore, 75% of genes associated with lower likelihood of response were co-expressed. Pharmacogenetic variation is associated with CBD response and influences expression of CBD targets in TRE. Implicated pathways, including cholesterol metabolism and glutathione conjugation, demonstrate potential interactions between CBD and common medications (e.g., statins and acetaminophen) that may require closer monitoring. These results highlight the role of pharmacogenes in fundamental biologic processes and potential genetic underpinnings of treatment-resistance.

Characterization of the blood-brain barrier in genetically diverse laboratory mouse strains.

BACKGROUND: Genetic variation in a population has an influence on the manifestation of monogenic as well as multifactorial disorders, with the underlying genetic contribution dependent on several interacting variants. Common laboratory mouse strains used for modelling human disease lack the genetic variability of the human population. Therefore, outcomes of rodent studies show limited relevance to human disease. The functionality of brain vasculature is an important modifier of brain diseases. Importantly, the restrictive interface between blood and brain-the blood-brain barrier (BBB) serves as a major obstacle for the drug delivery into the central nervous system (CNS). Using genetically diverse mouse strains, we aimed to investigate the phenotypic and transcriptomic variation of the healthy BBB in different inbred mouse strains. METHODS: We investigated the heterogeneity of brain vasculature in recently wild-derived mouse strains (CAST/EiJ, WSB/EiJ, PWK/PhJ) and long-inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, DBA/2J, NOD/ShiLtJ) using different phenotypic arms. We used immunohistochemistry and confocal laser microscopy followed by quantitative image analysis to determine vascular density and pericyte coverage in two brain regions-cortex and hippocampus. Using a low molecular weight fluorescence tracer, sodium fluorescein and spectrophotometry analysis, we assessed BBB permeability in young and aged mice of selected strains. For further phenotypic characterization of endothelial cells in inbred mouse strains, we performed bulk RNA sequencing of sorted endothelial cells isolated from cortex and hippocampus. RESULTS: Cortical vessel density and pericyte coverage did not differ among the investigated strains, except in the cortex, where PWK/PhJ showed lower vessel density compared to NOD/ShiLtJ, and a higher pericyte coverage than DBA/2J. The vascular density in the hippocampus differed among analyzed strains but not the pericyte coverage. The staining patterns of endothelial arteriovenous zonation markers were similar in different strains. BBB permeability to a small fluorescent tracer, sodium fluorescein, was also similar in different strains, except in the hippocampus where the CAST/EiJ showed higher permeability than NOD/ShiLtJ. Transcriptomic analysis of endothelial cells revealed that sex of the animal was a major determinant of gene expression differences. In addition, the expression level of several genes implicated in endothelial function and BBB biology differed between wild-derived and long-inbred mouse strains. In aged mice of three investigated strains (DBA/2J, A/J, C57BL/6J) vascular density and pericyte coverage did not change-expect for DBA/2J, whereas vascular permeability to sodium fluorescein increased in all three strains. CONCLUSIONS: Our analysis shows that although there were no major differences in parenchymal vascular morphology and paracellular BBB permeability for small molecular weight tracer between investigated mouse strains or sexes, transcriptomic differences of brain endothelial cells point to variation in gene expression of the intact BBB. These baseline variances might be confounding factors in pathological conditions that may lead to a differential functional outcome dependent on the sex or genetic polymorphism.

Genetic variants of PEAR1 and ischemic clinical outcomes in coronary artery disease patients: a systematic review and meta-analysis.

Aim: The aim of this study was to assess the association between PEAR1 polymorphisms and ischemic clinical outcomes. Materials & methods: We searched the electronic database for articles on the relationship of PEAR1 SNPs and ischemic events in patients with coronary artery disease (CAD) up to October 2020. Results: A total of 9914 patients with CAD from six studies focusing on 12 SNPs of PEAR1 were included in this study. The A allele of rs12041331 were associated with ischemic events (odds ratio: 1.40; 95% CI: 1.04-1.88; p = 0.03). The AA homozygotes of rs2768759 was related to a higher risk of ischemic events than carriers of the C allele (odds ratio: 2.08; 95% CI: 1.09-3.97; p = 0.03). Conclusion: PEAR1 rs12041331 and rs2768759 are significantly associated with ischemic events in patients with CAD.

Pharmacogenetics-Guided Advances in Antipsychotic Treatment.

Pharmacogenetics (PGx) research over the past 2 decades has produced extensive evidence for the influence of genetic factors on the efficacy and tolerability of antipsychotic treatment. However, the application of these findings to optimize treatment outcomes for patients in clinical practice has been limited. This paper presents a meta-review of key PGx findings related to antipsychotic response and common adverse effects, including antipsychotic-induced weight gain, tardive dyskinesia (TD), and clozapine-induced agranulocytosis (CIAG), and highlights advances and challenges in clinical implementation. Most robust findings from candidate gene and genomewide association studies were reported for associations between polymorphisms in CYP2D6 and exposure and response to specific antipsychotics. As a result, product labels and guidelines from various PGx expert groups have provided selection and dosing recommendations based on CYP2D6 metabolizer phenotypes for commonly prescribed antipsychotics. Other interesting genetic targets include DRD2 for antipsychotic response, SLC18A2 for TD, and the human leukocyte antigen (HLA) genes, HLA-DQB1 and HLA-B, for CIAG. Well-designed studies using large, well-characterized samples that leverages international collaborations are needed to validate previous findings, as well as discover new genetic variants involved in antipsychotic response and adverse effects.

Analysis Approaches to Identify Pharmacogenetic Associations With Pharmacodynamics.

Pharmacogenetics (PGx) seeks to enable selection of the right dose of the right drug for each patient to optimize therapeutic outcomes. Most PGx focuses on pharmacokinetics (PKs), due to our relatively advanced understanding of the genes involved in PKs and the causative effects of variants in those genes. Genetic variants can also affect pharmacodynamics (PDs), but relatively few PGx-PD associations have been identified. This is partially due to a more limited understanding of the relevant genes and the consequences of genetic variation, but is also due in part to the potential confounding of PK variability in assessments of clinical outcomes that have a contribution from both PKs and PDs. For example, it is challenging to confirm the effect of mu opioid receptor (OPRM1) genetic variation on opioid response due to the contribution of CYP2D6 genotype to bioactivation of some opioid drugs (i.e., codeine and tramadol). The objectives of this mini-review are to describe several recent efforts to discover and validate PGx-PD that disentangle the influence of PK variability and propose potential approaches that could be used in future PGx-PD analyses. We use the effect of OPRM1 genetics on opioid response to illustrate how these analyses could be conducted and conclude by discussing how PGx-PD could be translated into clinical practice to improve therapeutic outcomes.

Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia.

Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy.

Functional variability of rhesus macaque (Macaca mulatta) NAT2 gene for drug-metabolising arylamine N-acetyltransferase 2.

Human NAT2 is a polymorphic pharmacogene encoding for N-acetyltransferase 2, a hepatic enzyme active towards arylamine and arylhydrazine drugs, including the anti-tubercular antibiotic isoniazid. The isoenzyme also modulates susceptibility to chemical carcinogenesis, particularly of the bladder. Human NAT2 represents an ideal model for anthropological investigations into the demographic adaptation of worldwide populations to their xenobiotic environment. Its sequence appears to be subject to positive selection pressures that are population-specific and may be attributed to gene-environment interactions directly associated with exogenous chemical challenges. However, recent evidence suggests that the same evolutionary pattern may not be observed in other primates. Here, we report NAT2 polymorphism in 25 rhesus macaques (Macaca mulatta) and compare the frequencies and functional characteristics of 12 variants. Seven non-synonymous single nucleotide variations (SNVs) were identified, including one nonsense mutation. The missense SNVs were demonstrated to affect enzymatic function in a substrate-dependent manner, albeit more moderately than certain NAT1 SNVs recently characterised in the same cohort. Haplotypic and functional variability of NAT2 was comparable to that previously observed for NAT1 in the same population sample, suggesting that the two paralogues may have evolved under similar selective pressures in the rhesus macaque. This is different to the population variability distribution pattern reported for humans and chimpanzees. Recorded SNVs were also different from those found in other primates. The study contributes to further understanding of NAT2 functional polymorphism in the rhesus macaque, a non-human primate model used in biomedicine and pharmacology, indicating variability in xenobiotic acetylation that could affect drug metabolism.

Metabolomic Variation Aligns with Two Geographically Distinct Subpopulations of Brachypodium Distachyon before and after Drought Stress.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass that has been used to assess population level genomic variation. We have previously established a collection of 55 Brachypodium accessions that were sampled to reflect five different climatic regions of Turkey; designated 1a, 1c, 2, 3 and 4. Genomic and methylomic variation differentiated the collection into two subpopulations designated as coastal and central (respectively from regions 1a, 1c and the other from 2, 3 and 4) which were linked to environmental variables such as relative precipitation. Here, we assessed how far genomic variation would be reflected in the metabolomes and if this could be linked to an adaptive trait. Metabolites were extracted from eight-week-old seedlings from each accession and assessed using flow infusion high-resolution mass spectrometry (FIE-HRMS). Principal Component Analysis (PCA) of the derived metabolomes differentiated between samples from coastal and central subpopulations. The major sources of variation between seedling from the coastal and central subpopulations were identified. The central subpopulation was typified by significant increases in alanine, aspartate and glutamate metabolism and the tricarboxylic acid (TCA) cycle. Coastal subpopulation exhibited elevated levels of the auxin, indolacetic acid and rhamnose. The metabolomes of the seedling were also determined following the imposition of drought stress for seven days. The central subpopulation exhibited a metabolomic shift in response to drought, but no significant changes were seen in the coastal one. The drought responses in the central subpopulation were typified by changes in amino acids, increasing the glutamine that could be functioning as a stress signal. There were also changes in sugars that were likely to be an osmotic counter to drought, and changes in bioenergetic metabolism. These data indicate that genomic variation in our Turkish Brachypodium collection is largely reflected as distinctive metabolomes ("metabolotypes") through which drought tolerance might be mediated.

Comparison of the inhibitory effects of azole antifungals on cytochrome P450 3A4 genetic variants.

Cytochrome P450 (CYP) 3A4 is one of the major drug-metabolizing enzymes. Genetic variants of CYP3A4 with altered activity are one of the factors responsible for interindividual differences in drug metabolism. Azole antifungals inhibit CYP3A4 to cause clinically significant drug-drug interactions. In the present quantitative study, we investigated the inhibitory effects of three azole antifungals (ketoconazole, voriconazole, and fluconazole) on testosterone metabolism by recombinant CYP3A4 genetic variants (CYP3A4.1 (WT), CYP3A4.2, CYP3A4.7, CYP3A4.16, and CYP3A4.18) and compared them with those previously reported for itraconazole. The inhibition constants (Ki) of ketoconazole, voriconazole, and fluconazole for rCYP3A4.1 were 3.6 nM, 3.2 µM, and 16.1 µM, respectively. The Ki values of these azoles for rCYP3A4.16 were 13.9-, 13.6-, and 6.2-fold higher than those for rCYP3A4.1, respectively, whereas the Ki value of itraconazole for rCYP3A4.16 was 0.54-fold of that for rCYP3A4.1. The other genetic variants had similar effects on the Ki values of the three azoles, whereas a very different pattern was seen for itraconazole. In conclusion, itraconazole has unique characteristics that are distinct from those shared by the other azole anti-fungal drugs ketoconazole, voriconazole, and fluconazole with regard to the influence of genetic variations on the inhibition of CYP3A4.

Temporal evolution of sulfadoxine-pyrimethamine resistance genotypes and genetic diversity in response to a decade of increased interventions against Plasmodium falciparum in northern Ghana.

BACKGROUND: Anti-malarial drug resistance remains a key concern for the global fight against malaria. In Ghana sulfadoxine-pyrimethamine (SP) is used for intermittent preventive treatment of malaria in pregnancy and combined with amodiaquine for Seasonal Malaria Chemoprevention (SMC) during the high malaria season. Thus, surveillance of molecular markers of SP resistance is important to guide decision-making for these interventions in Ghana. METHODS: A total of 4469 samples from uncomplicated malaria patients collected from 2009 to 2018 was submitted to the Wellcome Trust Sanger Institute, UK for DNA sequencing using MiSeq. Genotypes were successfully translated into haplotypes in 2694 and 846 mono infections respectively for pfdhfr and pfdhps genes and the combined pfhdfr/pfdhps genes across all years. RESULTS: At the pfdhfr locus, a consistently high (> 60%) prevalence of parasites carrying triple mutants (IRNI) were detected from 2009 to 2018. Two double mutant haplotypes (NRNI and ICNI) were found, with haplotype NRNI having a much higher prevalence (average 13.8%) than ICNI (average 3.2%) across all years. Six pfdhps haplotypes were detected. Of these, prevalence of five fluctuated in a downward trend over time from 2009 to 2018, except a pfdhps double mutant (AGKAA), which increased consistently from 2.5% in 2009 to 78.2% in 2018. Across both genes, pfdhfr/pfdhps combined triple (NRNI + AAKAA) mutants were only detected in 2009, 2014, 2015 and 2018, prevalence of which fluctuated between 3.5 and 5.5%. The combined quadruple (IRNI + AAKAA) genotype increased in prevalence from 19.3% in 2009 to 87.5% in 2011 before fluctuating downwards to 19.6% in 2018 with an average prevalence of 37.4% within the nine years. Prevalence of parasites carrying the quintuple (IRNI + AGKAA or SGEAA) mutant haplotypes, which are highly refractory to SP increased over time from 14.0% in 2009 to 89.0% in 2016 before decreasing to 78.9 and 76.6% in 2017 and 2018 respectively. Though quintuple mutants are rising in prevalence in both malaria seasons, together these combined genotypes vary significantly within season but not between seasons. CONCLUSIONS: Despite high prevalence of pfdhfr triple mutants and combined pfdhfr/pfdhps quadruple and quintuple mutants in this setting SP may still be efficacious. These findings are significant as they highlight the need to continuously monitor SP resistance, particularly using deep targeted sequencing to ascertain changing resistance patterns.

Intrinsic and chemically-induced daughter number variations in cancer cell lines.

Multipolar mitosis was observed in cancer cells under mechanical stress or drug treatment. However, a comprehensive understanding of its basic properties and significance to cancer cell biology is lacking. In the present study, live-cell imaging was employed to investigate the division and nucleation patterns in four different cell lines. Multi-daughter divisions were observed in the three cancer cell lines HepG2, HeLa and A549, but not in the transformed non-cancer cell line RPE1. Multi-daughter mother cells displayed multi-nucleation, enlarged cell area, and prolonged division time. Under acidic pH or treatment with the anti-cancer drug 5-fluorouracil (5-FU) or the phytochemical compound wogonin, multi-daughter mitoses were increased to different extents in all three cancer cell lines, reaching as high as 16% of all mitoses. While less than 0.4% of the bi-daughter mitosis were followed by cell fusion events under the various treatment conditions, 50% or more of the multi-daughter mitoses were followed by fusion events at neutral, acidic or alkaline pH. These findings revealed a "Daughter Number Variation" (DNV) process in the cancer cells, with multi-daughter divisions in Stage 1 and cell fusions leading to the formation of cells containing up to five nuclei in Stage 2. The Stage 2-fusions were inhibited by 5-FU in A549 and HeLa, and by wogonin in A549, HeLa and HepG2. The parallel relationship between DNV frequency and malignancy among the different cell lines suggests that the inclusion of anti-fusion agents exemplified by wogonin and 5-FU could be beneficial in combinatory cancer chemotherapies.

Higher susceptibility to heme oxidation and lower protein stability of the rare α1C517Yß1 sGC variant associated with moyamoya syndrome.

NO sensitive soluble guanylyl cyclase (sGC) plays a key role in mediating physiological functions of NO. Genetic alterations of the GUCY1A3 gene, coding for the α1 subunit of sGC, are associated with several cardiovascular dysfunctions. A rare sGC variant with Cys517 â†’ Tyr substitution in the α1subunit, has been associated with moyamoya disease and achalasia. In this report we characterize the properties of this rare sGC variant. Purified α1C517Yß1 sGC preserved only ~25% of its cGMP-forming activity and showed an elevated Km for GTP substrate. However, the mutant enzyme retained a high affinity for and robust activation by NO, similar to wild type sGC. Purified α1C517Yß1 enzyme was more sensitive to specific sGC heme oxidizers and less responsive to heme reducing agents. When expressed in COS7 cells, α1C517Yß1 sGC showed a much stronger response to cinaciguat or gemfibrozil, which targets apo-sGC or sGC with ferric heme, as compared to its NO response or the relative response of the wild type sGC. A stronger response to cinaciguat was also observed for purified α1C517Yß1 in the absence of reducing agents. In COS7 cells, αCys517ß sGC was less stable than the wild type enzyme under normal conditions and exhibited accelerated degradation upon induction of cellular oxidative stress. We conclude that diminished cGMP-forming activity of this sGC variant is aggravated by its high susceptibility to oxidative stress and diminished protein stability. The combination of these deficiencies contributes to the severity of observed moyamoya and achalasia symptoms in human carriers of this rare α1C517Yß1 sGC variant.

Use of hiPSC to explicate genomic predisposition to anthracycline-induced cardiotoxicity.

The anticancer agents of the anthracycline family are commonly associated with the potential to cause severe toxicity to the heart. To solve the question of why particular a patient is predisposed to anthracycline-induced cardiotoxicity (AIC), researchers have conducted numerous pharmacogenomic studies and identified more than 60 loci associated with AIC. To date, none of these identified loci have been developed into US FDA-approved biomarkers for use in routine clinical practice. With advances in the application of human-induced pluripotent stem cell-derived cardiomyocytes, sequencing technologies and genomic editing techniques, variants associated with AIC can now be validated in a human model. Here, we provide a comprehensive overview of known genetic variants associated with AIC from the perspective of how human-induced pluripotent stem cell-derived cardiomyocytes can be used to help better explain the genomic predilection to AIC.

Association of Antibiotic Exposure With Survival and Toxicity in Patients With Melanoma Receiving Immunotherapy.

BACKGROUND: Gut microbial diversity is associated with improved response to immune checkpoint inhibitors (ICI). Based on the known detrimental impact that antibiotics have on microbiome diversity, we hypothesized that antibiotic receipt prior to ICI would be associated with decreased survival. METHODS: Patients with stage III and IV melanoma treated with ICI between 2008 and 2019 were selected from an institutional database. A window of antibiotic receipt within 3 months prior to the first infusion of ICI was prespecified. The primary outcome was overall survival (OS), and secondary outcomes were melanoma-specific mortality and immune-mediated colitis requiring intravenous steroids. All statistical tests were two-sided. RESULTS: There were 568 patients in our database of which 114 received antibiotics prior to ICI. Of the patients, 35.9% had stage III disease. On multivariable Cox proportional hazards analysis of patients with stage IV disease, the antibiotic-exposed group had statistically significantly worse OS (hazard ratio [HR] = 1.81, 95% confidence interval [CI] = 1.27 to 2.57; P <.001). The same effect was observed among antibiotic-exposed patients with stage III disease (HR = 2.78, 95% CI = 1.31 to 5.87; P =.007). When limited to only patients who received adjuvant ICI (n = 89), antibiotic-exposed patients also had statistically significantly worse OS (HR = 4.84, 95% CI = 1.09 to 21.50; P =.04). The antibiotic group had a greater incidence of colitis (HR = 2.14, 95% CI = 1.02 to 4.52; P =.046). CONCLUSION: Patients with stage III and IV melanoma exposed to antibiotics prior to ICI had statistically significantly worse OS than unexposed patients. Antibiotic exposure was associated with greater incidence of moderate to severe immune-mediated colitis. Given the large number of antibiotics prescribed annually, physicians should be judicious with their use in cancer populations likely to receive ICI.

The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease.

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.