Orally available nucleoside analog UMM-766 provides protection in a murine model of orthopox disease.

Although smallpox has been eradicated, other orthopoxviruses continue to be a public health concern as exemplified by the ongoing Mpox (formerly monkeypox) global outbreak. While medical countermeasures (MCMs) previously approved by the Food and Drug Administration for the treatment of smallpox have been adopted for Mpox, previously described vulnerabilities coupled with the questionable benefit of at least one of the therapeutics during the 2022 Mpox outbreak reinforce the need for identifying and developing other MCMs against orthopoxviruses. Here, we screened a panel of Merck proprietary small molecules and identified a novel nucleoside inhibitor with potent broad-spectrum antiviral activity against multiple orthopoxviruses. Efficacy testing of a 7-day dosing regimen of the orally administered nucleoside in a murine model of severe orthopoxvirus infection yielded a dose-dependent increase in survival. Treated animals had greatly reduced lesions in the lung and nasal cavity, particularly in the 10 µg/mL dosing group. Viral levels were also markedly lower in the UMM-766-treated animals. This work demonstrates that this nucleoside analog has anti-orthopoxvirus efficacy and can protect against severe disease in a murine orthopox model.IMPORTANCEThe recent monkeypox virus pandemic demonstrates that members of the orthopoxvirus, which also includes variola virus, which causes smallpox, remain a public health issue. While currently FDA-approved treatment options exist, risks that resistant strains of orthopoxviruses may arise are a great concern. Thus, continued exploration of anti-poxvirus treatments is warranted. Here, we developed a template for a high-throughput screening assay to identify anti-poxvirus small-molecule drugs. By screening available drug libraries, we identified a compound that inhibited orthopoxvirus replication in cell culture. We then showed that this drug can protect animals against severe disease. Our findings here support the use of existing drug libraries to identify orthopoxvirus-targeting drugs that may serve as human-safe products to thwart future outbreaks.

A multi-tissue gene expression dataset for hibernating brown bears.

OBJECTIVES: Complex physiological adaptations often involve the coordination of molecular responses across multiple tissues. Establishing transcriptomic resources for non-traditional model organisms with phenotypes of interest can provide a foundation for understanding the genomic basis of these phenotypes, and the degree to which these resemble, or contrast, those of traditional model organisms. Here, we present a one-of-a-kind gene expression dataset generated from multiple tissues of two hibernating brown bears (Ursus arctos). DATA DESCRIPTION: This dataset is comprised of 26 samples collected from 13 tissues of two hibernating brown bears. These samples were collected opportunistically and are typically not possible to attain, resulting in a highly unique and valuable gene expression dataset. In combination with previously published datasets, this new transcriptomic resource will facilitate detailed investigation of hibernation physiology in bears, and the potential to translate aspects of this biology to treat human disease.

Epigenetic inheritance of DNA methylation changes in fish living in hydrogen sulfide-rich springs.

Environmental factors can promote phenotypic variation through alterations in the epigenome and facilitate adaptation of an organism to the environment. Although hydrogen sulfide is toxic to most organisms, the fish Poecilia mexicana has adapted to survive in environments with high levels that exceed toxicity thresholds by orders of magnitude. Epigenetic changes in response to this environmental stressor were examined by assessing DNA methylation alterations in red blood cells, which are nucleated in fish. Males and females were sampled from sulfidic and nonsulfidic natural environments; individuals were also propagated for two generations in a nonsulfidic laboratory environment. We compared epimutations between the sexes as well as field and laboratory populations. For both the wild-caught (F0) and the laboratory-reared (F2) fish, comparing the sulfidic and nonsulfidic populations revealed evidence for significant differential DNA methylation regions (DMRs). More importantly, there was over 80% overlap in DMRs across generations, suggesting that the DMRs have stable generational inheritance in the absence of the sulfidic environment. This is an example of epigenetic generational stability after the removal of an environmental stressor. The DMR-associated genes were related to sulfur toxicity and metabolic processes. These findings suggest that adaptation of P. mexicana to sulfidic environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that become stable and are inherited by subsequent generations independent of the environment.

Antipsychotic Behavioral Phenotypes in the Mouse Collaborative Cross Recombinant Inbred Inter-Crosses (RIX).

Schizophrenia is an idiopathic disorder that affects approximately 1% of the human population, and presents with persistent delusions, hallucinations, and disorganized behaviors. Antipsychotics are the standard pharmacological treatment for schizophrenia, but are frequently discontinued by patients due to inefficacy and/or side effects. Chronic treatment with the typical antipsychotic haloperidol causes tardive dyskinesia (TD), which manifests as involuntary and often irreversible orofacial movements in around 30% of patients. Mice treated with haloperidol develop many of the features of TD, including jaw tremors, tongue protrusions, and vacuous chewing movements (VCMs). In this study, we used genetically diverse Collaborative Cross (CC) recombinant inbred inter-cross (RIX) mice to elucidate the genetic basis of antipsychotic-induced adverse drug reactions (ADRs). We performed a battery of behavioral tests in 840 mice from 73 RIX lines (derived from 62 CC strains) treated with haloperidol or placebo in order to monitor the development of ADRs. We used linear mixed models to test for strain and treatment effects. We observed highly significant strain effects for almost all behavioral measurements investigated (P < 0.001). Further, we observed strong strain-by-treatment interactions for most phenotypes, particularly for changes in distance traveled, vertical activity, and extrapyramidal symptoms (EPS). Estimates of overall heritability ranged from 0.21 (change in body weight) to 0.4 (VCMs and change in distance traveled) while the portion attributable to the interactions of treatment and strain ranged from 0.01 (for change in body weight) to 0.15 (for change in EPS). Interestingly, close to 30% of RIX mice exhibited VCMs, a sensitivity to haloperidol exposure, approximately similar to the rate of TD in humans chronically exposed to haloperidol. Understanding the genetic basis for the susceptibility to antipsychotic ADRs may be possible in mouse, and extrapolation to humans could lead to safer therapeutic approaches for schizophrenia.

Author Correction: Hibernation induces widespread transcriptional remodeling in metabolic tissues of the grizzly bear.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptomics of Tasmanian Devil (Sarcophilus Harrisii) Ear Tissue Reveals Homogeneous Gene Expression Patterns across a Heterogeneous Landscape.

In an era of unprecedented global change, exploring patterns of gene expression among wild populations across their geographic range is crucial for characterizing adaptive potential. RNA-sequencing studies have successfully characterized gene expression differences among populations experiencing divergent environmental conditions in a wide variety of taxa. However, few of these studies have identified transcriptomic signatures to multivariate, environmental stimuli among populations in their natural environments. Herein, we aim to identify environmental and sex-driven patterns of gene expression in the Tasmanian devil (Sarcophilus harrisii), a critically endangered species that occupies a heterogeneous environment. We performed RNA-sequencing on ear tissue biopsies from adult male and female devils from three populations at the extremes of their geographic range. There were no transcriptome-wide patterns of differential gene expression that would be suggestive of significant, environmentally-driven transcriptomic responses. The general lack of transcriptome-wide variation in gene expression levels across the devil's geographic range is consistent with previous studies that documented low levels of genetic variation in the species. However, genes previously implicated in local adaptation to abiotic environment in devils were enriched for differentially expressed genes. Additionally, three modules of co-expressed genes were significantly associated with either population of origin or sex.

Hibernation induces widespread transcriptional remodeling in metabolic tissues of the grizzly bear.

Revealing the mechanisms underlying the reversible physiology of hibernation could have applications to both human and animal health as hibernation is often associated with disease-like states. The present study uses RNA-sequencing to reveal the tissue and seasonal transcriptional changes occurring in grizzly bears (Ursus arctos horribilis). Comparing hibernation to other seasons, bear adipose has a greater number of differentially expressed genes than liver and skeletal muscle. During hyperphagia, adipose has more than 900 differentially expressed genes compared to active season. Hibernation is characterized by reduced expression of genes associated with insulin signaling, muscle protein degradation, and urea production, and increased expression within muscle protein anabolic pathways. Across all three tissues we find a subset of shared differentially expressed genes, some of which are uncharacterized, that together may reflect a common regulatory mechanism. The identified gene families could be useful for developing novel therapeutics to treat human and animal diseases.

Bacterial Diversity in Replicated Hydrogen Sulfide-Rich Streams.

Extreme environments typically require costly adaptations for survival, an attribute that often translates to an elevated influence of habitat conditions on biotic communities. Microbes, primarily bacteria, are successful colonizers of extreme environments worldwide, yet in many instances, the interplay between harsh conditions, dispersal, and microbial biogeography remains unclear. This lack of clarity is particularly true for habitats where extreme temperature is not the overarching stressor, highlighting a need for studies that focus on the role other primary stressors (e.g., toxicants) play in shaping biogeographic patterns. In this study, we leveraged a naturally paired stream system in southern Mexico to explore how elevated hydrogen sulfide (H2S) influences microbial diversity. We sequenced a portion of the 16S rRNA gene using bacterial primers for water sampled from three geographically proximate pairings of streams with high (> 20 µM) or low (~ 0 µM) H2S concentrations. After exploring bacterial diversity within and among sites, we compared our results to a previous study of macroinvertebrates and fish for the same sites. By spanning multiple organismal groups, we were able to illuminate how H2S may differentially affect biodiversity. The presence of elevated H2S had no effect on overall bacterial diversity (p = 0.21), a large effect on community composition (25.8% of variation explained, p < 0.0001), and variable influence depending upon the group-whether fish, macroinvertebrates, or bacteria-being considered. For bacterial diversity, we recovered nine abundant operational taxonomic units (OTUs) that comprised a core H2S-rich stream microbiome in the region. Many H2S-associated OTUs were members of the Epsilonproteobacteria and Gammaproteobacteria, which both have been implicated in endosymbiotic relationships between sulfur-oxidizing bacteria and eukaryotes, suggesting the potential for symbioses that remain to be discovered in these habitats.

Ancestry informative alleles captured with reduced representation library sequencing in Theobroma cacao.

As the source of chocolate, cacao has become one of the most important crops in the world. The identification of molecular markers to understand the demographic history, genetic diversity and population structure plays a pivotal role in cacao breeding programs. Here, we report the use of a modified genotyping-by-sequencing (GBS) approach for large-scale single nucleotide polymorphism (SNP) discovery and allele ancestry mapping. We identified 12,357 bi-allelic SNPs after filtering, of which, 7,009 variants were ancestry informative. The GBS approach proved to be rapid, cost-effective, and highly informative for ancestry assignment in this species.

The roles of plasticity and evolutionary change in shaping gene expression variation in natural populations of extremophile fish.

The notorious plasticity of gene expression responses and the complexity of environmental gradients complicate the identification of adaptive differences in gene regulation among populations. We combined transcriptome analyses in nature with common-garden and exposure experiments to establish cause-effect relationships between the presence of a physiochemical stressor and expression differences, as well as to test how evolutionary change and plasticity interact to shape gene expression variation in natural systems. We studied two evolutionarily independent population pairs of an extremophile fish (Poecilia mexicana) living in toxic, hydrogen sulphide (H2 S)-rich springs and adjacent nontoxic habitats and assessed genomewide expression patterns of wild-caught and common-garden-raised individuals exposed to different concentrations of H2 S. We found that 7.7% of genes that were differentially expressed between sulphidic and nonsulphidic ecotypes remained differentially expressed in the laboratory, indicating that sources of selection other than H2 S-or plastic responses to other environmental factors-contribute substantially to gene expression patterns observed in the wild. Concordantly differentially expressed genes in the wild and the laboratory were primarily associated with H2 S detoxification, sulphur processing and metabolic physiology. While shared, ancestral plasticity played a minor role in shaping gene expression variation observed in nature, we documented evidence for evolved population differences in the constitutive expression as well as the H2 S inducibility of candidate genes. Mechanisms underlying gene expression variation also varied substantially across the two ecotype pairs. These results provide a springboard for studying evolutionary modifications of gene regulatory mechanisms that underlie expression variation in locally adapted populations.

Genome-scale data reveal that endemic Poecilia populations from small sulphidic springs display no evidence of inbreeding.

Populations with limited ranges can be highly vulnerable to changes in their environment and are, thus, of high conservation concern. Populations that experience human-induced range reductions are often highly inbred and lack genetic diversity, but it is unknown whether this is also the case for populations with naturally small ranges. The fishes Poecilia sulphuraria (listed as critically endangered) and Poecilia thermalis, which are endemic to small hydrogen sulphide-rich springs in southern Mexico, are examples of such populations with inherently small habitats. We used geometric morphometrics and population genetics to quantify phenotypic and genetic variation within and among two populations of P. sulphuraria and one population of P. thermalis. Principal component analyses revealed phenotypic and genetic differences among the populations. Evidence for inbreeding was low compared to populations that have undergone habitat reduction. The genetic data were also used to infer the demographic history of these populations to obtain estimates for effective population sizes and migration rates. Effective population sizes were large given the small habitats of these populations. Our results imply that these three endemic extremophile populations should each be considered separately for conservation purposes. Additionally, this study suggests that populations in naturally small habitats may have lower rates of inbreeding and higher genetic diversity than expected, and therefore may be better equipped to handle environmental perturbations than anticipated. We caution, however, that the inferred lack of inbreeding and the large effective population sizes could potentially be a result of colonization by genetically diverse ancestors.

Sex-specific differences in transcriptome profiles of brain and muscle tissue of the tropical gar.

BACKGROUND: The tropical gar (Atractosteus tropicus) is the southernmost species of the seven extant species of gar fishes in the world. In Mexico and Central America, the species is an important food source due to its nutritional quality and low price. Despite its regional importance and increasing concerns about overexploitation and habitat degradation, basic genetic information on the tropical gar is lacking. Determining genetic information on the tropical gar is important for the sustainable management of wild populations, implementation of best practices in aquaculture settings, evolutionary studies of ancient lineages, and an understanding of sex-specific gene expression. In this study, the transcriptome of the tropical gar was sequenced and assembled de novo using tissues from three males and three females using Illumina sequencing technology. Sex-specific and highly differentially expressed transcripts in brain and muscle tissues between adult males and females were subsequently identified. RESULTS: The transcriptome was assembled de novo resulting in 80,611 transcripts with a contig N50 of 3,355 base pairs and over 168 kilobases in total length. Male muscle, brain, and gonad as well as female muscle and brain were included in the assembly. The assembled transcriptome was annotated to identify the putative function of expressed transcripts using Trinotate and SwissProt, a database of well-annotated proteins. The brain and muscle datasets were then aligned to the assembled transcriptome to identify transcripts that were differentially expressed between males and females. The contrast between male and female brain identified 109 transcripts from 106 genes that were significantly differentially expressed. In the muscle comparison, 82 transcripts from 80 genes were identified with evidence for significant differential expression. Almost all genes identified as differentially expressed were sex-specific. The differentially expressed transcripts were enriched for genes involved in cellular functioning, signaling, immune response, and tissue-specific functions. CONCLUSIONS: This study identified differentially expressed transcripts between male and female gar in muscle and brain tissue. The majority of differentially expressed transcripts had sex-specific expression. Expanding on these findings to other developmental stages, populations, and species may lead to the identification of genetic factors contributing to the skewed sex ratio seen in the tropical gar and of sex-specific differences in expression in other species. Finally, the transcriptome assembly will open future research avenues on tropical gar development, cell function, environmental resistance, and evolution in the context of other early vertebrates.

Deep Sequencing of 71 Candidate Genes to Characterize Variation Associated with Alcohol Dependence.

BACKGROUND: Previous genomewide association studies (GWASs) have identified a number of putative risk loci for alcohol dependence (AD). However, only a few loci have replicated and these replicated variants only explain a small proportion of AD risk. Using an innovative approach, the goal of this study was to generate hypotheses about potentially causal variants for AD that can be explored further through functional studies. METHODS: We employed targeted capture of 71 candidate loci and flanking regions followed by next-generation deep sequencing (mean coverage 78X) in 806 European Americans. Regions included in our targeted capture library were genes identified through published GWAS of alcohol, all human alcohol and aldehyde dehydrogenases, reward system genes including dopaminergic and opioid receptors, prioritized candidate genes based on previous associations, and genes involved in the absorption, distribution, metabolism, and excretion of drugs. We performed single-locus tests to determine if any single variant was associated with AD symptom count. Sets of variants that overlapped with biologically meaningful annotations were tested for association in aggregate. RESULTS: No single, common variant was significantly associated with AD in our study. We did, however, find evidence for association with several variant sets. Two variant sets were significant at the q-value <0.10 level: a genic enhancer for ADHFE1 (p = 1.47 × 10-5 ; q = 0.019), an alcohol dehydrogenase, and ADORA1 (p = 5.29 × 10-5 ; q = 0.035), an adenosine receptor that belongs to a G-protein-coupled receptor gene family. CONCLUSIONS: To our knowledge, this is the first sequencing study of AD to examine variants in entire genes, including flanking and regulatory regions. We found that in addition to protein coding variant sets, regulatory variant sets may play a role in AD. From these findings, we have generated initial functional hypotheses about how these sets may influence AD.

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance.

Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Because regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in further characterizing these mechanisms. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. Effects from these variants influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a new global allelic imbalance in expression favoring the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals.

The antipsychotic olanzapine interacts with the gut microbiome to cause weight gain in mouse.

The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an "obesogenic" bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.

Genetics of adverse reactions to haloperidol in a mouse diallel: a drug-placebo experiment and Bayesian causal analysis.

Haloperidol is an efficacious antipsychotic drug that has serious, unpredictable motor side effects that limit its utility and cause noncompliance in many patients. Using a drug-placebo diallel of the eight founder strains of the Collaborative Cross and their F1 hybrids, we characterized aggregate effects of genetics, sex, parent of origin, and their combinations on haloperidol response. Treating matched pairs of both sexes with drug or placebo, we measured changes in the following: open field activity, inclined screen rigidity, orofacial movements, prepulse inhibition of the acoustic startle response, plasma and brain drug level measurements, and body weight. To understand the genetic architecture of haloperidol response we introduce new statistical methodology linking heritable variation with causal effect of drug treatment. Our new estimators, "difference of models" and "multiple-impute matched pairs", are motivated by the Neyman-Rubin potential outcomes framework and extend our existing Bayesian hierarchical model for the diallel (Lenarcic et al. 2012). Drug-induced rigidity after chronic treatment was affected by mainly additive genetics and parent-of-origin effects (accounting for 28% and 14.8% of the variance), with NZO/HILtJ and 129S1/SvlmJ contributions tending to increase this side effect. Locomotor activity after acute treatment, by contrast, was more affected by strain-specific inbreeding (12.8%). In addition to drug response phenotypes, we examined diallel effects on behavior before treatment and found not only effects of additive genetics (10.2-53.2%) but also strong effects of epistasis (10.64-25.2%). In particular: prepulse inhibition showed additivity and epistasis in about equal proportions (26.1% and 23.7%); there was evidence of nonreciprocal epistasis in pretreatment activity and rigidity; and we estimated a range of effects on body weight that replicate those found in our previous work. Our results provide the first quantitative description of the genetic architecture of haloperidol response in mice and indicate that additive, dominance-like inbreeding and parent-of-origin effects contribute strongly to treatment effect heterogeneity for this drug.

Genome-wide association mapping of loci for antipsychotic-induced extrapyramidal symptoms in mice.

Tardive dyskinesia (TD) is a debilitating, unpredictable, and often irreversible side effect resulting from chronic treatment with typical antipsychotic agents such as haloperidol. TD is characterized by repetitive, involuntary, purposeless movements primarily of the orofacial region. In order to investigate genetic susceptibility to TD, we used a validated mouse model for a systems genetics analysis geared toward detecting genetic predictors of TD in human patients. Phenotypic data from 27 inbred strains chronically treated with haloperidol and phenotyped for vacuous chewing movements were subject to a comprehensive genomic analysis involving 426,493 SNPs, 4,047 CNVs, brain gene expression, along with gene network and bioinformatic analysis. Our results identified ~50 genes that we expect to have high prior probabilities for association with haloperidol-induced TD, most of which have never been tested for association with human TD. Among our top candidates were genes regulating the development of brain motor control regions (Zic4 and Nkx6-1), glutamate receptors (Grin1 and Grin2a), and an indirect target of haloperidol (Drd1a) that has not been studied as well as the direct target, Drd2.

No association of the serotonin transporter polymorphisms 5-HTTLPR and RS25531 with schizophrenia or neurocognition.

A promoter polymorphism in the serotonin transporter gene has been widely studied in neuropsychiatry. We genotyped the 5-HTTLPR/rs25531 triallelic polymorphism in 728 schizophrenia cases from the CATIE study and 724 control subjects. In a logistic regression with case/control status as dependent variable and 7 ancestry-informative principal components as covariates, the effect of 5-HTTLPR/rs25531 composite genotype was not significant (odds ratio = 1.008, 95% CI 0.868-1.172, P = 0.91). In cases only, 5-HTTLPR/rs25531 was not associated with neurocognition (summary neurocognitive index P = 0.21, working memory P = 0.32) or symptomatology (PANSS positive P = 0.67 and negative symptoms P = 0.46). We were unable to identify association of the triallelic 5-HTTLPR with schizophrenia, neurocognition, or core psychotic symptoms even at levels of significance unadjusted for multiple comparisons.