Questionnaire-based exposome-wide association studies for common diseases in the Personalized Environment and Genes Study.

The exposome collectively refers to all exposures, beginning in utero and continuing throughout life, and comprises not only standard environmental exposures such as point source pollution and ozone levels but also exposures from diet, medication, lifestyle factors, stress, and occupation. The exposome interacts with individual genetic and epigenetic characteristics to affect human health and disease, but large-scale studies that characterize the exposome and its relationships with human disease are limited. To address this gap, we used extensive questionnaire data from the diverse North Carolina-based Personalized Environment and Genes Study (PEGS, n = 9, 429) to evaluate exposure associations in relation to common diseases. We performed an exposome-wide association study (ExWAS) to examine single exposure models and their associations with 11 common complex diseases, namely allergic rhinitis, asthma, bone loss, fibroids, high cholesterol, hypertension, iron-deficient anemia, ovarian cysts, lower GI polyps, migraines, and type 2 diabetes. Across diseases, we found associations with lifestyle factors and socioeconomic status as well as asbestos, various dust types, biohazardous material, and textile-related exposures. We also found disease-specific associations such as fishing with lead weights and migraines. To differentiate between a replicated result and a novel finding, we used an AI-based literature search and database tool that allowed us to examine the current literature. We found both replicated findings, especially for lifestyle factors such as sleep and smoking across diseases, and novel findings, especially for occupational exposures and multiple diseases.

Interactive data sharing for multiple questionnaire-based exposome-wide association studies and exposome correlations in the Personalized Environment and Genes Study.

The correlations among individual exposures in the exposome, which refers to all exposures an individual encounters throughout life, are important for understanding the landscape of how exposures co-occur, and how this impacts health and disease. Exposome-wide association studies (ExWAS), which are analogous to genome-wide association studies (GWAS), are increasingly being used to elucidate links between the exposome and disease. Despite increased interest in the exposome, tools and publications that characterize exposure correlations and their relationships with human disease are limited, and there is a lack of data and results sharing in resources like the GWAS catalog. To address these gaps, we developed the PEGS Explorer web application to explore exposure correlations in data from the diverse North Carolina-based Personalized Environment and Genes Study (PEGS) that were rigorously calculated to account for differing data types and previously published results from ExWAS. Through globe visualizations, PEGS Explorer allows users to explore correlations between exposures found to be associated with complex diseases. The exposome data used for analysis includes not only standard environmental exposures such as point source pollution and ozone levels but also exposures from diet, medication, lifestyle factors, stress, and occupation. The web application addresses the lack of accessible data and results sharing, a major challenge in the field, and enables users to put results in context, generate hypotheses, and, importantly, replicate findings in other cohorts. PEGS Explorer will be updated with additional results as they become available, ensuring it is an up-to-date resource in exposome science.

Toxicological and pharmacokinetic properties of sucralose-6-acetate and its parent sucralose: in vitro screening assays.

The purpose of this study was to determine the toxicological and pharmacokinetic properties of sucralose-6-acetate, a structural analog of the artificial sweetener sucralose. Sucralose-6-acetate is an intermediate and impurity in the manufacture of sucralose, and recent commercial sucralose samples were found to contain up to 0.67% sucralose-6-acetate. Studies in a rodent model found that sucralose-6-acetate is also present in fecal samples with levels up to 10% relative to sucralose which suggest that sucralose is also acetylated in the intestines. A MultiFlow® assay, a high-throughput genotoxicity screening tool, and a micronucleus (MN) test that detects cytogenetic damage both indicated that sucralose-6-acetate is genotoxic. The mechanism of action was classified as clastogenic (produces DNA strand breaks) using the MultiFlow® assay. The amount of sucralose-6-acetate in a single daily sucralose-sweetened drink might far exceed the threshold of toxicological concern for genotoxicity (TTCgenotox) of 0.15 µg/person/day. The RepliGut® System was employed to expose human intestinal epithelium to sucralose-6-acetate and sucralose, and an RNA-seq analysis was performed to determine gene expression induced by these exposures. Sucralose-6-acetate significantly increased the expression of genes associated with inflammation, oxidative stress, and cancer with greatest expression for the metallothionein 1 G gene (MT1G). Measurements of transepithelial electrical resistance (TEER) and permeability in human transverse colon epithelium indicated that sucralose-6-acetate and sucralose both impaired intestinal barrier integrity. Sucralose-6-acetate also inhibited two members of the cytochrome P450 family (CYP1A2 and CYP2C19). Overall, the toxicological and pharmacokinetic findings for sucralose-6-acetate raise significant health concerns regarding the safety and regulatory status of sucralose itself.

The Genome of the Poecilogonous Annelid Streblospio benedicti.

Streblospio benedicti is a common marine annelid that has become an important model for developmental evolution. It is the only known example of poecilogony (where two distinct developmental modes occur within a single species) that is due to a heritable difference in egg size. The dimorphic developmental programs and life-histories exhibited in this species depend on differences within the genome, making it an optimal model for understanding the genomic basis of developmental divergence. Studies using S. benedicti have begun to uncover the genetic and genomic principles that underlie developmental uncoupling, but until now they have been limited by the lack of availability of genomic tools. Here, we present an annotated chromosomal-level genome assembly of S. benedicti generated from a combination of Illumina reads, Nanopore long reads, Chicago and Hi-C chromatin interaction sequencing, and a genetic map from experimental crosses. At 701.4 Mb, the S. benedicti genome is the largest annelid genome to date that has been assembled to chromosomal scaffolds. The complete genome of S. benedicti is valuable for functional genomic analyses of development and evolution, as well as phylogenetic comparison within the annelida and the Lophotrochozoa. Despite having two developmental modes, there is no evidence of genome duplication or substantial gene number expansions. Instead, lineage-specific repeats account for much of the expansion of this genome compared with other annelids.

Genome and transcriptome sequencing of the green bottle fly, Lucilia sericata, reveals underlying factors of sheep flystrike and maggot debridement therapy.

The common green bottle blow fly Lucilia sericata (family, Calliphoridae) is widely used for maggot debridement therapy, which involves the application of sterile maggots to wounds. The larval excretions and secretions are important for consuming necrotic tissue and inhibiting bacterial growth in wounds of patients. Lucilia sericata is also of importance as a pest of sheep and in forensic studies to estimate a postmortem interval. Here we report the assembly of a 565.3 Mb genome from long read PacBio DNA sequencing of genomic DNA. The genome contains 14,704 predicted protein coding genes and 1709 non-coding genes. Targeted annotation and transcriptional analyses identified genes that are highly expressed in the larval salivary glands (secretions) and Malpighian tubules (excretions) under normal growth conditions and following heat stress. The genomic resources will underpin future genetic studies and in development of engineered strains for genetic control of L. sericata and for biotechnology-enhanced maggot therapy.

High-resolution characterization of the structural features and genetic variation of six feline leukocyte antigen class I loci via single molecule, real-time (SMRT) sequencing.

Of the 12 full-length feline leukocyte antigen class I (FLAI) loci, 3 are presumed to be classical: FLAI-E, FLAI-H, and FLAI-K. As diversity is a class Ia hallmark, multi-allelism is an important surrogate supporting a classical designation, in the absence of direct demonstration of T-cell restriction. Conversely, limited polymorphism at an expressed locus suggests regulation of immune effectors with invariant receptors, and non-classical status. FLAI-A, FLAI-J, FLAI-L, and FLAI-O are putative class Ib genes in cats. For both classes, identifying prevalent variants across outbred populations can illuminate specific genotypes to be prioritized for immune studies, as shared alleles direct shared responses. Since variation is concentrated in exons 2 and 3, which encode the antigen-binding domains, partial-length cloning/sequencing can be used for allele discovery, but is laborious and occasionally ambiguous. Here we develop a targeted approach to FLAI genotyping, using the single-molecule real-time (SMRT) platform, which allows full-length (3.4-kb) reads without assembly. Consensus sequences matched full-length Sanger references. Thirty-one new class Ia genes were found in 17 cats. Alleles segregated strongly by loci, and the origins of formerly difficult-to-assign sequences were resolved. Although not targeted, FLAI-L and FLAI-J, and the pseudogene FLAI-F, were also returned. Eighteen class Ib alleles were identified. Diversity was restricted and outside hypervariable regions. Both class Ib genes were transcriptionally active. Novel alternative splicing of FLAI-L was observed. SMRT sequencing of FLAI amplicons is useful for full-length genotyping at feline class Ia loci. High-throughput sequencing could allow highly accurate allele surveys in large cat cohorts.

Complete Genome Sequences of Six Lactobacilli Isolated from American Quarter Horses.

We report the complete circular genome sequences of six Lactobacillus strains and their plasmids, if any, from the fecal material of quarter horses at different ages.

Complete Genome Sequences of Lactobacillus Strains C25 and P38, Isolated from Chicken Cecum.

We report the complete circular genome sequences of Lactobacillus crispatus strain C25, its plasmid, and Lactobacillus animalis strain P38; both strains were isolated from the cecum of 4-week-old chickens. These isolates represent potential probiotic strains for poultry.

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs.

The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

Pharmacometabolomics Informs About Pharmacokinetic Profile of Methylphenidate.

Carboxylesterase 1 (CES1) metabolizes methylphenidate and other drugs. CES1 gene variation only partially explains pharmacokinetic (PK) variability. Biomarkers predicting the PKs of drugs metabolized by CES1 are needed. We identified lipids in plasma from 44 healthy subjects that correlated with CES1 activity as determined by PK parameters of methylphenidate including a ceramide (q value = 0.001) and a phosphatidylcholine (q value = 0.005). Carriers of the CES1 143E allele had decreased methylphenidate metabolism and altered concentration of this phosphatidylcholine (q value = 0.040) and several high polyunsaturated fatty acid lipids (PUFAs). The half-maximal inhibitory concentration (IC50 ) values of chenodeoxycholate and taurocholate were 13.55 and 19.51 µM, respectively, consistent with a physiological significance. In silico analysis suggested that bile acid inhibition of CES1 involved both binding to the active and superficial sites of the enzyme. We initiated identification of metabolites predicting PKs of drugs metabolized by CES1 and suggest lipids to regulate or be regulated by this enzyme.

Elucidating Gene-by-Environment Interactions Associated with Differential Susceptibility to Chemical Exposure.

BACKGROUND: Modern societies are exposed to vast numbers of potentially hazardous chemicals. Despite demonstrated linkages between chemical exposure and severe health effects, there are limited, often conflicting, data on how adverse health effects of exposure differ across individuals. OBJECTIVES: We tested the hypothesis that population variability in response to certain chemicals could elucidate a role for gene-environment interactions (GxE) in differential susceptibility. METHODS: High-throughput screening (HTS) data on thousands of chemicals in genetically heterogeneous zebrafish were leveraged to identify a candidate chemical (Abamectin) with response patterns indicative of population susceptibility differences. We tested the prediction by generating genome-wide sequence data for 276 individual zebrafish displaying susceptible (Affected) vs. resistant (Unaffected) phenotypes following identical chemical exposure. RESULTS: We found GxE associated with differential susceptibility in the sox7 promoter region and then confirmed gene expression differences between phenotypic response classes. CONCLUSIONS: The results for Abamectin in zebrafish demonstrate that GxE associated with naturally occurring, population genetic variation play a significant role in mediating individual response to chemical exposure. https://doi.org/10.1289/EHP2662.

no blokes Is Essential for Male Viability and X Chromosome Gene Expression in the Australian Sheep Blowfly.

It has been hypothesized that the Drosophila 4th chromosome is derived from an ancient X chromosome [1]. In the Australian sheep blowfly, Lucilia cuprina, the heterochromatic X chromosome contains few active genes and orthologs of Drosophila X-linked genes are autosomal. Of 8 X-linked genes identified previously in L. cuprina, 6 were orthologs of Drosophila 4th-chromosome genes [2]. The X-linked genes were expressed equally in males and females. Here we identify an additional 51 X-linked genes and show that most are dosage compensated. Orthologs of 49 of the 59 X-linked genes are on the 4th chromosome in D. melanogaster. Because painting of fourth (Pof) is important for expression of Drosophila 4th-chromosome genes [3], we used Cas9 to make a loss-of-function knockin mutation in an L. cuprina Pof ortholog we call no blokes (nbl). Homozygous nbl males derived from homozygous nbl mothers die at the late pupal stage. Homozygous nbl females are viable, fertile, and live longer than heterozygous nbl females. RNA expression of most X-linked genes was reduced in homozygous nbl male pupae and to a lesser extent in nbl females compared to heterozygous siblings. The results suggest that NBL could be important for X chromosome dosage compensation in L. cuprina. NBL may also facilitate gene expression in the heterochromatic environment of the X chromosome in both sexes. This study supports the hypothesis on the origin of the Drosophila 4th chromosome and that a POF-like protein was required for normal gene expression on the ancient X chromosome.

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C16:1ω7c/C16:1ω6c, C16:0, and C18:1ω7c, and were consistent with the genus. Genomic G+C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3T as the type strain (=LMG 30136, =DSM 105008).

Population genetic diversity in zebrafish lines.

Toxicological and pharmacological researchers have seized upon the many benefits of zebrafish, including the short generation time, well-characterized development, and early maturation as clear embryos. A major difference from many model organisms is that standard husbandry practices in zebrafish are designed to maintain population diversity. While this diversity is attractive for translational applications in human and ecological health, it raises critical questions on how interindividual genetic variation might contribute to chemical exposure or disease susceptibility differences. Findings from pooled samples of zebrafish support this supposition of diversity yet cannot directly measure allele frequencies for reference versus alternate alleles. Using the Tanguay lab Tropical 5D zebrafish line (T5D), we performed whole genome sequencing on a large group (n = 276) of individual zebrafish embryos. Paired-end reads were collected on an Illumina 3000HT, then aligned to the most recent zebrafish reference genome (GRCz10). These data were used to compare observed population genetic variation across species (humans, mice, zebrafish), then across lines within zebrafish. We found more single nucleotide polymorphisms (SNPs) in T5D than have been reported in SNP databases for any of the WIK, TU, TL, or AB lines. We theorize that some subset of the novel SNPs may be shared with other zebrafish lines but have not been identified in other studies due to the limitations of capturing population diversity in pooled sequencing strategies. We establish T5D as a model that is representative of diversity levels within laboratory zebrafish lines and demonstrate that experimental design and analysis can exert major effects when characterizing genetic diversity in heterogeneous populations.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. METHODS: Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. RESULTS: Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. CONCLUSIONS: Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

Editor's Highlight: Comparative Dose-Response Analysis of Liver and Kidney Transcriptomic Effects of Trichloroethylene and Tetrachloroethylene in B6C3F1 Mouse.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are ubiquitous environmental contaminants and occupational health hazards. Recent health assessments of these agents identified several critical data gaps, including lack of comparative analysis of their effects. This study examined liver and kidney effects of TCE and PCE in a dose-response study design. Equimolar doses of TCE (24, 80, 240, and 800 mg/kg) or PCE (30, 100, 300, and 1000 mg/kg) were administered by gavage in aqueous vehicle to male B6C3F1/J mice. Tissues were collected 24 h after exposure. Trichloroacetic acid (TCA), a major oxidative metabolite of both compounds, was measured and RNA sequencing was performed. PCE had a stronger effect on liver and kidney transcriptomes, as well as greater concentrations of TCA. Most dose-responsive pathways were common among chemicals/tissues, with the strongest effect on peroxisomal ß-oxidation. Effects on liver and kidney mitochondria-related pathways were notably unique to PCE. We performed dose-response modeling of the transcriptomic data and compared the resulting points of departure (PODs) to those for apical endpoints derived from long-term studies with these chemicals in rats, mice, and humans, converting to human equivalent doses using tissue-specific dosimetry models. Tissue-specific acute transcriptional effects of TCE and PCE occurred at human equivalent doses comparable to those for apical effects. These data are relevant for human health assessments of TCE and PCE as they provide data for dose-response analysis of the toxicity mechanisms. Additionally, they provide further evidence that transcriptomic data can be useful surrogates for in vivo PODs, especially when toxicokinetic differences are taken into account.

A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2.

To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein µ2 from strain T1L, but not strain T3D, represses one antiviral response: alpha/beta interferon signaling. We report here that T1L, but not T3D, µ2 localizes to nuclear speckles, where it forms a complex with the mRNA splicing factor SRSF2 and alters its subnuclear localization. Reovirus replicates in cytoplasmic viral factories, and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L µ2 might target splicing of cell RNAs. Indeed, RNA sequencing revealed that reovirus T1L, but not T3D, infection alters the splicing of transcripts for host genes involved in mRNA posttranscriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L µ2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing.IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. We demonstrate here that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles, where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Our results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus.

Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny.

The taxonomy of the order Piroplasmida, which includes a number of clinically and economically relevant organisms, is a hotly debated topic amongst parasitologists. Three genera (Babesia, Theileria, and Cytauxzoon) are recognized based on parasite life cycle characteristics, but molecular phylogenetic analyses of 18S sequences have suggested the presence of five or more distinct Piroplasmida lineages. Despite these important advancements, a few studies have been unable to define the taxonomic relationships of some organisms (e.g. C. felis and T. equi) with respect to other Piroplasmida. Additional evidence from mitochondrial genome sequences and synteny should aid in the inference of Piroplasmida phylogeny and resolution of taxonomic uncertainties. In this study, we have amplified, sequenced, and annotated seven previously uncharacterized mitochondrial genomes (Babesia canis, Babesia vogeli, Babesia rossi, Babesia sp. Coco, Babesia conradae, Babesia microti-like sp., and Cytauxzoon felis) and identified additional ribosomal fragments in ten previously characterized mitochondrial genomes. Phylogenetic analysis of concatenated mitochondrial and 18S sequences as well as cox1 amino acid sequence identified five distinct Piroplasmida groups, each of which possesses a unique mitochondrial genome structure. Specifically, our results confirm the existence of four previously identified clades (B. microti group, Babesia sensu stricto, Theileria equi, and a Babesia sensu latu group that includes B. conradae) while supporting the integration of Theileria and Cytauxzoon species into a single fifth taxon. Although known biological characteristics of Piroplasmida corroborate the proposed phylogeny, more investigation into parasite life cycles is warranted to further understand the evolution of the Piroplasmida. Our results provide an evolutionary framework for comparative biology of these important animal and human pathogens and help focus renewed efforts toward understanding the phylogenetic relationships within the group.

Complete Genome Sequence of a Bacterium Representing a Deep Uncultivated Lineage within the Gammaproteobacteria Associated with the Degradation of Polycyclic Aromatic Hydrocarbons.

The bacterial strain TR3.2, representing a novel deeply branching lineage within the Gammaproteobacteria, was isolated and its genome sequenced. This isolate is the first cultivated representative of the previously described "Pyrene Group 2" (PG2) and represents a variety of environmental sequences primarily associated with petrochemical contamination and aromatic hydrocarbon degradation.

Complete Genome Sequence of NC983, a Live Attenuated Strain of Salmonella enterica Serovar Typhimurium.

Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate.