The circadian clock is disrupted in pancreatic cancer.

Disruption of the circadian clock is linked to cancer development and progression. Establishing this connection has proven beneficial for understanding cancer pathogenesis, determining prognosis, and uncovering novel therapeutic targets. However, barriers to characterizing the circadian clock in human pancreas and human pancreatic cancer-one of the deadliest malignancies-have hindered an appreciation of its role in this cancer. Here, we employed normalized coefficient of variation (nCV) and clock correlation analysis in human population-level data to determine the functioning of the circadian clock in pancreas cancer and adjacent normal tissue. We found a substantially attenuated clock in the pancreatic cancer tissue. Then we exploited our existing mouse pancreatic transcriptome data to perform an analysis of the human normal and pancreas cancer samples using a machine learning method, cyclic ordering by periodic structure (CYCLOPS). Through CYCLOPS ordering, we confirmed the nCV and clock correlation findings of an intact circadian clock in normal pancreas with robust cycling of several core clock genes. However, in pancreas cancer, there was a loss of rhythmicity of many core clock genes with an inability to effectively order the cancer samples, providing substantive evidence of a dysregulated clock. The implications of clock disruption were further assessed with a Bmal1 knockout pancreas cancer model, which revealed that an arrhythmic clock caused accelerated cancer growth and worse survival, accompanied by chemoresistance and enrichment of key cancer-related pathways. These findings provide strong evidence for clock disruption in human pancreas cancer and demonstrate a link between circadian disruption and pancreas cancer progression.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

duper is a null mutation of Cryptochrome 1 in Syrian hamsters.

The duper mutation is a recessive mutation that shortens the period length of the circadian rhythm in Syrian hamsters. These animals show a large phase shift when responding to light pulses. Limited genetic resources for the Syrian hamster (Mesocricetus auratus) presented a major obstacle to cloning duper. This caused the duper mutation to remain unknown for over a decade. In this study, we did a de novo genome assembly of Syrian hamsters with long-read sequencing data from two different platforms, Pacific Biosciences and Oxford Nanopore Technologies. Using two distinct ecotypes and a fast homozygosity mapping strategy, we identified duper as an early nonsense allele of Cryptochrome 1 (Cry1) leading to a short, unstable protein. CRY1 is known as a highly conserved component of the repressive limb of the core circadian clock. The genome assembly and other genomic datasets generated in this study will facilitate the use of the Syrian hamster in biomedical research.

Exon level machine learning analyses elucidate novel candidate miRNA targets in an avian model of fetal alcohol spectrum disorder.

Gestational alcohol exposure causes fetal alcohol spectrum disorder (FASD) and is a prominent cause of neurodevelopmental disability. Whole transcriptome sequencing (RNA-Seq) offer insights into mechanisms underlying FASD, but gene-level analysis provides limited information regarding complex transcriptional processes such as alternative splicing and non-coding RNAs. Moreover, traditional analytical approaches that use multiple hypothesis testing with a false discovery rate adjustment prioritize genes based on an adjusted p-value, which is not always biologically relevant. We address these limitations with a novel approach and implemented an unsupervised machine learning model, which we applied to an exon-level analysis to reduce data complexity to the most likely functionally relevant exons, without loss of novel information. This was performed on an RNA-Seq paired-end dataset derived from alcohol-exposed neural fold-stage chick crania, wherein alcohol causes facial deficits recapitulating those of FASD. A principal component analysis along with k-means clustering was utilized to extract exons that deviated from baseline expression. This identified 6857 differentially expressed exons representing 1251 geneIDs; 391 of these genes were identified in a prior gene-level analysis of this dataset. It also identified exons encoding 23 microRNAs (miRNAs) having significantly differential expression profiles in response to alcohol. We developed an RDAVID pipeline to identify KEGG pathways represented by these exons, and separately identified predicted KEGG pathways targeted by these miRNAs. Several of these (ribosome biogenesis, oxidative phosphorylation) were identified in our prior gene-level analysis. Other pathways are crucial to facial morphogenesis and represent both novel (focal adhesion, FoxO signaling, insulin signaling) and known (Wnt signaling) alcohol targets. Importantly, there was substantial overlap between the exomes themselves and the predicted miRNA targets, suggesting these miRNAs contribute to the gene-level expression changes. Our novel application of unsupervised machine learning in conjunction with statistical analyses facilitated the discovery of signaling pathways and miRNAs that inform mechanisms underlying FASD.

Genetic variation of major histocompatibility complex (MHC) in wild Red Junglefowl (Gallus gallus).

The major histocompatibility complex (MHC) is a multi-family gene cluster that encodes proteins with immuno-responsive function. While studies of MHC in domesticated poultry are relatively common, very little is known about this highly polymorphic locus in wild Red Junglefowl (Gallus gallus), the natural progenitor of domestic chickens. We investigated the diversity of MHC within and among four wild Red Junglefowl populations across diversified natural habitats in South Central Vietnam. Based on a SNP panel of 84 sites spanning 210 Kb of the MHC-B locus, we identified 310 unique haplotypes in 398 chromosomes. None of these haplotypes have been described before and we did not observe any of the wild Red Junglefowl haplotypes in domesticated chickens. Analysis of molecular variance (AMOVA) revealed that 94.51% of observed haplotype variation was accounted for at the within individual level. Little genetic variance was apportioned within and among populations, the latter accounting only for 0.83%. We also found evidence of increased recombination, including numerous hotspots, and limited linkage disequilibrium among the 84 SNP sites. Compared to an average haplotype diversity of 3.55% among seventeen lines of domestic chickens, our results suggest extraordinarily high haplotype diversity remains in wild Red Junglefowl and is consistent with a pattern of balancing selection. Wild Red Junglefowl in Vietnam, therefore, represent a rich resource of natural genomic variation independent from artificial selection.

Neural crest development in fetal alcohol syndrome.

Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Some affected individuals possess distinctive craniofacial deficits, but many more lack overt facial changes. An understanding of the mechanisms underlying these deficits would inform their diagnostic utility. Our understanding of these mechanisms is challenged because ethanol lacks a single receptor when redirecting cellular activity. This review summarizes our current understanding of how ethanol alters neural crest development. Ample evidence shows that ethanol causes the "classic" fetal alcohol syndrome (FAS) face (short palpebral fissures, elongated upper lip, deficient philtrum) because it suppresses prechordal plate outgrowth, thereby reducing neuroectoderm and neural crest induction and causing holoprosencephaly. Prenatal alcohol exposure (PAE) at premigratory stages elicits a different facial appearance, indicating FASD may represent a spectrum of facial outcomes. PAE at this premigratory period initiates a calcium transient that activates CaMKII and destabilizes transcriptionally active ß-catenin, thereby initiating apoptosis within neural crest populations. Contributing to neural crest vulnerability are their low antioxidant responses. Ethanol-treated neural crest produce reactive oxygen species and free radical scavengers attenuate their production and prevent apoptosis. Ethanol also significantly impairs neural crest migration, causing cytoskeletal rearrangements that destabilize focal adhesion formation; their directional migratory capacity is also lost. Genetic factors further modify vulnerability to ethanol-induced craniofacial dysmorphology and include genes important for neural crest development, including shh signaling, PDFGA, vangl2, and ribosomal biogenesis. Because facial and brain development are mechanistically and functionally linked, research into ethanol's effects on neural crest also informs our understanding of ethanol's CNS pathologies.

High-throughput transcriptome sequencing identifies candidate genetic modifiers of vulnerability to fetal alcohol spectrum disorders.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) is a leading cause of neurodevelopmental disability. Genetic factors can modify vulnerability to FASD, but these elements are poorly characterized. METHODS: We performed high-throughput transcriptional profiling to identify gene candidates that could potentially modify vulnerability to ethanol's (EtOH's) neurotoxicity. We interrogated a unique genetic resource, neuroprogenitor cells from 2 closely related Gallus gallus lines having well-characterized robust or attenuated EtOH responses with respect to intracellular calcium mobilization and CaMKII/ß-catenin-dependent apoptosis. Samples were not exposed to EtOH prior to analysis. RESULTS: We identified 363 differentially expressed genes in neuroprogenitors from these 2 lines. Kyoto Encyclopedia of Genes and Genomes analysis revealed several gene clusters having significantly differential enrichment in gene expression. The largest and most significant cluster comprised ribosomal proteins (38 genes, p = 1.85 × 10(-47) ). Other significantly enriched gene clusters included metabolism (25 genes, p = 0.0098), oxidative phosphorylation (18 genes, p = 1.10 × 10(-11) ), spliceosome (13 genes, p = 7.02 × 10(-8) ), and protein processing in the endoplasmic reticulum (9 genes, p = 0.0011). Inspection of gene ontogeny (GO) terms identified 24 genes involved in the calcium/ß-catenin signals that mediate EtOH's neurotoxicity in this model, including ß-catenin itself and both calmodulin isoforms. CONCLUSIONS: Four of the identified pathways with altered transcript abundance mediate the flow of cellular information from RNA to protein. Importantly, ribosome biogenesis also senses nucleolar stress and regulates p53-mediated apoptosis in neural crest. Human ribosomopathies produce craniofacial malformations and 11 known ribosomopathy genes were differentially expressed in this model of neural crest apoptosis. Rapid changes in ribosome expression are consistently observed in EtOH-treated mouse embryo neural folds, a model that is developmentally similar to ours. The recurring identification of ribosome biogenesis suggests it is a candidate modifier of EtOH vulnerability. These results highlight this approach's efficacy to formulate new, mechanistic hypotheses regarding EtOH's developmental damage.

Genetic population structure of the blister beetle, Gnathium minimum: core and peripheral populations.

Populations on the periphery of a species' range tend to contain lower genetic variation and increased genetic differentiation compared to populations at the core of a species range, although some exceptions to this generalization occur. The blister beetle Gnathium minimum (Say) exhibits a wide-ranging distribution in the western United States but has peripheral or disjunct populations in Mexico, Florida, and Wisconsin. We used amplified fragment length polymorphism (AFLP) to compare the genetic variation and magnitude of genetic differentiation of the Wisconsin peripheral population to western core populations (Colorado, Kansas, New Mexico, and Texas). The proportion of polymorphic loci was 53.6 and 54.3, and expected heterozygosity 0.1864 and 0.1933 for the Kansas/Colorado (n = 87) and New Mexico/Texas (n = 35) regions, respectively. Specimens from Wisconsin (n = 121) had a lower proportion of polymorphic loci (38.4) and expected heterozygosity (0.1475). Genetic cluster estimation with GENELAND and F ST values showed greater genetic differentiation among the sampling locations within Wisconsin compared to core regions. Significant isolation-by-distance (IBD) was also observed in Wisconsin but not within the core regions. Lower genetic variation and increased isolation may reduce the Wisconsin population's ability to respond to change, thereby increasing their susceptibility to extinction.

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing.

Complex regions in the human genome such as repeat motifs, pseudogenes and structural (SVs) and copy number variations (CNVs) present ongoing challenges to accurate genetic analysis, particularly for short-read Next-Generation-Sequencing (NGS) technologies. One such region is the highly polymorphic CYP2D loci, containing CYP2D6, a clinically relevant pharmacogene contributing to the metabolism of >20% of common drugs, and two highly similar pseudogenes, CYP2D7 and CYP2D8. Multiple complex SVs, including CYP2D6/CYP2D7-derived hybrid genes are known to occur in different configurations and frequencies across populations and are difficult to detect and characterize accurately. This can lead to incorrect enzyme activity assignment and impact drug dosing recommendations, often disproportionally affecting underrepresented populations. To improve CYP2D6 genotyping accuracy, we developed a PCR-free CRISPR-Cas9 based enrichment method for targeted long-read sequencing that fully characterizes the entire CYP2D6-CYP2D7-CYP2D8 loci. Clinically relevant sample types, including blood, saliva, and liver tissue were sequenced, generating high coverage sets of continuous single molecule reads spanning the entire targeted region of up to 52 kb, regardless of SV present (n = 9). This allowed for fully phased dissection of the entire loci structure, including breakpoints, to accurately resolve complex CYP2D6 diplotypes with a single assay. Additionally, we identified three novel CYP2D6 suballeles, and fully characterized 17 CYP2D7 and 18 CYP2D8 unique haplotypes. This method for CYP2D6 genotyping has the potential to significantly improve accurate clinical phenotyping to inform drug therapy and can be adapted to overcome testing limitations of other clinically challenging genomic regions.

Biologically Enhanced Genome-Wide Association Study Provides Further Evidence for Candidate Loci and Discovers Novel Loci That Influence Risk of Anterior Cruciate Ligament Rupture in a Dog Model.

Anterior cruciate ligament (ACL) rupture is a common condition that disproportionately affects young people, 50% of whom will develop knee osteoarthritis (OA) within 10 years of rupture. ACL rupture exhibits both hereditary and environmental risk factors, but the genetic basis of the disease remains unexplained. Spontaneous ACL rupture in the dog has a similar disease presentation and progression, making it a valuable genomic model for ACL rupture. We leveraged the dog model with Bayesian mixture model (BMM) analysis (BayesRC) to identify novel and relevant genetic variants associated with ACL rupture. We performed RNA sequencing of ACL and synovial tissue and assigned single nucleotide polymorphisms (SNPs) within differentially expressed genes to biological prior classes. SNPs with the largest effects were on chromosomes 3, 5, 7, 9, and 24. Selection signature analysis identified several regions under selection in ACL rupture cases compared to controls. These selection signatures overlapped with genome-wide associations with ACL rupture as well as morphological traits. Notable findings include differentially expressed ACSF3 with MC1R (coat color) and an association on chromosome 7 that overlaps the boundaries of SMAD2 (weight and body size). Smaller effect associations were within or near genes associated with regulation of the actin cytoskeleton and the extracellular matrix, including several collagen genes. The results of the current analysis are consistent with previous work published by our laboratory and others, and also highlight new genes in biological pathways that have not previously been associated with ACL rupture. The genetic associations identified in this study mirror those found in human beings, which lays the groundwork for development of disease-modifying therapies for both species.

Molecular and Functional Networks Linked to Sarcopenia Prevention by Caloric Restriction in Rhesus Monkeys.

Caloric restriction (CR) improves survival in nonhuman primates and delays the onset of age-related morbidities including sarcopenia, which is characterized by the age-related loss of muscle mass and function. A shift in metabolism anticipates the onset of muscle-aging phenotypes in nonhuman primates, suggesting a potential role for metabolism in the protective effects of CR. Here, we show that CR induced profound changes in muscle composition and the cellular metabolic environment. Bioinformatic analysis linked these adaptations to proteostasis, RNA processing, and lipid synthetic pathways. At the tissue level, CR maintained contractile content and attenuated age-related metabolic shifts among individual fiber types with higher mitochondrial activity, altered redox metabolism, and smaller lipid droplet size. Biometric and metabolic rate data confirm preserved metabolic efficiency in CR animals that correlated with the attenuation of age-related muscle mass and physical activity. These data suggest that CR-induced reprogramming of metabolism plays a role in delayed aging of skeletal muscle in rhesus monkeys.

Alcohol-mediated calcium signals dysregulate pro-survival Snai2/PUMA/Bcl2 networks to promote p53-mediated apoptosis in avian neural crest progenitors.

BACKGROUND: Prenatal alcohol exposure causes distinctive craniofacial anomalies that arise, in part, from the apoptotic elimination of neural crest (NC) progenitors that form the face. This vulnerability of NC to alcohol is puzzling as they normally express the transcriptional repressor Snail1/2 (in chick Snai2), which suppresses apoptosis and promotes their migration. Here, we investigate alcohol's impact upon Snai2 function. METHODS: Chick cranial NC cells were treated with acute alcohol (52 mM, 2 hr). We evaluated NC migration, gene expression, proliferation, and apoptosis thereafter. RESULTS: Transient alcohol exposure induced Snai2 (191% ± 23%; p = .003) and stimulated NC migration (p = .0092). An alcohol-induced calcium transient mediated this Snai2 induction, and BAPTA-AM blocked whereas ionomycin mimicked these pro-migratory effects. Alcohol suppressed CyclinD1 protein content (59.1 ± 12%, p = .007) and NC proliferation (19.7 ± 5.8%, p < .001), but these Snai2-enriched cells still apoptosed in response to alcohol. This was explained because alcohol induced p53 (198 ± 29%, p = .023), and the p53 antagonist pifithrin-α prevented their apoptosis. Moreover, alcohol counteracted Snai2's pro-survival signals, and Bcl2 was repressed (68.5 ± 6.0% of controls, p = .016) and PUMA was not induced, while ATM (1.32-fold, p = .01) and PTEN (1.30-fold, p = .028) were elevated. CONCLUSIONS: Alcohol's calcium transient uncouples the Snai2/p53 regulatory loop that normally prevents apoptosis during EMT. This represents a novel pathway in alcohol's neurotoxicity, and complements demonstrations that alcohol suppresses PUMA in mouse NC. We propose that the NCs migratory behavior, and their requirement for Snai2/p53 co-expression, makes them vulnerable to stressors that dysregulate Snai2/p53 interactions, such as alcohol.

Omics Technologies to Understand Activation of a Biosynthetic Gene Cluster in Micromonospora sp. WMMB235: Deciphering Keyicin Biosynthesis.

DNA sequencing of a large collection of bacterial genomes reveals a wealth of orphan biosynthetic gene clusters (BGCs) with no identifiable products. BGC silencing, for those orphan clusters that are truly silent, rather than those whose products have simply evaded detection and cluster correlation, is postulated to result from transcriptional inactivation of these clusters under standard laboratory conditions. Here, we employ a multi-omics approach to demonstrate how interspecies interactions modulate the keyicin producing kyc cluster at the transcriptome level in cocultures of kyc-bearing Micromonospora sp. and a Rhodococcus sp. We further correlate coculture dependent changes in keyicin production to changes in transcriptomic and proteomic profiles and show that these changes are attributable to small molecule signaling consistent with a quorum sensing pathway. In piecing together the various elements underlying keyicin production in coculture, this study highlights how omics technologies can expedite future efforts to understand and exploit silent BGCs.

Mhc-B haplotypes in "Campero-Inta" chicken synthetic line.

The major histocompatibility complex-B (MHC-B) in chickens is a cluster of genes located on chromosome 16. The chicken MHC-B is known to be highly associated with resistance to numerous diseases caused by viruses, bacteria, and parasitic pathogens. Since the level of resistance varies with MHC-B haplotypes, identification and classification of different haplotypes within lines is important for sustaining lines. The "Campero-INTA" chicken breed is a meat-type free-range poultry breed that was developed specifically for small producers in Argentina. Campero-INTA was started by selection in populations produced by crosses between a variety of established lines. MHC-B variation was examined in 65 samples obtained in 2002 using the VNTR marker LEI0258, a marker for MHC-B region. These samples plus and an additional 55 samples from 2018 were examined for variation using the MHC-B specific SNP panel that encompasses ∼230,000 bp of the MHC-B region. Eleven MHC-B SNP haplotypes with 6 LEI0258 alleles were identified in the 120 samples representing the Campero-INTA AH (male) line. Seven haplotypes originate from the breeds originally used in the development of Campero-INTA AH line. Two appear to be recombinant haplotypes. The origin of the remaining 2 is not known, but may be associated with genes introduced from crosses with the Fayoumi breed conducted more recently to sustain the line.

Genetic structure in Red Junglefowl (Gallus gallus) populations: Strong spatial patterns in the wild ancestors of domestic chickens in a core distribution range.

Red Junglefowl (Gallus gallus) are among the few remaining ancestors of an extant domesticated livestock species, the domestic chicken, that still occur in the wild. Little is known about genetic diversity, population structure, and demography of wild Red Junglefowl in their natural habitats. Extinction threats from habitat loss or genetic alteration from domestic introgression exacerbate further the conservation status of this progenitor species. In a previous study, we reported extraordinary adaptive genetic variation in the MHC B-locus in wild Red Junglefowl and no evidence of allelic introgression between wild and domestic chickens was observed. In this study, we characterized spatial genetic variation and population structure in naturally occurring populations of Red Junglefowl in their core distribution range in South Central Vietnam. A sample of 212 Red Junglefowl was obtained from geographically and ecologically diverse habitats across an area of 250 × 350 km. We used amplified fragment-length polymorphism markers obtained from 431 loci to determine whether genetic diversity and population structure varies. We found that Red Junglefowl are widely distributed but form small and isolated populations. Strong spatial genetic patterns occur at both local and regional scales. At local scale, population stratification can be identified to approximately 5 km. At regional scale, we identified distinct populations of Red Junglefowl in the southern lowlands, northern highlands, and eastern coastal portions of the study area. Both local and long-distance genetic patterns observed in wild Red Junglefowl may reflect the species' ground-dwelling and territorial characteristics, including dispersal barriers imposed by the Annamite Mountain Range. Spatially explicit analyses with neutral genetic markers can be highly informative and here elevates the conservation profile of the wild ancestors of domesticated chickens.

The Integrative Conjugative Element clc (ICEclc) of Pseudomonas aeruginosa JB2.

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds.

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis.

Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells' vulnerability. Gallus gallus embryos having 3-6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10-17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10-12, 60 genes), RNA Polymerase (P = 2.2 x 10-3, 15 genes) and Spliceosome (P = 2.6 x 10-2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work suggests ribosome biogenesis may be a novel target mediating alcohol's damage to developing neural crest. Our findings are consistent with observations that gene-environment interactions contribute to vulnerability in FASD.

Role of Cytochrome P450 Hydroxylase in the Decreased Accumulation of Vitamin E in Muscle from Turkeys Compared to that from Chickens.

Turkeys and chickens reared to 5 weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). Alpha tocopherol hydroxylase activity was greater in liver microsomes prepared from turkeys compared to that from chickens (p < 0.01). Alpha and gamma tocopherol metabolites were higher in turkey bile than in chicken when assessing the RRR AcT diet and the all-racemic AcT diet at 58 mg/kg feed (p < 0.01). Turkey cytochrome P450 2C29 was increased relative to its chicken ortholog on the basis of RNA-Seq transcript abundance (p < 0.001) and activity-based protein profiling (p < 0.01) of liver tissue. Alpha tocopherol concentrations in plasma, liver, and muscle from turkey were lower than the respective tissues from chicken (p < 0.05). Lipid oxidation was greater in turkey thigh than in chicken (p < 0.05). These results suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylase(s) in turkeys contributes to the decreased accumulation of alpha tocopherol in turkey tissues compared to that of chickens.

Genomic factors that shape craniofacial outcome and neural crest vulnerability in FASD.

Prenatal alcohol exposure (PAE) causes distinctive facial characteristics in some pregnancies and not others; genetic factors may contribute to this differential vulnerability. Ethanol disrupts multiple events of neural crest development, including induction, survival, migration, and differentiation. Animal models and genomic approaches have substantially advanced our understanding of the mechanisms underlying these facial changes. PAE during gastrulation produces craniofacial changes corresponding with human fetal alcohol syndrome. These result because PAE reduces prechordal plate extension and suppresses sonic hedgehog, leading to holoprosencephaly and malpositioned facial primordia. Haploinsufficiency in sonic hedgehog signaling increases vulnerability to facial deficits and may influence some PAE pregnancies. In contrast, PAE during early neurogenesis produces facial hypoplasia, preceded by neural crest reductions due to significant apoptosis. Factors mediating this apoptosis include intracellular calcium mobilization, elevated reactive oxygen species, and loss of trophic support from ß-catenin/calcium, sonic hedgehog, and mTOR signaling. Genome-wide SNP analysis links PDGFRA with facial outcomes in human PAE. Multiple genomic-level comparisons of ethanol-sensitive and - resistant early embryos, in both mouse and chick, independently identify common candidate genes that may potentially modify craniofacial vulnerability, including ribosomal proteins, proteosome, RNA splicing, and focal adhesion. In summary, research using animal models with genome-level differences in ethanol vulnerability, as well as targeted loss-and gain-of-function mutants, has clarified the mechanisms mediating craniofacial change in PAE. The findings additionally suggest that craniofacial deficits may represent a gene-ethanol interaction for some affected individuals. Genetic-level changes may prime individuals toward greater sensitivity or resistance to ethanol's neurotoxicity.