Single nucleus transcriptomics of ventral midbrain identifies glial activation associated with chronic opioid use disorder.

Dynamic interactions of neurons and glia in the ventral midbrain mediate reward and addiction behavior. We studied gene expression in 212,713 ventral midbrain single nuclei from 95 individuals with history of opioid misuse, and individuals without drug exposure. Chronic exposure to opioids was not associated with change in proportions of glial and neuronal subtypes, however glial transcriptomes were broadly altered, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes. Genes associated with activation of the immune response including interferon, NFkB signaling, and cell motility pathways were upregulated, contrasting with down-regulated expression of synaptic signaling and plasticity genes in ventral midbrain non-dopaminergic neurons. Ventral midbrain transcriptomic reprogramming in the context of chronic opioid exposure included 325 genes that previous genome-wide studies had linked to risk of substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

Proteomic insight into human directed selection of the domesticated chicken Gallus gallus.

Chicken domestication began at least 3,500 years ago for purposes of divination, cockfighting, and food. Prior to industrial scale chicken production, domestication selected larger birds with increased egg production. In the mid-20th century companies began intensive selection with the broiler (meat) industry focusing on improved feed conversion, rapid growth, and breast muscle yield. Here we present proteomic analysis comparing the modern broiler line, Ross 708, with the UIUC legacy line which is not selected for growth traits. Breast muscle proteome analysis identifies cellular processes that have responded to human directed artificial selection. Mass spectrometry was used to identify protein level differences in the breast muscle of 6-day old chicks from Modern and Legacy lines. Our results indicate elevated levels of stress proteins, ribosomal proteins and proteins that participate in the innate immune pathway in the Modern chickens. Furthermore, the comparative analyses indicated expression differences for proteins involved in multiple biochemical pathways. In particular, the Modern line had elevated levels of proteins affecting the pentose phosphate pathway, TCA cycle and fatty acid oxidation while proteins involved in the first phase of glycolysis were reduced compared to the Legacy line. These analyses provide hypotheses linking the morphometric changes driven by human directed selection to biochemical pathways. These results also have implications for the poultry industry, specifically Wooden Breast disease which is linked to rapid breast muscle growth.

Chicken pituitary transcriptomic responses to acute heat stress.

BACKGROUND: Poultry production is vulnerable to increasing temperatures in terms of animal welfare and in economic losses. With the predicted increase in global temperature and the number and severity of heat waves, it is important to understand how chickens raised for food respond to heat stress. This knowledge can be used to determine how to select chickens that are adapted to thermal challenge. As neuroendocrine organs, the hypothalamus and pituitary provide systemic regulation of the heat stress response. METHODS AND RESULTS: Here we report a transcriptome analysis of the pituitary response to acute heat stress. Chickens were stressed for 2 h at 35 °C (HS) and transcriptomes compared with birds maintained in thermoneutral temperatures (25 °C). CONCLUSIONS: The observations were evaluated in the context of ontology terms and pathways to describe the pituitary response to heat stress. The pituitaries of heat stressed birds exhibited responses to hyperthermia through altered expression of genes coding for chaperones, cell cycle regulators, cholesterol synthesis, transcription factors, along with the secreted peptide hormones, prolactin, and proopiomelanocortin.

A high-quality de novo genome assembly for clapper rail (Rallus crepitans).

The clapper rail (Rallus crepitans), of the family Rallidae, is a secretive marsh bird species that is adapted for high salinity habitats. They are very similar in appearance to the closely related king rail (R. elegans), but while king rails are limited primarily to freshwater marshes, clapper rails are highly adapted to tolerate salt marshes. Both species can be found in brackish marshes where they freely hybridize, but the distribution of their respective habitats precludes the formation of a continuous hybrid zone and secondary contact can occur repeatedly. This system, thus, provides unique opportunities to investigate the underlying mechanisms driving their differential salinity tolerance as well as the maintenance of the species boundary between the 2 species. To facilitate these studies, we assembled a de novo reference genome assembly for a female clapper rail. Chicago and HiC libraries were prepared as input for the Dovetail HiRise pipeline to scaffold the genome. The pipeline, however, did not recover the Z chromosome so a custom script was used to assemble the Z chromosome. We generated a near chromosome level assembly with a total length of 994.8 Mb comprising 13,226 scaffolds. The assembly had a scaffold N50 was 82.7 Mb, L50 of four, and had a BUSCO completeness score of 92%. This assembly is among the most contiguous genomes among the species in the family Rallidae. It will serve as an important tool in future studies on avian salinity tolerance, interspecific hybridization, and speciation.

Single Nucleus Transcriptomics Reveals Pervasive Glial Activation in Opioid Overdose Cases.

Dynamic interactions of neurons and glia in the ventral midbrain (VM) mediate reward and addiction behavior. We studied gene expression in 212,713 VM single nuclei from 95 human opioid overdose cases and drug-free controls. Chronic exposure to opioids left numerical proportions of VM glial and neuronal subtypes unaltered, while broadly affecting glial transcriptomes, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes, with prominent activation of the immune response including interferon, NFkB signaling, and cell motility pathways, sharply contrasting with down-regulated expression of synaptic signaling and plasticity genes in VM non-dopaminergic neurons. VM transcriptomic reprogramming in the context of opioid exposure and overdose included 325 genes with genetic variation linked to substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

CD99 and the Chicken Alloantigen D Blood System.

The chicken D blood system is one of 13 alloantigen systems found on chicken red blood cells. Classical recombinant studies located the D blood system on chicken chromosome 1, but the candidate gene was unknown. Multiple resources were utilized to identify the chicken D system candidate gene, including genome sequence information from both research and elite egg production lines for which D system alloantigen alleles were reported, and DNA from both pedigree and non-pedigree samples with known D alleles. Genome-wide association analyses using a 600 K or a 54 K SNP chip plus DNA from independent samples identified a strong peak on chicken chromosome 1 at 125-131 Mb (GRCg6a). Cell surface expression and the presence of exonic non-synonymous SNP were used to identify the candidate gene. The chicken CD99 gene showed the co-segregation of SNP-defined haplotypes and serologically defined D blood system alleles. The CD99 protein mediates multiple cellular processes including leukocyte migration, T-cell adhesion, and transmembrane protein transport, affecting peripheral immune responses. The corresponding human gene is found syntenic to the pseudoautosomal region 1 of human X and Y chromosomes. Phylogenetic analyses show that CD99 has a paralog, XG, that arose by duplication in the last common ancestor of the amniotes.

The Chicken A and E Blood Systems Arise from Genetic Variation in and around the Regulators of Complement Activation Region.

The tightly linked A and E blood alloantigen systems are 2 of 13 blood systems identified in chickens. Reported herein are studies showing that the genes encoding A and E alloantigens map within or near to the chicken regulator of complement activation (RCA) gene cluster, a region syntenic with the human RCA. Genome-wide association studies, sequence analysis, and sequence-derived single-nucleotide polymorphism information for known A and/or E system alleles show that the most likely candidate gene for the A blood system is C4BPM gene (complement component 4 binding protein, membrane). Cosegregation of single-nucleotide polymorphism-defined C4BPM haplotypes and blood system A alleles defined by alloantisera provide a link between chicken blood system A and C4BPM. The best match for the E blood system is the avian equivalent of FCAMR (Fc fragment of IgA and IgM receptor). C4BPM is located within the chicken RCA on chicken microchromosome 26 and is separated from FCAMR by 89 kbp. The genetic variation observed at C4BPM and FCAMR could affect the chicken complement system and differentially guide immune responses to infectious diseases.

Opioid deaths involving concurrent benzodiazepine use: Assessing risk factors through the analysis of prescription drug monitoring data and postmortem toxicology.

BACKGROUND: A high proportion of opioid drug deaths involve concurrent benzodiazepine use. To reduce the risk of drug overdose, various prescription drug monitoring programs have been implemented. This study examined the impact of concurrent benzodiazepine use on opioid-related deaths, and the utility of the Michigan Automated Prescription System (MAPS) in predicting risk of opioid death. METHODS: Wayne County Medical Examiner's Office cases from 2018 were examined in terms of MAPS data and MAPS-derived drug risk scores, as well as postmortem toxicology. Opioid death cases with concurrent benzodiazepine use were compared to non-drug deaths. RESULTS: For cases with a MAPS history for 6 months preceding death, the incidence of opioid prescriptions filled did not differ between groups. In contrast, significantly more opioid death cases had filled a benzodiazepine prescription; alprazolam prescription was the single best predictor of opioid drug death. Groups differed in MAPS-calculated drug risk scores, though these were less predictive of opioid death than some individual measures of prescription drug use. In terms of postmortem toxicology, fentanyl was the best discriminator between cohorts, with significant associations seen for morphine, benzodiazepine, or cocaine use. Similar results were obtained in the subset of subjects filling a prescription within a month of death, except that MAPS risk scores no longer predicted drug deaths. CONCLUSION: MAPS scores did not adequately predict risk of opioid-related death. Contrary to expectations, prescription opioid use was not correlated with opioid-related death, whereas concurrent use of opioids and benzodiazepines represented a highly significant risk factor.

Transcriptomic and metabolomic characterization of post-hatch metabolic reprogramming during hepatic development in the chicken.

BACKGROUND: Artificial selection of modern meat-producing chickens (broilers) for production characteristics has led to dramatic changes in phenotype, yet the impact of this selection on metabolic and molecular mechanisms is poorly understood. The first 3 weeks post-hatch represent a critical period of adjustment, during which the yolk lipid is depleted and the bird transitions to reliance on a carbohydrate-rich diet. As the liver is the major organ involved in macronutrient metabolism and nutrient allocatytion, a combined transcriptomics and metabolomics approach has been used to evaluate hepatic metabolic reprogramming between Day 4 (D4) and Day 20 (D20) post-hatch. RESULTS: Many transcripts and metabolites involved in metabolic pathways differed in their abundance between D4 and D20, representing different stages of metabolism that are enhanced or diminished. For example, at D20 the first stage of glycolysis that utilizes ATP to store or release glucose is enhanced, while at D4, the ATP-generating phase is enhanced to provide energy for rapid cellular proliferation at this time point. This work has also identified several metabolites, including citrate, phosphoenolpyruvate, and glycerol, that appear to play pivotal roles in this reprogramming. CONCLUSIONS: At Day 4, metabolic flexibility allows for efficiency to meet the demands of rapid liver growth under oxygen-limiting conditions. At Day 20, the liver's metabolism has shifted to process a carbohydrate-rich diet that supports the rapid overall growth of the modern broiler. Characterizing these metabolic changes associated with normal post-hatch hepatic development has generated testable hypotheses about the involvement of specific genes and metabolites, clarified the importance of hypoxia to rapid organ growth, and contributed to our understanding of the molecular changes affected by decades of artificial selection.

Utility of CDC Screening Guidelines and Autopsy Findings in Identifying Decedents Who Die of SARS-CoV-2 Infection.

ABSTRACT: We assess the utility of a Centers for Disease Control and Prevention (CDC) guidelines-based coronavirus disease 2019 (COVID-19) screening checklist for postmortem severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance, detailing the relationship between the histologic findings at autopsy and attribution of death to COVID-19.SARS-CoV-2 nasopharyngeal swabs were collected at the time of autopsy in all "checklist-positive" decedents. Additional "checklist-negative" decedents were randomly tested daily. Lung slides were blindly reviewed by 3 pathologists, assessing for the presence of diffuse alveolar damage (DAD) and other findings. Sixteen decedents had positive postmortem SARS-CoV-2 nasopharyngeal swabs and underwent complete autopsies. Seven decedents had positive screening checklists. Of these, 4 had DAD and 1 had COVID-19-associated thromboembolic disease. Of the 9 decedents with negative screening checklists, 2 had DAD, but only 1 was attributed to COVID-19; the other was likely drug related. Acute bronchopneumonia was the second most common finding, and aspiration was the likely etiology in cases without concomitant DAD. COVID-19-related DAD was identified more commonly in decedents who screened positive by CDC checklist, but false-negatives did occur. Medical examiner offices should maintain a low threshold for random testing of decedents even when COVID-19 is not suspected.

Severe Acute Respiratory Syndrome Coronavirus 2 Surveillance in Decedents in a Large, Urban Medical Examiner's Office.

BACKGROUND: Given the challenges in implementing widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is increasing interest in alternative surveillance strategies. METHODS: We tested nasopharyngeal swabs from 1094 decedents in the Wayne County Medical Examiner's Office for SARS-CoV-2. All decedents were assessed using a coronavirus disease 2019 (COVID-19) checklist, and decedents flagged using the checklist (298) were preferentially tested. A random sample of decedents not flagged using the checklist were also tested (796). We statistically analyzed the characteristics of decedents (age, sex, race, and manner of death), differentiating between those flagged using the checklist and not and between those SARS-CoV-2-positive and not. RESULTS: A larger percentage of decedents overall were male (70% vs 48%) and black (55% vs 36%) compared with the catchment population. Seven-day average percent positivity among flagged decedents closely matched the trajectory of percent positivity in the catchment population, particularly during the peak of the outbreak (March and April 2020). After a lull in May to mid-June, new positive tests in late June coincided with increased case detection in the catchment. We found large racial disparities in test results; SARS-CoV-2-positive decedents were substantially more likely to be black than SARS-CoV-2-negative decedents (82% vs 51%). SARS-CoV-2-positive decedents were also more likely to be older and to have died of natural causes, including of COVID-19 disease. CONCLUSIONS: Disease surveillance through medical examiners and coroners could supplement other forms of surveillance and serve as a possible early outbreak warning sign.

Medical Examiner Review of the Characteristics of Fire-Related Homicides.

While fire-related deaths are regularly encountered by medical examiners, fire-related homicides are relatively uncommon. Although some large retrospective studies of fire-related deaths have been performed, few large studies have specifically reviewed fire-related homicides. Autopsy, scene investigation, and ancillary studies were reviewed for 38 fire-related homicides evaluated at the Wayne County Medical Examiner's Office in Detroit, Michigan. The largest proportion of cases were inhalation-related deaths in dwelling fires (n = 21, 55%), followed by deaths from thermal injury after immolation (n = 8, 21%) and traumatic death with contemporaneous or subsequent immolation (n = 8, 21%). There was one case of postmortem immolation. Although carboxyhemoglobin (COHb) levels played a significant role in evaluation of these cases, no single factor was diagnostic of a particular cause or manner of death. Fire-related homicides present unique diagnostic challenges because multiple insults frequently contribute to the cause death. Death at the scene and COHb level above 10% are the most useful factors in establishing smoke and soot inhalation as the cause of death. Some autopsy findings are helpful in establishing or ruling out smoke and soot inhalation as contributing to or sole cause of death, but an evaluation of the entire circumstances and autopsy findings is necessary.

Dysbiosis in the Dead: Human Postmortem Microbiome Beta-Dispersion as an Indicator of Manner and Cause of Death.

The postmortem microbiome plays an important functional role in host decomposition after death. Postmortem microbiome community successional patterns are specific to body site, with a significant shift in composition 48 h after death. While the postmortem microbiome has important forensic applications for postmortem interval estimation, it also has the potential to aid in manner of death (MOD) and cause of death (COD) determination as a reflection of antemortem health status. To further explore this association, we tested beta-dispersion, or the variability of microbiomes within the context of the "Anna Karenina Principle" (AKP). The foundational principle of AKP is that stressors affect microbiomes in unpredictable ways, which increases community beta-dispersion. We hypothesized that cases with identified M/CODs would have differential community beta-dispersion that reflected antemortem conditions, specifically that cardiovascular disease and/or natural deaths would have higher beta-dispersion compared to other deaths (e.g., accidents, drug-related deaths). Using a published microbiome data set of 188 postmortem cases (five body sites per case) collected during routine autopsy in Wayne County (Detroit), MI, we modeled beta-dispersion to test for M/COD associations a priori. Logistic regression models of beta-dispersion and case demographic data were used to classify M/COD. We demonstrated that beta-dispersion, along with case demographic data, could distinguish among M/COD - especially cardiovascular disease and drug related deaths, which were correctly classified in 79% of cases. Binary logistic regression models had higher correct classifications than multinomial logistic regression models, but changing the defined microbial community (e.g., full vs. non-core communities) used to calculate beta-dispersion overall did not improve model classification or M/COD. Furthermore, we tested our analytic approach on a case study that predicted suicides from other deaths, as well as distinguishing MOD (e.g., homicides vs. suicides) within COD (e.g., gunshot wound). We propose an analytical workflow that combines postmortem microbiome indicator taxa, beta-dispersion, and case demographic data for predicting MOD and COD classifications. Overall, we provide further evidence the postmortem microbiome is linked to the host's antemortem health condition(s), while also demonstrating the potential utility of including beta-dispersion (a non-taxon dependent approach) coupled with case demographic data for death determination.

Abusive Pediatric Thoracolumbar Fracture Due to Forced Hyperextension: Case Report, Biomechanical Considerations, and Review of the Literature.

Pediatric thoracolumbar fractures are rare due to the physiological differences which afford greater resilience to the immature spine. Most pediatric thoracolumbar fractures occur as the result of high energy trauma, such as motor vehicle accidents, and modes of reasonable accidental injuries are limited by age and developmental capabilities of the child. These fractures can occur as the result of inflicted blunt force trauma and child abuse, and in most cases, the mechanism of injury to the spine is not known. We report the death of a 29-month-old man due to blunt force trauma to the back and forced hyperextension of the thoracolumbar spine causing fracture of the fourth lumbar (L4) vertebral body. A complete forensic examination revealed a previous healing fracture of the anterior aspect of the L4 vertebral body, with acute disruption of the anterior longitudinal ligament overlying the fracture site, complete fracture of the vertebral body, and fatal retroperitoneal hemorrhage. We present a review of the biomechanical considerations of the pediatric spine, a survey of pediatric spinal fractures, and a review of the literature on pediatric abusive thoracolumbar fractures. In this case, there was never a provided explanation for how the injury occurred; however, understanding the biomechanics of the pediatric spine allowed for the determination of the mechanism, force required to produce this specific pattern of abusive spinal injury, and the manner of death.

Diffuse alveolar damage (DAD) resulting from coronavirus disease 2019 Infection is Morphologically Indistinguishable from Other Causes of DAD.

AIMS: Diffuse alveolar damage (DAD) is a ubiquitous finding in inpatient coronavirus disease 2019 (COVID-19)-related deaths, but recent reports have also described additional atypical findings, including vascular changes. An aim of this study was to assess lung autopsy findings in COVID-19 inpatients, and in untreated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive individuals who died in the community, in order to understand the relative impact of medical intervention on lung histology. Additionally, we aimed to investigate whether COVID-19 represents a unique histological variant of DAD by comparing the pathological findings with those of uninfected control patients. METHODS AND RESULTS: Lung sections from autopsy cases were reviewed by three pulmonary pathologists, including two who were blinded to patient cohort. The cohorts included four COVID-19 inpatients, four cases with postmortem SARS-CoV-2 diagnoses who died in the community, and eight SARS-CoV-2-negative control cases. DAD was present in all but one SARS-CoV-2-positive patient, who was asymptomatic and died in the community. Although SARS-CoV-2-positive patients were noted to have more focal perivascular inflammation/endothelialitis than control patients, there were no significant differences in the presence of hyaline membranes, fibrin thrombi, airspace organisation, and 'acute fibrinous and organising pneumonia'-like intra-alveolar fibrin deposition between the cohorts. Fibrinoid vessel wall necrosis, haemorrhage and capillaritis were not features of COVID-19-related DAD. CONCLUSIONS: DAD is the primary histological manifestation of severe lung disease in COVID-19 patients who die both in hospital and in the community, suggesting no contribution of hyperoxaemic mechanical ventilation to the histological changes. There are no distinctive morphological features with which to confidently differentiate COVID-19-related DAD from DAD due to other causes.

fRNAkenseq: a fully powered-by-CyVerse cloud integrated RNA-sequencing analysis tool.

BACKGROUND: Decreasing costs make RNA sequencing technologies increasingly affordable for biologists. However, many researchers who can now afford sequencing lack access to resources necessary for downstream analysis. This means that even as algorithms to process RNA-Seq data improve, many biologists still struggle to manage the sheer volume of data produced by next generation sequencing (NGS) technologies. Scalable bioinformatics tools that exploit multiple platforms are needed to democratize bioinformatics resources in the sequencing era. This is essential for equipping many research groups in the life sciences with the tools to process the increasingly unwieldy datasets they produce. METHODS: One strategy to address this challenge is to develop a modern generation of sequence analysis tools capable of seamless data sharing and communication. Such tools will provide interoperability through offerings of interlinked resources. Systems of interlinked, scalable resources, which often incorporate cloud data storage, are broadly referred to as cyberinfrastructure. Cyberinfrastructure integrated tools will help researchers to robustly analyze large scale datasets by efficiently sharing data burdens across a distributed architecture. Additionally, interoperability will allow emerging tools to cross-adapt features of existing tools. It is important that these tools are designed to be easy to use for biologists. RESULTS: We introduce fRNAkenseq, a powered-by-CyVerse RNA sequencing analysis tool that exhibits interoperability with other resources and meets the needs of biologists for comprehensive, easy to use RNA sequencing analysis. fRNAkenseq leverages a complex set of Application Programming Interfaces (APIs) associated with the NSF-funded cyberinfrastructure project, CyVerse, to execute FASTQ-to-differential expression RNA-Seq analyses. Integrating across bioinformatics platforms, fRNAkenseq also exploits cloud integration and cross-talk with another CyVerse associated tool, CoGe. fRNAkenseq offers novel features for the biologist such as more robust and comprehensive pipelines for enrichment than those currently available by default in a single tool, whether they are cloud-based or local installation. Importantly, cross-talk with CoGe allows fRNAkenseq users to execute RNA-Seq pipelines on an inventory of 47,000 archived genomes stored in CoGe or upload their own draft genome.

Evaluating Bioinformatic Pipeline Performance for Forensic Microbiome Analysis*,†,‡.

Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories. These differences can potentially affect microbial community composition and downstream analyses. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, varying minimum library size or minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that three open-source bioinformatic pipelines, MG-RAST, mothur, and QIIME2, had significant differences in relative abundance, alpha-diversity, and beta-diversity, despite the same input data. Increasing minimum library size and sample size increased the number of low-abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice affect results in important ways. Given the growing potential application of forensic microbiology to the criminal justice system, continued research on standardizing computational methodology will be important for downstream applications.

Effects of acute and chronic heat stress on the performance, egg quality, body temperature, and blood gas parameters of laying hens.

The goal of this experiment was to measure the physiological response of individual laying hens exposed to heat stress (HS). Performance, egg quality, body temperature (BT), and blood chemistry of laying hens were individually recorded before and after various intervals of daily cyclic HS. In total, 407 18-week-old W-36 parent-line laying hens (Hy-Line International, Dallas Center, IA) were housed individually in battery cages. After an acclimation period, baseline data were collected from 22 to 24-wk before the hens were subjected to a daily cyclic HS consisting of 7 h at 35°C returning to 30°C for the remaining 17 h/D from 24 to 28-wk of age. Eggs were collected and individually weighed daily. Feed intake (FI), egg production (EP), egg weights, egg mass, BW, and feed efficiency (FE) (g egg/kg FI) were calculated over 2-wk time periods. Eggs were collected for quality assessment the day before HS began, the 2nd day of HS, and on a weekly basis throughout the 4-wk HS. Blood was collected and BT measured the day before heat HS was initiated, on the first day of HS, and again at 2 and 4-wk of HS. Blood PCO2 and iCa decreased, and blood pH increased within 4 to 6 h of HS (P ≤ 0.01). Shell weights decreased with acute HS, possibly due to the reduction in blood iCa (P ≤ 0.01). After 4-wk of HS the blood pH returned to pre-HS levels but iCa remained decreased (P ≤ 0.01). Shell weights remained low and Haugh units decreased after 2 and 4-wk of HS (P ≤ 0.01). Feed efficiency was increased and FI, EP, and BW decreased by 2-wk of HS and remained low through 4-wk (P ≤ 0.01). The cyclic HS had a significant effect on the performance, egg quality, and blood chemistry over the 4-wk HS.

Variant analysis pipeline for accurate detection of genomic variants from transcriptome sequencing data.

The wealth of information deliverable from transcriptome sequencing (RNA-seq) is significant, however current applications for variant detection still remain a challenge due to the complexity of the transcriptome. Given the ability of RNA-seq to reveal active regions of the genome, detection of RNA-seq SNPs can prove valuable in understanding the phenotypic diversity between populations. Thus, we present a novel computational workflow named VAP (Variant Analysis Pipeline) that takes advantage of multiple RNA-seq splice aware aligners to call SNPs in non-human models using RNA-seq data only. We applied VAP to RNA-seq from a highly inbred chicken line and achieved high accuracy when compared with the matching whole genome sequencing (WGS) data. Over 65% of WGS coding variants were identified from RNA-seq. Further, our results discovered SNPs resulting from post transcriptional modifications, such as RNA editing, which may reveal potentially functional variation that would have otherwise been missed in genomic data. Even with the limitation in detecting variants in expressed regions only, our method proves to be a reliable alternative for SNP identification using RNA-seq data. The source code and user manuals are available at https://modupeore.github.io/VAP/.

Genetic lines respond uniquely within the chicken thymic transcriptome to acute heat stress and low dose lipopolysaccharide.

Exposure to high temperatures is known to impair immune functions and disease resistance of poultry. Characterizing changes in the transcriptome can help identify mechanisms by which immune tissues, such as the thymus, respond to heat stress. In this study, 22-day-old chickens from two genetic lines (a relatively resistant Fayoumi line and a more susceptible broiler line) were exposed to acute heat stress (35 °C) and/or immune simulation with lipopolysaccharide (LPS; 100 µg/kg). Transcriptome responses in the thymus were identified by RNA-sequencing (RNA-seq). Expression of most genes was unaffected by heat and/or LPS in the Fayoumi line, whereas these treatments had more impact in the broiler line. Comparisons between the broiler and Fayoumi transcriptomes identified a large number of significant genes both at homeostasis and in response to treatment. Functional analyses predicted that gene expression changes impact immune responses, apoptosis, cell activation, migration, and adhesion. In broilers, acute heat stress changed thymic expression responses to LPS and could impact thymocyte survival and trafficking, and thereby contribute to the negative effects of high temperatures on immune responses. Identification of these genes and pathways provides a foundation for testing targets to improve disease resistance in heat-stressed chickens.