Differential Transcriptome Responses to Aflatoxin B1 in the Cecal Tonsil of Susceptible and Resistant Turkeys

The nearly-ubiquitous food and feed-borne mycotoxin aflatoxin B1 (AFB1) is carcinogenic and mutagenic, posing a food safety threat to humans and animals. One of the most susceptible animal species known and thus a good model for characterizing toxicological pathways, is the domesticated turkey (DT), a condition likely due, at least in part, to deficient hepatic AFB1-detoxifying alpha-class glutathione S-transferases (GSTAs). Conversely, wild turkeys (Eastern wild, EW) are relatively resistant to the hepatotoxic, hepatocarcinogenic and immunosuppressive effects of AFB1 owing to functional gene expression and presence of functional hepatic GSTAs. This study was designed to compare the responses in gene expression in the gastrointestinal tract between DT (susceptible phenotype) and EW (resistant phenotype) following dietary AFB1 challenge (320 ppb for 14 days); specifically in cecal tonsil which functions in both nutrient absorption and gut immunity. RNAseq and gene expression analysis revealed significant differential gene expression in AFB1-treated animals compared to control-fed domestic and wild birds and in within-treatment comparisons between bird types. Significantly upregulated expression of the primary hepatic AFB1-activating P450 (CYP1A5) as well as transcriptional changes in tight junction proteins were observed in AFB1-treated birds. Numerous pro-inflammatory cytokines, TGF-β and EGF were significantly down regulated by AFB1 treatment in DT birds and pathway analysis suggested suppression of enteroendocrine cells. Conversely, AFB1 treatment modified significantly fewer unique genes in EW birds; among these were genes involved in lipid synthesis and metabolism and immune response. This is the first investigation of the effects of AFB1 on the turkey gastro-intestinal tract. Results suggest that in addition to the hepatic transcriptome, animal resistance to this mycotoxin occurs in organ systems outside the liver, specifically as a refractory gastrointestinal tract

Altered Gene Response to Aflatoxin B\u3csub\u3e1\u3c/sub\u3e in the Spleens of Susceptible and Resistant Turkeys

Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1exposure

Expression of miRNAs in turkey muscle satellite cells and differential response to thermal challenge

Thermal stress alters the transcriptome and subsequent tissue physiology of poultry; thus, it can negatively impact poultry production through reduced meat quality, egg production, and health and wellbeing. The modulation of gene expression is critical to embryonic development and cell proliferation, and growing evidence suggests the role of non-coding RNAs (RNA:RNA interaction) in response to thermal stress in animals. MicroRNAs (miRNAs) comprise a class of small regulatory RNAs that modulate gene expression through posttranscriptional interactions and regulate mRNAs, potentially altering numerous cellular processes. This study was designed to identify and characterize the differential expression of miRNAs in satellite cells (SCs) from the turkey pectoralis major muscle and predict important miRNA:mRNA interactions in these developing SCs under a thermal challenge. Small RNA sequencing was performed on RNA libraries prepared from SCs cultured from 1-week-old male Nicholas commercial turkeys (NCTs) and non-selected Randombred Control Line 2 turkeys during proliferation and differentiation at the control temperature (38°C) or under a thermal challenge (33°C or 43°C). A total of 353 miRNAs (161 known and 192 novel) were detected across the sequenced libraries. Expression analysis found fewer differentially expressed miRNAs in the SCs of NCT birds, suggesting that the miRNA response to heat stress has been altered in birds selected for their modern commercial growth traits. Differentially expressed miRNAs, including those with described roles in muscle development, were detected both among temperature treatments and between genetic lines. A prominent differential expression of miR-206 was found in proliferating turkey SCs with a significant response to thermal challenges in both lines. In differentiating SCs, isoforms of miR-1 had significant differential responses, with the expression of miR-206 being mainly affected only by cold treatment. Target gene predictions and Gene Ontology analysis suggest that the differential expression of miRNAs during thermal stress could significantly affect cellular proliferation and differentiation

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues

Altered Gene Response to Aflatoxin B₁ in the Spleens of Susceptible and Resistant Turkeys

Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1 exposure

Differential Transcriptome Responses to Aflatoxin B1 in the Cecal Tonsil of Susceptible and Resistant Turkeys

The nearly-ubiquitous food and feed-borne mycotoxin aflatoxin B1 (AFB1) is carcinogenic and mutagenic, posing a food safety threat to humans and animals. One of the most susceptible animal species known and thus a good model for characterizing toxicological pathways, is the domesticated turkey (DT), a condition likely due, at least in part, to deficient hepatic AFB1-detoxifying alpha-class glutathione S-transferases (GSTAs). Conversely, wild turkeys (Eastern wild, EW) are relatively resistant to the hepatotoxic, hepatocarcinogenic and immunosuppressive effects of AFB1 owing to functional gene expression and presence of functional hepatic GSTAs. This study was designed to compare the responses in gene expression in the gastrointestinal tract between DT (susceptible phenotype) and EW (resistant phenotype) following dietary AFB1 challenge (320 ppb for 14 days); specifically in cecal tonsil which functions in both nutrient absorption and gut immunity. RNAseq and gene expression analysis revealed significant differential gene expression in AFB1-treated animals compared to control-fed domestic and wild birds and in within-treatment comparisons between bird types. Significantly upregulated expression of the primary hepatic AFB1-activating P450 (CYP1A5) as well as transcriptional changes in tight junction proteins were observed in AFB1-treated birds. Numerous pro-inflammatory cytokines, TGF-β and EGF were significantly down regulated by AFB1 treatment in DT birds and pathway analysis suggested suppression of enteroendocrine cells. Conversely, AFB1 treatment modified significantly fewer unique genes in EW birds; among these were genes involved in lipid synthesis and metabolism and immune response. This is the first investigation of the effects of AFB1 on the turkey gastro-intestinal tract. Results suggest that in addition to the hepatic transcriptome, animal resistance to this mycotoxin occurs in organ systems outside the liver, specifically as a refractory gastrointestinal tract

Response of Turkey Muscle Satellite Cells to Thermal Challenge. II. Transcriptome Effects in Differentiating Cells

Background: Exposure of poultry to extreme temperatures during the critical period of post-hatch growth can seriously affect muscle development and thus compromise subsequent meat quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells by thermal challenge during differentiation. Our goal is to better define how thermal stress alters breast muscle ultrastructure and subsequent development.Results: Skeletal muscle satellite cells previously isolated from the Pectoralis major muscle of 7-wk-old male turkeys (Meleagris gallopavo) from two breeding lines: the F-line (16 wk body weight-selected) and RBC2 (randombred control line) were used in this study. Cultured cells were induced to differentiate at 38°C (control) or thermal challenge temperatures of 33 or 43°C. After 48 h of differentiation, cells were harvested and total RNA was isolated for RNAseq analysis. Analysis of 39.9 Gb of sequence found 89% mapped to the turkey genome (UMD5.0, annotation 101) with average expression of 18,917 genes per library. In the cultured satellite cells, slow/cardiac muscle isoforms are generally present in greater abundance than fast skeletal isoforms. Statistically significant differences in gene expression were observed among treatments and between turkey lines, with a greater number of genes affected in the F-line cells following cold treatment whereas more differentially expressed (DE) genes were observed in the RBC2 cells following heat treatment. Many of the most significant pathways involved signaling, consistent with ongoing cellular differentiation. Regulation of Ca2+ homeostasis appears to be significantly affected by temperature treatment, particularly cold treatment.Conclusions: Satellite cell differentiation is directly influenced by temperature at the level of gene transcription with greater effects attributed to selection for fast growth. At lower temperature, muscle-associated genes in the satellite cells were among the genes with the greatest down regulation consistent with slower differentiation and smaller myotubes. Fewer expression differences were observed in the differentiating cells than previously observed for proliferating cells. This suggests the impact of temperature on satellite cells occurs primarily at early points in satellite cell activation

Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B1

The food-borne mycotoxin aflatoxin B1 (AFB1) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M.g. silvestris) are more resistant. AFB1 toxicity entails bioactivation by hepatic cytochrome P450s to the electrophilic exo-AFB1-8,9-epoxide (AFBO). Domesticated turkeys lack functional hepatic GST-mediated detoxification of AFBO, and this is largely responsible for the differences in resistance between turkey types. This study was designed to characterize transcriptional changes induced in turkey livers by AFB1 , and to contrast the response of domesticated (susceptible) and wild (more resistant) birds. Gene expression responses to AFB1 were examined using RNA-sequencing. Statistically significant differences in gene expression were observed among treatment groups and between turkey types. Expression analysis identified 4621 genes with significant differential expression (DE) in AFB1 -treated birds compared to controls. Characterization of DE transcripts revealed genes dis-regulated in response to toxic insult with significant association of Phase I and Phase II genes and others important in cellular regulation, modulation of apoptosis, and inflammatory responses. Constitutive expression of GSTA3 was significantly higher in wild birds and was significantly higher in AFB1-treated birds when compared to controls for both genetic groups. This pattern was also observed by qRT-PCR in other wild and domesticated turkey strains. Results of this study emphasize the differential response of these genetically distinct birds, and identify genes and pathways that are differentially altered in aflatoxicosis

Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey

The hepatic transcriptome of the turkey poult (Meleagris gallopavo) is minimally altered by high inorganic dietary selenium.

There is interest in supplementing animals and humans with selenium (Se) above Se-adequate levels, but the only good biomarker for toxicity is tissue Se. We targeted liver because turkeys fed 5 μg Se/g have hepatic Se concentrations 6-fold above Se-adequate (0.4 μg Se/g) levels without effects on growth or health. Our objectives were (i) to identify transcript biomarkers for high Se status, which in turn would (ii) suggest proteins and pathways used by animals to adapt to high Se. Turkey poults were fed 0, 0.025, 0.4, 0.75 and 1.0 μg Se/g diet in experiment 1, and fed 0.4, 2.0 and 5.0 μg Se/g in experiment 2, as selenite, and the full liver transcriptome determined by RNA-Seq. The major effect of Se-deficiency was to down-regulate expression of a subset of selenoprotein transcripts, with little significant effect on general transcript expression. In response to high Se intake (2 and 5 μg Se/g) relative to Se-adequate turkeys, there were only a limited number of significant differentially expressed transcripts, all with only relatively small fold-changes. No transcript showed a consistent pattern of altered expression in response to high Se intakes across the 1, 2 and 5 μg Se/g treatments, and there were no associated metabolic pathways and biological functions that were significant and consistently found with high Se supplementation. Gene set enrichment analysis also found no gene sets that were consistently altered by high-Se and supernutritional-Se. A comparison of differentially expressed transcript sets with high Se transcript sets identified in mice provided high Se (~3 μg Se/g) also failed to identify common differentially expressed transcript sets between these two species. Collectively, this study indicates that turkeys do not alter gene expression in the liver as a homeostatic mechanism to adapt to high Se