Dramatic genome-wide reprogramming of mRNA in hypometabolic muscle.

In response to seasonal droughts, the green striped burrowing frog Cyclorana alboguttata enters a reversible hypometabolic state called aestivation where heart rate and oxygen consumption can be reduced despite warm (>25C°) ambient temperatures. With a view to understanding molecular mechanisms we profiled aestivating versus control gastrocnemius muscle using mRNA sequencing. This indicated an extensive metabolic reprogramming, with nearly a quarter of the entire transcriptome (3996 of 16,960 mRNA) exhibiting a nominal >2-fold change. Consistent with a physiological adaptation to spare carbohydrate reserves, carbohydrate catabolism was systemically downregulated. A 630-fold downregulation of ENO3 encoding the enolase enzyme was most striking. The 590 frog orthologs of mRNA encoding the mitoproteome were, viewed as a population, significantly downregulated during aestivation, although not to the same extent as mRNA encoding carbohydrate catabolism. Prominent examples include members of the TCA cycle (IDH2), electron transport chain (NDUFA6), the ATP synthase complex (ATP5F1B) and ADP/ATP intracellular transport (SLC25A4). Moreover, mRNA derived from the mt genome itself (e.g. mt-ND1) were also downregulated. Most prominent among the upregulated mRNA are those encoding aspects of regulated proteolysis including the proteosome (e.g. PSME4L), peptidases (USP25), atrogins (FBXO32) and ubiquitination (VCP). Finally, we note the ∼5-fold upregulation of the mRNA EIFG3 that encodes part of the EIF4F complex. This possesses global control of protein synthesis. Given protein synthesis is repressed in aestivating frogs this indicates the skeletal musculature is poised for accelerated translation of mRNA upon emergence, supporting a strategy to rapidly restore function when the summer rains come.

'Spikeopathy': COVID-19 Spike Protein Is Pathogenic, from Both Virus and Vaccine mRNA.

The COVID-19 pandemic caused much illness, many deaths, and profound disruption to society. The production of 'safe and effective' vaccines was a key public health target. Sadly, unprecedented high rates of adverse events have overshadowed the benefits. This two-part narrative review presents evidence for the widespread harms of novel product COVID-19 mRNA and adenovectorDNA vaccines and is novel in attempting to provide a thorough overview of harms arising from the new technology in vaccines that relied on human cells producing a foreign antigen that has evidence of pathogenicity. This first paper explores peer-reviewed data counter to the 'safe and effective' narrative attached to these new technologies. Spike protein pathogenicity, termed 'spikeopathy', whether from the SARS-CoV-2 virus or produced by vaccine gene codes, akin to a 'synthetic virus', is increasingly understood in terms of molecular biology and pathophysiology. Pharmacokinetic transfection through body tissues distant from the injection site by lipid-nanoparticles or viral-vector carriers means that 'spikeopathy' can affect many organs. The inflammatory properties of the nanoparticles used to ferry mRNA; N1-methylpseudouridine employed to prolong synthetic mRNA function; the widespread biodistribution of the mRNA and DNA codes and translated spike proteins, and autoimmunity via human production of foreign proteins, contribute to harmful effects. This paper reviews autoimmune, cardiovascular, neurological, potential oncological effects, and autopsy evidence for spikeopathy. With many gene-based therapeutic technologies planned, a re-evaluation is necessary and timely.

Seasonal resource selection of an arboreal habitat specialist in a human-dominated landscape: A case study using red panda.

Human-dominated landscapes provide heterogeneous wildlife habitat. Conservation of habitat specialists, like red pandas Ailurus fulgens, inhabiting such landscapes is challenging. Therefore, information on resource use across spatial and temporal scales could enable informed-decision making with better conservation outcomes. We aimed to examine the effect of geo-physical, vegetation, and disturbance variables on fine-scale habitat selection of red pandas in one such landscape. We equipped 10 red pandas with GPS collars in eastern Nepal in 2019 and monitored them for 1 year. Our analysis was based on a generalized-linear-mixed model. We found the combined effect of geo-physical, vegetation, and disturbance variables resulted in differences in resource selection of red pandas and that the degree of response to these variables varied across seasons. Human disturbances, especially road and cattle herding activities, affected habitat utilization throughout the year whereas other variables were important only during restricted periods. For instance, geo-physical variables were influential in the premating and cub-rearing seasons while vegetation variables were important in all seasons other than premating. Red pandas selected steeper slopes with high solar insolation in the premating season while they occupied elevated areas and preferred specific aspects in the cub-rearing season. Furthermore, the utilized areas had tall bamboo in the birthing and cub-rearing seasons while they also preferred diverse tree species and high shrub cover in the latter. Our study demonstrates the significance of season-specific management, suggests the importance of specific types of vegetation during biologically crucial periods, and emphasizes the necessity to minimize disturbances throughout the year.

Multi-breed genomic predictions and functional variants for fertility of tropical bulls.

Worldwide, most beef breeding herds are naturally mated. As such, the ability to identify and select fertile bulls is critically important for both productivity and genetic improvement. Here, we collected ten fertility-related phenotypes for 6,063 bulls from six tropically adapted breeds. Phenotypes were comprised of four bull conformation traits and six traits directly related to the quality of the bull's semen. We also generated high-density DNA genotypes for all the animals. In total, 680,758 single nucleotide polymorphism (SNP) genotypes were analyzed. The genomic correlation of the same trait observed in different breeds was positive for scrotal circumference and sheath score on most breed comparisons, but close to zero for the percentage of normal sperm, suggesting a divergent genetic background for this trait. We confirmed the importance of a breed being present in the reference population to the generation of accurate genomic estimated breeding values (GEBV) in an across-breed validation scenario. Average GEBV accuracies varied from 0.19 to 0.44 when the breed was not included in the reference population. The range improved to 0.28 to 0.59 when the breed was in the reference population. Variants associated with the gene HDAC4, six genes from the spermatogenesis-associated (SPATA) family of proteins, and 29 transcription factors were identified as candidate genes. Collectively these results enable very early in-life selection for bull fertility traits, supporting genetic improvement strategies currently taking place within tropical beef production systems. This study also improves our understanding of the molecular basis of male fertility in mammals.

Effect of disturbances and habitat fragmentation on an arboreal habitat specialist mammal using GPS telemetry: a case of the red panda.

CONTEXT: Habitat specialists residing in human-modified landscapes are likely to be more vulnerable to disturbance because of a functional reliance on very particular habitat features. However, there have been few studies designed to specifically address that issue. OBJECTIVES: This study aimed to explore how the red panda, an iconic endangered habitat specialist, behaves when faced with disturbances and habitat fragmentation. In particular, we attempted to examine the effect of anthropogenic disturbances and fragmentation on home-range size, activity patterns, and recursion. METHODS: Using GPS telemetry we monitored 10 red pandas and documented disturbances using camera trapping for one year in eastern Nepal. We performed spatial analysis, analysed activity patterns and evaluated the effect of habitat fragmentation and disturbances on home-range size and residence time using Linear Mixed Models. RESULTS: Home-range size increased in areas with low availability of forest cover whilst home ranges were smaller in areas with a high road density. Red pandas spent more time in large habitat patches away from roads and cattle stations. Crossing rates suggested that roads acted as a barrier for movement across their habitat. Red pandas also partitioned their activity to minimize interactions with disturbances. CONCLUSIONS: Red pandas seem to make a trade-off to co-exist in human-dominated landscapes which may have adverse long-term effects on their survival. This indicates that current patterns of habitat fragmentation and forest exploitation may be adversely affecting red panda conservation efforts and that landscape-scale effects should be considered when planning conservation actions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10980-021-01357-w.

Movement and dispersal of a habitat specialist in human-dominated landscapes: a case study of the red panda.

BACKGROUND: Habitat specialists living in human-dominated landscapes are likely to be affected by habitat fragmentation and human disturbances more than generalists. But there is a paucity of information on their response to such factors. We examined the effect of these factors on movement patterns of red pandas Ailurus fulgens, a habitat and diet specialist that inhabits the eastern Himalaya. METHODS: We equipped 10 red pandas (six females, four males) with GPS collars and monitored them from September 2019 to March 2020 in Ilam, eastern Nepal. We collected habitat and disturbance data over four seasons. We considered geophysical covariates, anthropogenic factors and habitat fragmentation metrics, and employed linear -mixed models and logistic regression to evaluate the effect of those variables on movement patterns. RESULTS: The median daily distance travelled by red pandas was 756 m. Males travelled nearly 1.5 times further than females (605 m). Males and sub-adults travelled more in the mating season while females showed no seasonal variation for their daily distance coverage. Red pandas were relatively more active during dawn and morning than the rest of the day, and they exhibited seasonal variation in distance coverage on the diel cycle. Both males and females appeared to be more active in the cub-rearing season, yet males were more active in the dawn in the birthing season. Two sub-adult females dispersed an average of 21 km starting their dispersal with the onset of the new moon following the winter solstice. The single subadult male did not disperse. Red pandas avoided roads, small-habitat patches and large unsuitable areas between habitat patches. Where connected habitat with high forest cover was scarce the animals moved more directly than when habitat was abundant. CONCLUSIONS: Our study indicates that this habitat specialist is vulnerable to human disturbances and habitat fragmentation. Habitat restoration through improving functional connectivity may be necessary to secure the long-term conservation of specialist species in a human-dominated landscape. Regulation of human activities should go in parallel to minimize disturbances during biologically crucial life phases. We recommend habitat zonation to limit human activities and avoid disturbances, especially livestock herding and road construction in core areas.

Improved Trapping and Handling of an Arboreal, Montane Mammal: Red Panda Ailurus fulgens.

It is sometimes essential to have an animal in the hand to study some of their ecological and biological characteristics. However, capturing a solitary, cryptic, elusive arboreal species such as the red panda in the wild is challenging. We developed and successfully tested a protocol for tracking, trapping, immobilization, and handling of red pandas in the wild in eastern Nepal. We established a red panda sighting rate of 0.89 panda/day with a capture success rate of 0.6. We trapped and collared one animal in 3.7 days. On average, we took nearly 136 (range 50-317) min to capture an animal after spotting it. Further processing was completed in 38.5 (21-70) min. Before capture, we found it difficult to recognize the sex of the red panda and to differentiate sub-adults above six months from adults. However, body weight, body length, tail length, shoulder height, and chest girth can be used for diagnosis, as these attributes are smaller in sub-adults. Our method is a welfare-friendly way of trapping and handling wild red pandas. We report new morphometric data that could serve as a guide for field identification.

Upstream Regulator Analysis of Wooden Breast Myopathy Proteomics in Commercial Broilers and Comparison to Feed Efficiency Proteomics in Pedigree Male Broilers.

In an effort to understand the apparent trade-off between the continual push for growth performance and the recent emergence of muscle pathologies, shotgun proteomics was conducted on breast muscle obtained at ~8 weeks from commercial broilers with wooden breast (WB) myopathy and compared with that in pedigree male (PedM) broilers exhibiting high feed efficiency (FE). Comparison of the two proteomic datasets was facilitated using the overlay function of Ingenuity Pathway Analysis (IPA) (Qiagen, CA, USA). We focused on upstream regulator analysis and disease-function analysis that provides predictions of activation or inhibition of molecules based on (a) expression of downstream target molecules, (b) the IPA scientific citation database. Angiopoeitin 2 (ANGPT2) exhibited the highest predicted activation Z-score of all molecules in the WB dataset, suggesting that the proteomic landscape of WB myopathy would promote vascularization. Overlaying the FE proteomics data on the WB ANGPT2 upstream regulator network presented no commonality of protein expression and no prediction of ANGPT2 activation. Peroxisome proliferator coactivator 1 alpha (PGC1α) was predicted to be inhibited, suggesting that mitochondrial biogenesis was suppressed in WB. PGC1α was predicted to be activated in high FE pedigree male broilers. Whereas RICTOR (rapamycin independent companion of mammalian target of rapamycin) was predicted to be inhibited in both WB and FE datasets, the predictions were based on different downstream molecules. Other transcription factors predicted to be activated in WB muscle included epidermal growth factor (EGFR), X box binding protein (XBP1), transforming growth factor beta 1 (TGFB1) and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). Inhibitions of aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT) and estrogen related receptor gamma (ESRRG) were also predicted in the WB muscle. These findings indicate that there are considerable differences in upstream regulators based on downstream protein expression observed in WB myopathy and in high FE PedM broilers that may provide additional insight into the etiology of WB myopathy.

A low-density SNP genotyping panel for the accurate prediction of cattle breeds.

Genomic tools to better define breed composition in agriculturally important species have sparked scientific and commercial industry interest. Knowledge of breed composition can inform multiple scientifically important decisions of industry application including DNA marker-assisted selection, identification of signatures of selection, and inference of product provenance to improve supply chain integrity. Genomic tools are expensive but can be economized by deploying a relatively small number of highly informative single-nucleotide polymorphisms (SNP) scattered evenly across the genome. Using resources from the 1000 Bull Genomes Project we established calibration (more stringent quality criteria; N = 1,243 cattle) and validation (less stringent; N = 864) data sets representing 17 breeds derived from both taurine and indicine bovine subspecies. Fifteen successively smaller panels (from 500,000 to 50 SNP) were built from those SNP in the calibration data that increasingly satisfied 2 criteria, high differential allele frequencies across the breeds as measured by average Euclidean distance (AED) and high uniformity (even spacing) across the physical genome. Those SNP awarded the highest AED were in or near genes previously identified as important signatures of selection in cattle such as LCORL, NCAPG, KITLG, and PLAG1. For each panel, the genomic breed composition (GBC) of each animal in the validation dataset was estimated using a linear regression model. A systematic exploration of the predictive accuracy of the various sized panels was then undertaken on the validation population using 3 benchmarking approaches: (1) % error (expressed relative to the estimated GBC made from over 1 million SNP), (2) % breed misassignment (expressed relative to each individual's breed recorded), and (3) Shannon's entropy of estimated GBC across the 17 target breeds. Our analyses suggest that a panel of just 250 SNP represents an adequate balance between accuracy and cost-only modest gains in accuracy are made as one increases panel density beyond this point.

Genome-Wide Co-Expression Distributions as a Metric to Prioritize Genes of Functional Importance.

Genome-wide gene expression analysis are routinely used to gain a systems-level understanding of complex processes, including network connectivity. Network connectivity tends to be built on a small subset of extremely high co-expression signals that are deemed significant, but this overlooks the vast majority of pairwise signals. Here, we developed a computational pipeline to assign to every gene its pair-wise genome-wide co-expression distribution to one of 8 template distributions shapes varying between unimodal, bimodal, skewed, or symmetrical, representing different proportions of positive and negative correlations. We then used a hypergeometric test to determine if specific genes (regulators versus non-regulators) and properties (differentially expressed or not) are associated with a particular distribution shape. We applied our methodology to five publicly available RNA sequencing (RNA-seq) datasets from four organisms in different physiological conditions and tissues. Our results suggest that genes can be assigned consistently to pre-defined distribution shapes, regarding the enrichment of differential expression and regulatory genes, in situations involving contrasting phenotypes, time-series, or physiological baseline data. There is indeed a striking additional biological signal present in the genome-wide distribution of co-expression values which would be overlooked by currently adopted approaches. Our method can be applied to extract further information from transcriptomic data and help uncover the molecular mechanisms involved in the regulation of complex biological process and phenotypes.

Dynamics of Gene Co-expression Networks in Time-Series Data: A Case Study in Drosophila melanogaster Embryogenesis.

Co-expression networks tightly coordinate the spatiotemporal patterns of gene expression unfolding during development. Due to the dynamic nature of developmental processes simply overlaying gene expression patterns onto static representations of co-expression networks may be misleading. Here, we aim to formally quantitate topological changes of co-expression networks during embryonic development using a publicly available Drosophila melanogaster transcriptome data set comprising 14 time points. We deployed a network approach which inferred 10 discrete co-expression networks by smoothly sliding along from early to late development using 5 consecutive time points per window. Such an approach allows changing network structure, including the presence of hubs, modules and other topological parameters to be quantitated. To explore the dynamic aspects of gene expression captured by our approach, we focused on regulator genes with apparent influence over particular aspects of development. Those key regulators were selected using a differential network algorithm to contrast the first 7 (early) with the last 7 (late) developmental time points. This assigns high scores to genes whose connectivity to abundant differentially expressed target genes has changed dramatically between states. We have produced a list of key regulators - some increasing (e.g., Tusp, slbo, Sidpn, DCAF12, and chinmo) and some decreasing (Rfx, bap, Hmx, Awh, and mld) connectivity during development - which reflects their role in different stages of embryogenesis. The networks we have constructed can be explored and interpreted within Cytoscape software and provide a new systems biology approach for the Drosophila research community to better visualize and interpret developmental regulation of gene expression.

The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro.

Anti-angiogenic agents, such as the multi-tyrosine kinase inhibitor sunitinib, are key first line therapies for metastatic clear cell renal cell carcinoma (ccRCC), but their mechanism of action is not fully understood. Here, we take steps towards validating a computational prediction based on differential transcriptome network analysis that phosphorylated adapter RNA export protein (PHAX) is associated with sunitinib drug treatment. The regulatory impact factor differential network algorithm run on patient tissue samples suggests PHAX is likely an important regulator through changes in genome-wide network connectivity. Immunofluorescence staining of patient tumours showed strong localisation of PHAX to the microvasculature consistent with the anti-angiogenic effect of sunitinib. In normal kidney tissue, PHAX protein abundance was low but increased with tumour grade (G1 vs. G3/4; p < 0.01), consistent with a possible role in cancer progression. In organ culture, ccRCC cells had higher levels of PHAX protein expression than normal kidney cells, and sunitinib increased PHAX protein expression in a dose dependent manner (untreated vs. 100 µM; p < 0.05). PHAX knockdown in a ccRCC organ culture model impacted the ability of sunitinib to cause cancer cell death (p < 0.0001 untreated vs. treated), suggesting a role for PHAX in mediating the efficacy of sunitinib.

Association analysis of loci implied in "buffering" epistasis.

The existence of buffering mechanisms is an emerging property of biological networks, and this results in the buildup of robustness through evolution. So far, there are no explicit methods to find loci implied in buffering mechanisms. However, buffering can be seen as interaction with genetic background. Here we develop this idea into a tractable model for quantitative genetics, in which the buffering effect of one locus with many other loci is condensed into a single statistical effect, multiplicative on the total additive genetic effect. This allows easier interpretation of the results and simplifies the problem of detecting epistasis from quadratic to linear in the number of loci. Using this formulation, we construct a linear model for genome-wide association studies that estimates and declares the significance of multiplicative epistatic effects at single loci. The model has the form of a variance components, norm reaction model and likelihood ratio tests are used for significance. This model is a generalization and explanation of previous ones. We test our model using bovine data: Brahman and Tropical Composite animals, phenotyped for body weight at yearling and genotyped at high density. After association analysis, we find a number of loci with buffering action in one, the other, or both breeds; these loci do not have a significant statistical additive effect. Most of these loci have been reported in previous studies, either with an additive effect or as footprints of selection. We identify buffering epistatic SNPs present in or near genes reported in the context of signatures of selection in multi-breed cattle population studies. Prominent among these genes are those associated with fertility (INHBA, TSHR, ESRRG, PRLR, and PPARG), growth (MSTN, GHR), coat characteristics (KIT, MITF, PRLR), and heat resistance (HSPA6 and HSPA1A). In these populations, we found loci that have a nonsignificant statistical additive effect but a significant epistatic effect. We argue that the discovery and study of loci associated with buffering effects allow attacking the difficult problems, among others, of the release of maintenance variance in artificial and natural selection, of quick adaptation to the environment, and of opposite signs of marker effects in different backgrounds. We conclude that our method and our results generate promising new perspectives for research in evolutionary and quantitative genetics based on the study of loci that buffer effect of other loci.

Gene expression identifies metabolic and functional differences between intramuscular and subcutaneous adipocytes in cattle.

BACKGROUND: This study used a genome-wide screen of gene expression to better understand the metabolic and functional differences between commercially valuable intramuscular fat (IMF) and commercially wasteful subcutaneous (SC) fat depots in Bos taurus beef cattle. RESULTS: We confirmed many findings previously made at the biochemical level and made new discoveries. The fundamental lipogenic machinery, such as ACACA and FASN encoding the rate limiting Acetyl CoA carboxylase and Fatty Acid synthase were expressed at 1.6-1.8 fold lower levels in IMF, consistent with previous findings. The FA elongation pathway including the rate limiting ELOVL6 was also coordinately downregulated in IMF compared to SC as expected. A 2-fold lower expression in IMF of ACSS2 encoding Acetyl Coenzyme A synthetase is consistent with utilisation of less acetate for lipogenesis in IMF compared to SC as previously determined using radioisotope incorporation. Reduced saturation of fat in the SC depot is reflected by 2.4 fold higher expression of the SCD gene encoding the Δ9 desaturase enzyme. Surprisingly, CH25H encoding the cholesterol 25 hydroxylase enzyme was ~ 36 fold upregulated in IMF compared to SC. Moreover, its expression in whole muscle tissue appears representative of the proportional representation of bovine marbling adipocytes. This suite of observations prompted quantification of a set of oxysterols (oxidised forms of cholesterol) in the plasma of 8 cattle exhibiting varying IMF. Using Liquid Chromatography-Mass Spectrometry (LC-MS) we found the levels of several oxysterols were significantly associated with multiple marbling measurements across the musculature, but (with just one exception) no other carcass phenotypes. CONCLUSIONS: These data build on our molecular understanding of ruminant fat depot biology and suggest oxysterols represent a promising circulating biomarker for cattle marbling.

Visual Opsin Diversity in Sharks and Rays.

The diversity of color vision systems found in extant vertebrates suggests that different evolutionary selection pressures have driven specializations in photoreceptor complement and visual pigment spectral tuning appropriate for an animal's behavior, habitat, and life history. Aquatic vertebrates in particular show high variability in chromatic vision and have become important models for understanding the role of color vision in prey detection, predator avoidance, and social interactions. In this study, we examined the capacity for chromatic vision in elasmobranch fishes, a group that have received relatively little attention to date. We used microspectrophotometry to measure the spectral absorbance of the visual pigments in the outer segments of individual photoreceptors from several ray and shark species, and we sequenced the opsin mRNAs obtained from the retinas of the same species, as well as from additional elasmobranch species. We reveal the phylogenetically widespread occurrence of dichromatic color vision in rays based on two cone opsins, RH2 and LWS. We also confirm that all shark species studied to date appear to be cone monochromats but report that in different species the single cone opsin may be of either the LWS or the RH2 class. From this, we infer that cone monochromacy in sharks has evolved independently on multiple occasions. Together with earlier discoveries in secondarily aquatic marine mammals, this suggests that cone-based color vision may be of little use for large marine predators, such as sharks, pinnipeds, and cetaceans.

MicroRNA profiling associated with muscle growth in modern broilers compared to an unselected chicken breed.

BACKGROUND: Genetically selected modern broiler chickens have acquired outstanding production efficiency through rapid growth and improved feed efficiency compared to unselected chicken breeds. Recently, we analyzed the transcriptome of breast muscle tissues obtained from modern pedigree male (PeM) broilers (rapid growth and higher efficiency) and foundational Barred Plymouth Rock (BPR) chickens (slow growth and poorer efficiency). This study was designed to investigate microRNAs that play role in rapid growth of the breast muscles in modern broiler chickens. RESULTS: In this study, differential abundance of microRNA (miRNA) was analyzed in breast muscle of PeM and BPR chickens and the results were integrated with differentially expressed (DE) mRNA in the same tissues. A total of 994 miRNA were identified in PeM and BPR chicken lines from the initial analysis of small RNA sequencing data. After filtering and statistical analyses, the results showed miR-2131-5p, miR-221-5p, miR-126-3p, miR-146b-5p, miR-10a-5p, let-7b, miR-125b-5p, and miR-146c-5p up-regulated whereas miR-206 down-regulated in PeM compared to BPR breast muscle. Based on inhibitory regulations of miRNAs on the mRNA abundance, our computational analysis using miRDB, an online software, predicated that 118 down-regulated mRNAs may be targeted by the up-regulated miRNAs, while 35 up-regulated mRNAs appear to be due to a down-regulated miRNA (i.e., miR-206). Functional network analyses of target genes of DE miRNAs showed their involvement in calcium signaling, axonal guidance signaling, and NRF2-mediated oxidative stress response pathways suggesting their involvement in breast muscle growth in chickens. CONCLUSION: From the integrated analyses of differentially expressed miRNA-mRNA data, we were able to identify breast muscle specific miRNAs and their target genes whose concerted actions can contribute to rapid growth and higher feed efficiency in modern broiler chickens. This study provides foundation data for elucidating molecular mechanisms that govern muscle growth in chickens.

RAPID COMMUNICATION: A haplotype information theory method reveals genes of evolutionary interest in European vs. Asian pigs.

Asian and European wild boars were independently domesticated ca. 10,000 yr ago. Since the 17th century, Chinese breeds have been imported to Europe to improve the genetics of European animals by introgression of favorable alleles, resulting in a complex mosaic of haplotypes. To interrogate the structure of these haplotypes further, we have run a new haplotype segregation analysis based on information theory, namely compression efficiency (CE). We applied the approach to sequence data from individuals from each phylogeographic region (n = 23 from Asia and Europe) including a number of major pig breeds. Our genome-wide CE is able to discriminate the breeds in a manner reflecting phylogeography. Furthermore, 24,956 nonoverlapping sliding windows (each comprising 1,000 consecutive SNP) were quantified for extent of haplotype sharing within and between Asia and Europe. The genome-wide distribution of extent of haplotype sharing was quite different between groups. Unlike European pigs, Asian pigs haplotype sharing approximates a normal distribution. In line with this, we found the European breeds possessed a number of genomic windows of dramatically higher haplotype sharing than the Asian breeds. Our CE analysis of sliding windows captures some of the genomic regions reported to contain signatures of selection in domestic pigs. Prominent among these regions, we highlight the role of a gene encoding the mitochondrial enzyme LACTB which has been associated with obesity, and the gene encoding MYOG a fundamental transcriptional regulator of myogenesis. The origin of these regions likely reflects either a population bottleneck in European animals, or selective targets on commercial phenotypes reducing allelic diversity in particular genes and/or regulatory regions.

Molecular regulation of high muscle mass in developing Blonde d'Aquitaine cattle foetuses.

The Blonde d'Aquitaine (BA) is a French cattle breed with enhanced muscularity, partly attributable to a MSTN mutation. The BA m. Semitendinosus has a faster muscle fibre isoform phenotype comprising a higher proportion of fast type IIX fibres compared to age-matched Charolais (CH). To better understand the molecular network of modifications in BA compared to CH muscle, we assayed the transcriptomes of the m. Semitendinosus at 110, 180, 210 and 260 days postconception (dpc). We used a combination of differential expression (DE) and regulatory impact factors (RIF) to compare and contrast muscle gene expression between the breeds. Prominently developmentally regulated genes in both breeds reflected the replacement of embryonic myosin isoforms (MYL4, MYH3) with adult isoforms (MYH1) and the upregulation of mitochondrial metabolism (CKMT2, AGXT2L1) in preparation for birth. However, the transition to a fast, glycolytic muscle phenotype in the MSTN mutant BA is detectable through downregulation of various slow twitch subunits (TNNC1, MYH7, TPM3, CSRP3) beyond 210 dpc, and a small but consistent genome-wide reduction in mRNA encoding the mitoproteome. Across the breeds, NRIP2 is the regulatory gene possessing a network change most similar to that of MSTN.

Proteogenomics Reveals Enriched Ribosome Assembly and Protein Translation in Pectoralis major of High Feed Efficiency Pedigree Broiler Males.

Background: In production animal agriculture, the cost of feed represents 60-70% of the total cost of raising an animal to market weight. Thus, development of viable biomarkers for feed efficiency (FE, g gain/g feed) to assist in genetic selection of breeding stock remains an important goal in commercial breeding programs. Methods: Global gene (cDNA microarray, RNAseq) and protein expression (shotgun proteomics) analyses have been conducted on breast muscle samples obtained from pedigree broiler males (PedM) exhibiting high and low FE phenotypes. Using the entire datasets (i.e., no cutoffs for significance or fold difference in expression) the number of genes or proteins that were expressed numerically higher or lower in the high FE compared to the low FE phenotype for key terms or functions, e.g., ribosomal, mitochondrial ribosomal, tRNA, RNA binding motif, RNA polymerase, small nuclear ribonucleoprotein, and protein tyrosine phosphatase, were determined. Bionomial distribution analysis (exact) was then conducted on these datasets to determine significance between numerically up or down expression. Results: Processes associated with mitochondrial proteome expression (e.g., mitochondrial ribosomal proteins, mitochondrial transcription, mitochondrial tRNA, and translation) were enriched in breast muscle from the high FE compared to the low FE pedigree male broiler phenotype. Furthermore, the high FE phenotype exhibited enrichment of ribosome assembly (e.g., RNA polymerase, mitochondrial and cytosolic ribosomes, small, and heterogeneous nuclear ribonucleoproteins), as well as nuclear transport and protein translation processes compared to the low FE phenotype. Quality control processes (proteosomes and autophagy) were also enriched in the high FE phenotype. In contrast, the low FE phenotype exhibited enrichment of cytoskeletal proteins, protein tyrosine phosphatases, and tyrosine kinases compared to the high FE phenotype. These results suggest that processes of mitochondrial and cytosolic ribosomal construction, activity, and protein translation would be enhanced in high FE breast muscle, and that phosphorylation of tyrosine moieties of proteins could be prolonged in the high compared to low FE phenotype. The results indicate the presence of a proteogenomic architecture that could enhance ribosome construction, protein translation, and quality control processes and contribute to the phenotypic expression of feed efficiency in this PedM broiler model.

Progesterone signalling in broiler skeletal muscle is associated with divergent feed efficiency.

BACKGROUND: We contrast the pectoralis muscle transcriptomes of broilers selected from within a single genetic line expressing divergent feed efficiency (FE) in an effort to improve our understanding of the mechanistic basis of FE. RESULTS: Application of a virtual muscle model to gene expression data pointed to a coordinated reduction in slow twitch muscle isoforms of the contractile apparatus (MYH15, TPM3, MYOZ2, TNNI1, MYL2, MYOM3, CSRP3, TNNT2), consistent with diminishment in associated slow machinery (myoglobin and phospholamban) in the high FE animals. These data are in line with the repeated transition from red slow to white fast muscle fibres observed in agricultural species selected on mass and FE. Surprisingly, we found that the expression of 699 genes encoding the broiler mitoproteome is modestly-but significantly-biased towards the high FE group, suggesting a slightly elevated mitochondrial content. This is contrary to expectation based on the slow muscle isoform data and theoretical physiological capacity arguments. Reassuringly, the extreme 40 most DE genes can successfully cluster the 12 individuals into the appropriate FE treatment group. Functional groups contained in this DE gene list include metabolic proteins (including opposing patterns of CA3 and CA4), mitochondrial proteins (CKMT1A), oxidative status (SEPP1, HIG2A) and cholesterol homeostasis (APOA1, INSIG1). We applied a differential network method (Regulatory Impact Factors) whose aim is to use patterns of differential co-expression to detect regulatory molecules transcriptionally rewired between the groups. This analysis clearly points to alterations in progesterone signalling (via the receptor PGR) as the major driver. We show the progesterone receptor localises to the mitochondria in a quail muscle cell line. CONCLUSIONS: Progesterone is sometimes used in the cattle industry in exogenous hormone mixes that lead to a ~20% increase in FE. Because the progesterone receptor can localise to avian mitochondria, our data continue to point to muscle mitochondrial metabolism as an important component of the phenotypic expression of variation in broiler FE.