TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

Transcriptome Analysis of Campylobacter jejuni and Campylobacter coli during Cold Stress.

Campylobacter spp. are known to cause campylobacteriosis, a bacterial disease that remains a public health threat. Campylobacter spp. are prevalent in retail meat and liver products, and the prolonged survival of Campylobacter in the low temperatures needed for storage is a challenge for food safety. In this study, RNA-seq was used for the analysis of the C. coli HC2-48 (Cc48) and C. jejuni OD2-67 (Cj67) transcriptomes at 4 °C in a nutrient-rich medium (chicken juice, CJ) and Mueller-Hinton broth (MHB) for 0 h, 0.5 h, 24 h and 48 h. Differentially expressed genes (DEGs) involved in flagellar assembly were highly impacted by low temperatures (4 °C) in C. coli HC2-48, whereas genes related to the ribosome and ribonucleoprotein complex were modulated for C. jejuni OD2-67 at 4 °C. Most of the DEGs in cells grown at 4 °C in the two medium formulations were not significantly expressed at different incubation times. Although more DEGs were observed in CJ as compared to MHB in both Campylobacter strains, the absence of common genes expressed at all incubation times indicates that the food matrix environment is not the sole determinant of differential expression in Campylobacter spp. at low temperatures.

Autoantibodies identify primary Sjögren's syndrome in patients lacking serum IgG specific for Ro/SS-A and La/SS-B.

OBJECTIVE: Identify autoantibodies in anti-Ro/SS-A negative primary Sjögren's syndrome (SS). METHODS: This is a proof-of-concept, case-control study of SS, healthy (HC) and other disease (OD) controls. A discovery dataset of plasma samples (n=30 SS, n=15 HC) was tested on human proteome arrays containing 19 500 proteins. A validation dataset of plasma and stimulated parotid saliva from additional SS cases (n=46 anti-Ro+, n=50 anti-Ro-), HC (n=42) and OD (n=54) was tested on custom arrays containing 74 proteins. For each protein, the mean+3 SD of the HC value defined the positivity threshold. Differences from HC were determined by Fisher's exact test and random forest machine learning using 2/3 of the validation dataset for training and 1/3 for testing. Applicability of the results was explored in an independent rheumatology practice cohort (n=38 Ro+, n=36 Ro-, n=10 HC). Relationships among antigens were explored using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) interactome analysis. RESULTS: Ro+ SS parotid saliva contained autoantibodies binding to Ro60, Ro52, La/SS-B and muscarinic receptor 5. SS plasma contained 12 novel autoantibody specificities, 11 of which were detected in both the discovery and validation datasets. Binding to ≥1 of the novel antigens identified 54% of Ro- SS and 37% of Ro+ SS cases, with 100% specificity in both groups. Machine learning identified 30 novel specificities showing receiver operating characteristic area under the curve of 0.79 (95% CI 0.64 to 0.93) for identifying Ro- SS. Sera from Ro- cases of an independent cohort bound 17 of the non-canonical antigens. Antigenic targets in both Ro+ and Ro- SS were part of leukaemia cell, ubiquitin conjugation and antiviral defence pathways. CONCLUSION: We identified antigenic targets of the autoantibody response in SS that may be useful for identifying up to half of Ro seronegative SS cases.

Ancestral origins are associated with SARS-CoV-2 susceptibility and protection in a Florida patient population.

COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The severity of COVID-19 is highly variable and related to known (e.g., age, obesity, immune deficiency) and unknown risk factors. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, e.g., whether individual differences in immunity play a role in patient symptomatology and how much human leukocyte antigen (HLA) contributes to this. To reveal genetic determinants of susceptibility to COVID-19 severity in the population and further explore potential immune-related factors, we performed a genome-wide association study on 284 confirmed COVID-19 patients (cases) and 95 healthy individuals (controls). We compared cases and controls of European (EUR) ancestry and African American (AFR) ancestry separately. We identified two loci on chromosomes 5q32 and 11p12, which reach the significance threshold of suggestive association (p<1x10-5 threshold adjusted for multiple trait testing) and are associated with the COVID-19 susceptibility in the European ancestry (index rs17448496: odds ratio[OR] = 0.173; 95% confidence interval[CI], 0.08-0.36 for G allele; p = 5.15× 10-5 and index rs768632395: OR = 0.166; 95% CI, 0.07-0.35 for A allele; p = 4.25×10-6, respectively), which were associated with two genes, PPP2R2B at 5q32, and LRRC4C at 11p12, respectively. To explore the linkage between HLA and COVID-19 severity, we applied fine-mapping analysis to dissect the HLA association with mild and severe cases. Using In-silico binding predictions to map the binding of risk/protective HLA to the viral structural proteins, we found the differential presentation of viral peptides in both ancestries. Lastly, extrapolation of the identified HLA from the cohort to the worldwide population revealed notable correlations. The study uncovers possible differences in susceptibility to COVID-19 in different ancestral origins in the genetic background, which may provide new insights into the pathogenesis and clinical treatment of the disease.

Dysregulated long non-coding RNA in Sjögren's disease impacts both interferon and adaptive immune responses.

OBJECTIVE: Sjögren's disease (SjD) is an autoimmune disease characterised by inflammatory destruction of exocrine glands. Patients with autoantibodies to Ro/SSA (SjDRo+) exhibit more severe disease. Long non-coding RNAs (lncRNAs) are a functionally diverse class of non-protein-coding RNAs whose role in autoimmune disease pathology has not been well characterised. METHODS: Whole blood RNA-sequencing (RNA-seq) was performed on SjD cases (n=23 Ro/SSA negative (SjDRo-); n=27 Ro/SSA positive (SjDRo+) and healthy controls (HCs; n=27). Bioinformatics and pathway analyses of differentially expressed (DE) transcripts (log2 fold change ≥2 or ≤0.5; padj<0.05) were used to predict lncRNA function. LINC01871 was characterised by RNA-seq analyses of HSB-2 cells with CRISPR-targeted LINC01871 deletion (LINC01871-/ -) and in vitro stimulation assays. RESULTS: Whole blood RNA-seq revealed autoantibody-specific transcription profiles and disproportionate downregulation of DE transcripts in SjD cases relative to HCs. Sixteen DE lncRNAs exhibited correlated expression with the interferon (IFN)-regulated gene, RSAD2, in SjDRo+ (r≥0.65 or ≤-0.6); four antisense lncRNAs exhibited IFN-regulated expression in immune cell lines. LINC01871 was upregulated in all SjD cases. RNA-seq and pathway analyses of LINC01871-/ - cells implicated roles in cytotoxic function, differentiation and IFNγ induction. LINC01871 was induced by IFNγ in a myeloid cell line and regulated by calcineurin/NFAT pathway and T cell receptor (TCR) signalling in primary human T cells. CONCLUSION: LINC01871 influences expression of many immune cell genes and growth factors, is IFNγ inducible, and regulated by calcineurin signalling and TCR ligand engagement. Altered LINC01871 expression may influence the dysregulated T cell inflammatory pathways implicated in SjD.

Genome-wide association study identifies Sjögren's risk loci with functional implications in immune and glandular cells.

Sjögren's disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjögren's cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands.

Blood Plasma Biomarkers for Woody Breast Disease in Commercial Broilers.

Woody breast (WB) myopathy results in poor muscle quality. The increasing incidence of WB over the last several years indicates a need for improved prediction or early diagnosis. We hypothesized that the use of body fluids, including blood, may be more suitable than breast muscle tissue in developing a minimally invasive diagnostic tool for WB detection. To identify potential early-age-biomarkers that may represent the potential onset of WB, blood samples were collected from 100, 4 wks old commercial male broilers. At 8 wks of age, WB conditions were scored by manual palpation. A total of 32 blood plasma samples (eight for each group of WB and non-WB control birds at two time points, 4 wks and 8 wks) were subjected to shotgun proteomics and untargeted metabolomics to identify differentially abundant plasma proteins and metabolites in WB broilers compared to non-WB control (Con) broilers. From the proteomics assay, 25 and 16 plasma proteins were differentially abundant (p < 0.05) in the 4 and 8 wks old samples, respectively, in WB compared with Con broilers. Of those, FRA10A associated CGG repeat 1 (FRAG10AC1) showed >2-fold higher abundance in WB compared with controls. In the 8 wks old broilers, 4 and 12 plasma proteins displayed higher and lower abundances, respectively, in WB compared with controls. Myosin heavy chain 9 (MYH9) and lipopolysaccharide binding protein (LBP) showed more than 2-fold higher abundances in WB compared with controls, while transferrin (TF) and complement C1s (C1S) showed more than 2-fold lower abundances compared with controls. From the untargeted metabolomics assay, 33 and 19 plasma metabolites were differentially abundant in birds at 4 and 8 wks of age, respectively, in WB compared with controls. In 4 wks old broilers, plasma 3-hydroxybutyric acid (3-HB) and raffinose concentrations showed the highest and lowest fold changes, respectively, in WB compared with controls. The blood plasma 3-HB and raffinose concentrations were confirmed with targeted biochemical assays. Blood biomarkers, such as 3-HB and raffinose, may be suitable candidate targets in the prediction of WB onset at early ages.

Identification of SNPs Associated with Stress Response Traits within High Stress and Low Stress Lines of Japanese Quail.

Mitigation of stress is of great importance in poultry production, as chronic stress can affect the efficiency of production traits. Selective breeding with a focus on stress responses can be used to combat the effects of stress. To better understand the genetic mechanisms driving differences in stress responses of a selectively bred population of Japanese quail, we performed genomic resequencing on 24 birds from High Stress (HS) and Low Stress (LS) lines of Japanese quail using Illumina HiSeq 2 × 150 bp paired end read technology in order to analyze Single Nucleotide Polymorphisms (SNPs) within the genome of each line. SNPs are common mutations that can lead to genotypic and phenotypic variations in animals. Following alignment of the sequencing data to the quail genome, 6,364,907 SNPs were found across both lines of quail. 10,364 of these SNPs occurred in coding regions, from which 2886 unique, non-synonymous SNPs with a SNP% ≥ 0.90 and a read depth ≥ 10 were identified. Using Ingenuity Pathway Analysis, we identified genes affected by SNPs in pathways tied to immune responses, DNA repair, and neurological signaling. Our findings support the idea that the SNPs found within HS and LS lines of quail could direct the observed changes in phenotype.

In vitro characterization of chicken gut bacterial isolates for probiotic potentials.

Probiotics often play an important role in improving gut health in chickens through multiple mechanisms, including enhancement of tight junctions, nutrient acquisition, niche colonization, or coaggregation with enteric pathogens. The objective of this study was to characterize lactic acid bacteria (LAB) isolated from the gut of healthy broiler chickens for a number of phenotypes that might be indicative of good probiotic potentials. A total 40 bacterial isolates were isolated from 3-week-old chickens using Man, Rogosa and Sharpe (MRS) agar plates. The bacterial isolates were evaluated in vitro for motility, autoaggregation, pathogen inhibition, pH of overnight culture, growth on different agar plates, and their impact on gut integrity. Selected isolates were genotyped by sequencing the 16S-23S rRNA gene intergenic region. Based on the phenotype and genotype, we identified 20 potential probiotic (PP) isolates that belong to LAB. Multivariate analysis showed that PP isolates were positively correlated with parameters such as growth on MRS agar plate (pH 5.5), pathogen inhibition, and autoaggregation. However, growth on MacConkey agar plates, supernatant pH, motility, and transepithelial electrical resistance were negatively correlated with the PP isolates. Furthermore, in vivo study needs to be performed for evaluation of the utility of these probiotic candidates in poultry production.

Transcriptomics and proteomics reveal a cooperation between interferon and T-helper 17 cells in neuromyelitis optica.

Type I interferon (IFN-I) and T helper 17 (TH17) drive pathology in neuromyelitis optica spectrum disorder (NMOSD) and in TH17-induced experimental autoimmune encephalomyelitis (TH17-EAE). This is paradoxical because the prevalent theory is that IFN-I inhibits TH17 function. Here we report that a cascade involving IFN-I, IL-6 and B cells promotes TH17-mediated neuro-autoimmunity. In NMOSD, elevated IFN-I signatures, IL-6 and IL-17 are associated with severe disability. Furthermore, IL-6 and IL-17 levels are lower in patients on anti-CD20 therapy. In mice, IFN-I elevates IL-6 and exacerbates TH17-EAE. Strikingly, IL-6 blockade attenuates disease only in mice treated with IFN-I. By contrast, B-cell-deficiency attenuates TH17-EAE in the presence or absence of IFN-I treatment. Finally, IFN-I stimulates B cells to produce IL-6 to drive pathogenic TH17 differentiation in vitro. Our data thus provide an explanation for the paradox surrounding IFN-I and TH17 in neuro-autoimmunity, and may have utility in predicting therapeutic response in NMOSD.

Copy number variation study in Japanese quail associated with stress related traits using whole genome re-sequencing data.

Copy number variation (CNV) is a major driving factor for genetic variation and phenotypic diversity in animals. To detect CNVs and understand genetic components underlying stress related traits, we performed whole genome re-sequencing of pooled DNA samples of 20 birds each from High Stress (HS) and Low Stress (LS) Japanese quail lines using Illumina HiSeq 2×150 bp paired end method. Sequencing data were aligned to the quail genome and CNVnator was used to detect CNVs in the aligned data sets. The depth of coverage for the data reached to 41.4x and 42.6x for HS and LS birds, respectively. We identified 262 and 168 CNV regions affecting 1.6 and 1.9% of the reference genome that completely overlapped 454 and 493 unique genes in HS and LS birds, respectively. Ingenuity pathway analysis showed that the CNV genes were significantly enriched to phospholipase C signaling, neuregulin signaling, reelin signaling in neurons, endocrine and nervous development, humoral immune response, and carbohydrate and amino acid metabolisms in HS birds, whereas CNV genes in LS birds were enriched in cell-mediated immune response, and protein and lipid metabolisms. These findings suggest CNV genes identified in HS and LS birds could be candidate markers responsible for stress responses in birds.

Transcriptomic analysis of the development of skeletal muscle atrophy in cancer-cachexia in tumor-bearing mice.

Cancer-cachexia (CC) is a wasting condition directly responsible for 20-40% of cancer-related deaths. The mechanisms controlling development of CC-induced muscle wasting are not fully elucidated. Most investigations focus on the postcachectic state and do not examine progression of the condition. We recently demonstrated mitochondrial degenerations precede muscle wasting in time course progression of CC. However, the extent of muscle perturbations before wasting in CC is unknown. Therefore, we performed global gene expression analysis in CC-induced muscle wasting to enhance understanding of intramuscular perturbations across the development of CC. Lewis lung carcinoma (LLC) was injected into the hind-flank of C57BL6/J mice at 8 wk of age with tumor allowed to develop for 1, 2, 3, or 4 wk and compared with PBS-injected control. Muscle wasting was evident at 4 wk LLC. RNA sequencing of gastrocnemius muscle samples showed widespread alterations in LLC compared with PBS animals with largest differences seen in 4 wk LLC, suggesting extensive transcriptomic alterations concurrent to muscle wasting. Commonly altered pathways included: mitochondrial dysfunction and protein ubiquitination, along with other less studied processes in this condition regulating transcription/translation and cytoskeletal structure. Current findings present novel evidence of transcriptomic shifts and altered cellular pathways in CC-induced muscle wasting.

Whole Genome Resequencing of Arkansas Progressor and Regressor Line Chickens to Identify SNPs Associated with Tumor Regression.

Arkansas Regressor (AR) chickens, unlike Arkansas Progressor (AP) chickens, regress tumors induced by the v-src oncogene. To better understand the genetic factors responsible for this tumor regression property, whole genome resequencing was conducted using Illumina Hi-Seq 2 × 100 bp paired-end read method (San Diego, CA, USA) with AR (confirmed tumor regression property) and AP chickens. Sequence reads were aligned to the chicken reference genome (galgal5) and produced coverage of 11× and 14× in AR and AP, respectively. A total of 7.1 and 7.3 million single nucleotide polymorphisms (SNPs) were present in AR and AP genomes, respectively. Through a series of filtration processes, a total of 12,242 SNPs were identified in AR chickens that were associated with non-synonymous, frameshift, nonsense, no-start and no-stop mutations. Further filtering of SNPs based on read depth ≥ 10, SNP% ≥ 0.75, and non-synonymous mutations identified 63 reliable marker SNPs which were chosen for gene network analysis. The network analysis revealed that the candidate genes identified in AR chickens play roles in networks centered to ubiquitin C (UBC), phosphoinositide 3-kinases (PI3K), and nuclear factor kappa B (NF-kB) complexes suggesting that the tumor regression property in AR chickens might be associated with ubiquitylation, PI3K, and NF-kB signaling pathways. This study provides an insight into genetic factors that could be responsible for the tumor regression property.

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.

Identification and Differential Abundance of Mitochondrial Genome Encoding Small RNAs (mitosRNA) in Breast Muscles of Modern Broilers and Unselected Chicken Breed.

Background: Although small non-coding RNAs are mostly encoded by the nuclear genome, thousands of small non-coding RNAs encoded by the mitochondrial genome, termed as mitosRNAs were recently reported in human, mouse and trout. In this study, we first identified chicken mitosRNAs in breast muscle using small RNA sequencing method and the differential abundance was analyzed between modern pedigree male (PeM) broilers (characterized by rapid growth and large muscle mass) and the foundational Barred Plymouth Rock (BPR) chickens (characterized by slow growth and small muscle mass). Methods: Small RNA sequencing was performed with total RNAs extracted from breast muscles of PeM and BPR (n = 6 per group) using the 1 × 50 bp single end read method of Illumina sequencing. Raw reads were processed by quality assessment, adapter trimming, and alignment to the chicken mitochondrial genome (GenBank Accession: X52392.1) using the NGen program. Further statistical analyses were performed using the JMP Genomics 8. Differentially expressed (DE) mitosRNAs between PeM and BPR were confirmed by quantitative PCR. Results: Totals of 183,416 unique small RNA sequences were identified as potential chicken mitosRNAs. After stringent filtering processes, 117 mitosRNAs showing >100 raw read counts were abundantly produced from all 37 mitochondrial genes (except D-loop region) and the length of mitosRNAs ranged from 22 to 46 nucleotides. Of those, abundance of 44 mitosRNAs were significantly altered in breast muscles of PeM compared to those of BPR: all mitosRNAs were higher in PeM breast except those produced from 16S-rRNA gene. Possibly, the higher mitosRNAs abundance in PeM breast may be due to a higher mitochondrial content compared to BPR. Our data demonstrate that in addition to 37 known mitochondrial genes, the mitochondrial genome also encodes abundant mitosRNAs, that may play an important regulatory role in muscle growth via mitochondrial gene expression control.

Cancer cachexia-induced muscle atrophy: evidence for alterations in microRNAs important for muscle size.

Muscle atrophy is a hallmark of cancer cachexia resulting in impaired function and quality of life and cachexia is the immediate cause of death for 20-40% of cancer patients. Multiple microRNAs (miRNAs) have been identified as being involved in muscle development and atrophy; however, less is known specifically on miRNAs in cancer cachexia. The purpose of this investigation was to examine the miRNA profile of skeletal muscle atrophy induced by cancer cachexia to uncover potential miRNAs involved with this catabolic condition. Phosphate-buffered saline (PBS) or Lewis lung carcinoma cells (LLC) were injected into C57BL/6J mice at 8 wk of age. LLC animals were allowed to develop tumors for 4 wk to induce cachexia. Tibialis anterior muscles were extracted and processed to isolate small RNAs, which were used for miRNA sequencing. Sequencing results were assembled with mature miRNAs, and functions of miRNAs were analyzed by Ingenuity Pathway Analysis. LLC animals developed tumors that contributed to significantly smaller tibialis anterior muscles (18.5%) and muscle cross-sectional area (40%) compared with PBS. We found 371 miRNAs to be present in the muscle above background levels. Of these, nine miRNAs were found to be differentially expressed. Significantly altered groups of miRNAs were categorized into primary functionalities including cancer, cell-to-cell signaling, and cellular development among others. Gene network analysis predicted specific alterations of factors contributing to muscle size including Akt, FOXO3, and others. These results create a foundation for future research into the sufficiency of targeting these genes to attenuate muscle loss in cancer cachexia.

RNA sequencing for global gene expression associated with muscle growth in a single male modern broiler line compared to a foundational Barred Plymouth Rock chicken line.

BACKGROUND: Modern broiler chickens exhibit very rapid growth and high feed efficiency compared to unselected chicken breeds. The improved production efficiency in modern broiler chickens was achieved by the intensive genetic selection for meat production. This study was designed to investigate the genetic alterations accumulated in modern broiler breeder lines during selective breeding conducted over several decades. METHODS: To identify genes important in determining muscle growth and feed efficiency in broilers, RNA sequencing (RNAseq) was conducted with breast muscle in modern pedigree male (PeM) broilers (n = 6 per group), and with an unselected foundation broiler line (Barred Plymouth Rock; BPR). The RNAseq analysis was carried out using Ilumina Hiseq (2 x 100 bp paired end read) and raw reads were assembled with the galgal4 reference chicken genome. With normalized RPM values, genes showing >10 average read counts were chosen and genes showing <0.05 p-value and >1.3 fold change were considered as differentially expressed (DE) between PeM and BPR. DE genes were subjected to Ingenuity Pathway Analysis (IPA) for bioinformatic functional interpretation. RESULTS: The results indicate that 2,464 DE genes were identified in the comparison between PeM and BPR. Interestingly, the expression of genes encoding mitochondrial proteins in chicken are significantly biased towards the BPR group, suggesting a lowered mitochondrial content in PeM chicken muscles compared to BPR chicken. This result is inconsistent with more slow muscle fibers bearing a lower mitochondrial content in the PeM. The molecular, cellular and physiological functions of DE genes in the comparison between PeM and BPR include organismal injury, carbohydrate metabolism, cell growth/proliferation, and skeletal muscle system development, indicating that cellular mechanisms in modern broiler lines are tightly associated with rapid growth and differential muscle fiber contents compared to the unselected BPR line. Particularly, PDGF (platelet derived growth factor) signaling and NFE2L2 (nuclear factor, erythroid 2-like 2; also known as NRF2) mediated oxidative stress response pathways appear to be activated in modern broiler compared to the foundational BPR line. Upstream and network analyses revealed that the MSTN (myostatin) -FST (follistatin) interactions and inhibition of AR (androgen receptor) were predicted to be effective regulatory factors for DE genes in modern broiler line. PRKAG3 (protein kinase, AMP-activated, gamma 3 non-catalytic subunit) and LIPE (lipase E) are predicted as core regulatory factors for myogenic development, nutrient and lipid metabolism. CONCLUSION: The highly upregulated genes in PeM may represent phenotypes of subclinical myopathy commonly observed in the commercial broiler breast tissue, that can lead to muscle hardening, named as woody breast. By investigating global gene expression in a highly selected pedigree broiler line and a foundational breed (Barred Plymouth Rock), the results provide insight into cellular mechanisms that regulate muscle growth, fiber composition and feed efficiency.