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.

Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

BACKGROUND: The Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo. RESULTS: Illumina sequencing technology and reference based assembly on Red Jungle Fowl genome sequences were used. Results of genome resequencing of pooled DNA of each 10 BL and SL chickens reached 5.1x and 7.0x coverage, respectively. The total number of SNPs was 4.8 and 5.5 million in BL and SL genome, respectively. Through a series of filtering processes, a total of ~1 million unique SNPs were found in the SL alone. Eventually of the 156 reliable marker SNPs, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, 139 genes were chosen for further analysis. Of these, 14 randomly chosen SNPs were examined for SNP verification by PCR and Sanger sequencing to detect SNP positions in 20 BL and 70 SL chickens. The results of the analysis of the 14 SNPs clearly showed differential frequencies of nucleotide bases in the SNP positions between BL and SL chickens. Bioinformatic analysis showed that the 156 most reliable marker SNPs included genes involved in dermatological diseases/conditions such as ADAMTS13, ASPM, ATP6V0A2, BRCA2, COL12A1, GRM5, LRP2, OBSCN, PLAU, RNF168, STAB2, and XIRP1. Intermolecular gene network analysis revealed that candidate genes identified in SLV play a role in networks centered on protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP). CONCLUSIONS: Various potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the SLV chicken through genome resequencing. The genetic markers and bioinformatic interpretations of amino acid mutations found in SLV chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility.

Early regulation of viral infection reduces inflammation and rescues mx-positive mice from lethal avian influenza infection.

Differing sensitivity of influenza A viruses to antiviral effects of the Myxovirus resistance (Mx) protein implies varying global gene expression profiles in the host. The role of Mx protein during lethal avian influenza (AI) virus infection was examined using Mx1-deficient C57BL/6 (B6-Mx1(-/-)) and congenic Mx1-expressing (B6-Mx1(+/+)) mice infected with a virulent, mouse-adapted avian H5N2 Ab/Korea/ma81/07 (Av/ma81) virus. After infection, B6-Mx1(+/+) mice were completely protected from lethal AI-induced mortality, and exhibited attenuated clinical disease and reduced viral titers and pathology in the lungs, compared with B6-Mx1(-/-) mice. Transcriptional profiling of lung tissues revealed that most of the genes up-regulated after infection are involved in activation of the immune response and host defense. Notably, more abundant and sustained expression of cytokine/chemokine genes was observed up to 3 dpi in B6-Mx1(-/-) mice, and this was associated with excessive induction of cytokines and chemokines. Consequently, massive infiltration of macrophages/monocytes and granulocytes into lung resulted in severe viral pneumonia and potentially contributed to decreased survival of B6-Mx1(-/-) mice. Taken together, our data show that dysregulated gene transcriptional activity corresponded to persistent induction of cytokine/chemokines and recruitment of cytokine-producing cells that promote inflammation in B6-Mx1(-/-) mouse lungs. Thus, we provide additional evidence of the interplay of genetic, molecular, and cellular correlates governed by the Mx1 protein that critically determine disease outcome during lethal AI virus infection.

Bioenergetics in chicken embryo fibroblast cells: evidence of lower proton leak in spontaneously immortalized chicken embryo fibroblasts compared to young and senescent primary chicken embryo fibroblast cells.

A spontaneously immortalized chicken embryo fibroblast (CEF) cell line (DF-1) is known to exhibit faster growth rate and greater sensitivity to oxidative stress compared to the primary parent CEF (pCEF1°) cells. Thus, major objectives of this study were to assess cell bioenergetics in pCEF1° and DF-1 cells under control conditions and in response to 4-hydroxy 2-nonenal (4-HNE) induced oxidative challenge. Cell bioenergetics were assessed by flux analysis of oxygen consumption rate (OCR). Under control conditions, DF-1 cells had higher OCR associated with ATP synthase activity and mitochondrial oxygen reserve capacity as well as lower OCR due to proton leak and non-mitochondrial cytochrome c oxidase activity. In response to 4-HNE (0 to 30 µM), DF-1 cells were more sensitive to oxidant challenge than both young (passage 8) and senescent (passage 19) pCEF1° cells. Both passages 8 and 19 pCEF1° cells exhibited higher proton leak in response to 4-HNE, but this was not observed in DF-1 cells. Inducible proton leak occurs by 4-HNE stimulated activation of uncoupling protein (UCP) and adenine nucleotide translocase (ANT). From mRNA expression data indicated that ANT and avian UCP were down-regulated and up-regulated, respectively, in DF-1 compared to pCEF1° cells. Thus, we hypothesize that DF-1 cells are unable to increase proton leak due to lower expression of ANT, but not avian UCP, and this inability to increase proton leak contributes to greater susceptibility to oxidative stress of DF-1 cells compared to pCEF1° cells.

Microarray analysis of early and late passage chicken embryo fibroblast cells.

Primary cultured cells derived from normal tissue have a limited lifespan due to replicative senescence and show distinct phenotypes such as irreversible cell cycle arrest and enlarged morphology. Studying senescence-associated genetic alterations in chicken cells will provide valuable knowledge of cellular growth characteristics, when compared with normal and rapidly growing cell lines. Microarray analysis of early- and late-passage (passage 4 and 18, respectively) primary chicken embryo fibroblast (CEF) cells was performed with a 4X44K chicken oligo microarray. A total of 1,888 differentially expressed genes were identified with a 2-fold level cutoff that included 272 upregulated and 1,616 downregulated genes in late-passage senescent CEF cells. Bioinformatic analyses were performed using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com). Of the 1,888 differentially expressed genes in senescent CEF cells, 458 were identified as functionally known genes and only 61 genes showed upregulation. Because senescent cells generally showed the deactivated states of most cellular mechanisms for proliferation and energy metabolism, intensified analysis on upregulated genes revealed that the molecular mechanisms in senescent CEF cells are characterized by the suppression of cell cycle and proliferation, progression of cell death including apoptosis, and increased expression of various secreting factors. These regulatory pathways may be opposite to those found in the immortal CEF cell line, such as the DF-1 immortal line. Further comparison of differentially expressed genes between senescent and immortal DF-1 CEF cells showed that 35 genes overlapped and were oppositely regulated. The global gene expression profiles may provide insight into the cellular mechanisms that regulate cellular senescence and immortalization of CEF cells.

Establishment of an immortal chicken embryo liver-derived cell line.

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

Genome-wide host responses against infectious laryngotracheitis virus vaccine infection in chicken embryo lung cells.

BACKGROUND: Infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) infection causes high mortality and huge economic losses in the poultry industry. To protect chickens against ILTV infection, chicken-embryo origin (CEO) and tissue-culture origin (TCO) vaccines have been used. However, the transmission of vaccine ILTV from vaccinated- to unvaccinated chickens can cause severe respiratory disease. Previously, host cell responses against virulent ILTV infections were determined by microarray analysis. In this study, a microarray analysis was performed to understand host-vaccine ILTV interactions at the host gene transcription level. RESULTS: The 44 K chicken oligo microarrays were used, and the results were compared to those found in virulent ILTV infection. Total RNAs extracted from vaccine ILTV infected chicken embryo lung cells at 1, 2, 3 and 4 days post infection (dpi), compared to 0 dpi, were subjected to microarray assay using the two color hybridization method. Data analysis using JMP Genomics 5.0 and the Ingenuity Pathway Analysis (IPA) program showed that 213 differentially expressed genes could be grouped into a number of functional categories including tissue development, cellular growth and proliferation, cellular movement, and inflammatory responses. Moreover, 10 possible gene networks were created by the IPA program to show intermolecular connections. Interestingly, of 213 differentially expressed genes, BMP2, C8orf79, F10, and NPY were expressed distinctly in vaccine ILTV infection when compared to virulent ILTV infection. CONCLUSIONS: Comprehensive knowledge of gene expression and biological functionalities of host factors during vaccine ILTV infection can provide insight into host cellular defense mechanisms compared to those of virulent ILTV.

Understanding mechanisms of vitiligo development in Smyth line of chickens by transcriptomic microarray analysis of evolving autoimmune lesions.

BACKGROUND: The Smyth line (SL) of chicken is an excellent avian model for human autoimmune vitiligo. The etiology of vitiligo is complicated and far from clear. In order to better understand critical components leading to vitiligo development, cDNA microarray technology was used to compare gene expression profiles in the target tissue (the growing feather) of SL chickens at different vitiligo (SLV) states. RESULTS: Compared to the reference sample, which was from Brown line chickens (the parental control), 395, 522, 524 and 526 out of the 44 k genes were differentially expressed (DE) (P ≤ 0.05) in feather samples collected from SL chickens that never developed SLV (NV), from SLV chickens prior to SLV onset (EV), during active loss of pigmentation (AV), and after complete loss of melanocytes (CV). Comparisons of gene expression levels within SL samples (NV, EV, AV and CV) revealed 206 DE genes, which could be categorized into immune system-, melanocyte-, stress-, and apoptosis-related genes based on the biological functions of their corresponding proteins. The autoimmune nature of SLV was supported by predominant presence of immune system related DE genes and their remarkably elevated expression in AV samples compared to NV, EV and/or CV samples. Melanocyte loss was confirmed by decreased expression of genes for melanocyte related proteins in AV and CV samples compared to NV and EV samples. In addition, SLV development was also accompanied by altered expression of genes associated with disturbed redox status and apoptosis. Ingenuity Pathway Analysis of DE genes provided functional interpretations involving but not limited to innate and adaptive immune response, oxidative stress and cell death. CONCLUSIONS: The microarray results provided comprehensive information at the transcriptome level supporting the multifactorial etiology of vitiligo, where together with apparent inflammatory/innate immune activity and oxidative stress, the adaptive immune response plays a predominant role in melanocyte loss.

Genome sequence comparison of two United States live attenuated vaccines of infectious laryngotracheitis virus (ILTV).

This study was conducted to identify unique nucleotide differences in two U.S. chicken embryo origin (CEO) vaccines [LT Blen (GenBank accession: JQ083493) designated as vaccine 1; Laryngo-Vac(®) (GenBank accession: JQ083494) designated as vaccine 2] of infectious laryngotracheitis virus (ILTV) genomes compared to an Australian Serva vaccine reference ILTV genome sequence [Gallid herpesvirus 1 (GaHV-1); GenBank accession number: HQ630064]. Genomes of the two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end reads. Results revealed that few nucleotide differences (23 in vaccine 1; 31 in vaccine 2) were found and indicate that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only eight amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, and US8 in vaccine 1. Similarly, in vaccine 2, eight amino acid differences were found in UL54, UL27, UL28, UL36, UL1, ICP4, US10, and US8. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically close to both the Serva vaccine and 63140/C/08/BR (GenBank accession: HM188407) and are distinct from the two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), which showed close similarity to each other. These data demonstrate the potential of high-throughput sequencing technology to yield insight into the sequence variation of different ILTV strains. This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line.

BACKGROUND: When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. RESULTS: A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. CONCLUSIONS: The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells.

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.

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus.

BACKGROUND: Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 x 44 K Agilent chicken custom oligo microarrays. RESULTS: Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-kappaB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. CONCLUSION: The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections.

Identification and expression analysis of infectious laryngotracheitis virus encoding microRNAs.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a pivotal role in the regulation of gene expression at the post transcriptional level. Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus belonging to the herpesviridae family. It causes an acute respiratory disorder in chicken resulting in high mortality rates. ILTV encoding miRNAs have been identified in cell cultures infected by ILTV. Seven candidates ILTV encoding miRNAs were identified by the 454 FLX genome sequencing method. Five ILTV miRNAs identified in this study were identical to those previously reported by Waidner et al. (Virology 388:128, 2009). Two unique ILTV miRNAs, iltv-miR-I1-3p and iltv-miR-I7-3p, were identified in this study. The iltv-miR-I1-3p is the passenger strand of I1-5p, which was previously known. The iltv-miR-I7-3p showed a perfect match with the complementary passenger strand in contrast to other miRNA species showing imperfect complementarity with the passenger strand. The I7-3p was mapped in the replication origin (oriL) of the palindrome stem loop sequence of the ILTV genome. Expression of all ILTV miRNAs were confirmed by the end point PCR using small RNA libraries generated from either ILTV infected or uninfected control chicken embryo kidney (CEK) cells.

A method for the rapid isolation of virus from cultured cells.

A simple and efficient collection method using hypotonic burst to isolate virions from infected cultured cells is described. Distilled water treatment of avian metapneumovirus (AMPV)-infected cells with thorough mixing and repeated pipeting was considerably faster for virion collection in avian cells compared to the widely used freeze-thaw (F-T) method (30 min vs. 3-4 h). This method was also more effective for virion collection. The total number of virions recovered from AMPV-infected immortal turkey turbinate cells by the novel water lysis method was 3-fold higher than by the F-T method. This simple water lysis method can be applied to virion collection for other RNA viruses such as the paramyxoviruses that are used to infect cultured cells.

Species-specific deletion of the viral attachment glycoprotein of avian metapneumovirus.

The avian metapneumovirus (AMPV) genome encodes the fusion (F), small hydrophobic (SH), and attachment glycoprotein (G) as envelope glycoproteins. The F and G proteins mainly function to allow viral entry into host cells during the early steps of the virus life cycle. The highly variable AMPV G protein is a major determinant for distinguishing virus subtypes. Sequence analysis was used to determine if any differences between avian or mammalian cell propagated subtype C AMPV could be detected for the 1.8kb G gene. As a result, the complete 1.8kb G gene was found to be present when AMPV was propagated in our immortal turkey turbinate (TT-1) cell line regardless of passage number. Surprisingly, AMPV propagated for 15 or more passages in mammalian Vero cells revealed an essentially deleted G gene in the viral genome, resulting in no G gene mRNA expression. Although the Vero cell propagated AMPV genome contained a small 122 nucleotide fragment of the G gene, no other mRNA variants were detected from either mammalian or avian propagated AMPV. The G gene truncation might be caused by cellular molecular mechanisms that are species-specific. The lack of viral gene deletions suggests that avian cell propagated AMPV will provide a better alternative host for live recombinant vaccine development based on a reverse genetics system.

Enzymatic engineering of the porcine genome with transposons and recombinases.

BACKGROUND: Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs. RESULTS: Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons. CONCLUSION: We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.

Establishment of an immortal turkey turbinate cell line suitable for avian metapneumovirus propagation.

Until recently, there has not been a homologous avian cellular substrate which could continuously produce high titer avian metapneumovirus (AMPV); development of such a cell line should provide an excellent model system for studying AMPV infection. We have established a non-tumorigenic immortal turkey turbinate cell line (TT-1) to propagate sufficiently high AMPV titers. Currently, immortal TT-1 cells are growing continuously at 1.2-1.4 population doublings per day and are at passage 160. Kinetic analysis suggests that AMPV can infect and replicate more rapidly in TT-1 compared to Vero cells, although both cell types undergo apoptosis upon infection. The non-tumorigenic, reverse transcriptase negative TT-1 cell line can serve as an excellent homologous cellular substrate for virus propagation.

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.

Acute Heat Stress Alters the Expression of Orexin System in Quail Muscle.

Accumulating evidences indicate that the hypothalamic neuropeptide orexins or hypocretins are involved in stress-induced responses in mammals. Recently, we found that orexin is expressed and secreted from avian muscle cells, however its regulation is still unknown. In this study, we investigated the effect of heat and oxidative stress, the most challenging stressors in poultry production, on the expression of orexin system in quail muscle tissues and myoblast cell lines. Four week-old genetically selected susceptible and resistant Japanese quail (Coturnix coturnix Japonica) lines were exposed to acute heat stress (HS, 37°C for 1.5 h) or maintained at thermoneutral conditions (24°C). Quail myoblast (QM7) cell line was exposed to heat stress (45°C) for 0.5, 1, 2, or 4 h. The control cells were maintained at 37°C. The cells were also treated with several doses of hydrogen peroxide (H2O2, 10-200 µM) or 4-Hydroxynonenal (4-HNE, 10-30 µM) as oxidative stress. Untreated cells were used as controls. Acute HS significantly induced the expression of HSP70 and down-regulated orexin system in both quail muscle tissue and QM7 cells. Similarly, H2O2 but not 4-HNE treatment significantly increased HSP70 protein levels and dysregulated the expression of orexin and its related receptors in a dose-dependent manner in QM7 cells. Transient overexpression of HSP70 down-regulated the expression of orexin system in QM7 cells. Taken together, these data indicate that orexin may be a key player in stress response in avian muscle by demonstrating that heat and oxidative stress alter the expression of orexin system in quail muscle. This effect might be mediated through HSP70. Unraveling the upstream regulators and downstream effectors of orexin in avian muscle merits further in depth investigations.