Transcriptome Analysis of Effects of Folic Acid Supplement on Gene Expression in Liver of Broiler Chickens.

Folic acid is a water-soluble B vitamin, and plays an important role in regulating gene expression and methylation. The liver is the major site of lipid biosynthesis in the chicken. Nevertheless, how gene expression and regulatory networks are affected by folic acid in liver of broilers are poorly understood. This paper conducted the RNA-seq technology on the liver of broilers under folic acid challenge investigation. First, 405 differentially expressed genes (DEGs), including 157 significantly upregulated and 248 downregulated, were detected between the control group (C) and the 5 mg folic acid group (M). Second, 68 upregulated DEGs and 142 downregulated DEGs were determined between C group and 10 mg folic acid group (H). Third, there were 165 upregulated genes and 179 downregulated genes between M and H groups. Of these DEGs, 903 DEGs were successfully annotated in the public databases. The functional classification based on GO and KEEGG showed that "general function prediction only" represented the largest functional classes, "cell cycle" (C vs. M; M vs. H), and "neuroactive ligand-receptor interaction" (C vs. H) were the highest unique sequences among three groups. SNP analysis indicated that numbers of C, M and H groups were 145,450, 146,131, and 123,004, respectively. Total new predicted alternative splicing events in C, M, and H groups were 9,521, 9,328, and 8,929, respectively. A protein-protein interaction (PPI) network was constructed, and the top 10 hub genes were evaluated among three groups. The results of real time PCR indicated that mRNA abundance of PPARγ and FAS in abdominal fat of M and H groups were reduced compared with the C group (P < 0.05). Ultramicroscopy results showed that folic acid could reduce lipid droplets in livers from chickens. Finally, contents of LPL, PPARγ, and FAS in abdominal fat were decreased with the folic acid supplmented diets (P < 0.01). These findings reveal the effects of folic acid supplemention on gene expression in liver of broilers, which can provide information for understanding the molecular mechanisms of folic acid regulating liver lipid metabolism.

Effect of Maternal Folate Deficiency on Growth Performance, Slaughter Performance, and Serum Parameters of Broiler Offspring.

In this study, we aimed to investigate the influence of maternal folate deficiency on the production performance and serum parameters of broiler offspring. The 120 healthy female broilers (30-week-old) were randomly allotted into two groups. The groups were either fed a basal diet supplemented with 2.0 mg/kg folate (NF) or basal diet (FD). The experiment lasted 12 weeks, and 120 fertilized eggs were collected from each group for hatching. In total, 80 chicks were selected from each group and fed under the same conditions. No significant difference was observed in the average daily feed intake, average daily gain, and feed to gain ratio of 21- and 42-day-old broilers between NF and FD groups (P>0.05). Moreover, slaughter performance of 21- and 42-day-old broiler offspring were not affected by the maternal FD. The subcutaneous fat thickness at the age of 21 days increased significantly by maternal FD (P<0.01); however, there was no significant difference between the two groups at 42 days of age (P>0.05). Similarly, no significant differences were detected in the intermuscular fat width, lipid percentage in the liver, breast muscle, and thigh muscle between the NF and FD groups at 21- and 42-days of age (P>0.05). Serum concentrations of MTHFR, DHFR, LEP, IGF2, LPL and HCY in the 21-day-old broilers were not affected by maternal FD (P>0.05), but those of HSL at 21 days of age was enhanced by maternal FD (P<0.05). These findings indicated that maternal folate deficiency had no influence on production performance, slaughter performance, most fat traits of 21- and 42-day-old broiler offspring, and serum parameters of 21-day-old broiler offspring except HSL.

Single nucleotide polymorphism rs737028527 (G>A) affect miR-1b-3p biogenesis and effects on chicken egg-laying traits.

The abundance of miR-1b-3p in the chicken ovary is greater after sexual maturation. In the present study there was assessment of whether a single nucleotine polymorphism (SNP) led to an alteration in expression of reproductive traits. The miR-1b-3p abundance was greatest in ovarian follicles The SNP site of rs737028527 (G > A), located in the 734 bp upstream region of pre-miR-1b-3p, was identified in three different chicken breeds. Results from an association analysis of chicken egg-laying traits indicated the SNP was associated with age at first egg production (AFE) and egg number at 32 and 48 weeks (E32, E48; P < 0.01). Hens with genotype AA had an earlier AFE and greater E32 and E48 than hens with other genotypes. The abundance of mature miR-1b-3p in the hens with the GG genotype was larger than those with the AA genotype (P < 0.01), and the luciferase activity of GG genotype promoter was also greater in birds with the AA than GG genotype (P < 0.05). There was inhibition of the production of the transcription factor bound by the specificity protein 1 (Sp1) as a result of the G-to-A mutation, and the luciferase activity of the GG, but not AA, genotype was markedly increased by Sp1. In conclusion, the SNP, rs737028527 (G> A), affected the abundance of mature miR-1b-3p by Sp1 and was associated with chicken egg-laying traits. Data from the present study allow for an increased understanding of the functions and regulation of miR-1b-3p in ovarian follicle development of hens.

Classification of thyroid hormone receptor agonists and antagonists using statistical learning approaches.

In silico models are presented for modeling and predicting thyroid hormone receptor (TR) agonists and antagonists. A data set consisting of 258 compounds is used in the present work. The C4.5, random forest (RF) and support vector machine (SVM) statistical methods were used for evaluation. The performance of the quantitative structure-activity relationships was further validated with fivefold cross-validation and an independent external test set. The C4.5 model is slightly weak, and the prediction accuracies of the agonists and antagonists are 93.2 and 57.8% for cross-validation, respectively, averaging 83.1% of correctly classified compounds in the test set. The RF model possesses an average prediction accuracy of 84.0 and 87.1% for the cross-validation and external validation, respectively. Furthermore, the overall prediction accuracy and the external prediction accuracy are 96.6 and 97.2%, respectively, for the SVM model. The results would validate the reliability of the derived models, further demonstrating that RF and SVM models are useful tools capable of classifying TR-binding ligands as agonists or antagonists.

Identification of differentially expressed genes in broiler offspring under maternal folate deficiency.

Folate plays an important role in DNA and RNA synthesis by donating methyl groups. To investigate the effects of maternal folate deficiency (FD) on the abdominal adipose transcriptome and on the accumulation of lipid droplets in the liver tissue of chicken offspring, differentially expressed genes (DEGs) of FD were identified with digital gene expression tag profiling. Ultramicroscopy suggested that the size of lipid droplets in hepatocytes increased with FD, while the lipid droplets population number was largely not affected. The serum parameters assay showed that the concentrations of MTHFR (476.57 vs. 395.27), DHFR (45.056 vs. 38.952), LPL (50.408 vs. 48.677), HCY (4.354 vs. 3.836), LEP (9.951 vs. 8.673), and IGF2 (1209.4 vs. 1027.7) in offspring serum of the FD group were significantly higher than those of the normal folate (NF) group ( P < 0.01). The 442 DEGs between NF and FD groups were identified by digital gene expression profiling. Considering the DEGs in the FD groups vs. NF groups, 179 genes were upregulated while 263 downregulated, and in particular, 145 upregulated and 214 downregulated DEGs were successfully annotated with the nonredundant database. Gene Ontology analysis showed that FD mainly affected cellular processes, cell part and binding, cell killing, virions, and receptor regulator activity. With pathway analysis, it indicated that 123 unigenes were assigned to 115 KEGG pathways, but only five of 115 these pathways were significantly enriched with P values ≤ 0.05. Taken together, these results provide a foundation for further studying the responses of offspring to maternal FD in breeding chickens.

Genome-wide survey reveals dynamic effects of folate supplement on DNA methylation and gene expression during C2C12 differentiation.

Folic acid supplements taken during pregnancy can prevent neural tube defects and other developmental abnormalities. Here, we explored the effects of folate supplementation on gene expression and DNA methylation during C2C12 differentiation. Based on the folic acid concentration, this study comprised three groups: low folate (L), normal folate (N), and high-folate supplement (H). Our analyses revealed that differentiation and the mRNA expression of the gene myogenin in C2C12 cell were enhanced by folic acid; however, the overall methylation percentage in myogenin promoter between different treatment groups was not significantly different ( P > 0.05). The results of MeDIP-chip showed that hundreds of differentially methylated regions (DMRs) were identified between every two groups in both promoter and CpG islands, respectively. Genes with DMRs between N and L groups were mainly enriched in the processes of cell differentiation and cell development, whereas those with DMRs between H and N groups were frequently enriched in cellular process/cycle and cell metabolic processes. In addition, correlation analysis between methylation profile and expression profile revealed that some genes were regulated by methylation status directly. Together, these analyses suggest that folate deficiency and supplementation can influence the differentiation, genome-wide DNA methylation level and the expression of myogenesis-related genes including myogenin in the C2C12 cell line.

Epistatic adaptive evolution of human color vision.

Establishing genotype-phenotype relationship is the key to understand the molecular mechanism of phenotypic adaptation. This initial step may be untangled by analyzing appropriate ancestral molecules, but it is a daunting task to recapitulate the evolution of non-additive (epistatic) interactions of amino acids and function of a protein separately. To adapt to the ultraviolet (UV)-free retinal environment, the short wavelength-sensitive (SWS1) visual pigment in human (human S1) switched from detecting UV to absorbing blue light during the last 90 million years. Mutagenesis experiments of the UV-sensitive pigment in the Boreoeutherian ancestor show that the blue-sensitivity was achieved by seven mutations. The experimental and quantum chemical analyses show that 4,008 of all 5,040 possible evolutionary trajectories are terminated prematurely by containing a dehydrated nonfunctional pigment. Phylogenetic analysis further suggests that human ancestors achieved the blue-sensitivity gradually and almost exclusively by epistasis. When the final stage of spectral tuning of human S1 was underway 45-30 million years ago, the middle and long wavelength-sensitive (MWS/LWS) pigments appeared and so-called trichromatic color vision was established by interprotein epistasis. The adaptive evolution of human S1 differs dramatically from orthologous pigments with a major mutational effect used in achieving blue-sensitivity in a fish and several mammalian species and in regaining UV vision in birds. These observations imply that the mechanisms of epistatic interactions must be understood by studying various orthologues in different species that have adapted to various ecological and physiological environments.

Genome-wide gene expression profiles in lung tissues of pig breeds differing in resistance to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, typical respiratory symptoms in piglets, and high mortality rate of piglets. In this study, we employed an Affymetrix microarray chip to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited a range of clinical features that typify the disease, whereas the DPL pigs showed only mild signs of the disease. Overall, the DPL group had a lower percentage of CD4(+) cells and lower CD4(+)/CD8(+)ratios than the DLY group (p<0.05). For both IL-10 and TNF-α, the DLY pigs had significantly higher levels than the DPL pigs (p<0.01). The DLY pigs have lower serum IFN-γ levels than the DPL pigs (p<0.01). The serum IgG levels increased slightly from 0 dpi to 7 dpi, and peaked at 14 dpi (p<0.0001). Microarray data analysis revealed 16 differentially expressed (DE) genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The mRNA expression levels of 10 out of the 16 DE genes were validated by real-time quantitative RT-PCR and their fold change was consistent with the result of microarray data analysis. We further analyzed the mRNA expression level of 8 differentially expressed genes between the DPL and DLY pigs for both uninfected and infected groups, and found that TF and USP18 genes were important in underlying porcine resistance or susceptibility to PRRSV.

Molecular characterization and expression analysis of fat mass and obesity-associated gene in rabbit.

Fat mass and obesity-associated (FTO) gene codes for a nuclear protein of the AlkB related nonhaem iron and 2-oxoglutaratedependent oxygenase superfamily, and is involved in animal fat deposition and human obesity. In this work, the molecular characterization and expression features of rabbit (Oryctolagus cuniculus) FTO cDNA were analysed. The rabbit FTO cDNA with a size of 2158 bp was cloned, including 1515 bp of the open reading frame that encoded a basic protein of 504 amino acids. Homologous comparison indicated that the rabbit FTO shared 36.36-91.88% identity with those from other species and phylogenetic analysis showed that the rabbit FTO is closely related to human, but more distantly related to zebrafish. The New Zealand rabbit FTO mRNA was detected in all tissues examined, with the highest levels found in the spleen and the lowest found in the kidney. However, no significant differences were seen in cerebellum, corpora quadrigemina, medulla oblongata and cerebral cortex of commercial adult rabbits. Moreover, mRNA levels of FTO in liver tissues were significantly increased in lactating New Zealand rabbits compared with 70-day-old, 90-day-old and gestating rabbits (P < 0.05). In contrast, FTO mRNA levels were significantly lower in longissimus dorsi muscle of 90-day-old New Zealand rabbits than in 70-day-old rabbits (P <0.05). However, the expression levels of FTO in mammary gland and ovary of gestating and lactating rabbits were not significantly different (P > 0.05).

Different expression patterns of PRRSV mediator genes in the lung tissues of PRRSV resistant and susceptible pigs.

Porcine reproductive and respiratory syndrome (PRRS) has caused severe economic loss in most swine-producing countries. The resistance to PRRS virus (PRRSV) infection varies among pig breeds and lines. In this study, we found that the Chinese Dapulian pigs (DPL) were more resistant to PRRSV than commercial Duroc×Landrace×Yorkshire (DLY) crossbred pigs in that lower rectal temperature and lower PRRSV copy number in the serum were detected in the former. Analysis of the mRNA expression of five PRRSV mediator genes (SIGLEC1, NMMHC-IIA, CD163, VIM and HSPG2) in the lung tissues indicated differences in expression between DLY and DPL pigs. In uninfected porcine lung tissues, the levels of SIGLEC1, NMMHC-IIA, CD163 and VIM genes were significantly higher in DLY than in DPL pigs (P<0.05); in PRRSV-infected pigs, the expression levels of NMMHC-IIA and CD163 mRNA were significantly higher in DPL pigs compared to uninfected ones (P<0.05), whereas these levels were not different in DLY pigs or between infected DPL and DLY pigs. Thus, the different expression of PRRSV mediator genes is likely related to pig resistance to PRRSV.

Effect of dietary betaine supplementation on lipogenesis gene expression and CpG methylation of lipoprotein lipase gene in broilers.

Experiments were conducted to investigate the effect of betaine supplementation on mRNA expression levels of lipogenesis genes and CpG methylation of lipoprotein lipase gene (LPL) in broilers. From 22 days of age, 78 broilers were feed basal diet without betaine and basal diet supplemented with 0.1% betaine, respectively, and at 56 and 66 days of age, the traits of 15 chickens (7 males and 8 females) of each group were recorded and abdominal fat pads were collected. The mRNA expression levels of several lipogenesis gene were analyzed by semi-quantitative RT-PCR and real-time quantitative RT-PCR (qPCR), respectively. The CpG methylation profile at the promoter region of LPL gene in 66-day-old broilers was determined by bisulfite sequencing. The average daily gain and percent abdominal fat traits were slightly improved in 56-day-old and 66-day-old broilers after dietary supplementation of betaine to diet. After adding 0.1% betaine to diet, the mRNA levels of fatty acid synthase (FAS) and adipocyte-type fatty acid-binding protein genes in abdominal adipose were significantly decreased in 56-day-old broilers, and those of LPL and FAS genes in abdominal adipose were significantly decreased in 66-day-old broilers comparing with the control group (P < 0.05 and P < 0.001). Moreover, in 66-day-old broilers fed 0.1% betaine diet, a different CpG methylation pattern was observed: the CpG dinucleotides of 1st, 6th, 7th, 8th and from 10th to 50th were less methylated; however, those of 2nd, 5th and 9th were more heavily methylated. The results suggest that transcription of some lipogenesis genes was decreased by betaine supplementation and betaine may decrease LPL mRNA expression by altering CpG methylation pattern on LPL promoter region.

Molecular characterization and chromosomal mapping of porcine brain and muscle Arnt-like protein-1 gene.

As a transcription factor regulating circadian rhythm, brain and muscle Arnt-like protein-1 (BMAL1) plays an important role in lipid homeostasis. The Chinese indigenous and western pig breeds show marked difference in fat deposition, the structure and function of porcine BMAL1 (pBMAL1) between them might be different. In present study, the molecular characteristics and chromosomal location of pBMAL1 were analyzed. The results indicated that pBMAL1 cDNA had a coding region of 1,878 bp and shared 94.36, 89.85 and 89.79% identity with human, mouse and rat BMAL1, respectively, and the pBMAL1 protein had 99.20, 98.24 and 97.92% identity to those of human BMAL1b, mouse BMAL1b and rat BMAL1b, respectively. Compared with other mammals, pBMAL1 was more closely related to human BMAL1. The expression of pBMAL1 was detected in kidney, stomach, spleen, bladder, gallbladder, lumbar spinal cord, medulla oblongata, heart, longissimus dorsi muscle, liver, small intestine, large intestine, lung and backfat tissues. In adipose tissues, it was detected in mesentery fat, leaf fat, caul fat, backfat and cardiac fat, however, the expression level was not significantly different. Alternative usage of exon 2 was revealed to result in two pBMAL1 transcripts. Finally, by using a whole genome porcine radiation hybrid (RH) panel (IMpRH), the pBMAL1 gene was mapped to SSC 2p11-q21.