HIF-1α and Bcl-2 expression in lung tissue under hypoxia and normoxia conditions.

<p>Data were obtained from hT, hW, nT, and nW chicken tissues at E16, E18, E19, and E20. (A) Quantitative expression analysis of HIF-1α mRNA in embryonic lung at E16, E18, E19, and E20. The stress reaction in hW was robust as compared with the smooth response in hT. Data are expressed as the mean ± SEM for each group. (B) Quantitative expression analysis of <i>bcl-2</i> mRNA in the embryonic lung, heart, brain and liver at E16 and E19. There were no changes in the expression of <i>bcl-2</i> mRNA between the E16 and E19 in each kind of embryonic tissues. Data are expressed as the mean ± SEM for each group. (C) Analysis of HIF-1α and Bcl-2 expression at the protein level in lung tissue (shown in western blot and densitometry value). HIF-1α protein expression increased from E16 to E19, however in different level in hT, hW, nT, and nW group. Bcl-2 protein did show different levels of expression across different time points and different groups. (* <i>P</i><0.05; ** P<0.01; ns  =  not significant).</p

<i>bcl-2</i> is a target gene of miR-15a, but not miR-16 in chicken lung.

<p>(A) miR-15a regulates the translation of <i>bcl-2</i> mRNA through a sequence that is not conserved with human sequence. miR-15a and the binding site in the gga-<i>bcl-2</i> 3′-UTR are shown, but miR-16 shows no target site in this part of the sequence. (B) miR-15a/16 have a consistent target site in human <i>bcl-2</i> 3′-UTR. The miR-15a binding site in the <i>bcl-2</i> 3′-UTR sequence mediates translation repression by miR-15a. (C) The luciferase reporter vector contains two luciferase cDNAs, Renilla luciferase (hRluc) and firefly luciferase (hluc). The <i>bcl-2</i> 3′-UTR was fused to the hRluc cDNA downstream sequence. In co-transfected cells, the miR-15a mimic decreased the expression of hRluc and miR-15a mimic inhibitor rescued hRluc activity; no differences were seen for miR-16. Data are expressed as the mean ± SEM. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Expression of miRNAs in chicken lung tissues.

<p>(A) Total RNA from tissues from E19 chicken embryos was blotted with probes for miR-15a, miR-144 and U6 (loading control). miR-15a and miR-144 were identified in late-stage embryonic lung tissue. In each sample, the total RNA was mixed with samples from 9 chickens. (B) Quantitative expression analysis of miR-15a showed hypoxia-related expression that was affected by both the species and environmental conditions. The hW chicken group was most sensitive relative to other three groups. In the nW chicken group, there was a response at E19. At E19 of the nW chicken group, the expression of miR-15a remained relatively high in the hT chicken group compared with the nT, nW chicken groups and was largely unchanged in the nT chicken group through the whole embryo stages. Data are expressed as the mean ± SEM for each group. (C) Quantitative expression analysis of miR-15a and miR-16 in the embryonic lung, heart, brain and liver at E16 and E19 tissues and were expressed as the mean ± SEM for each group. Under hypoxia stress, miR-15a was more highly expressed at E19 than at E16 in the brain, heart and lung for the hW group and in the lung and brain for the hT group. miR-16 showed a weak response to stress in the embryonic lung (hW). Result statistically different are indicated with an asterisk/s (* <i>P</i><0.05; ** P<0.01; ns : not significant). E16-20  =  embryonic d13-20, respectively. d1, d2, d3  =  the 1^st day after hatching, the 2^nd day after hatching, the 3^rd day after hatching.</p

Analysis of apoptosis in lung specimens from chickens incubated under normal and hypoxic conditions.

<p>From E16 to E18, no TUNEL staining was identified (A–H). At E19, apoptotic cells were localized in the mesenchyme surrounding the atrias and infundibula of the chicken lungs (L, M, N). There was no obvious staining in nT chicken at E19 (K). At higher magnification, staining was clearly seen in the regions between ACs and not in the parabronchi, atrias, or infundibula (arrows in L’, M’, N’). hW staining at E19 (arrow in N’) was clearly darker than that observed in hT or nW lung sections at this stage (arrows in M’, N’). The tube density in hW at E19 was also higher than that in sections from those other two groups. Scale bar  =  200 µm.</p

Immunohistochemical staining (dark brown coloring) for Bcl-2 protein in lung specimens from chickens incubated under normal and hypoxic conditions.

<p>Bcl-2 protein expression in lung samples from nW, hT, and hW gradually increased moderately from E16 to E18 before the onset of lung functioning (E–H), and disappeared at E19. By E18 in nW lungs, weaker Bcl-2 expression was observed relative to hT (G) and hW (H) chicken groups. In nT chicken sections, Bcl-2 protein staining was nearly unchanged throughout the developmental stages examined (A, E, K). To see details of the E18 lung structure, higher-magnification photomicrographs were taken. Bcl-2 staining was detected in the mesenchyme (arrows) around the ACs and not in the infundibula or atrias of chicken lung (F’, G’, H’)”. In the hW section at E18, Bcl-2 staining was strong (arrow in H’), but weaker in hT and nW at this stage (arrows in G’, F’). Scale bar  =  200 µm.</p

Data_Sheet_2_Complete genome sequences of classical swine fever virus: Phylogenetic and evolutionary analyses.PDF

The classical swine fever virus (CSFV) outbreaks cause colossal losses of pigs and drastic economic impacts. The current phylogenetic CSFV groups were determined mainly based on the partial genome. Herein, 203 complete genomic sequences of CSFVs collected worldwide between 1998 and 2018 available on the GenBank database were retrieved for re-genotyping and recombination analysis. The maximum likelihood phylogenetic tree determined two main groups, GI and GII, with multiple sub-genotypes. The “strain 39” (GenBank ID: AF407339), previously identified as belonging to sub-genotypes 1.1 or 2.2 based on the partial sequences, is found to be genetically distinct and independent, forming a new lineage depicted as GI-2.2b. Ten potential natural recombination events were identified, seven of which were collected in China and found involved in the genetic diversity of CSFVs. Importantly, the vaccine strains and highly virulent strains were all involved in the recombination events, which would induce extra challenges to vaccine development. These findings alarm that attenuated vaccines should be applied with discretion and recommend using subunit vaccines in parallel with other preventive strategies for better management of CSFVs.</p

Summary of RNA sequence (RNA-Seq) mapping data.

<p>A: Venn diagrams of the number of genes expressed in each sample. B: The number of detected genes with different expression levels against the range of fragments per kilobase of exon length million mapped reads (FPKM) values.</p

Identification of Genes Related to Growth and Lipid Deposition from Transcriptome Profiles of Pig Muscle Tissue

<div><p>Transcriptome profiles established using high-throughput sequencing can be effectively used for screening genome-wide differentially expressed genes (DEGs). RNA sequences (from RNA-seq) and microRNA sequences (from miRNA-seq) from the tissues of <i>longissimus dorsi</i> muscle of two indigenous Chinese pig breeds (Diannan Small-ear pig [DSP] and Tibetan pig [TP]) and two introduced pig breeds (Landrace [LL] and Yorkshire [YY]) were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to muscle growth and lipid deposition. We obtained 27.18 G clean data through the RNA-seq and detected that 18,208 genes were positively expressed and 14,633 of them were co-expressed in the muscle tissues of the four samples. In all, 315 DEGs were found between the Chinese pig group and the introduced pig group, 240 of which were enriched with functional annotations from the David database and significantly enriched in 27 Gene Ontology (GO) terms that were mainly associated with muscle fiber contraction, cadmium ion binding, response to organic substance and contractile fiber part. Based on functional annotation, we identified 85 DEGs related to growth traits that were mainly involved in muscle tissue development, muscle system process, regulation of cell development, and growth factor binding, and 27 DEGs related to lipid deposition that were mainly involved in lipid metabolic process and fatty acid biosynthetic process. With miRNA-seq, we obtained 23.78 M reads and 320 positively expressed miRNAs from muscle tissues, including 271 known pig miRNAs and 49 novel miRNAs. In those 271 known miRNAs, 20 were higher and 10 lower expressed in DSP-TP than in LL-YY. The target genes of the 30 miRNAs were mainly participated in MAPK, GnRH, insulin and Calcium signaling pathway and others involved cell development, growth and proliferation, etc. Combining the DEGs and the differentially expressed (DE) miRNAs, we drafted a network of 46 genes and 18 miRNAs for regulating muscle growth and a network of 15 genes and 16 miRNAs for regulating lipid deposition. We identified that <i>CAV2</i>, <i>MYOZ2</i>, <i>FRZB</i>, miR-29b, miR-122, miR-145-5p and miR-let-7c, etc, were key genes or miRNAs regulating muscle growth, and <i>FASN</i>, <i>SCD</i>, <i>ADORA1</i>, miR-4332, miR-182, miR-92b-3p, miR-let-7a and miR-let-7e, etc, were key genes or miRNAs regulating lipid deposition. The quantitative expressions of eight DEGs and seven DE miRNAs measured with real-time PCR certified that the results of differential expression genes or miRNAs were reliable. Thus, 18,208 genes and 320 miRNAs were positively expressed in porcine <i>longissimus dorsi</i> muscle. We obtained 85 genes and 18 miRNAs related to muscle growth and 27 genes and 16 miRNAs related to lipid deposition, which provided new insights into molecular mechanism of the economical traits in pig.</p></div

Summary of sequencing reads aligned with the <i>Sus scrofa</i> genome and annotated genes.

<p>DSP, Diannan Small-ear pig; TP, Tibetan pig; LL, Landrace; YY, Yorkshire.</p><p>Summary of sequencing reads aligned with the <i>Sus scrofa</i> genome and annotated genes.</p

Comparison of transcript expression levels between Diannan Small-ear pig-Tibetan pig (DSP-TP) and Landrace-Yorkshire (LL-YY) groups.

<p>The vertical axis represents the Log2 fragments per kilobase of exon per million fragments mapped (FPKM) in the DSP-TP and the horizontal axis represents the Log2 FPKM in the LL-YY. Differentially expressed (DE) miRNAs were filtered using P ≤0.01 and |log2ratio| ≥1 as a threshold. The red points represent upregulated miRNAs, and the green points indicate downregulated miRNAs. The gray spots represent no significant differences between the DSP-TP and LL-YY samples.</p