Protective Effect and Mechanism of Placenta Extract on Liver.

The placenta contains multiple biologically active substances, which exert antioxidation, anti-inflammatory, immunomodulatory, and delayed aging effects. Its extract can improve hepatic morphology and function: on the one hand, it can reduce liver interstitial collagen deposition, lipogenesis, and inflammatory cell infiltration and improve fibrosis; on the other hand, it can prevent hepatocellular degeneration by scavenging reactive oxygen species (ROS) and inhibiting inflammatory cytokine production, further improve hepatocyte apoptosis and necrosis, and promote hepatocyte regeneration, making it a promising liver-protective agent. Current research on placenta extract (PE) mainly focuses on treating a specific type of liver injury, and there are no systematic reports. Therefore, this review comprehensively summarizes the treatment reports of PE on liver injury and analyzes its mechanism of action.

Effect of anemoside B4 on milk whey in clinical mastitis-affected cows elucidated using tandem mass tag (TMT)-based quantitative proteomics.

Intramuscular injection of anemoside B4 (AB4) has a superior therapeutic effect on clinical mastitis in lactating cows. Here, we explored AB4's effect on milk whey in clinical mastitis-affected cows using proteomics. Among fifty clinical mastitis cows received AB4 administration (0.05 ml/kg/day, for 7 days), twelve healed cows were selected and marked as group T. Twelve clinically heathy cows received the same dose of saline for 7 days, marked as group C. Collected milk whey of group T before and after AB4 administration marked as T1 and T2, respectively. The milk whey of group C after saline injection marked as C1. Milk whey protein changes were detected using tandem mass tag-based quantitative proteomic. We identified 872 quantifiable proteins in the samples. Among them, 511 proteins between T1 and C1, and 361 proteins between T2 and T1 were significantly altered. T1 than C1 had significantly more proteins associated with inflammatory damage and trans-endothelial migration of leukocytes, whereas these proteins were reduced in T2 treated with AB4. Compared with C, proteins associated with fibrin clot degradation and complement system activation were downregulated in T1 but upregulated in T2. In summary, AB4 can exert its therapeutic effect on clinical mastitis in cows mainly by reducing inflammatory damage, activating the complement system, inhibiting trans-endothelial migration of leukocytes, and promoting degradation of milk fibrin clots.

Antioxidant Capacity and Protective Effect of Cow Placenta Extract on D-Galactose-Induced Skin Aging in Mice.

Placental extract has been used for skin care and delaying skin aging. Cow placenta is an abundant resource with a large mass, which has not been harnessed effectively. Cow placenta extract (CPE) has the functions of antioxidation, anti-inflammatory, promoting growth and development, and promoting hair growth. However, little is known about the effect of oral administration of cow placenta extract on skin conditions. Therefore, the present study aimed to investigate the antioxidant capacity of CPE in vitro and in vivo and its protective effect on d-galactose (D-gal) induced skin aging in mice. The results showed that CPE had strong free radical scavenging, reducing and metal chelating activities. CPE can increase the activity of catalase (CAT), glutathione peroxidase (GSH-Px), peroxidase (POD), superoxide dismutase (SOD), and the content of glutathione (GSH), decrease the content of malondialdehyde (MDA). Moreover, CPE can decrease the gene and protein expression of matrix metalloproteinase 1a (MMP-1a) and matrix metalloproteinase 3 (MMP-3) and increase the expression of transforming growth factor-ß (TGF-ß) and tissue inhibitor of metalloproteinase 1 (TIMP-1) of mouse skin. Histopathological analysis showed CPE reduced the collagen damage caused by D-gal, increased collagen synthesis and reduced its degradation to delay skin aging.

Comparative iTRAQ Proteomics Reveals Multiple Effects of Selenium Yeast on Dairy Cows in Parturition.

The effects of prepartum dietary supplementation with selenium yeast on low abundant plasma proteins in postpartum dairy cows are not known. In this study, 24 healthy parturient dairy cows were divided into two groups (group C, a control group, and group T, a selenium treatment group). Low abundance proteins were extracted from plasma samples of calving cows, and 542 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis. Dietary supplementation with selenium yeast caused differential abundance of 48 proteins with a fold change of more than 1.2 or less than 0.83 (p < 0.05); 14 proteins were upregulated and 34 were downregulated. The top five gene ontology (GO) enrichment terms for the differentially expressed proteins were protein homotetramerization (or tetramerization), defense response to bacteria or fungus, acute-phase reactions, nucleotide catabolic process, and positive regulation of lipid metabolic process. All proteins involved in acute-phase reactions were downregulated, indicating that selenium ameliorates systemic inflammation. The vast majority of proteins involved in the defense response to microorganisms were downregulated, thereby affecting innate immunity. The decreased abundance of apolipoprotein A-I and apolipoprotein C-II, critical proteins for positive regulation of lipid metabolism, indicated that selenium may optimize lipid metabolism. The iTRAQ results showed that prenatal supplementation with yeast selenium can relieve systemic inflammation after parturition. Moreover, selenium may reduce the effects of metabolic diseases, which can improve glyconeogenesis and prevent ketosis and fatty liver.

New genotypes and molecular characterization of Enterocytozoon bieneusi in pet birds in Southwestern China.

Enterocytozoon bieneusi, a unicellular enteric microsporidian parasite, can infect humans and a wide range of animals throughout the world. Although E. bieneusi has been identified in many animals, there is no information regarding the genotypes of E. bieneusi in pet birds in China. Birds are important sources of emerging infectious diseases that affect humans, and immunosuppressed individuals can be exposed to potential zoonotic agents shed by birds. The aim of the present study was to determine the prevalence and genotypic diversity of E. bieneusi in pet birds, as well as assessed its zoonotic potential. A total of 387 fecal samples were collected from Psittaciformes (n = 295), Passeriformes (n = 67), and Galliformes (n = 16) from four pet markets in Sichuan province, Southwestern China. The overall prevalence of E. bieneusi in pet birds was 25.1% based on nested polymerase chain reaction analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene (Psittaciformes, 21.7%; Passeriformes, 37.3%; Galliformes, 50.0%). Eight genotypes of E. bieneusi were identified, including five known genotypes (D, SC02, BEB6, CHB1, and MJ5) and three novel genotypes (SCB-I, SCB-II, and SCB-III). In phylogenetic analysis, genotypes D and SC02 and one novel genotype SCB-II were clustered within group 1, genotype BEB6 was classified within group 2, and the remaining genotypes (CHB1, MJ5, SCB-I, and SCB-III) clustered with group 10. To the best of our knowledge, this is the first report of E. bieneusi infection in pet birds in China. Genotypes D, SC02, and BEB6 that have been previously identified in humans, were found in pet birds in this study, suggesting that these pet birds can be a potential source of human microsporidiosis in China.

Genome-wide DNA methylation profile of prepubertal porcine testis.

The biological structure and function of the mammalian testis undergo important developmental changes during prepuberty and DNA methylation is dynamically regulated during testis development. In this study, we generated the first genome-wide DNA methylation profile of prepubertal porcine testis using methyl-DNA immunoprecipitation (MeDIP) combined with high-throughput sequencing (MeDIP-seq). Over 190 million high-quality reads were generated, containing 43642 CpG islands. There was an overall downtrend of methylation during development, which was clear in promoter regions but less so in gene-body regions. We also identified thousands of differentially methylated regions (DMRs) among the three prepubertal time points (1 month, T1; 2 months, T2; 3 months, T3), the majority of which showed decreasing methylation levels over time. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that many genes in the DMRs were linked with cell proliferation and some important pathways in porcine testis development. Our data suggest that DNA methylation plays an important role in prepubertal development of porcine testis, with an obvious downtrend of methylation levels from T1 to T3. Overall, our study provides a foundation for future studies and gives new insights into mammalian testis development.

Transcriptome profiling indicating canine parvovirus type 2a as a potential immune activator.

Canine parvovirus type 2a (CPV-2a) is a variant of CPV-2, which is a highly contagious pathogen causing severe gastroenteritis and death in young dogs. However, how CPV-2 participates in cell regulation and immune response remains unknown. In this study, persistently infected MDCK cells were generated through culture passage of the CPV-2a-infected cells for ten generations. Our study showed that CPV-2a induces cell proliferation arrest and cell morphology alternation before the fourth generation, whereas, the cell morphology returns to normal after five times of passages. PCR detection of viral VP2 gene demonstrated that CPV-2a proliferate with cell passage. An immunofluorescence assay revealed that CPV-2a particles were mainly located in the cell nuclei of MDCK cell. Then transcriptome microarray revealed that gene expression pattern of MDCK with CPV-2a persistent infection is distinct compared with normal cells. Gene ontology annotation and Kyoto Encyclopedia of Genes and Genome pathway analysis demonstrated that CPV-2a infection induces a series of membrane-associated genes expression, including many MHC protein or MHC-related complexes. These genes are closely related to signaling pathways of virus-host interaction, including antigen processing and presentation pathway, intestinal immune network, graft-versus-host disease, and RIG-I-like helicases signaling pathway. In contrast, the suppressed genes mediated by CPV-2a showed low enrichment in any category, and were only involved in pathways linking to synthesis and metabolism of amino acids, which was confirmed by qPCR analysis. Our studies indicated that CPV-2a is a natural immune activator and has the capacity to activate host immune responses, which could be used for the development of antiviral strategy and biomaterial for medicine.

Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase.

Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na(+)/H(+) exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses.

[Effects of retinol on expressions of epidermal growth factor, stem cell factor, colony-stimulating factor 1 and leukemia inhibitory factor in human umbilical cord-derived mesenchymal stem cells].

OBJECTIVE: To investigate effects of retinol on the expressions of epidermal growth factor (EGF), stem cell factor (SCF), colony-stimulating factor 1 (CSF1) and leukemia inhibitory factor (LIF) in cultured human umbilical-derived mesenchymal stem cells (UCMSCs). METHODS: Human UCMSCs were isolated from human umbilical cord and identified for immunophenotypes. The cells were then cultured in DMEM/F12 media supplemented with 12% fetal bovine serum (FBS), 12% FBS+1 µmol/L retinol, 15% knockout serum replacement (KSR) and 15% KSR+ 1 µmol/L retinol. The expressions of the cytokines EGF, SCF, CSF1 and LIF in the cells were detected using RT-PCR and ELISA. RESULTS: The isolated cells exhibited characteristic immunophenotypes of human UCMSCs and expressed EGF, CSF1 and SCF at both mRNA and protein levels but not LIF protein. Retinol (1 µmol/L) significantly promoted the expressions of SCF and CSF1 at both mRNA and protein levels but did not result in changes of EGF and LIF expressions in human UCMSCs. CONCLUSION: Retinol at the concentration of 1 µmol/L can promote expression of SCF and CSF1 in human UCMSCs in vitro.

CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals.

CRISPR/Cas system, which uses CRISPR RNAs (crRNAs) to guide Cas nuclease to silence invading nucleic acids, is self-defense system against exogenous virus or plasmid in bacteria and archaea. Through molecular modification, the typeⅡCRISPR/Cas system has become a highly efficient site-directed genome editing technique, which is simpler than zinc-finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) and easier to be designed and applied. In this review, we summarize the evolutionary history of CRISPR/Cas9 system, the working principle and modification process of type Ⅱ CRISPR/Cas and its application in animal genome modification. We also analyze the existing problems and improvement program of the CRISPR/Cas9 system as well as its application prospect combined with successful cases, which may provide innovative perspectives on improving animal traits and establishing animal models of human diseases.

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca(2+) concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China.

Canine parvovirus (CPV) can cause acute hemorrhagic diarrhea and fatal myocarditis in young dogs. Currently, most studies have focused on the evolution of the VP2 gene, whereas the full-length genome of CPV has been rarely reported. In this study, the whole genomes of CPV-LZ1 and CPV-LZ2 strains prevalent in Northwest China were determined and analyzed in comparison with those of the reference CPVs. The genome sequences of both LZ strains consisted of 5053 nucleotides. CPV-LZ1 and CPV-LZ2 strains were designated as new CPV-2a and CPV-2b, respectively. Sequence alignment analysis results revealed that these two new strains underwent specific unique variations during the process of local adaption. The left non-translated regions of these strains formed a Y-shaped hairpin structure, whereas the right non-translated regions lacked the reiteration of DNA sequence. A phylogenetic tree constructed from 33 whole coding regions of CPVs showed a strong spatial clustering, and these two strains belonged to the Chinese strain cluster lineage. This study provides a method to obtain the full-length genome of CPV. The isolation and characterization of these viruses adds incrementally to the knowledge of the full-length genome of CPV. The results from this study also provide insight into the molecular epidemiology and genetic diversity of the CPV field isolates from Northwest China and can be useful in preventing and controlling CPV infection in this region.

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

[Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases].

Disruption of myostatin (MSTN) gene in pigs may improve porcine lean meat percentage (LMP), and create an animal model for certain human diseases. Using zinc-finger nucleases (ZFNs) technology, MSTN gene was deleted in Wuzhishan miniature pig fibroblasts by transfection of either ZFNs plasmids or ZFNs mRNA in high efficiency. Strikingly, ZFNs encoding mRNA could produce MSTN+/-and MSTN-/- cell colonies with single or double allele deletion by single transfection. Sequencing results demonstrated that 92.18% of the mutations were short fragment deletions or insertions (≤10 bp). Prediction of amino acids sequences indicated that more than half of the mutations cause premature transla-tional-termination codon. MSTN+/+, MSTN+/-, and MSTN-/- cell colonies were used as nuclear donor for somatic cell nuclear transfer (SCNT), and developmental potential of SCNT embryos were measured by the blastocyst rate. The results revealed no significant difference in development competence among the three kinds of reconstructed embryos (14.29% vs. 19.64% vs. 16.13%), which provides the possibility of making myostatin knock out pigs in the future.