Investigation of circulating infectious agents in experimental Beagle dogs of a production colony and three research facilities in China from June 2021 to May 2022.

To understand the epizootiologic characteristics of pathogens and opportunistic infections in one Beagle dog production colony and three research facilities, viruses and mycoplasma were detected in 1777 samples collected from Beagle dogs in China by polymerase chain reaction/reverse transcription polymerase chain reaction, and bacteria were isolated and identified by 16S rRNA sequence analysis. In addition, genotyping of the major circulating viruses was carried out by amplification of gene fragments and homology analysis. Canine coronavirus (CCoV), Escherichia coli, canine parvovirus (CPV), Bordetella bronchiseptica, Clostridium perfringens, Mycoplasma cynos, Klebsiella pneumoniae, Streptococcus canis, canine astrovirus (CaAstV), canine kobuvirus (CaKV), Pseudomonas aeruginosa, Proteus mirabilis, Macrococcus canis, Pasteurella canis, canine bocavirus (CBoV) and canine adenovirus (CAdV) were detected in the samples. Single, double, triple and quadruple infections accounted for 6.6%, 1.4%, 1.2% and 0.96% of samples, respectively. CCoV strains in 81 samples included three genotypes, CCoV-I, CCoV-IIa and CCoV-IIb, by analysis of S gene. The rate of single infection of CCoV-I, CCoV-IIa or CCoV-IIb was 19%, 38% or 7.4% respectively. The double and triple infection rates of CCoV were 32.8% and 2.5% respectively. All CPV strains in 36 samples belonged to CPV-2c. There were three amino acid differences in the Fiber protein of CAdV-positive sample QD2022, compared with the reference strain Toronto A26/61 and the vaccine strain YCA-18. These results suggest that CCoV and CPV are primary infectious agents, and that these two viruses were often identified in mixed infections, or coinfections alongside mycoplasma or other bacteria. These results will provide the basis for improvements in prevention and control of naturally occurring infectious diseases in Beagle dog production colonies and research facilities.

A novel splice variant of human XRN2 gene is mainly expressed in blood leukocyte.

The XRN2 gene (XRN2a) is the human homologue of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for RNA metabolism and cell viability. Xrn2p/Rat1p, product of XRN2/RAT1 gene, functions in the mRNA degradation and processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Here we describe the cloning and characterization of a novel splice variant of the human XRN2 gene (XRN2b). The 3271-bp cDNA encodes a putative protein with 907 amino acid residues, which shares high homology with mouse DHM1 protein. RT-PCR analysis showed that XRN2b was mainly expressed in blood leukocyte tissue, while XRN2a was detected in several human tissues and in human tumor tissues.

Expression pattern and preliminary functional analysis of human CREB4 gene.

cAMP response element-binding (CREB) proteins are a family of mammalian transcription activators that mediate cAMP and calcium-dependent gene expression through the cAMP response element (CRE). CREB4 is a novel member of the human CREB family. RT-PCR showed CREB4 transcripts were found in lung carcinoma LX-1, colon adenocarcinoma CX-1, prastatic adenocarcinoma PC-3, colon carcinoma G1-112, and pancreatic adenocarcinoma G1-103. Constructing CREB4 and CREB(215-395aa) fusion protein with the entire prokaryotic LexA protein respectively disclosed that CREB4 protein functioned as a transcription activator and its N-terminal accounted for the activation ability. Furthermore,a fusion protein of GFP and full-length CREB4 was localized in cytoplasm,whereas the fusion protein of GFP and a deletion mutant lacking the C-terminal putative transmembrane domain was translocated in nucleus. Our results suggested that putative transmembrane domain of CREB4 protein was associated with modulation of its function for the transcriptional activation.

Preparation and cytocompatibility of PLGA scaffolds with controllable fiber morphology and diameter using electrospinning method.

Poly(lactide-co-glycolide) (PLGA) scaffolds prepared by the electrospinning technology were paid more attention in tissue engineering, but detailed investigation on the influence of process parameters on scaffold morphology and fiber diameters as well as distribution remains to be conducted. In the current study, PLGA concentration, electric field strength, and feeding rate were varied to prepare different PLGA scaffolds. It was shown that with the varying of the above parameters, the electrospun porous PLGA scaffolds exhibited fibrous morphology either with/out beads or bead-fiber string. The diameter of nanofibers increased with the PLGA concentration and feeding rate, whereas the electric field strength exerted only minor effects on the average diameter of the nanofibers. The cytocompatibility of the scaffold prepared from optimizing fabrication parameters was evaluated using human fibroblasts. Good cell attachment, fast proliferation rate, characteristic fibroblast morphology with homogeneous distribution was observed on the scaffold. As a whole, this study demonstrated that the fiber morphology and diameter of the electrospun PLGA scaffolds could be tailored by controlling fabrication parameters. And nanofibrous PLGA could act as a biologically preferred scaffold for fibroblasts growth with maintenance of characteristic morphology.

[PDGF-BB initiates vascular smooth muscle-like phenotype differentiation of human bone marrow mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the feasibility of human bone marrow mesenchymal stem cells (hBMSCs) in vitro differentiation into vascular smooth muscle cells with induction of platelet-derived growth Factor BB (PDGF-BB). METHODS: Bone marrow mesenchymal stem cells of adult healthy donors were separated from iliac crest aspiration and expanded in DMEM-LG medium. Cells at passage 1 were transferred to EGM-2 medium containing PDGF-BB (20 ng/ml) and cultured for 14 days. The expression of SM alpha-actin, SM calponin, SMMHC and SM 22alpha were detected by immunofluorescence and observed with fluorescence microscope. mRNA expression of SMalpha-actin, SM calponin, SMMHC as well as SM 22alpha was analyzed by RT-PCR. The method of Western-Blot was applied to determine protein expression of SM 22alpha. Cells with induction were observed for the expression of SM alpha-actin,SM calponin,SMMHC by FACs analysis. RESULTS: With the induction of PDGF-BB, the morphology of cells changed to a spindle fibroblastic appearance. By fluorescence microscope observation, expression of SM alpha-actin, SM calponin and SMMHC was found intracellularly in PDGF-BB treated hBMSCs at 14 days. Western-Blot detection confirmed SM 22alpha expression by 14 days induction. RT-PCR of characteristic vascular smooth muscle cells related genes, such as SM alpha-actin, SM calponin, SMMHC and SM 22alpha revealed differentiation of vascular smooth muscle cells phenotype in monolayer culture upon stimulation with PDGF-BB for 14 days. The positive expression of SM alpha-actin, SM calponin and SMMHC in induced cells was significantly higher than that in non-induced cells (P < 0.05, n=3). CONCLUSION: These results suggested hBMSCs could be differentiated into vascular smooth muscle cell phenotype with PDGF-BB induction in vitro.