Cardiac Disease Associated Genetic Variants in Yi Nationality in Regions with High Incidence of Yunnan Sudden Unexplained Death.

ABSTRACT: Objective To explore the association of cardiac disease associated genetic variants and the high incidence of Yunnan sudden unexplained death （YNSUD） in Yi nationality. Methods The genomic DNA was extracted from peripheral blood samples collected from 205 Yi villagers from YNSUD aggregative villages （inpatient group） and 197 healthy Yi villagers from neighboring villages （control group）. Fifty-two single nucleotide variants （SNVs） of 25 cardiac disease associated genes were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. The SPSS 17.0 was used to analyze data. The pathogenicities of variants with differences between the two groups that have statistical significance were predicted by protein function prediction software PolyPhen-2 and SIFT. All villagers from inpatient group were given electrocardiogram （ECG） examination using a 12-lead electrocardiograph. Results The allele frequency and the genotype frequency of missense mutation DSG2 （rs2278792, c.2318G>A, p.R773K） of pathogenic genes of arrhythmogenic right ventricular cardiomyopathy （ARVC） in inpatient group was higher than that in control group （P<0.05）. Abnormal ECG changes were detected in 71 individuals （34.6%） in the inpatient group, among which 54 individuals carried R773K mutation, including clockwise （counterclockwise） rotation, left （right） axis deviation, ST segment and T wave alteration and heart-blocking. Conclusion Definite pathogenic mutations have not been found in the 52 cardiac disease genes associated SNVs detected in Yi nationality in regions with high incidence of YNSUD. The cause of high incidence of YNSUD in Yi nationality needs further study.

Biological mechanisms related to differences in residual feed intake in dairy cows.

Residual feed intake (RFI), defined as the difference between an animal's actual feed intake and expected feed intake over a specific period, is an inheritable character of feed conversion efficiency in dairy cows. Research has shown that a lower RFI could improve the profitability of milk production. This study explored variation in RFI by comparing the differences in body size, milk performance, feeding behavior, and serum metabolites in 29 Holstein cows in mid lactation. The cows were selected from a total of 84 animals based on their RFI following feedlot tests. Selected cows were ranked into high RFI (RFI >1 SD above the mean, n=14) and low RFI (RFI<1 SD below the mean, n=15). The low RFI cows (more efficient) consumed 1.59 kg/day less dry matter than the high RFI group (P<0.01), while they produced nearly equal 4% fat-corrected milk. The milk : feed ratio was higher for the low RFI group than for the high RFI group (P<0.05). The levels of milk protein (P<0.01), total solids (P<0.05), and nonfat solids (P<0.05) were also higher for the low RFI group, whereas milk urea nitrogen was lower (P<0.01). The daily feeding duration was shorter for the low RFI group than for the high RFI group (P<0.01). No significant differences were found in levels of glucose, ß-hydroxybutyrate, prolactin, insulin, IGF-1, growth hormone or ghrelin, but the level of neuropeptide Y was higher (P<0.01) and levels of leptin and non-esterified fatty acid (P<0.05) were lower for the low RFI group than for the high RFI group. There were substantial differences between cows with different RFI, which might affect the efficiency of milk protein metabolism and fat mobilization.

Gene expression profiling of hormonal regulation related to the residual feed intake of Holstein cattle.

An accumulation of over a decade of research in cattle has shown that genetic selection for decreased residual feed intake (RFI), defined as the difference between an animal's actual feed intake and its expected feed intake, is a viable option for improving feed efficiency and reducing the feed requirements of herds, thereby improving the profitability of cattle producers. Hormonal regulation is one of the most important factors in feed intake. To determine the relationship between hormones and feed efficiency, we performed gene expression profiling of jugular vein serum on hormonal regulation of Chinese Holstein cattle with low and high RFI coefficients. 857 differential expression genes (from 24683 genes) were found. Among these, 415 genes were up-regulated and 442 genes were down-regulated in the low RFI group. The gene ontology (GO) search revealed 6 significant terms and 64 genes associated with hormonal regulation, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) selected the adipocytokine signaling pathway, insulin signaling pathway. In conclusion, the study indicated that the molecular expression of genes associated with hormonal regulation differs in dairy cows, depending on their RFI coefficients, and that these differences may be related to the molecular regulation of the leptin-NPY and insulin signaling pathways.

Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells.

Bone morphogenetic protein-7 (BMP-7) and SOX9 are important transcription factors in chondrogenesis. In this study, we examined the biological function of the adeno-associated virus (AAV)-mediated BMP-7 and SOX9 double gene in vitro co-transfection of nucleus pulposus cells of human degenerative intervertebral disc. Human intervertebral disc nucleus pulposus cells were cultured in vitro and subcultured for 5 generations. Using rAAV-BMP-7 and rAAV-SOX9 AAV2-type AAV viruses, the cells were divided into 4 groups: blank normal, BMP-7 transfection, SOX9 transfection, and BMP-7 and SOX9 co-transfection. After 48 h, expression of type II collagen and its mRNA in nucleus pulposus cells was determined. The expression of type II collagen in BMP-7 transfection, SOX9 transfection, and co-transfection groups was up-regulated to varying degrees compared to the blank control group. The type II collagen mRNA level expression in the co-transfection group was significantly higher than in other groups (P < 0.05). AAV-mediated BMP-7 and SOX9 in vitro co-transfection can promote the synthesis of type II collagen in nucleus pulposus cells in the human degenerative intervertebral disc. Double-gene therapy has a synergistic effect in the treatment of intervertebral disc degeneration.

Effect of phytosterols on rumen fermentation in vitro.

We investigated the effect of phytosterols on rumen fermentation in vitro using gas syringes as incubators. Phytosterols were dissolved in ethyl acetate (8.3%) and added at various concentrations to the common diet in rumen fluid. In vitro gas production (GP) was recorded after 3, 6, 12, 18, and 24 h incubation. Incubation was stopped at 6, 12, and 24 h and the inoculants were then tested for pH, dry matter digestibility (DMD), microbial protein yield (MCP), lactic acid, NH3-N, and volatile fatty acids (VFAs). GP was consistently higher than the control; particularly, treatments at 12, 18, and 24 h reached extremely significant levels (P < 0.01). Compared to the control group, the pH of ruminal fluid was slightly lower after incubation, and DMD and MCP increased with increasing phytosterol level except for the content of MCP at 6 h, which changed only minimally. Lactate was significantly lower after treatment compared to the control at 12 h (P < 0.01) and 24 h (P < 0.05), while NH3-N at 12 h (P < 0.05) and 24 h (P < 0.01) after treatment decreased significantly. Acetate, propionate, butyrate, and total VFA for all treatments were higher than those of the control, particularly for butyrate at 6 h (P < 0.01). These results suggest that phytosterols modify rumen fermentation by inhibiting released harmful products and promoting the release of beneficial product, which may be useful for improving nutrient utilization and animal health.

RNAi-mediated knockdown of relaxin decreases in vitro proliferation and invasiveness of osteosarcoma MG-63 cells by inhibition of MMP-9.

PURPOSE: The purpose of this study is to determine the role of relaxin knowdown by siRNA transfection in cellular growth and invasion of osteosarcoma MG-63 cells, and discusses the molecular mechanisms of this action. MATERIALS AND METHODS: The expression of relaxin in MG-63 cell was examined by western blot or RT-PCR. To evaluate the biological role of relaxin, proliferation assay (MTT) and invasion assay (BD Matrigel™), apoptosis assay (TUNEL and ELISA) and cell cycle analysis (flow cytometer) were performed after silencing relaxin using siRNA. MMP-9 expressions were analyzed using RT-PCR, western blot and zymography after silencing relaxin. RESULTS: Results showed that the downregulation of relaxin expression by siRNA in human osteosarcoma MG-63 cells significantly inhibited cell proliferation and invasion in vitro. Furthermore, relaxin knockdown led to cell arrest in the G1/G0 phase of the cell cycle, and eventual apoptosis enhancement in MG-63 cells. We provide evidence in our cell model that the relaxin siRNA down-regulated the expression of MMP-9 and the MMP-9 activity, suggesting that relaxin may promote the proliferation, invasion and metastasis of osteosarcoma cells by regulating the expression of MMP-9 and facilitating ECM degradation. CONCLUSIONS: Therefore, siRNA-directed knockdown of relaxin may represent a viable clinical therapy for osteosarcoma.

Co-transfection of adeno-associated virus-mediated human vascular endothelial growth factor165 and transforming growth factor-ß1 into annulus fibrosus cells of rabbit degenerative intervertebral discs.

Intervertebral disc degeneration is a common condition that may lead to low back pain and radiculopathy. Understanding the pathophysiology and cellular and molecular events of degenerative disc disease has resulted in the proposal of a gene therapy approach to halt and reverse disc degeneration. We explored the feasibility of reversing intervertebral disc degeneration using human vascular endothelial growth factor165 (hVEGF165) and transforming growth factor-ß1 (TGF-ß1) gene therapy. hVEGF165 complementary DNA was obtained from pcDNA3(+)-hVEGF165 and cloned into adeno-associated virus (AAV)-pSNAV plasmids to construct the recombinant plasmid, AAV-pSNAV-hVEGF165. After identification through restriction enzyme digestion and DNA sequencing, the AAV-pSNAV-hVEGF165 was transfected into HEK293 cells and vascular endothelial cells. Protein expression of hVEGF165 was detected using a fluorescent immunohistochemical assay, and the effect of hVEGF165 on vascular endothelial cell proliferation was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Packaged AAV-hVEGF165 and AAV-TGF-ß1 were co-transfected into the annulus fibrosus cells of degenerative intervertebral discs. hVEGF165 and TGF-ß1 expression by annulus fibrosus cells and the effect of the co-transfection on the level of collagen type I protein expression by annulus fibrosus cells were detected with Western blot. The results of restriction enzyme digestion and DNA sequencing confirmed that AAV-pSNAV-hVEGF165 plasmids were constructed. The fluorescent immunohistochemical results confirmed hVEGF165 protein expression. The MTT results showed that the hVEGF165 protein promoted vascular endothelial cell proliferation. Biologically active AAV-hVEGF165 and AAV-TGF-ß1 were successfully constructed. Western blot confirmed hVEGF165 and TGF-ß1 expression in annulus fibrosus cells and demonstrated that the level of collagen type I protein expression was significantly higher in annulus fibrosus cells co-transfected with both AAV-hVEGF165 and AAV-TGF-ß1 compared with that in cells transfected with AAV-hVEGF165 or AAV-TGF-ß1 alone. hVEGF165 has a synergistic effect with TGF-ß1 that promotes the expression of collagen type I protein in annulus fibrosus cells from degenerative intervertebral discs.

Investigation of characteristics of feather follicle stem cells and their regeneration potential.

Feather follicles have the extraordinary ability to regenerate and undergo molting cycles. Being tissue-specific stem cells, feather follicle stem cells (FFSCs) have a strong capacity for proliferation and are presumed to be progenitor cells for various epidermal organs. In order to characterize FFSCs and to understand how the feather epidermis and FFSCs produce such a reliable differentiation program resulting in the formation of complex feathers, We developed a culture scheme to select and expand FFSCs from chick feather follicles. FFSCs were examined with cell profiles, mutilpotential differentiation and immunocytochemical staining. FFSCs from a single clone were capable of self-renewal and proliferation. These cells expressed integrin ß1, CD49c, cytokeratin 15 (K15), cytokeratin 19 (K19) and a neural-genic cell marker, nestin, but not a teminal differentiation-related keratinocyte marker, cytokeratin 10 (K10). FFSCs could trans-differentiate into adipocytes, neurocytes and keratinocytes. The formation of micro-feather like structures ex-vivo also revealed the potential of regeneration. These results demonstrate that FFSCs possess the properties of stem/progenitor cells and may therefore serve as a useful model for studying mechanisms of stem cell differentiation and their involvement in organ regeneration.

Proteomic analysis of human bone marrow mesenchymal stem cells transduced with human telomerase reverse transcriptase gene during proliferation.

OBJECTIVES: Previous studies have reported immortalization and tumorigenicity of human mesenchymal stem cells (hMSCs) transduced with exogenous human telomerase reverse transcriptase (hTERT). We also have established a line of hMSCs transduced with hTERT (hTERT-hMSCs) and we have cultured these cells for 290 population doublings (PDs) during which they demonstrated a large proliferation potential but with no tumorigenicity. The aim of this study was to investigate the protein expression profile of hTERT-hMSCs with two-dimensional gel electrophoresis and peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to be able to analyse the effects of exogenous hTERT on protein expression in hMSCs. MATERIALS AND METHODS: We generated proteome maps of primary hMSCs and hTERT-hMSCs at PD 95 and PD 275. RESULTS: A total of 1543 +/- 145 protein spots in gels of primary MSCs at PD 12, 1611 +/- 186 protein spots in gels of hTERT-hMSCs at PD 95 and 1451 +/- 126 protein spots in gels of hTERT-hMSCs at 275 PD were detected. One hundred of these were successfully identified, including 20 which were differentially expressed. CONCLUSIONS: The results suggest that sustaining levels of prohibitin and p53 expression along with differential expression of proteins in hTERT-hMSCs provide an insight into lack of transforming activity of hTERT-hMSCs during cell proliferation.

Comparative study on semen quality of one- and two-year-old ganders during the entire reproductive season.

AIM: To evaluate the characteristics of semen produced by one- and two-years old White Italian ganders during the entire reproductive season, in order to clarify whether the young ganders are responsible for a low fertility rate in young geese. METHODS: Males were kept individually in cages under natural light. Semen was collected by dorso-abdominal massage three times a week and routine examination was performed. RESULTS: The mean ejaculate volume (2.1 and 1.6 mL, respectively) and sperm concentration (323 and 281 x 10(6)/mL, respectively) in one-year-old ganders were higher than those of two-year-old ones. The percentages viable spermatozoa of one- and two-year-old ganders were similar (91.4 and 92.3%, respectively), but the percentage of normally formed viable spermatozoa was significantly higher in the older ganders than in the younger (47.8 and 42.9%, respectively, P < 0.05). CONCLUSION: The semina from one- or two-year-old Ganders were similar in regard to volume, sperm density and sperm motility, but the percentage of normally formed viable spermatozoa, which is critical for fertilization, was significantly higher in the older ganders. It appears that the ganders are responsible for the low fertility rate in young geese.