This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H2O2) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 microM H2O2, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H2O2 treatment. H2O2 damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over-expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.
Animals ; Animals, Newborn ; Blotting, Western/veterinary ; Caspase 3/*genetics/metabolism ; Cattle ; Cattle Diseases/etiology/*genetics/metabolism ; Duodenum/metabolism ; Enteritis/etiology/genetics/metabolism/*veterinary ; Epithelial Cells/metabolism ; *Gene Expression Regulation ; Glycoproteins/*genetics/metabolism ; Hydrogen Peroxide/pharmacology ; Male ; Proto-Oncogene Proteins c-bcl-2/*genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Real-Time Polymerase Chain Reaction/veterinary

OBJECTIVECarnitine, the only carrier for fatty acid to enter mitochondria for oxidation, has significant effects on serum lipids metabolism and male fertility. This study aimed to investigate the correlation of serum carnitine levels with sex and age among healthy Chinese adults in Nanjing.

METHODSWe obtained fasting blood samples from 347 healthy Chinese adults in Nanjing (182 males, 165 females, aged from 20 to 89), determined the levels of serum free, acylcarnitine and total carnitine by high performance liquid chromatography with pre-column ultraviolet derivatization, and analyzed the correlation of serum carnitine levels with the age and sex of the subjects.

RESULTSTotal and free carnitine levels were significantly higher in the males than in the females (P < 0.05) while no statistically significant differences were found in the acylcarnitine level (P > 0.05). Among those aged between 20 to 50 years, levels of serum free and total carnitine were significantly higher in men than in women (P < 0.05) and they increased with age in females, but with no significant differences between males and females older than 50 years. The acylcarnitine level showed no obvious differences in any age and sex groups.

CONCLUSIONSerum free and total carnitine levels are correlated with sex and age in healthy adults.

Adult ; Age Factors ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; Carnitine ; blood ; Chromatography, High Pressure Liquid ; Female ; Humans ; Male ; Middle Aged ; Sex Factors ; Young Adult

Moxibustion therapy is a non-invasive and natural therapy that is accepted by modern society with advantages of effectiveness and low-cost. However, the traditional process of producing hand-made moxa cone is time consuming, complicated, inefficient and difficult for mass production. Consequently, it hardly meets the clinical demands. This seriously reduces the popularization and promotion of traditional therapy with moxa cone. To deal with this problem, a new type of convenient moxa cone maker, which can easily and quickly produce multi-model moxa cones in mass production with constant specifications and uniform density. The mass production process is saving in material and low cost with high production efficiency. Application of the convenient moxa cone maker meets the needs of moxa cone in the clinical and scientific research and contributes to the popularization and promotion of traditional therapy with moxa cone.
Equipment Design ; Moxibustion ; economics ; instrumentation

Objective To assess the impact of postnatal exposure to antibiotics on intestinal microbiome in preterm infants with 16S rDNA sequencing technology.Methods This study was conducted on 19 preterm infants admitted to the neonatal intensive care unit (NICU) at Tongji Hospital immediately after birth from September 2015 to February 2016.Two groups were set up according to the duration of antibiotic exposure (＜3 d,n=10;＞7 d,n=9).Fecal samples were collected from each infant within the first day and 2 or 3 weeks after bitrth.High-throughput sequencer (Hiseq 2500) was used for sequencing,from which information on composition and abundance of species,phylogenetic evolution and bacterial community diversity was obtained.Intergroup differences was analyzed with independent samples t-test or Fisher's exact test.Results (1) No statistically significant difference was found in general information about the infants between the two groups.(2) The intestinal flora in preterm infants was mainly composed of Lactococcus,Enterococcus and Bacillus for both groups before antibiotic treatment (36.41％,23.40％ and 14.98％).The proportions of Lactococcus and Bacillus were decreased significantly (1.73％ and 1.25％,P＜0.01) with Enterococcus becoming the predomiant genus (16.73％) after antibiotic treatment,while the relative proportions of Staphylococcus,Clostridium and Bifidobacterium were raised.(3) The Shannon index was decreased after antibiotic exposure [(2.34±0.84) vs (1.06±0.96) in ＜3 d group,and (2.64± 1.04) vs (0.35±0.36) in ＞7 d group,both P＜0.05],and the other three Alpha diversity indexes,including observed species,Chaol and PD whole tree indexes,were also decreased within each group (all P＜0.05).(4) Bacterial assemblages showed high beta diversity in both groups before the usage of antibiotics,but antibiotic therapy reduced the diversity.(5) Anoism analysis showed significant differences in the composition of intestinal flora within each group before and after antibiotic exposure (R=0.555and 0.733,both P=0.001),but no difference was found between the two groups after antibiotic exposure (R=0.060,P=0.138).Conclusions Antibiotic exposure,even short-term (＜3 d) administration,may significantly change the distribution of intestinal microbiota in preterm infants.Prolonged usage of antibiotics could have detrimental influence on intestinal flora.Therefore,for preterm babies,prescription of antibiotics should be cautious,even short-term empirical usage.

OBJECTIVESix1 and Six4 are expressed in several tumors, and associated with tumor progress and poor prognosis. The aim of this study was to investigate the expression of Six1 and Six4 in esophageal squamous cell carcinoma (ESCC), and to evaluate their correlation with the clinicopathological factors and prognosis.

METHODSTissue microarray technology and immunohistochemical method (EnVision) were used to detect the expression of Six1 and Six4 in the tumor tissues and corresponding adjacent normal epithelium of esophagus from 292 ESCC patients.

RESULTSAmong the 292 ESCC patients, the positive rates of Six1 and Six4 protein expression in tumor tissues were 72.9% (213/292) and 56.2% (164/292), respectively, significantly higher than the expression rate of 33.2% (97/292) and 32.5% (95/292) in adjacent normal epithelium of esophagus (P < 0.05). Chi square test showed that the expression of Six1 protein was related to tumor size, depth of tumor invasion and patient survival status; higher Six4 protein expression level was related to poor differentiation and increased depth of invasion. Single factor Log-rank analysis revealed that gender, TNM stage, Six1 protein expression level were related to the overall survival of ESCC patients (P < 0.05), while the five-year survival rate was significantly higher in the Six1-negative group than the Six1-positive group [51.9% (41/79) vs. 43.7% (93/213)]. Multi-factor Cox proportional risk model analysis showed that TNM stage and positive expression of Six1 were independent prognostic factors for ESCC patients (P < 0.05).

CONCLUSIONSSix1 and Six4 are highly expressed in ESCC. Their expression levels are closely related to the progress and prognosis of ESCC. Over-expression of Six1 is related to poor prognosis in ESCC patients. Thus, Six1 could be used as an important prognostic indicator for ESCC patients.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; surgery ; Esophageal Neoplasms ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Homeodomain Proteins ; metabolism ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Prognosis ; Proportional Hazards Models ; Risk Factors ; Survival Rate ; Trans-Activators ; metabolism ; Tumor Burden

Objective To investigate the effect of different perfusion flow rates on proliferation and osteoblastic differentiation of human mesenchymal stem cells (hMSCs) in large scale β-TCP (tricalcium phosphate) scaffold at perfusion bioreactor. Methods hMSCs isolated from iliac bone marrow aspiration were loaded into large scale β-TCP scaffold and cultured in perfusion bioreactor at the perfusion flow rate of 3, 6 or 9 mL/min for 15 days. The culture media were collected for D-glucose consumption assay every 3 days. After perfusion culture for 15 days, the cell-scaffold composites were harvested for assessment of cell viability by MTT colorimetric method, SEM observation and osteogenic gene expression by real-time PCR. Results The proliferation of hMSCs assayed by daily glucose consumption showed that at early stage of culture, cells proliferated faster at flow rate of 9 mL/min than at 3 or 6 mL/min (P<0.001); while at late stage of culture, cells proliferated faster at flow rate of 6 mL/min (P<0.05). The cell viability indicated that the cell-scaffold composites at flow rate of 6 mL/min exhibited the most viable cells (P<0.001). SEM indicated that all the macropores of the scaffold at different flow rates were filled with cellular layers. All cellular layers at flow rate of 3 mL/min were incompact, but that at 9 mL/min were compact; at flow rate of 6 mL/min, the cellular layers were either compact or incompact. Real-time PCR revealed that after perfusion culture for 15 days, the mRNA expression of osteobalstic genes including ALP and OP, were enhanced significantly at flow rate of 6 and 9 mL/min as compared to that at 3 mL/min (P<0.01); however, the 9 mL/min group presented the higher OC expression than 3 and 6 mL/min group (P<0.001). Conclusions At early stage of perfusion culture, the proliferation of hMSCs was promoted at flow rate of 9 mL/min, while at late stage, there was more viable cells in scaffolds at flow rate of 6 mL/min. The osteoblastic differentiation of hMSCs was facilitated with the increase of perfusion flow rate, which was attributed to the increased flow shear stress.

Objective: To analyze the migration of the HIV/AIDS cases and related factors in Liangshan Yi autonomous prefecture (Liangshan). Methods: According to HIV/AIDS Comprehensive Response Information Management System of China Information System for Disease Control and Prevention, a total of 28 772 HIV/AIDS cases who had follow-up records in Liangshan in 2020 were included in the survey. The migration of the HIV/AIDS cases was described and the related factors were analyzed using multiple logistic regression models, and the migration destinations of the HIV/AIDS cases were mapped. Results: Among the 28 772 HIV/AIDS cases, 20.89% (6 010/28 772) had migration in 2020. Multivariate logistic regression analysis showed that among the HIV/AIDS cases, the migration related factors included being aged 15-24 years (compared with being aged 0-14 years, OR=2.74, 95%CI:2.04-3.69) and ethnic group (compared with Han ethnic group, OR=2.44, 95%CI:2.19-2.72), having education level of junior high school (compared with having education level of primary school or below, OR=1.25, 95%CI:1.14-1.38), being unmarried (compared with being married, OR=1.29, 95%CI:1.20-1.39), being engaged in business services (compared with being engaged in farming, OR=1.96, 95%CI:1.31-2.92), receiving antiviral treatment <1 year (compared with receiving antiviral treatment >3 years, OR=1.42, 95%CI:1.26-1.61), having recent CD4⁺T lymphocytes (CD4) counts >500 cells/μl (compared with having recent CD4 counts <200 cells/μl, OR=1.15, 95%CI:1.03-1.29). The geographical distribution maps showed that among all cities in Sichuan, Xichang (13.26%, 797/6 010) and Chengdu (10.12%,608/6 010) were the main migration destinations of the HIV/AIDS cases, and the provinces outside Sichuan where the HIV/AIDS cases would like to migrate to were mainly Guangdong (18.19%, 1 093/6 010) and Zhejiang provinces (7.67%, 461/6 010) in 2020. The HIV/AIDS cases who migrated where Liangshan, within Sichuan province, and to other provinces accounted for 27.67% (1 663/6 010), 15.34% (922/6 010) and 56.99% (3 425/6 010), respectively. Conclusions: More attention should be paid to the mobility characteristics and the classification management of HIV/AIDS cases according to their characteristics in Liangshan. Timely access to information on changes in the place of work and residence of HIV/AIDS cases should be warranted when they have migration. Good referrals and management for mobility of HIV/AIDS cases in different places should be made to reduce loss to follow-up and improving interventions.
Acquired Immunodeficiency Syndrome/epidemiology* ; Adolescent ; Adult ; Child ; Child, Preschool ; China/epidemiology* ; Ethnicity ; HIV Infections/epidemiology* ; Humans ; Infant ; Infant, Newborn ; Logistic Models ; Marriage ; Young Adult