Smu_195c is a protein with 86 amino acids in Streptococcus mutans, a primary pathogen for human dental caries. The specific function of Smu_195c is still unknown and there are no conserved domains in it. In order to find out its function, the gene encodes Smu_195c was cloned and expressed in E. coli as N-terminally 6*His tagged recombinant protein. Two crystal forms were obtained by the hanging drop method. Form Ⅰ belongs to space group P6122 or P6522 with the unit cell parameters a = b = 62.93 (A), c= 90.63 (A), γ=120° and form Ⅱ belongs to the space group P41212 or P43212 with the unit cell parameters a =b=57.97 (A), c = 103.51 (A).Crystals from the protein with His-tag belong to form Ⅰ, however, crystals from the protein without His-tag belong to both.

Objective: To assess the status of handgrip strength of elderly population from longevity areas in China, and to analyze the correlative factors of handgrip strength of elderly people. Methods: Data from Chinese Longitudinal Healthy Longevity Survey (CLHLS) in 2012 was used, from which1 967 participants aged ≥60 years old with valid data of grip strength value from 8 Chinese longevity areas were included. Information on demographics characteristic, life style and health status was collected using questionnaires. The handgrip strength of both left and right hands were measured by grip dynamometer. The different characteristics of group of participants with different grip strength were compared and then analyzed by adopting the Cumulative odds Logistic regression model to identify main factors associated with hand grip strength. Results: The P₅₀ (P₂₅-P₅₀) of hand grip strength of elderly people from the eight longevity areas was 20 (11-28) kg; The hand grip strength of males was 26 (18-34) kg, which was higher than that of females(14 (9-20) kg) (P<0.001). Cumulative odds Logistic regression model showed that the hand grip strength of females was lower than males, whose β value (95%CI) was-1.22 (-1.43--1.00). The elderly who was at a higher age, smoking, drinking or with anemia, had a comparatively lower handgrip strength, whose β (95%CI) value were separately-0.08(-0.09~-0.07),-0.29(-0.56~-0.02),-0.54(-0.80~-0.28), and-0.41(-0.62~-0.20). And the elderly who had a higher boby mass index, drinking tea and outdoor activities, had a comparatively higher handgrip strength, whose β(95%CI) value were separately 0.28 (0.15-0.40), 0.25(0.03-0.47) and 0.51(0.30-0.71). Conclusion Age and gender were the main correlative factors, lifestyles and physical conditions might also be correlative factors of hand grip strength of the elderly from longevity areas in China.

Deoxycytidylate (dCMP) deaminase is an enzyme belonged to dCMP cyt deam family. The dCMP deaminase from Streptococcus mutans UA159 was cloned and expressed in E. coli, and purified to homogeneity. The FPLC size exclusion chromatography analysis reveals that the S. mutans dCMP deaminase forms hexamer in solution. The protein was crystallized using hanging drop vapour-diffusion method and diffracted to a resolution of 3.1 ?. The diffraction data were collected at BSRF beamline 3W1A. The crystals belong to P213 space group, with unit cell parameters a = b = c = 113.2 ?, ? = ? = ? = 90?. Assuming there are two subunits per asymmetric unit, the Matthews coefficient is 3.6 ?3?Da-1. This is the first crystallization report of the wild-type deoxycytidylate deaminase.

Deoxycytidylate (dCMP) deaminase is an enzyme belonged to dCMP cyt deam family. The dCMP deaminase from Streptococcus mutans UA159 was cloned and expressed in E. coli, and purified to homogeneity. The FPLC size exclusion chromatography analysis reveals that the S. mutans dCMP deaminase forms hexamer in solution. The protein was crystallized using hanging drop vapour-diffusion method and diffracted to a resolution of 3.1 (A). The diffraction data were collected at BSRF beamline3W1A. The crystals belong to P213 space group, with unit cell parameters a = b = c = 113.2(A), α =β = γ = 90°. Assuming there are two subunits per asymmetric unit, the Matthews coefficient is 3.6 (A)3 ·Da-1. This is the first crystallization report of the wild-type deoxycytidylate deaminase.

Objective:To study the effect of the injected bone marrow mesenchymal stem cells (BMSC) on rats with pulmonary fibrosis induced by paraquat (PQ) during different poisoning periods and explore the potential mechanism.Methods:From October to December 2018, BMSCs of SPF SD rats were isolated and purified by whole-bone marrow adherent culture method and cultured to the Third Generation (P3) . The surface antigens CD29, CD90, CD45 and CD34 of P3 BMSC were detected by Flow cytometry, the formation of alkaline phosphatase (ALP) , calcium nodules and fat droplets were observed by ALP, Alizarin Red staining and oil red O staining. At the same time, 36 SPF male rats were randomly divided into 6 groups: NC Group (Blank Control Group, injected with the same amount of saline) and PQ group (PQ model group, injected with 20% PQ solution 18 mg/kg intraperitoneally) , bMSC-A group, BMSC-B group, BMSC-C group and BMSC-D group were injected with BMSC suspension 1×10 6 cells/mice at 3 h、3 d、7 d and 14 d after PQ poisoning. After 28 days, the rats were killed, the lung organ coefficients were calculated, the hydroxyproline (HYP) content in lung tissue was calculated by alkaline hydrolysis, and the lung injury and fibrosis were observed by HE and Masson staining, serum TGF-1、TNF-α、MMP-9 and TIMP-1 were detected by Elisa. Results:High Purity BMSCs were successfully isolated and obtained. The P3 BMSC generation was positive expression of CD29、CD90、and negative expression of CD34、CD45, and had the potential of osteogenic and adipogenic differentiation. The results of HE staining and Masson staining showed that the alveolar structure in NC group was intact and homogeneous, in PQ group, the alveolar structure was severely damaged and a lot of collagen fibers and fibroblasts were deposited, and the degrees of lung injury in each BMSC intervention group were obviously less than in PQ group, in BMSC-A group and BMSC-B group, the degrees of reduction were obvious. Compared with NC group, the Lung organ coefficient, HYP content in lung tissue and TGF-β1, TIMP-1 levels in serum were significantly higher in PQ group ( P<0.05) , while TNF-α and MMP-9 had no significant difference ( P>0.05) . Compared with PQ group, the lung organ Coefficients, HYP, TGF-1 and TIMP-β1 in BMSC-A and BMSC-B groups were lower than those in PQ group ( P<0.05) . The Lung organ coefficients, TGF-β1 and TIMP-1 in BMSC-C and BMSC-D groups were lower than those in PQ group, there was no significant difference ( P>0.05) . Conclusion:Early BMSC injecting can alleviate pulmonary fibrosis induced by PQ. The mechanism may be that BMSC can reduce pulmonary fibrosis through reducing the level of TGF-β1 and regulating the balance of TIMP-1/MMP-9, threrby reducing inflammatory damage and increasing the degradation of extracellular matrix (ECM) .

Objective:To study the effect of the injected bone marrow mesenchymal stem cells (BMSC) on rats with pulmonary fibrosis induced by paraquat (PQ) during different poisoning periods and explore the potential mechanism.Methods:From October to December 2018, BMSCs of SPF SD rats were isolated and purified by whole-bone marrow adherent culture method and cultured to the Third Generation (P3) . The surface antigens CD29, CD90, CD45 and CD34 of P3 BMSC were detected by Flow cytometry, the formation of alkaline phosphatase (ALP) , calcium nodules and fat droplets were observed by ALP, Alizarin Red staining and oil red O staining. At the same time, 36 SPF male rats were randomly divided into 6 groups: NC Group (Blank Control Group, injected with the same amount of saline) and PQ group (PQ model group, injected with 20% PQ solution 18 mg/kg intraperitoneally) , bMSC-A group, BMSC-B group, BMSC-C group and BMSC-D group were injected with BMSC suspension 1×10 6 cells/mice at 3 h、3 d、7 d and 14 d after PQ poisoning. After 28 days, the rats were killed, the lung organ coefficients were calculated, the hydroxyproline (HYP) content in lung tissue was calculated by alkaline hydrolysis, and the lung injury and fibrosis were observed by HE and Masson staining, serum TGF-1、TNF-α、MMP-9 and TIMP-1 were detected by Elisa. Results:High Purity BMSCs were successfully isolated and obtained. The P3 BMSC generation was positive expression of CD29、CD90、and negative expression of CD34、CD45, and had the potential of osteogenic and adipogenic differentiation. The results of HE staining and Masson staining showed that the alveolar structure in NC group was intact and homogeneous, in PQ group, the alveolar structure was severely damaged and a lot of collagen fibers and fibroblasts were deposited, and the degrees of lung injury in each BMSC intervention group were obviously less than in PQ group, in BMSC-A group and BMSC-B group, the degrees of reduction were obvious. Compared with NC group, the Lung organ coefficient, HYP content in lung tissue and TGF-β1, TIMP-1 levels in serum were significantly higher in PQ group ( P<0.05) , while TNF-α and MMP-9 had no significant difference ( P>0.05) . Compared with PQ group, the lung organ Coefficients, HYP, TGF-1 and TIMP-β1 in BMSC-A and BMSC-B groups were lower than those in PQ group ( P<0.05) . The Lung organ coefficients, TGF-β1 and TIMP-1 in BMSC-C and BMSC-D groups were lower than those in PQ group, there was no significant difference ( P>0.05) . Conclusion:Early BMSC injecting can alleviate pulmonary fibrosis induced by PQ. The mechanism may be that BMSC can reduce pulmonary fibrosis through reducing the level of TGF-β1 and regulating the balance of TIMP-1/MMP-9, threrby reducing inflammatory damage and increasing the degradation of extracellular matrix (ECM) .

Objective: To explore the association between biomarkers and activities of daily living (ADL) in the elderly over 65 years old from longevity areas in China. Methods: A total of 2 439 people from 8 longevity areas were included in our baseline survey in 2012. Using questionnaires, body measurements, and blood biochemical examinations, information on demographics characteristic, life style, ADL, blood pressure and biomarkers were collected. Based on these six items of ADL (bathing, dressing, indoor activities, toileting, eating, bowel and bladder control), we constructed a dichotomous indicator for ADL. A respondent was defined as ADL disabled if any difficulty in one or more of the above six activities was reported. Information were collected in the follow-up in 2014 using the same questionnaires and examinations. We excluded information on the elderly who lacked ADL or biomarkers test results or with ADL disability at baseline study. Finally 938 elderly people over 65 years old were included in this analysis. Multivariate logistic regression model was used to analyze the influence factors of ADL disability. Results: During the 2-year follow-up, 100 (10.7%) participants developed into ADL disability, with a rate at 10.7%. Multivariate logistic regression analysis indicated that each year increase in age or each 1 mmHg (1 mmHg=0.133 kPa) increase in systolic blood pressure (SBP) would cause the risk of ADL disability to increase 9% or 1%, whose OR (95%CI) were separately 1.09 (1.06-1.12), 1.01 (1.00-1.02). Han nationality or cognitive impairment increased the risk of ADL disability, whose OR (95%CI) values were separately 4.90 (1.13-21.24), 2.47 (1.44-4.25), while increased lymphocyte count (>1.60×10⁹/L), being married, or participating in recreational activities decreased the risk of ADL disability, whose OR (95%CI) values were separately 0.51 (0.31-0.82), 0.52 (0.28-0.96), 0.43 (0.23-0.80). Conclusion In the elderly elevated lymphocyte count was associated with lower risk of ADL disability. In addition, incresed age, increased SBP, unmarried, Han nationality or cognitive impairment were associated with the increasing risk of ADL disability in older people, while participating in recreational activities would reduce the risk.

Objective: To investigate the influence factors of survival outcome among elderly aged ≥80 years old. Methods: In baseline survey in 2009, 930 participants aged ≥80 years old were enrolled from 7 longevity areas, to collect the information of socioeconomic factors, life style, cognitive function, activities of daily living and diseases, as well as physical examination to test biomarkers of blood and urine. The survival status was followed up at 2012 and 2014 survey. Stepwise Cox proportional hazards models were used to screen influence factors of 5-year survival. Results: During 5 years of follow-up, 571 participants died, 133 participants were lost to follow up, and the all-cause mortality was 63.4%. In stepwise Cox proportional hazards models, male, unmarried, self-reported poor life quality, disability in daily life, cognitive impairment, cardiovascular and cerebrovascular diseases, chronic kidney diseases were risk factors for elderly survival outcome, with the HR (95%CI) at 1.75 (1.40-2.12), 1.49 (1.10-2.03), 1.40 (1.16-1.69), 1.37 (1.11-1.70), 1.51 (1.22-1.88), 1.62 (1.18-2.23) and 1.48 (1.23-1.77) respectively. Each 1 year increase in age corresponded to 4% increase in mortality risk (HR (95%CI)=1.04 (1.02-1.05)); each 1 kg/m² increase in BMI corresponded to 5% increase in mortality risk (HR (95%CI)=0.95 (0.93-0.98)); each 1.0×10⁹/L increase in total lymphocyte count (TLC) corresponded to 13% increase in mortality risk (HR (95%CI)=0.87 (0.76-0.99)). Additionally, the mortality risk decreased 19% (HR (95%CI)=0.81 (0.66-0.98)) in participants with regularly physical exercise compared to those without; and the mortality risk decreased 41% (HR (95% CI)=0.59 (0.40-0.88)) in participants with elevated triglycerides (TG, ≥2.26 mmol/L) compared to those without. Conclusion In Chinese longevity areas, some nutritional and immune indices such as relatively higher level of BMI, TLC and TG were independent protective factors for 5-year survival outcome, which was different from general adults and younger elderly.

Objective:A nested-PCR assay is developed to detect and identify the genomic DNA of Brucella vaccine A19 strain. Methods:The whole genomic sequences of Brucella vaccine A19 strain and other Brucella spp. strains were compared and analyzed. The primers were designed by nucleotide difference sites. The nested-PCR assay was established to detect and identify Brucella vaccine A19 strain. The genomic DNA of Brucella vaccine A19 strain was extracted and diluted. The diluted template DNA was tested for sensitivity of using nested-PCR assay. And the specificity of nested-PCR assay was tested for the genomic DNA of other Brucella spp. strains and non- Brucella spp. strains. Results:The minimum detection limit of the nested-PCR assay was 3.43 fg. The nested-PCR assay established for amplification of Brucella vaccine A19 strain showed 246 bp electrophoresis bands, while other Brucella spp. strains showed 314 bp electrophoresis bands, and non- Brucella spp. strains did not produce electrophoresis bands. Conclusions:The nested-PCR assay established has the characteristics of high sensitivity and specificity. It can be detected when there is one copy of Brucella vaccine A19 strain genomic DNA in the reaction system. This method is particularly suitable for the detection and identification of trace genomic DNA of Brucella vaccine A19 strain in sample.

Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.