The culture conditions of Saccharomyces cerevisiae sp.strain by 1.1 b were optimized for the production of D-(-)-mandelate dehydrogenase which is useful for the asymmetric bioreduction of benzoylformate to form D-(-)-mandelate.The optimum medium(per liter)consistes of 60 g peptone,30 g maltose, 0.5 g MgSO_4,0.01 g ZnSO_4,1.0 g KCl.After optimization of the culture medium,the enzyme production in shake flasks is enhanced from 2.56 to 20.21 U/L.The optimum fermentation conditions were determined as follows:medium volume 100 mL(i.e.,40%for a 250-mL shake flask),pH 6.5,inoculum size 10%,temperature 30℃,and cultivation time 25 h.

Objective To investigate the risk factors associated with feeding intolerance in very low birth weight infants (VLBWI) and to investigate the beneficial factors for preventing feeding intolerance.Methods Retrospectively analysis was carried out in 151 VLBWI cases in our hospital from January 2005 to November 2010.Logistic regression analysis was performed to find the risk factors of feeding intolerance.Results The overall rate of feeding intolerance in VLBWI was 47.7 ％ (72/151).Dysporia and infection were associated with feeding intolerance in VLBWI (Wald x2 =7.748 and 4.555,P =0.05 and 0.033 respectively).Gender,multifetation were not associated with feeding intolerance(Ps ＞0.05).Conclusion The feeding intolerance is associated with dysporia and infection in VLBWI.

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by LipofectamineTM 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

Objective To study the effect of repairment of articular cartilage defects in non weight-bearing area of rabbit with bone marrow-derived mesenchymal stem cells (BMSCs) seeded on human acellular amniotic membrane (HAAM).Methods From July 2012 to March 2013,bone marrow-derived mesenchymal stem cells were isolated and purified from rabbit in vitro.The cells were seeded on human acellular anniotic membrane at the concentration of 1.63 × l05/cm2.From 7 days to 8 days after cultured,the complexes of BMSC and HAAM were examined under electronmicroscope,light microscope and by HE stain.Full thickness empty defects measuring 4 mm in diameter by 3 mm depth were prepared in femoral intercondylar fossa of 24 rabbits.The rabbits were randomized into two groups:group A and group B with 12 each group.The defects of right knees were served as control and the left as experimental group.BMSCs/HAAM composite was cultured and then transplanted into the defect of left knee joint in group A as group BMSCs/HAAM and HAAM into group B as group HAAM.These rabbits were killed at 4 and 12 weeks after surgery in each group and the newly cartilage samples were evaluated grossly,histologically are graded.Results In the 4th and 12th week after the operation,the regenerated tissue were white,soft and smooth.Chondrocytes were found in the tissue In the 12th week,the morphology,distribution and arrangement of the regenerated tissues were similar to normal cartilage in the knees with HAAM-BMSCs transplantation.The regenerated tissues grew to be integrated with the surrounding normal cartilage with obscure boundary between them.Chondrocytes were found in all layer of the tissue,surrounding normal cartilage with obscure boundary between them.In the HAAM transplantation,the rough surface of regenerated tissue sunk obviously and the fibmblasts in all layer were found.While there were no regenerated tissue in the control side.Conclusion BMSCs seeded on HAAM could repair the articular cartilage defects of femoral intercondylar fossa from rabbits.

Objective To evaluate whether second-look endoscopy could prevent endoscopic submucosal dissection complicated with delayed bleeding and to clarify what kind of lesions that need second-look endoscopy. Methods 98 patients with early gastric cancer by histological diagnosis from Oct. 2014 to Sep. 2016 were included in this study. Mucosal damage related bleeding within 24 h after ESD was considered delayed bleeding. Clinical data of patients, lesions, surgical factors, and the incidences of bleeding before and after the second endoscopy examination were retrospectively studied. Results The en bloc resection rate of 98 patients was 100.0%, and all incisal margins were negative. No gastrointestinal perforation, death or severe complications occurred. 5.1%of the patients (5/98) occurred delayed bleeding after ESD and had been successfully stopped in a second-look endoscopy, and none received operations. No delayed bleeding occurred in follow-ups of patients with negative delayed bleeding. 40.0 % of the patients (2/5) with delayed bleeding received blood transfusion. The median time of second-look endoscopy after ESD was the second day after operation (1 ~ 3 d). The median bleeding time of 5 patients with delayed bleeding after ESD was the first day after operation (1 ~ 10 d). The median duration of surgery was 75 min (60~150 min), and the prediction success rate was 94.9 %. The results of univariate analysis showed that age [(69.6 ± 7.9) vs (60.9 ± 10.1) years old, P = 0.003] and operation time [(90.0 ± 41.0) vs (66.0 ± 42.0) min, P = 0.000] were both the risk factors in delayed bleeding and non-bleeding group. Binary Logistic regression analysis showed that operation time (OR^ =1.07; 95%CI:0.73 ~ 14.63, P = 0.010) was the only predictor of delayed bleeding after ESD. Conclusions Second-look endoscopy might be effective in preventing delayed bleeding after gastric ESD, especially within 48 hours after ESD. The operation time was an independent risk factor for delayed bleeding after gastric ESD.

Objective To investigate the effect of physical training on plasma N-terminal pro-brain natri- uretic peptide(NT-proBNP)levels in patients with chronic heart failure(CHF).Methods Eighty NYHAⅡ-ⅢCHF patients were randomly divided into a training group(n=42)and a control group(n=38).A 6-minute walk- ing test was performed within 24 hours after the patients were admitted.The 6-minute walking distance and plasma NT-proBNP levels were determined before and after 8 weeks of programmed physical training.The patients of both groups were treated with routine drugs for heart failure.6-minute walk training was only performed in the training group twice a day for 8 weeks.Results Physical training could significantly reduce plasma NT-proBNP levels and improve performance on the 6-minute walking test.Conclusions Physical training could significantly reduce plas- ma NT-proBNP levels and improve the motor function of patients with CHF,and could be helpful in delaying the de- velopment of CHF.

Present research of bone marrow mesenchymal stem cells (BMSCs) mainly contains the studies of the characteristics of biology, the induced differentiation, the mechanism of regulation and so on. The research of the characteristics of biology and the osteogenic and adopocitic induction are comparatively mature, showing many biological characteristics such as adherence, plasticity, heterology, self-renewal, rapid clone formation and transplantation. Adherence combined with density gradient centrifugation method is widely used in the isolation and purification of BMSCs. As to neural induction, there are many uncertainties, such as morphous of typical neurons, specific labeling, excitability, synaptic connection with other neurons, synaptic potential, and whether the induction of fully functional neurons from BMSCs is possible. In addition, the research of the regulation mechanism is mostly uncertain, and there are still many safety problems.

Objective To explore the effects of gossypol acetate on the morphological features and the gene expression in the femoral head of Sprague-Dawley rat in vivo after treated with dexamethasone .Methods Dexamethasone(Dex) was injected into the abdominal cavity of SD rats at an dose of 10 mg/kg ,twice a week ,and feed gossypol acetate 5 mg · kg -1 · d-1 .The controls re-ceived saline 2 mL injection .The treatment lasted for 12 and 20 weeks .The slices of the femoral head were made for HE and immu-nohistochemical study .The total mRNA was extracted for RT-PCR assessment .Results The cancellous bone trabecular became sparse ,trabecular bone area ratio decreased ,bone marrow fat tissue increased .These changes were fitted for pathological character of bone necrosis .The gossypol acetate could not affect the pathological changes .The proportion of the positive stained osteoblasts increased ,adipocytes decreased .PPARγ,C/EBPα,11β-HSD1 expression enhanced ,Runx2 down regulated in the treatment groups and GAA group .Conclusion Dex can induce evident pathological changes conform to the characters of femoral head necrosis .They may have closed correlation between 11β-HSD1 and the gene expression .But GAA could not affected the pathological changes and abnormality of the gene expression .

ObjectiveTo evaluate the effectiveness of transcatheter Amplatzer device closure on patent ductus arteriosus(PDA),and to give some evidences for the clinical application.MethodsAll studies in the world regard to the controlled trials(CT) about transcatheter Amplatzer device closure and cardiac surgery on PDA were searched and made synthetic evaluation by means of Meta-analysis.RevMan 4.2.2 software was used for statistical analysis.Cases relative risk(RR)and its 95% confidence interval(CI)of procedure failure,the incidence of complication and residual shunt were calculated.ResultsTotally 5 studies including 349 cases were analyzed.Operation failure of Amplatzer device occlusion was higher than cardiac surgery [5 CT,349 cases,3.0% vs 0,RR=4.29,95%CI(0.77,23.95)](P=0.10).Incidence of complication of Amplatzer device occlusion was lower than cardiac surgery[5 CT,343 cases,3.1% vs 38.0%,RR=0.11,95%CI(0.05,0.23)](P