Objective To study the expression of tight junction factors in human placental tissues derived from assisted reproductive technology (ART) and natural pregnancy and its role in placental barrier.Methods Ten placental samples were collected from the women who had undergone ART treatment and 11 placenta were collected from control group.Transmission electron microscope (TEM) examination was utilized to detect the morphology of placental tight junctions.The mRNA of claudin (CLDN) 1,CLDN4,CLDN5,CLDN8,zonula occudens (ZO) 1 was detected by real-time PCR and the protein of CLDN4,CLDN8 and occludin (OCLN) were measured by western blot.Results TEM microscopy results showed that placenta samples derived both ART and control placenta had normal microscopic histological features of tight junctions,localized in the apical part of the syncytium and also between the cell-cell contacts of fetal blood vessel endothelial.The expression level of CLDN4 mRNA were 0.87 ±0.17 in ART group and 1.18 ± 0.30 in control group,respectively.The expression level of CLDN8 mRNA were 3.25 ± 2.32 in ART group and 1.08±0.41 in control group,respectively.The mRNA level of CLDN4 and CLDN8 were significantly differentially expressed in ART derived placenta when compared with control groups.The expression level of CLDN1,CLDN5,OCLN and ZO1 mRNA were 0.49 ± 0.44,0.80 ± 0.20,0.92 ± 0.18 in ART group and 1.09±0.82,1.21 ±0.78,0.80± 0.27 in control group,respectively,in which there were no significant differences between two groups.Western Blot analysis showed the protein levels of tight junctions CLDN4,CLDN8 and OCLN did not differ between groups.Conclusions Tight junction factors were expressed in human placental tissues.Tight junction derived from ATR platenta might have mild dysfunction.

Objective To investigate the role of phosphatidylinositol 3?kinase∕protein kinase B∕glycogen synthase kinase?3β ( PI3K∕Akt∕GSK?3β) signaling pathway in hydrogen?induced inhibition of neuronal ap?optosis induced by focal cerebral ischemia?reperfusion ( I∕R) in rats. Methods One hundred and twenty
healthy adult male Sprague?Dawley rats, weighing 200-220 g, were divided into 5 groups ( n=24 each) u?sing a random number table: sham operation group ( group S); cerebral I∕R group ( group I∕R); hydrogen group (group H2); LY294002 (specific PI3K inhibitor) group (group LY); LY294002+hydrogen group ( group LY+H2 ) . Focal cerebral I∕R was induced by occlusion of the middle cerebral artery for 1 h followed by 24 h reperfusion. In H2 and H2+LY groups, the animals inhaled 67% H2+33% O2 for 2 h starting from onset of reperfusion, and then inhaled H2 for 2 h every 6 h. In LY and LY+ H2 groups, LY294002 ( 10 mmol∕L) 10 μl was injected into the lateral cerebral ventricle at 10 min before reperfusion. Neurologic defi?cit was evaluated and scored ( NDS) at 24 h of reperfusion. The rats were then sacrificed, and the brains were removed for measurement of the cerebral infarct size ( by TTC staining) and apoptosis in cortical neu?rons ( by TUNEL) and for determination of the expression of Akt in the ischemic cerebral cortex, phospho?rylated Akt ( p?Akt) , GSK?3β and phosphorylated GSK?3β ( p?GSK?3β) and Bcl?2 and Bax positive cell count in the ischemic cerebral cortex ( by immuno?histochemistry) . The apoptosis index ( AI) , p?Akt∕Akt ratio and p?GSK?3β∕GSK3?β ratio were calculated. Results Compared with group S, the NDS, cerebral infarct size, AI, p?Akt∕Akt ratio, p?GSK?3β∕GSK?3β ratio and Bax positive cell count were significantly increased, and the Bcl?2 positive cell count was significantly decreased in group I∕R ( P<0?05) . Compared with group I∕R, the NDS, cerebral infarct size, AI and Bax positive cell count were significantly de?creased, and the p?Akt∕Akt ratio, p?GSK?3β∕GSK?3β ratio and Bcl?2 positive cell count were significantly increased in group H2 ( P <0?05) , and no significant changes were found in the parameters mentioned a?bove in LY and LY+H2 groups ( P>0?05) . Compared with group H2 , the NDS, cerebral infarct size, AI and Bax positive cell count were significantly increased, and the p?Akt∕Akt ratio, p?GSK?3β∕GSK?3βratio and Bcl?2 positive cell count were significantly decreased in group LY+H2 ( P<0?05) . Conclusion The mechanism by which hydrogen inhibits focal cerebral I∕R?induced neuronal apoptosis is associated with the activation of PI3K∕Akt∕GSK?3β signaling pathway in rats.

Objective To investigate the effects of inhaling high concentration of hydrogen gas on the expressions of endoplasmic reticulum stress(ERS) related protein glucose regulated protein 78 (GRP78),Caspase-12 and the neural cell apoptosis and related proteins Bcl-2 and Bax in the rats with focal cerebral ischemia reperfusion(I/R) injury.Methods Seventy-two healthy SPF male Sprague-Dawley rats were selected and then randomly divided into the control group(Ⅰ:without any treatment),sham operation group (Ⅱ),cerebral IRI group (Ⅲ) and hydrogen gas treatment group (Ⅳ),18 cases in each group.Focal cerebral ischemia reperfusion injury (IRI) model was induced by using the suture-occluded method.The neurological deficits score (NDS) was assessed at 24 h after cerebral reperfusion in four groups.The cerebral infarction severity and size were detected by TTC staining and neuronal apoptosis of brain cortex were tested by TUNEL technique.The apoptosis index (AI) was calculated.Then the expressions of GRP78,Caspase-12,Bcl-2 and Bax were assessed by Western blot and immunohistochemistry.Results As compared with the group Ⅰ and Ⅱ,NDS score,cerebral infarction size,AI and the expressions of GRP78,Caspase-12 and Bax in cerebral cortex in the group Ⅲl and Ⅳ were significantly increased,while the expression of Bcl-2 in cerebral cortex was markedly decreased(P＜0.05);compared with the group Ⅲ,NDS score,brain infarction size,AI and the expression of Caspase-12 and Bax in cerebral cortex in the group Ⅳ were markedly decreased,while the expressions of GRP78 and Bcl-2 were dramatically increased (P＜0.05).Conclusion Inhaling high concentration of hydrogen gas has a certain protective effect on cerebral IRI in rats through increasing endoplasmic reticulum GRP78 protein expression after IRI and inhibiting Caspase-12 activation,thus inhibiting ERS and promoting the repair function of endoplasmic reticulum.

Objective To evaluate the role of phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) signaling pathway in hydrogen-induced inhibition of cell apoptosis during myocardial ischemia/reperfusion (I/R) in rats.Methods Forty healthy male Sprague-Dawley rats,weighing 300-350 g,were randomly allocated into 4 groups (n =10 each) using a random number table:sham operation group (S group),I/R group,hydrogen group (group H),and hydrogen + LY294002 group (group HL).Myocardial I/R was induced by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion.In H and HL groups,99.6 ％ hydrogen 5 ml/kg was injected intraperitoneally immediately after beginning of reperfusion,and in addition LY294002 (0.3 mg/kg) was injected through the caudal vein before hydrogen injection in group HL.Arterial blood samples were collected at the end of 120 min reperfusion for determination of serum creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH) activities.The rats were then sacrificed.Myocardial apoptosis was detected by TUNEL and apoptosis index (AI) was calculated.The expression of Bcl-2,Bax and caspase-3 was detected by immuno-histochemistry.Bcl-2/Bax ratio was calculated.Results The serum CK-MB and LDH activities and AI were significantly increased,the expression of myocardial Bcl-2,Bax and caspase-3 was upregulated,and the ratio of Bcl-2/Bax was decreased in group I/R as compared with group S.Compared with group I/R,the serum CK-MB and LDH activities and AI were significantly decreased,the expression of myocardial Bcl2 was up-regulated,while the expression of myocardial Bax and caspase-3 was down-regulated,and the ratio of Bcl-2/Bax was increased in group H,and no significant changes were found in the parameters mentioned above in group HL.The serum CK-MB and LDH activities and AI were significantly increased,the expression of myocardial Bcl-2 was down-regulated,while the expression of myocardial Bax and caspase-3 was up-regulated,and Bcl-2/Bax ratio was decreased in group HL as compared with group H.Conclusion Hydrogen can activate the PI3K/Akt signaling pathway,and further up-regulates Bcl-2 expression and down-regulates Bax and Caspase-3 expression,thus inhibiting cell apoptosis during myocardial I/R in rats.

Objective To investigate the effect of high concentration hydrogen gas on neurons in the rat hippocampus CA1 region during global cerebral ischemic/reperfusion injury (GCIR) Methods Four-vessel occlusion was used to establish rat model with GCIR injury. One hundred and five healthy male Sprague-Dawley rats were randomly divided into sham operation group(SH group, n = 15), model group(4-VO group, n = 45) and treatment group(4-VO+H2 group,n = 45). After 72 h and 9 d reperfusion, hippocampal CA1 region pyramidal neurons in every group were detected with Nissle staining , immunohistochemical neuron-specific nuclear protein (NeuN), specific protein antibody microglial cells (Iba1) staining and the relationship of position between neurons and microglia was observed through fluorescence double staining. We used Morris water maze to test the space orientation ability and the learning and memory ability in rats after 9 d reperfusion. Results Compared with those of 4-VO group,the neurons of hippocampus CA1 region were closer to normal in 72 h and 9 d in 4-VO+H2 group and neuron form and the number of neuron survival were increased significantly (P < 0.05);immunohistochemical staining showed that the number of neuron survival in 4-VO+H 2 group was obviously higher than that in 4-VO group (P < 0.05) and the number of microglia in 4-VO group was obviously higher than that in 4-VO+H2 group (P < 0.05). Water maze experiment showed that the swimming time in quadrant Ⅳ in 4-VO+H2 group was longer than that in 4-VO group (P < 0.05). Conclusion Inhalation of high concentration hydrogen gas has prominent protective effect on neurons of rat hippocampal CA1 region during reperfusion. The mechanism may be related with inhibiting the microglia excitation and activation during GCIR.

Pdx-1, an important transcription factor highlighting in the early pancreatic development,islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research.Our aim was to explore the role of pdx-1 in pan-creatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse tran-scriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this re-search, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 (siPDX-1) or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hy-bridization and immunofluorescent staining results showed that siPDX-I disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.

Objective To explore the risk factors of cognitive impairment of elderly patients with cerebral infarction in order to provide the theoretical basis for the clinical intervention. Methods A total of 237 cases with senile cerebral infarction were selected as ours subjects who were hospitalized from Mar. 2010 to Jun. 2013 in Kailuan General Hospital Affiliated to Hebei United University. The general condition and medical history were recorded. The auxiliary examination was performed. Cerebral infarction was diagnosed based on the onset to diagnosis standard and MoCA scores of within 2 weeks. The patients with less than 26 MoCA score were diagnosed as cognitive dysfunction and otherwise were thought as normal. Single factor analysis methods and non conditional Logistic regression were applied to analyze the analysis. Results There was no significant difference in terms of incidence between patients with different gender. Patients with age more than 75 years old and lower education levels had the high incidence rate than those with younger age and high education levels( χ2=16. 661,5. 453;P﹤0. 05). The cognitive dysfunction incidence of patients with white collar was lower than those with blue collar(χ2 = 5. 458,P ﹤ 0. 05 ). And the cognitive dysfunction incidence of patients with hypertention,diabetes,heart disease and leukoaraiosis were higher than those without the above diseases(χ2 =28. 423,5. 621,7. 768,6. 070;P﹤0. 05). The incidence of patients smoking more was significantly higher than that of smoking less or no(χ2 =5. 045,P ﹤0. 05 ). Multiple factors and non conditional Logistic regression analysis showed that,67 Senile cerebral infarction patients occurred cognitive impairment within 2 weeks. The independent risk factors for its occurrence included age greater than 75 years( P=0. 000 ),diabetes mellitus( P=0. 043),hypertension(P=0. 000)and leukoaraiosis(P=0. 041). Conclusion There are many risk factors related to cognitive impairment after cerebral infarction occurred in the elderly. The intervention should take in many aspects and the risk factors should early found.

Objective To evaluate the stability of U6 and let-7a as internal reference genes of miRNAs in RTqPCR by using femoral head samples of cartilage tissue from inbred DA rats.Methods Total RNA was extracted from femoral head cartilage tissues of female DA rats at three different time points,i.e.at birth (D0),ablactation (D21) and maturation (D42).The expressions of different miRNAs (miR-1,-25,-26a,-140,-146a,-150,-181a,-195,-223 and-337) were detected by RT-qPCR using U6 or let-7a as the internal reference.The two sets of miR expression were compared with the results from Solexa sequencing in our pioneer work to evaluate the stability of the two internal references.Results The relative values of U6 (P =0.045) and let-7a (P =0.021 5) revealed significant difference in the D42 sample.Both in U6 and let-7a systems,miR-26a,-140,-223,and-337 showed a similar tendency in expression and quantification but miR-1 and-146a did not have significant differences.miR-25,-150,-181a and-195 differed significantly (P＜0.05).Comparison of absolute quantification results between the two generations' sequencing showed that let-7a is more stable than U6.Conclusion Let-7a is more suitable to be used as the internal reference gene in RT-qPCR for miRNAs in cartilage tissue.

Objective To assess the relationship between HIV genetic subtypes and HIV resistance in China.Methods The literature retrieval was conducted by using Chinese ScienceTechnology Journal Database (VIP),Wanfang Data,Chinese Journal Full-text Database (CNKI),PubMed and Web of Science to select the papers on the relationship between HIV subtypes and HIV drug resistance in China during 2005-2015.Eligible papers were included according to the inclusion.Meta-analysis was performed by using software Stata 12.0.Results A total of 43 papers were selected and the pooled rate of drug resistance was 15.1％ and the rate of primary drug resistance was 9.5％,the subtypes associated drug resistance were CRF01_AE,CRF07 BC,CRF08_ BC,B/B'and C.The pooled rates of drug resistance of each subtype were 12.8％,7.4％,14.3％,25.7％ and 34.9％ and the rates of primary drug resistance of each subtype were 7.3％,5.7％,11.5％,15.5％ and 23.9％,respectively.Subgroup analysis showed that both treated and area subgroup showed a significant difference among groups (P＜0.05).The rates of primary resistance of each subtype in northern China and southwestern China were higher than that in southern China.Conclusion The distribution of HIV genotypes in China was complex and the prevalence of primary drug resistance of each subtype was high,together with a significant difference among subtypes.It is necessary to strengthen the monitoring of different subtypes of drug resistant strains in China to prevent the recombination and spreading of resistant strains.

Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; genetics ; secretion ; Helianthus ; metabolism ; Kluyveromyces ; genetics ; Metabolic Engineering ; methods ; Plant Tubers ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; enzymology ; genetics