Recombinant plasmids pHY-PA, pBBR-PA were constructed in which genes pdc and adhB were placed under the control of tac promoter, respectively, and had successfully expressed in Escherichia coli. Then these recombinant plasmids were electroporated into Lactobacillus strains for ethanol production. Preliminary ethanol fermentation using these Lactobacillus strains and their recombinants was carried out using 42℃ as fermentation temperature. The results indicated that introducing pdc and adhB, ethanologenic pathway was successfully constructed in L.plantanum CICIM B0080. 0.4% (V/V) ethanol was detected at the end of fermentation with 6.7% glucose, and that is 67-fold as the wild-type B0080. Two-fold of ethanol production was detected in L.amylovorus B0112 (pHY-PA) and L.acidophilus B0068 (pBBR-PA). Introducing both pdc and adhB, and meanwhile knock-outing the lactate dehydrogrnase gene may better convert carbon flux to ethanologenic direction.

ObjectiveTo explore the effect and mechanism of Toll-like receptor 4(TLR4) ligand LPS-mediated inhibition hepatitis B virus (HBV) replication in Bewo cells.MethodsFirst of all,2 μg 1.3-fold HBV recombinant vector pcDNA3.1 ( + )-HBV1.3 were transfected into Bewo cells,after 12 h,the cells were treated with LPS for 3 d.To observe the kinetics of IFN-β and TNF-α expression in Bewo cells,the Bewo cells were exposed to TLR4 ligand LPS.And the effect of pyrrolidine dithiocarbamate ( PDTC),an inhibitor of NF-κB,on LPS-induced cytokines was also observed.The HBsAg,HBeAg and HBV DNA level in the culture supernatant were detected by Microparticle Enzyme Immunoassay (MEIA) and fluorescence quantitative PCR,respectively,and the expression of IFN-β,TNF-α,TRIF and MyD88 was detected by ELISA and RT-PCR,respectively.ResultsCompared with control group,LPS could significantly suppress HBV replication in Bewo cells ( P ＜0.01 ),and it could induce the production of TNFα in Bewo cells ( P ＜ 0.05 ),in time-and dose-dependent manners.PDTC strongly inhibited LPS and induced TNF-α production,but had no much effect on IFN-β in Bewo cells ( P ＜ 0.001 ).Compared with control group,the mRNA levels of MyD88 were significantly induced by LPS in the Bewo cells transfected with this recombinant vector( P ＜ 0.001 ).ConclusionsTLR4 ligand LPS could significantly suppress HBV replication by inducing TNF-α production in Bewo cells mainly via the MyD88/ NF-κB signal pathway.

Central composite design (CCD) is one of the most commonly used design methods in response surface optimization and has been widely applied in the field of pharmaceutics to optimize preparations. On the 20th anniversary of the introduction of CCD into China, the paper reviews its application in domestic pharmaceutical researches. Based on the brief introduction of basic principle and operation steps of CCD, the mistakes emerging in the application of CCD are summarized, including conceptual confusion with Box-Behnken design and face-centered CCD as well as wrong designs. Besides, the issues concerning the selection of factors and responses are discussed. The article is helpful for researchers to comprehensively understand the CCD and facilitates the rational application of this method.

BACKGROUNDIn tumors the process of apoptosis occurs over an interval of time after chemotherapy. It is important to determine the best time for detecting apoptosis by in vivo imaging. In this study, we evaluated the dynamics and feasibility of imaging non-small cell lung cancer (NSCLC) apoptosis induced by paclitaxel treatment using a (99)Tc(m)-labeled Annexin V recombinant with ten consecutive histidines (His10-Annexin V) in a mouse model.

METHODS(99)Tc(m)-His10-Annexin V was prepared by one step direct labeling; radio-chemical purity (RCP) and radio-stability was tested. The binding of (99)Tc(m)-His10-Annexin V to apoptotic cells was validated in vitro using camptothecin-induced Jurkat cells. In vivo bio-distribution was determined in mice by dissection. The human H460 NSCLC tumor cell line (H460) tumor-bearing mice were treated with intravenous paclitaxel 24, 48 and 72 hours later. (99)Tc(m)-His10-Annexin V was injected intravenously, and planar images were acquired at 2, 4 and 6 hours post-injection on a dual-head gamma camera fitted with a pinhole collimator. Tumor-to-normal tissue ratios (T/NT) were calculated by ROI analysis and they reflected specific binding of (99)Tc(m)-His10-Annexin V. Mice were sacrificed after imaging. Caspase-3, as the apoptosis detector, was determined by flow cytometry, and DNA fragmentation was analyzed by the terminal deoxynucleotidytransferase mediated dUTP nick-end labeling (TUNEL) assay. Nonspecific accumulation of protein was estimated using bovine serum albumin (BSA). The imaging data were correlated with TUNEL-positive nuclei and caspase-3 activity.

RESULTS(99)Tc(m)-His10-Annexin V had a RCP > 98% and high stability 2 hours after radio-labeling, and it could bind to apoptotic cells with high affinity. Bio-distribution of (99)Tc(m)-His10-Annexin V showed predominant uptake in kidney, relatively low uptake in myocardium, liver and gastrointestinal tract, and rapid clearance from blood and kidney was observed. The T/NT was significantly increased after paclitaxel treatment, whereas it was low in untreated tumors (T/NT = 1.43 ± 0.18). The %ID/g activity in Group 2 (24 hours), Group 3 (48 hours) and Group 4 (72 hours) after treatment was 2.55 ± 0.73, 3.35 ± 1.10, and 3.4 ± 0.96, respectively. Whereas in the non-treated group, Group 1, %ID/g was 1.10 ± 0.18. The radiotracer uptake was positively correlated to the apoptotic index (r = 0.852, P < 0.01), as well as caspase-3 activity (r = 0.816, P < 0.01).

CONCLUSIONThis study addresses the dynamics and feasibility of imaging non-small cell lung tumor apoptosis using (99)Tc(m)- His10-Annexin V.

Animals ; Annexin A5 ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Histidine ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Organotechnetium Compounds ; Paclitaxel ; therapeutic use ; Radiopharmaceuticals

The present study aimed to clarify the smoking cessation motivations,challenges and coping strategies among pregnant couples.A qualitative design using a grounded theory approach was applied.Data were collected by individual semi-structured interviews with 39 married individuals (21 non-smoking pregnant women and 18 smoking or ever-smoking men with a pregnant wife) and 3 imams in an ethnically diverse region of far westem China.The most common theme for smoking cessation motivation was "embryo quality" (i.e.,a healthier baby),followed by family's health.Most interviewees reported that husband's withdrawal symptoms were the greatest challenge to smoking cessation,followed by the Chinese tobacco culture.Coping strategies given by the pregnant women typically involved combining emotional,behavioral and social interventions.Social interventions showed advantages in helping to quit smoking.Pregnancy appears to be a positive stimulus for pregnant couples' smoking cessation.Our results suggest that pregnancy,a highly important life event,may help to reduce barriers to smoking cessation at the social level (e.g.,limiting access to cigarettes,avoiding temptation to smoke),but does little to help with the withdrawal symptoms.Professional guidance for smoking cessation is still necessary.

The present condition in the acupoint term translation was analyzed and its existent problems in this area were discussed in this paper. The authors suggested that in translating the terms of acupoints, the translation on the meaning of the acupoints should be added, in this way, it can not only keep the integrity in acupoint translation, but also make the inheritance of the Chinese precious culture of Traditional Chinese Medicine further available.
Acupuncture Points ; Animals ; Culture ; Humans ; Medicine, Chinese Traditional ; Translating

OBJECTIVETo explore the inhibitory effect of combination of lamivudine with thymosin alpha1 (Talpha1) on the replication of duck hepatitis B virus (DHBV).

METHODSPeking ducks of 1 d old were challenged with DHBV-positive serum and used as a duck hepatitis B model. After treated with lamivudine for three months, the ducks were randomly grouped and treated with or without Talpha1 for 8 d. Serum DHBV titrate was observed by semi-quantitative PCR, and inflammation and degeneration of hepatocytes were observed by pathology examination.

RESULTSThe serum DHBV titrate was significantly reduced (4483.2+/-5193.4 compared with 9351.8+/-5059.6) after lamivudine treatment, and it was reduced more significantly(1692.2+/-589.2) after combination treatment with Talpha1. Lamivudine reduced the degeneration degree of hepatocytes (3.2+/-0.8 compared with 4.6+/-0.5) and the inflammation degree of liver (6.2+/-3.3 compared with 8.6+/-2.8). The combination treatment with Talpha1 increased liver inflammation degree (9.0+/-5.2).

CONCLUSIONBoth Talpha1 and lamivudine may reduce the replication of DHBV in Peking ducks and combination treatment may have the better anti-virus effect and enhance immune response in liver.

Animals ; Animals, Newborn ; Antiviral Agents ; therapeutic use ; Cells, Cultured ; Drug Therapy, Combination ; Ducks ; Hepadnaviridae Infections ; drug therapy ; Hepatitis B Virus, Duck ; drug effects ; genetics ; Hepatitis, Viral, Animal ; drug therapy ; Hepatocytes ; virology ; Lamivudine ; therapeutic use ; Thymosin ; analogs & derivatives ; therapeutic use ; Virus Replication ; drug effects

A fermentation system composed of four airlift suspended-bed bioreactors in series and with a total working volume of 4800 mL was established. Continuous ethanol fermentation using self-flocculating yeast SPSC01, a fusant from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two-stage enzymatic hydrolyte of dry milling corn powder, was continuously run for 120 days. All of the backset distillage collected after distilling the final beer was used to mix the corn powder and no any other wastes except the solid residue of corn powder was discharged from the fermentation system, which guaranteed the distillage to be recycled at its maximum. The experimental results revealed that both ethanol and residual sugar in the final beer could be maintained relatively stable with their average levels of 93.6 and 7.9 g/L, respectively when the fermentation system was operated at the dilution rate of 0.05 h(-1). Parameter oscillations reported previously were also observed for the first and second bioreactors, but were effectively attenuated thereafter, which indicated that high yeast cell concentrations resulted from the self-immobilization of this special self-flocculating strain contributed to damp these oscillations. The monitoring of residual nitrogen and phosphor indicated that the accumulations of these nutritional elements occurred and the amount of these inorganic salts supplemented in the substrate should be decreased properly.
Bioreactors ; microbiology ; Ethanol ; metabolism ; Fermentation ; Flocculation ; Immobilization ; Saccharomyces cerevisiae ; metabolism ; Schizosaccharomyces ; metabolism ; Zea mays ; metabolism

Objective To investigate the effect of telmisartan on the expression of peroxisome proliferator activated receptor gamma(PPARγ)and its regulation in myocardial remodeling in spontaneously type 2 diabetic male Otsuka Long-Evans Tokushima Fatty(OLETF)rats fed with high-fat diet.Methods Twenty-eight four-week-old male OLETF rats were fed with high-fat diet. From 22-week of age, the pre-diabetic OLETF rats were randomly assigned to three groups:telmisartan-treated group[O-T group,5 mg/(kg·d),n=10],pioglitazone-treated group[O-P group,10 mg/(kg·d),n=8]and untreated group ( O-C group, equal volume of normal saline, n=10), continuously administration for 22-week. Twelve sex and age matched Long-Evans Tokushima Otsuka(LETO)rats were used as control(LETO group).At 22 and 48-week of age, the glucose tolerance of the rats was assessed by the oral glucose tolerance test(OGTT).At 48 weeks of age,five rats were randomly selected from each group,and clamp experiments were carried out.The glucose infusion rate from 60 min to 120 min(GIR60-120)was measured.All rats were sacrificed and the myocardial tissues were dissected.The ratio of heart weight to body weight(HW/BW)was calculated.Blood samples were collected,and serum PPARγ,tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and adiponectin were measured using ELISA and radioimmunoassay.The mRNA expressions of IL-6,PPARγ1 and PPARγ2 were measured by real-time PCR.The protein expression levels of PPARγ,adiponectin,IL-6 and NF-κB were determined by Western blot assay.The myocardial pathological changes were observed under light microscope (HE staining, Masson staining and PAS staining), and ultrastructural changes were observed under transmission electron microscope.Results At 22-week of age,neither type 2 diabetes mellitus(T2DM)nor IGT were found in three groups.At 48-week of age,T2DM was found in seven rats of O-C group,and IGT was found in two rats.T2DM was found in one rat of O-C group,and IGT was found in three rats.Neither T2DM nor IGT was found in the other two groups.GIR60-120and HW/BW were all significantly lower in the O-P group and O-T group than those of the O-C group at 48-week of age (P<0.05). Systolic blood pressure(SBP)and diastolic blood pressure(DBP)were significantly lower in O-T group than those in other three groups(P<0.05).Compared with the O-C group,the serum levels of PPARγ and adiponectin were significantly up-regulated,whereas the serum levels of IL-6 and TNF-α were down-regulated by telmisartan administration in O-T group (P<0.05).There were no significant differences in the above indexes between O-P and O-T groups.The results of real-time PCR and Westen blot assay showed that the mRNA expression of PPARγ1 was increased,and IL-6 expression decreased in O-T group compared with those of O-C group. The protein expressions of PPARγ and adiponectin were increased, and protein expressions of NF-κB and IL-6 were significantly decreased in O-P group and O-T group compared with those of O-C group(P<0.05).In the O-C group,the arrangernent of myocardial cells was irregular,myocardial fibers were swollen,a large amount of fibrotic tissue in the myocardial interstitium, and glycogen accumulation under light and electron microscope. Besides, myofibril breakage and perinuclear space expansion, myocardial mitochondria were apparently damaged or even dissolved. Compared with the O-C group, myocardial fibers arranged neatly, no obvious glycogen deposition and the ultrastructural changes of myocardium were obviously reduced in O-T group and O-P group. Conclusion Telmisartan can increase the expression level of PPARγ in the serum and myocardial tissue, reduce myocardial fibrosis and alleviate cardiac remodeling in the high-fat-diet OLETF rats.

Fluorescence intraoperative cholangiography (IOC) is a potential alternative for identifying anatomical variation and preventing iatrogenic bile duct injuries by using the near-infrared probe indocyanine green (ICG).However,the dynamic process and mechanism of fluorescenceIOC have not been elucidated in previous publications.Herein,the optical properties of the complex of ICG and bile,dynamic fluorescence cholangiography and iatrogenic bile duct injuries were investigated.The emission spectrum of ICG in bile peaked at 844 nm and ICG had higher tissue penetration.Extrahepatic bile ducts could fluoresce 2 min after intravenous injection,and the fluorescence intensity reached a peak at 8 min.Inaddition,biliary dynamics were observed owing to ICG excretion from the bile ducts into the duodenum.Quantitative analysis indicated that ICG-guided fluorescence IOC possessed a high signal to noise ratio compared to the surrounding peripheral tissue and the portal vein.Fluorescence IOC was based on rapid uptake of circulating ICG in plasma by hepatic cells,excretion of ICG into the bile and then its interaction with protein molecules in the bile.Moreover,fluorescence IOC was sensitive to detect bile duct ligation and acute bile duct perforation using ICG in rat models.All of the results indicated that fluorescence IOC using ICG is a valid alternative for the cholangiography of extrahepatic bile ducts and has potential for measurement of biliary dynamics.