Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Bioreactors ; Catalysis ; Escherichia coli ; genetics ; Glucose ; Glucosyltransferases ; Industrial Microbiology ; Organisms, Genetically Modified ; Trehalose ; biosynthesis

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Objective To comparative analysis the intracytoplasmic sperm injection (ICSI)result and rate of sperm DNA in-tegrity (DNA fragmentation index,DFI)about testicular and epididymis sperm.Methods Totally 183 obstructed azoospermia pa-tients were choosed to use ICSI.80 cycles by PESA and 103 cycles by TESA,compared two groups of sperm DNA integrity rate and ICSI outcome.Results Sperm DNA integrity rate,fertilization rate,cleavage rate,good-qualityembryo rate and pregnancy rate com-pared with no difference by ICSI(P >0.05).Conclusion DNA integrity rate and ICSI outcomes of the testis and epididymis sperm have no significant differences,clinicians can be based on personal experiences or patients,wills to select sperm for ICSI.

Objective To evaluate the feasibility and effectiveness of four closure techniques ,large incision with plastic distal at‐tachment and clip with suction ,1 .1 cm small incision with plastic distal attachment and clip with suction in natural orifice translu‐minal endoscopic surgery(NOTES) .Methods Forty‐one ex vivo porcine stomachs were involved in this research .According to the size of incision and different methods of incision closure ,all ex vivo porcine stomachs were divided into four groups .Group A in‐volved 8 ex vivo porcine stomachs ,their size of incision were 1 .2-2 .0 cm ,and their incisions were closured by a clip closure direct‐ly ;group B involved 10 ex vivo porcine stomachs ,their size of incision were 1 .2-2 .0 cm ,and their incisions were closed by plastic end attachment with suction and clip enclosure technique;group C involved 10 ex vivo porcine stomachs ,their size of incision were smaller than 1 .1 cm ,and their incisions were by a clip closure directly ;group D involved 13 ex vivo porcine stomachs ,their size of incision were smaller than 1 .1 cm ,and closured by plastic end attachment with suction and clip enclosure technique .Stomach leaks were evaluated by leaking studies after the procedure .Results In group A ,1 incision was closed successfully ,7 incisions were clo‐sured unsuccessfully ;in group B ,2 incisions were closed successfully ,8 incisions were closed unsuccessfully ;in group C ,2 incisions were closed successfully ,8 incisions were closured unsuccessfully ;in group D ,11 incisions were closed successfully ,2 incisions were Closured unsuccessfully .Fisher′s exact test showed that group D was significantly related to the success of incision closure .The were significant differences between group A and D ,group B and group D(P<0 .05) .Conclusion Small incision(smaller than 1 cm) and plastic end attachment with suction and clip enclosure technique are optimal in NOTES procedure in this ex vivo porcine stomachs study .

UNLABELLEDObjective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).

METHODSThis retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.

RESULTSNo significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.

CONCLUSIONRescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.

Acrosome ; pathology ; Acrosome Reaction ; DNA Fragmentation ; Female ; Fertilization ; Fertilization in Vitro ; Humans ; Infertility ; Male ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

Objective The optimal access for natural orifice transluminal endoscopic surgery is still uncertain .This study was designed to compare the practicability and maneuverability of transgastric ,transunmbilical ,and transrectal approach in abdominal surgery in a canine model .Methods Three dogs were used in this research .Three approach :trangastric ,transunmbilical and tran-srectal approach were carried out for abdominal exploration ,liver biopsy ,bladder biopsy and an attempted cholecystectomy .The ma-neuverability ,endoscopic image ,performer′s perception ,and spatial orientation were evaluated .Results The maneuverability of trangastric ,and transrectal approach NOTES were better than transunmbilical NOTES .Abdominal exploration ,live biopsy ,and bladder biopsy were completed successfully .The cholecystectomy was failed because of poor exposure and difficulty of separating the around tissure .Conclusion The optimal approach for upper abdomen NOTES is transrectal route .For lower abdomen NOTES , the trangastric approach is superior to other accesses .Further study is needed to develop more flexible and precise equipment for NOTES and to evaluate more feasible access approach .