To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.
Animals ; Egg Proteins ; genetics ; metabolism ; Electroporation ; Fermentation ; Immunization ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; secretion ; Swine ; Zona Pellucida Glycoproteins

OBJECTIVETo identify associated proteins involved in the molecular response of ischemic preconditioning (IPC) against intestinal ischemia/reperfusion (II/R) in the intestinal mucosa of rats.

METHODSSixteen SD rats were randomly divided into II/R and IPC groups. II/R injury in rats was produced by clamping superior mesenteric artery for 60 min followed by 60 min reperfusion. IPC was elicited by 20 min ischemia and 5 min reperfusion before index ischemia. The intestinal mucosa was scratched immediately after 60 min of reperfusion and total proteins were separated by immobilized pH gradient (IPG) based on two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using Image Master 2D Elite 5.0 image analysis software and identified by MALDI-TOF-MS. The biological information of these proteins was searched in the database of these peptide mass finger-printing (PMF). Western blotting and RT-PCR were used to validate the differentially expressed proteins.

RESULTSImage analysis revealed that averages of 1404+/-20 and 1338+/-20 were detected in II/R and IPC groups. A total of 10 spots yielded good spectra, and 8 spots matched with known proteins after database searching. These proteins were mainly involved in anti-oxidation, inhibiting apoptosis and energy metabolism. Western blot confirmed up-regulation of aldehyde dehydrogenase and RT-PCR confirmed up-regulation of aldose reductase in IPC group.

CONCLUSIONThe clues provided by comparative proteome strategy will shed light on molecular mechanisms of IPC against II/R injury.

Animals ; Intestinal Diseases ; metabolism ; pathology ; Intestinal Mucosa ; metabolism ; pathology ; Ischemic Preconditioning ; Male ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology

Objective The internationalization of modern scientific research and its high degree of complexity have showed the importance of scientific research cooperation.How to promote cooperation more effectively is an important research topic.Methods This article analyzes the research cooperation of Institute of Hematology & Blood Diseases Hospital Chinese Academy of Medical Sciences during the past 12 years.Results Scientific research cooperation is a big trigger for the output of papers,especially high-level output,international and native institutions' cross-cooperation can be used as an important way for expanding scientific research cooperation and provides a reference for decision-making.Conclusions Strengthen the awareness of cooperation,increasing scientific research funding,establishing common and hot research directions and constructing highlevel platform can expand scientific research cooperation.

AIM:To describe the outcomes of corneal stromal lenticules in repairing of corneal ulcer and/or perforation.METHODS:This was a retrospective chart review of 6 eyes of 6 patients from January to June 2017,who underwent corneal ulcer repair with the corneal,stromal lenticules harvested from femtosecond laser refractive surgery and kept in pure glycerin for use.Three cases of infectious corneal ulcers were bacterial,fungal,and infection with foreign bodies in corneal deep layer,one each.The other 3 were corneal ulcer perforation.Making sure no air bubble between donor graft and Descemet membrane.The mean follow-up time was 3.71 ±1.56mo (range 1-6mo).RESULTS:All eyes were successfully treated under control of infection without intra-operative complications,and early postoperative evaluation showed a clear graft in all cases.The last follow-up visit showed the mean best corrected visual acuity (VA) significantly improved after surgery.There was significant difference from 0.48±0.12 to 1.50±0.08 (P＜0.01).CONCLUSION:The preliminary results suggest that the use of corneal stromal lenticules may be a safe and effective surgical alternative for corneal ulcer,even though the long-term outcome of the graft needs to be further observed.

【Objective】To investigate the lncRNA expression profile and potential roles in mouse intestinal mucosa after I/R treatment and explore the co-expression relationship between dysregulated lncRNA and apoptotic mRNA at the early stage of reperfusion. 【Methods】 The expression profiles of lncRNA were obtained using microarray and some lncRNA were further validated by quantitative real- time polymerase chain reaction （qRT- PCR）. Gene ontology（GO）analyses were performed to determine closely related biological functions，especially apoptosis-related functions. Finally， the known apoptosis- related mRNA with obviously changes were selected to construct the co-expression network of the dysregulated lncRNA and their correlated apoptotic mRNA， and were analyzed by CNC analysis to calculate the significant correlation of IncRNA-mRNA pairs.【Results】Compared with sham operation group，the expression profile of lncRNA in intestinal epithelium of mice after intestinal I/R was significantly changed ，including 1 503 up- regulated lncRNAs and 2 099 down- regulated lncRNA （Fold change≥2，P<0.05）. At the same time，1 528 mRNA were up- regulated in I/R group，while 1 630 mRNA were down- regulated（fold change≥2.0，P<0.05）. GO enrichment analysis showed that the main functions involved in regulation were lipid metabolism，redox reaction，stress reaction，apoptosis process，programmed cell death，cell cycle，inflammatory response，endothelial cell differentiation and proliferation， tissue remodeling，MAPK，Wnt，vascular endothelial growth factor signaling pathway and so on. Apoptosis-related subitems were enriched in the up- and down-regulated annotations of GO molecular function in different forms ，which were in the forefront. There was a significant co-expression relationship between apoptosis- related mRNA and dysregulated lncRNA. 【Conclusion】 In this study，we established and preliminarily validated the expression profiles of the differentially expressed lncRNA at the early stage of reperfusion in mouse intestinal ischemia injury. Bioinformatics analysis showed that the important biological function of dysregulated lncRNA was the regulation of apoptosis-related processes，and a large number of those lncRNA were indeed highly coexpressed with apoptotic genes ，which would provide a basis and direction for future research.

In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.
Acute Lung Injury ; chemically induced ; drug therapy ; genetics ; metabolism ; Adaptor Proteins, Vesicular Transport ; genetics ; metabolism ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Cardamine ; chemistry ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Flowers ; chemistry ; Humans ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Plant Extracts ; administration & dosage ; chemistry ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Objective:To study the effect of combined decoction and single decoction of Houpu Wenzhongtang on rats with deficiency-cold of spleen and stomach from the perspective of metabonomics, to find out the relevant potential biomarkers and metabolic pathways, and to explore the similarities and differences between the combined decoction and single decoction, so as to provide reference for the feasibility analysis of replacing traditional decoction with single dispensing granule of this formula. Method:SD rats were randomly divided into normal group, model group, Houpu Wenzhongtang combined decoction group and simgle decoction group. Rats in the normal group were given distilled water by intragastric administration, rats in the other three groups were given cold vinegar at 4 ℃ in the morning and refined lard in the afternoon for 10 days (the dosage of 10 mL·kg^-1). After the model was successfully established, rats in the combined decoction group and the single decoction group were given corresponding decoction with dosage of 1.8 g·kg^-1 (according to the amount of crude drugs), once a day for 7 days. Ultra-high liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) technique was used to analyze the small molecular endogenous metabolites in urine. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and principal component analysis (PCA) were used to compare the changes of differential metabolites among the normal group, model group, Houpu Wenzhongtang combined decoction group and single decoction group, and the differential metabolites were introduced into Kyoto Encyclopedia of Genes and Genomes (KEGG) for metabolic pathway analysis. Result:Compared with the model group, the Houpu Wenzhongtang combined decoction group and single decoction group jointly regulated 13 potential biomarkers, including phosphatidylcholine(PC), lysophosphatidic acid(LysoPA) and cholic acid, etc. They played a role in treating deficiency-cold of spleen and stomach by influencing metabolic pathways such as glycerophospholipid metabolism, glycerolipid metabolism, phosphatidylinositol signaling system and so on. The combined decoction and single decoction of Houpu Wenzhongtang could obviously restore the body weight, motilin and gastrin contents of rats with deficiency-cold of spleen and stomach to normal levels. Conclusion:According to biochemical indexes, there is no obvious difference between combined decoction and single decoction of Houpu Wenzhongtang, but according to metabonomics, the combined decoction may be slightly better than the single decoction. The research shows that it is feasible to replace traditional decoction with single dispensing granule of Houpu Wenzhongtang in clinical application.