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Objective:To study the effect of senescence gene silent information regulator 6 (Sirt6) knockout on the brain of aged mice.Methods:Sirt6-flox transgenic mice were constructed, and the mouse brain tissue was specifically knocked out by Emx1-Cre tool mice.According to genotyping, 11 wild-type mice were selected as control group(WT group) and 10 Sirt6 gene konckout mice were selected as conditional knockout group(cKO group). Body size and body weight of the aged mice were measured and cerebral cortex thickness was measured by HE staining.Brain neurogenesis was analyzed with EdU markers.The expression of RNA-binding protein HuR and apoptosis-related protein Caspase-3 were detected by Western blot.Meanwhile, histone acetylation levels in the cortex were detected.Results:Sirt6 brain tissue-specific knocked out mice were successfully constructed.Compared with the brain tissue area((2.07±0.22) cm 2)and cortical thickness ((970.56±80.91) μm) of WT mice in the 12-month-old group, the brain tissue area ((1.61±0.14)cm 2) and cortical thickness ((822.88±53.94) μm) in Sirt6 cKO group were smaller, and the differences were statistically significant (both P<0.05). EdU incorporation into nerve cells showed that the number of EdU incorporation into periventricular nerve cells in cKO group was lower ((4.75±1.48)) than that in WT group ((10.29±1.93)). The difference was statistically significant ( P<0.001). In the experiment of 17 months age group, mice in cKO group were smaller in body size, lower in body weight ((29.00±1.08) g) and smaller in brain area ((1.54±0.55)cm 2)compared with WT group in body size, body weight ((35.25±4.17) g) and brain tissue area ((1.98±0.18) cm 2)(both P<0.05). The expression of Caspase-3 and HuR in cortical proteins of these two age groups decreased( t=2.95, 5.38, both P<0.05), and the expression of H3K9ac and H3K56ac increased( t=3.53, 2.78, both P<0.05), but the expression of Sirt1 homologous gene remained unchanged( t=1.26, P>0.05). Conclusion:The specific deletion of Sirt6 in brain tissue can lead to the decrease of brain neurogenesis in aged mice, and the aggravation of aging and the increase of apoptosis, which may be the reason for the thinning of cerebral cortex and brain tissue atrophy.The molecular mechanism is speculated to be related to the increase of acetylation level after Sirt6 knockout
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Objective To determine the effect of NS3 and NS4A proteins of Zika virus on the neuronal migration in vivo.Methods To identify the coding sequence of NS3 and NS4A,the genome of Zika SZ01 was sequenced by rapid amplification of cDNA ends (RACE) and reverse-transcription PCR,then NS3 and NS4A was constructed in pCIG vector fused with Flag-tag to express these proteins.And then these plasmids was transfected into the embryo brain of E13.5 mice by in utero electroporation,the distribution of the cells which express these proteins in the cortex was detected by Flag,eGFP and TBR1 fluorescence in E18.5 mice through immunohistochemistry so as to assess the influence of viral proteins on the neuronal migration of mouse cortex.Results 1) Sequence results showed that the amino acid sequence of NS4A is consistent with NCBI data,while NS3 has 1 amino acid mutant.2) As the fluorescence of Flag and eGFP can co-localization,the eGFP fluorescence signal marks the cells that have expressed these virus proteins in cortex.3) TBR1 fluorescence shows the distribution of the cells that express NS4A in vivo are significantly different from pCIG control and NS3 (P＜0.001).Conclusions The NS4A protein of Zika virus may affect the neuronal migration in vivo.
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Objective To observe the clinic efficacy of open transforaminal lumbar interbody fusion (TLIF) compared with minimally invasive operation in treating lumbar spinal stenosis and instability among obese and non‐obese patients .Methods A ret‐rospective analysis was performed in these cases of mono‐segmental lumbar spinal stenosis and instability between January 2011 and January 2013 .Perioperative index ,clinical efficacy ,and imaging results were observed and compared between different groups .Re‐sults Thirty‐four obese cases and 105 non‐obese cases were divided into two groups ,including conventional posterior open TLIF and minimally invasive TLIF operation ,to compare the results .Perioperative indexes of obese patients were more than non‐obese patients undergone open TLIF operation way and there was significant difference(P0 .05) .No cases of slippage or breakage of implants were found among all these patients after 6 months of follow up .Postoperative VAS and ODI among these four groups were better than before(P0 .05);undergoing minimally invasive postoperative VAS in obese group and in non‐obese group ,there was not significant difference(P>0 .05) .Conclusion Therefore ,obese may be risk factor in treating lumbar spinal stenosis and instability .
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GROβis a member of the CXC chemokine superfamily.It plays an important role in inflammation and wound healing process.As extensive research continued, researchers realized that the gro gene was one of onco-genes.And its expression product, GROβ, was also found to be very important in angiogenesis, tumorigeness, me-tastasis, and interaction between tumor and immune cells.
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BACKGROUND:Recently, minimal y invasive techniques obtained more attention. Some new minimal y invasive methods have been used in the treatment of spine fracture and provide new chal enges for conventional open surgery. OBJECTIVE:To discuss the clinical efficacy of conventional posterior open pedicle screw fixation versus minimal y invasive operation (using Mast Quadrant System and Sextant percutaneous pedicle screw fixation) for treating single-segment thoracolumbar fractures without neurological damages. METHODS:A total of 94 cases of single-segment thoracolumbar fracture without neurological damages, who were treated in Department of Spine Surgery, Liuzhou Worker’s Hospital in China from January 2012 to January 2013, were enrol ed in this study. According to patients’ conditions and wil ing, they were divided into open fixation group, Quadrant fixation group and percutaneous Sextant fixation group. Perioperative index, clinical efficacy, and imaging results were observed and compared among different groups. RESULTS AND CONCLUSION:Intraoperative blood loss, incision length and length of stay were better in the Quadrant fixation group and percutaneous Sextant fixation group than in the posterior open fixation group (P0.05). Postoperative Visual Analog Scale scores and Oswestry Disability Index were better in the two minimal y invasive groups than in the conventional open fixation group (P<0.05). These results suggested that compared with conventional open operation, minimal y invasive operation (Mast Quadrant System and Sextant percutaneous pedicle screw fixation) in the treatment of thoracolumbar fractures not only can achieve similar imaging result, but has smal incision, less blood loss, quick recovery, high safety, and obtains good clinical therapeutic outcomes. In the case of strict surgical indications, minimal y invasive method is an ideal choice in treating thoracolumbar fractures without neurological damages.
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Objective To explore correlates of health-care seeking behavior in patients with irritable bowel syndrome (IBS).Methods Four thousand permanent residents were recruited from eight urban communities and rural villages in Guangzhou and Huizhou, Guangdong province during 2009 by cluster stratified sampling for face-to-face questionnaire survey, including symptoms of bowel disease,behavior of seeking for health-care, demographic characteristics, coping style, life events and medical history.IBS was identified based on the Rome Ⅱ Criteria.Patient with IBS were divided into two groups,one seeking health-care at hospitals or clinics and the other non-seeking health-care.Univariate and multivariate logistic regression analysis was used to compare difference between the two groups and explore its related factors.Results A total of 237 IBS patients were identified based on the Rome Ⅱ Criteria, 53 of them (22.4% ) had sought health-care due to their symptoms.Results of multivariate logistic regression analysis showed that preference in seeking for health-care, abdominal pain lasting for more than one hour in each episode and extra-gastrointestinal symptoms were main factors related to their seeking for health-care,adjusted for age and gender, with odds ratios (ORs) of 1.81 (95% CI: 1.27 -2.58), 1.41 (95% CI:1.01 - 5.14 ) and 2.14 ( 95% CI: 1.06 - 4.33 ), respectively.Conclusions Extra-gastrointestinal symptoms and abdominal pain lasting for more than one hour in each episode correlate their health-care seeking behavior in patients with IBS, as well as their preferences in seeking for health-care.
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Objective To express the human recombinant SUMO1 protein and prepare monoclonal antibody(mAb) against it.Methods The recombinant expression plasmid pET32a-HIS-SUMO1 was made and transformed into E.coli(BL21),then the recombinant fusion protein HIS-SUMO1 was expressed and purified.The BALB/c mice were immuned with pure protein HIS-SUMO1 as antigen.Monoclonal antibody against SUMO1 was prepared with standard hybridoma technology.The hybridoma cell lines were obtained by ELISA and Western blot screening procedure,the isotype of the mAbs were further identified by immune-double diffusion.Ascites were collected from one propagated hybridoma cell line and mAbs were purified by using the Kit of Millipore.The valence of mAb was detected by Western Blot.Results The recombinant protein HIS-SUMO1 is expressed and purified.Three hybfidmas producing antibodies against SUMO1 were obtained,the isotypes of three mAbs are IgG1,Western blot showed that the antibodies were specific for SUMO1.The antibody purified from the ascites has better specificity.Conclusion The SUMO1 mAb prepared by using recombinant SUMO1 protein as antigen can be used for detectingthe protein sumoylation.
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Objective To prepare the single chain antibody against N protein of SARS-CoV. Methods With N protein of SARS-CoV expressed in E.coli as antigen, we obtained the single chain antibody against N protein by screening the phage display library of human single chain antibodies. Results The anti-N protein antibody didn’t cross-interacte with BSA and the short peptide containing 6 histidines. The specific interaction between the antibody and N protein was inhibited by the anti-N protein monoclone antibody from immunized mice. ConclusionThe single chain antibody we got is specific to N protein of SARS-CoV,it can be a candidate antibody for fast detection of N protein of SARS-CoV and SARS virus particles in clinical trial study of SARS pathogenesis.
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Post-translational modification by ubiquitin and ubiquitin-like modifiers(Ubls) is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes.Previous research showed that,through covalently modification by ubiquitin or ubls,the substrate proteins can be regulated in many different ways like stability,subcellular localization,enzymatic activity,protein-protein interaction and so on.Therefore,we believe,that ubiquitin and ubls play very important roles in cellular and biological processes by modifying plenty of proteins.To better understand the ubiquitin and ubls system,proteomic approaches have been developed to purify and identify more protein substrates.Large-scale idendification of ubiquitin/ubls-modification sites by mass spectrometry is particularly important for understanding the molecular mechanism and function of ubiquitin/ubls modification.Upto the present,more and more scientists are getting interested and participating in proteomics research of ubiquitin/Ubl modifications.This review summarizes the rencent results in this field.
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It's a multi-protein complex and not single protein that accomplish most of cellular activity.Therefore,to identify and analyze the composition of protein complex is necessary for studying the function of proteins.Tandem affinity purification technique(TAP) enables the effective purification of complex under physiological conditions without knowledge of the complex composition and function.Now,it has been adapted to analyze the composition of the protein complexes in yeast.Combined with mass spectrometry,TAP can identify the interacting proteins of target protein and offer great help for opening protein interacting network and function out.
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Objective To study the effect of PcG member NSPc1 on proliferation of HeLa cells.Methods Using bioinfomatic analysis to design the siRNA sequence to knockdown NSPc1.Detecting the expression level of NSPc1 in HeLa cell line using semi-quantitative RT-PCR,Real-time PCR and Western blot after transfection of the designed siRNA.Transient transfecting pSUPER-NSPc1 into Hela cells and performing BrdU incorporation assay.Establishing NSPc1 stably knockdown cell line,comparing proliferation abilities with the control cells.Results(1)The designed siRNA did efficiently knockdown the expression of NSPc1;(2)Transient knockdown of NSPc1 could repress BrdU incorporation;(3) The established NSPc1-knockdown cell lines had a significantly lower proliferation rate than that of control cells.Conclusion The expression of NSPc1 is necessary for the normal proliferation of HeLa cells.The NSPc1 stably knockdown cell pool is a useful model for further study of pathway related to NSPc1.
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<p><b>OBJECTIVE</b>To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern.</p><p><b>METHODS</b>According to the sequence of human EST, which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple-tissue Northern blot.</p><p><b>RESULTS</b>Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified. Both containing the ubiquitin conjugating domain, the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence. They belong to a highly conserved and widely expressed E2 enzyme family. Northern blot shows that they are expressed exclusively in adult human heart, placenta, and pancreas but no transcripts can be detected in brain, lung, liver, skeletal muscle or kidney.</p><p><b>CONCLUSIONS</b>The gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.</p>
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Animals , Female , Humans , Mice , Rats , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary , Genetics , Molecular Sequence Data , Myocardium , Metabolism , Pancreas , Metabolism , Placenta , Metabolism , Sequence Alignment , Sequence Analysis, DNA , Ubiquitin , Genetics , Ubiquitin-Conjugating Enzymes , Chemistry , GeneticsABSTRACT
0.05). Conclusions Gastric hypersensitivity, impaired proximal gastric accommodation and delayed gastric emptying may be important but independent pathophysiological factors of FD. Different pathophysiological factors can coexist in one patient with FD.