OBJECTIVE To construct an insulin-resistant(IR)small intestinal organoid model of mice and study the protective effect of flavanomarein(FM)on the intestinal mucosal barrier in the model.METHODS ①Small intestinal organoid models of C57BL/6J and db/db of mice were constructed.The expressions of Ki-67,E-cadherin(E-cad),lysozyme(Lyz)and mucin-2(Muc-2)in small intestinal organ-oids were detected by 3D immunofluorescence.RT-qPCR was used to detect the expressions of fibro-nectin(Fn),glucagon-like peptide-1(GLP-1)and peotide YY(PYY)mRNA while Western blotting was used to detect the expressions of Fn,GLP-1 and PYY protein.The Lyz secretion level was detected by ELISA.② Small intestinal organoids were divided into five groups:C57BL/6J mice 'small intestinal organ-oids as the normal control group,db/db mice' intestinal organoids as the IR model group,db/db mice small intestinal organoids with flavanomarein 25,50 and 100 μmol·L-1 intervention for 48 h as IR model+ FM groups.RT-qPCR was used to detect the expression of Lyz mRNA while Western blotting was used to detect the expression of Lyz protein.RESULTS ① On the 6th day of small intestinal organoid culture,a ring structure with a clear luminal structure was formed and an IR mouse small intestinal organoid model was established.3D Immunofluorescence detection showed that the established small intestinal organoids all expressed Ki-67,E-cad,Lyz and MUC-2.Compared with the normal control group,the expres-sion of Fn mRNA in the IR model group was significantly increased(P<0.05)while the expressions of GLP-1 and PYY mRNA were significantly decreased(P<0.05).Compared with the normal control group,the expression of Fn protein in the IR model group was significantly decreased(P<0.05)while the expressions of GLP-1 and PYY protein were significantly increased(P<0.05).ELISA results showed that compared with the normal control group,the secretion levels of Lyz in the IR model group were signifi-cantly decreased(P<0.01).② RT-qPCR results showed that compared with the normal control group,the expression of Lyz mRNA in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz mRNA in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).Western blotting results showed that compared with the normal control group,the expression of Lyz protein in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz protein in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).CONCLUSION The constructed IR mouse small intestinal organoid model provides a more complete in vitro research model for exploring the pathophysiological mechanism by which drug interventions help repair the intestinal mucosal barrier.FM may maintain the intestinal mucosal barrier by reversing the decrease in Lyz expression levels in IR mice,thereby improving IR.

Heme oxygenase-1(HO-1)is an inducible heme oxygenase and a catalytic enzyme for heme decomposition reactions,which can catalyze the heme decomposition into CO,biliverdin and Fe2+.HO-1 and its metabolites have anti-inflammatory,antioxidant and anti-apoptotic effects in human body,and play an important role in neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease,amyotrophic lateral sclerosis,and Huntington's disease.This article will review the production,distribution,and gene structure of HO-1,the biological characteristics of its metabolites,and the role and mechanism of HO-1 in neurodegenerative diseases,in order to provide a theoretical basis for the clinical application of HO-1.

Objective To investigate the expression and significance of the adaptor protein epsin 3 (EPN3) in colorectal cancer in order to provide reference for further stud)' of EPN3. Methods GEPIA and GEDS were used to analyze the expression of EPN3 in colorectal cancer tissues and cells. SMART and cBioPortal databases were used to analyze the relationship between EPN3 gene metfrylation and cop)' number variation and its expression level. Metascape was used to complete analysis of gene ontology functional annotation and related pathways of EPN3 related genes and BioPlex was applied to construct a protein network in HCT116 cell. Thirteen pairs of colorectal cancer adjacent tissue and cancer tissue specimens were collected, and EPN3 mRNA expression were detected by Real-time PCR. The effect of abilities of cell proliferation, clone formation and migration via silencing EPN3 in HCT116 and HT29 were observed. Results GEPIA, GEDS, SMART and cBioPortal analyses showed that EPN3 was highly expressed in colorectal tumor tissues (P<0. 01), and was related to methylation and copy number variation. The enrichment result of EPN3 related genes showed that it was mainly related to cell adhesion. And a protein interaction network constructed by CCDC130, TNFAIP1, PHGDH, EPN2, etc. was related to protein ubiquitination. Real-time PCR result showed that EPN3 was highly expressed in tumor tissues (P<0. 05). Silencing EPN3 inhibited the proliferation, clony formation and migration abilities of HCT116 and HT29 cells. Conclusion EPN3 is highly expressed in colorectal cancer tissues and is related to cell adhesion and protein ubiquitination. Down-regulated EPN3 can inhibit abilities of proliferation, clony formation and migration of HCT116 and HT29 cells, and this could provide a reference for further research on EPN3.

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.
Animals ; Cyclization ; Endo-1,4-beta Xylanases ; chemistry ; metabolism ; Enzyme Stability ; Industrial Microbiology ; methods ; Microbiota ; Protein Engineering ; Rumen ; enzymology ; microbiology ; Temperature

Objective :To explore therapeutic effect of emergency percutaneous coronary intervention (PCI) combined ticagrelor on patients with acute ST elevation myocardial infarction (STEMI ) and its influence on level of tumor necrosis factor (TNF)—α.Methods : A total of 98 patients with acute STEMI treated in our hospital from Jan 2016 to Jan 2017 were selected .Patients were randomly and equally divided into clopidogrel group and ticagrelor group , each group received corresponding medication before PCI .LVEF ,LVEDd ,TNF—α level before and one month after treatment and TIMI grade before and after PCI ,recanalization time ,postoperative corrected TIMI frame (CTFC) , percentages of thrombus aspiration ,auxiliary IABP and no—reflow ,and incidence of adverse events during six—month follow—up after PCI were observed and compared between two groups .Results : Compared with before PCI and treatment ,there were significant rise in LVEF on one month after treatment and TIMI grade after PCI ,and signifi—cant reductions in LVEDd and TNF—α level in two groups on one month after treatment , P＝0.001 all.Compared with clopidogrel group on one month after treatment ,there was significant rise in LVEF [ (49.80 ± 4.17 )% vs. (57.32 ± 5.10)%] ,and significant reductions in LVEDd [ (57. 94 ± 4. 70) mm vs .(47.11 ± 3.49) mm] ,TNF—α level [ (17. 82 ± 2.84) pg／ml vs .(8.40 ± 2. 04 ) pg／ml] and postoperative percentage of no—reflow (24.44% vs. 4.44%) in ticagrelor group , P＜0. 01 all ,there were no significant difference in other operative related indexes be—tween two groups , P＞0.05 all.After six—month follow—up ,incidence rate of composite endpoint events in ticagrelor group was significantly lower than that of clopidogrel group (11. 11% vs.31. 11%) , P＝0.021 ,there were no sig—nificant difference in percentages of in—stent thrombus ,infarct related artery revascularization and recurrent unsta—ble angina pectoris between two groups , P＞0.05 all .Conclusion : Short—term therapeutic effect of emergency PCI combined ticagrelor is significant .It can significantly improve prognosis ,which is worth extending .

Objective To investigate the role of bone marrow mesenchymal stem cells (BM-MSCs) in the invasion and metastasis of gastric cancer cells and to explore its mechanism.Methods SGC7901 and KATO-Ⅲ gastric cancer cells were co-cultured with BM-MSCs respectively,and the invasion ability of SGC7901 and KATO-Ⅲ gastric cancer cells were detected by Transwell assay.Secondly,CD133 + and CD133-cells were sorted from KATO-Ⅲ gastric cancers and co-cultured with BM-MSCs respectively to compare their changes in invasiveness.Meanwhile,the expressions of p-AKT and epithelial-mesenchymal transition (EMT) relative factors in gastric cancer cells were detected by Western-blot.The role of CD133 in BM-MSCs affecting the ability of invasion of gastric cancer cells was further vertified by the overexpression of CD133 in SGC7901 cells.SPSS17.0 software was used for statistical processing,and the stand deviation of the measurement data were expressed as the standard deviation,independent sample t test was conducted.Results The invasiveness of co-cultured SGC7901 and KATO-Ⅲ cells was significantly enhanced.The invasive ability of KATO-Ⅲ CD133+ cells co-cultured with BM-MSCs tended to increase more significantly than that of co-cultured CD133 cells[(259.0 ± 24.0)vs (58.0 ±5.6),P ＜ 0.001].The expressions of p-AKT,Snail and N-cadherin were significantly increased in co-cultured CD133+ cells (P =0.003,P =0.003,P =0.002),while the expression of E-cadherin was reduced (P =0.021).After co-cultured with BM-MSCs,the expression of E-cadherin was also reduced in CD133-cells (P =0.005),but the expressions of p-AKT,Snail and N-cadherin were no significantly changes (P =0.744,P =0.277,P =0.295).SGC7901 co-cultured with BM-MSC after overexpression of CD133 showed higher i nvasiveness than blank control group[(239.3 ± 24.0) vs (103.3 ± 15.5),P ＜ 0.001].The expressions of p-AKT,Snail and N-cadherin were significantly increased when co-cultured with BM-MSCs in the group of CD133 overexpression (P =0.001,P =0.001,P =0.001),while the expression of E-cadherin was significantly decreased(P =0.003).The expressions of Snail and N-cadherin were also significantly increased after co-cultured with BM-MSCs in the blank control group (P =0.001,P =0.004),and the expression of E-cadherin was significantly decreased (P =0.018),while the expression of p-AKT was not significantly changed (P =0.193).Conclusions BM-MSCs can enhance the invasion and metastasis of gastric cancer cells by promoting the EMT of gastric cancer cells.CD133 may be involved in the regulation of EMT in gastric cancer cells through the PI3K/AKT signaling pathway.

Objective     To explore the effect of 16F gastric tube on pain relief in postoperative lung cancer patients. Methods     A total of 118 lung cancer patients were treated with radical resection of lung cancer in our hospital between January 2015 and May 2016. The patients were assigned into two groups: a 16F gastric tube group (16F group, 60 patients, 30 males and 30 females at age of 41-73 (52.13±7.83) years and a 28F drainage tube group (28F group, 58 patients, 25 males and 33 females at age of 45-75 (55.62±4.27) years. Clinical effects were compared between the two groups. Results     There was no statistical difference in drainage time (4.47±1.03 d vs. 4.24±1.16 d, P=0.473), drainage amount (560.37±125.00 ml vs. 656.03±132.45 ml, P=0.478), incidences of pneumothorax (5/60 vs. 2/58, P=0.439), pleural effusion (6/60 vs. 3/58, P=0.522), and subcutaneous emphysema (3/60 vs. 1/58, P=0.635) between the two groups (P>0.05). The pain caused by the drainage tube in the16F group was less than that in the 28F drainage tube group with a statistical difference (F=4 242.996, P<0.001). The frequency of taking analgesics in the 16F group was significantly less than that in the 28F group (12/60 vs. 26/58, P<0.001). Conclusion     The effects of draining pleural effusions and promoting lung recruitment are similar between the 16F group and the 28F group. However, the wound pain caused by 16F gastric tube is significantly less than that by 28F drainage tube.

Objective To investigate the correlation between the subjective well-being,optimism and coping mode of patients with inflammatory bowel disease(IBD). Methods A total of 159 IBD patients were surveyed and analyzed by the general information questionnaire, General Well-Being Schedule, Revised The Life Orientation Test, and Simplified Coping Style Questionnaire. Results The subjective well-being scores of IBD patients were (75.04 ± 14.32). And the Pearson analysis showed that subjective well-being scores were positively correlated with optimism scores and positive coping scores(r=0.408, P＜0.01; r=0.429, P＜0.01). Negative coping was negatively correlated with subjective well-being scores(r=-0.174, P＜0.05). Optimism had a direct positive predictive effect on subjective well-being with a path coefficient of 0.22, and coping mode was an intermediary variable of optimism and subjective well-being. Conclusion The subjective well-being of patients with IBD needs to be further improved, which is both related to optimism and coping mode. Coping mode takes effects as a mediator in optimism and subjective well-being.

BACKGROUNDMedullary cystic kidney disease (MCKD) is clinically indistinguishable from several other autosomal-dominant renal diseases; thus, molecular genetic testing is needed to establish a definitive diagnosis. A specific type of single cytosine insertion in the variable number tandem repeat (VNTR) of the mucin 1 (MUC1) gene is the only known cause of MCKD1; however, genetic analysis of this mutation is difficult and not yet offered routinely. To identify the causative mutation/s and establish a definitive diagnosis in a Chinese family with chronic kidney disease, clinical assessments and genetic analysis were performed, including using a modified genotyping method to identify the MUC1- VNTR single cytosine insertion.

METHODSClinical data from three patients in a Chinese family with chronic kidney disease were collected and evaluated. Linkage analysis was used to map the causative locus. Mutation analysis of uromodulin (UMOD) gene was performed using polymerase chain reaction (PCR) and direct sequencing. For MUC1 genotyping, the mutant repeat units were enriched by MwoI restriction, and then were amplified and introduced into pMD-18T vectors. The 192 clones per transformant were picked up and tested by colony PCR and second round of MwoI digestion. Finally, Sanger sequencing was used to confirm the MUC1 mutation.

RESULTSClinical findings and laboratory results were consistent with a tubulointerstitial lesion. Linkage analysis indicated that the family was compatible with the MCKD1 locus. No mutations were found in UMOD gene. Using the modified MUC1 genotyping method, we detected the MUC1-VNTR single cytosine insertion events in three patients of the family; and mutation-containing clones were 12/192, 14/192, and 5/96, respectively, in the three patients.

CONCLUSIONSClinical and genetic findings could support the MCKD1 diagnosis. The modified strategy has been demonstrated to be a practical way to detect MUC1 mutation.

Objective To investigate the value of blood culture result in guiding clinical rational use of drugs in clinic.Methods The medical records of 100 children patients with blood culture positive in the four departments of our hospital were retrospectively analyzed.Results The empirical treatment accounted for 98%,the goodness of fit in the medication before culture with the appropriate antibacterial drugs according to final detected bacteria was only 51.6%;after issuing the level 1 report,the adding and substituting drugs in adjusting the antibacterial drugs accounted for about 20% each,60% of antibacterial drugs were not adjusted;after issuing the level 2 report,about 5% of children patients added the antibacterial drugs,the proportion for replacing antibacterial drugs reached 32%,meanwhile the proportion for reducing accounted for 3%;after issuing the level 3 report,the consistency of antibacterial drugs use with the final drug susceptibility test results reached 83%,the inconsistency was only about 5%.Conclusion Clinical pediatric doctors in this hospital still heavily rely on the results of blood culture 3 levels reports,executing antibacterial agents from the experience treatment to the target treatment is also very active.