Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase (PAF-AH) isoform I , and study the enzyme activity by different substrates. Methods The (3 subunit of PAF-AH isoform I was cloned and expressed in E. coli. Exogenously expressed recombinant protein was purified to SDS-PAGE homogeneity, and its activity was identified by arylesterase detection. Phenylacetate, 1-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine ( 2-Thio PAF) and l-myristoy1-2-( 4-nitrophenylsuccinyl) phosphatidylcholine (the latter two were commercial plasma PAF-AH substrates) were used for the substrate identification. The plasma type PAF-AH was served as positive control. Results Recombinant protein of β subunit of PAF-AH isoform I was successfully constructed and expressed in E. coli after purification. Compared with positive control, the recombinant protein could hydrolyze phenylacetate and 2-Thio PAF, but could not hydrolyze l-myristoyl-2-( 4-nitrophenylsuccinyl) phosphatidylcholine. Conclusion Recombinant protein of β subunit of PAF-AH isoform I can be successfully constructed. There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoform I and plasma type PAF-AH, which provides a quick method to differentiate PAF-AH isoform I from plasma type PAF-AH.

Objective:To explore the effect of inherent depression on chronic visceral hypersensitivity. The differences of visceral sensitivity, colitis, and brain activation between Fawn-Hooded ( FH/Wjd) and Sprague-Dawley( SD) rats were identified after neonatal colon acetate stimulation.Methods:The specific pathogen free Fawn-Hooded (FH/Wjd) and Sprague-Dawley(SD) rats were used to establish irritable bowel syndrome (IBS) model.The visceral sensitivity was measured by colorectal distension (CRD). The expression of 5-hydroxytryptamine (5-HT), mast cell (MC), indoleamine 2,3-dioxygenase (IDO) in colon and IDO in specific cerebral regions were detected through immunohistochemistry.Results:Ab-dominal withdrawal reflex ( AWR) scores showed that visceral sensitivity of acetate-enema groups was sig-nificantly higher than that of saline-enema groups ( FH/Wjd:2.44 ±0.04 vs.1.96 ±0.07, P<0.05;SD:1.75 ±0.13 vs.1.32 ±0.05, P<0.05).Furthermore, FH/Wjd rats of IBS group scored signifi-cantly higher than SD rats of IBS group (2.44 ±0.04 vs.1.75 ±0.13, P<0.05).The MC amounts of both SD and FH/Wjd IBS group rats were significantly more than those of their control groups ( FH/Wjd:43.24 ±1.72 vs.24.92 ±1.38, P <0.01.SD:23.80 ±1.28 vs.14.24 ±0.92, P <0.01).
Besides, the MC amounts of control and IBS group of FH/Wjd rats were significantly more than that of SD IBS group rats ( P<0 .01 ) .The IDO and 5-HT positive cells in colonic mucosa of IBS group of both SD and FH/Wjd rats were significantly more than those of their control groups, respectively(P <0.01). The IDO, 5-HT positive cells in colonic mucosa of both control and IBS group of FH/Wjd rats were significantly more than those of both control and IBS group of SD rats ( control:IDO,24.64 ±2.22 vs. 15.52 ±1.39;5-HT,21.32 ±1.26 vs.12.72 ±1.12.IBS: IDO,44.92 ±2.31 vs.20.85 ±1.72;5-HT, 31.84 ±1.57 vs.19.65 ±1.09.P <0.01).The expression of IDO in prelimbic cortex (PrL) areas of FH/Wjd IBS rats was significantly higher than that of IBS group of SD rats (49.60 ±4.31 vs. 35.60 ±2.42, P <0.01) , and the expression of IDO in rostral anterior cingulate cortex ( rACC) areas of FH/Wjd IBS rats was significantly more than that of FH/Wjd control rats (45.44 ±1.16 vs.34.08 ± 2.76, P <0.01) .Conclusion:Inherent depressive FH/Wjd rats were more sensitive to neonatal colon acetate stimulation, presenting as visceral hypersensitivity which maybe associated with increased MC amounts and over-expression of 5-HT and IDO in colon, suggesting that depression disorder may aggra-vate functional disturbance of gastrointestinal tract by regulating the response to inflammatory stimulation.

Objective:To investigate the association of surrogate indexes of insulin resistance with the risk of nonalcoholic fatty liver disease(NAFLD)and advanced liver fibrosis in newly diagnosed patients with type 2 mellitus.Methods:A total of 429 newly diagnosed type 2 diabetic patients hospitalized in the Department of Endocrinology and Metabolism, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine from June 2018 to June 2020 were included. The patients were divided into NAFLD group( n=263)and non-NAFLD group( n=166)according to the results of abdominal ultrasound, NAFLD patients were divided into advanced liver fibrosis group( n=33)and non-advanced liver fibrosis group( n=230)according to the NAFLD fibrosis score(NFS). The correlation of the triglyceride/high density lipoprotein-cholesterol ratio(TG/HDL-C), triglyceride glucose index(TyG), visceral adiposity index(VAI), triglyceride glucose-body mass index(TyG-BMI), homeostasis model assessment of insulin resistance index(HOMA-IR)with NAFLD and the occurrence of advanced liver fibrosis were analyzed. Results:The prevalence of NAFLD in newly diagnosed type 2 diabetic patients was 61.3%, and the prevalence of advanced liver fibrosis was 12.5%. After adjusting the potential confounders, multivariate logistic regression analysis showed that TG/HDL-C, TyG, VAI, TyG-BMI, and HOMA-IR were independently and positively correlated with NAFLD, and only TyG-BMI was independently and positively correlated with advanced liver fibrosis( OR=1.021, 95% CI 1.009-1.034, P<0.01). The receiver operating characteristic analysis showed that the area under the curue of TyG-BMI was more than those of TG/HDL-C, TyG, VAI and HOMA-IR for predicting the occurrence of NAFLD in newly diagnosed type 2 diabetic patients(0.83 vs 0.73, 0.74, 0.73, 0.67, P<0.01), but had no predictive value for advanced liver fibrosis. Conclusion:TyG-BMI is superior to TG/HDL-C, TyG, VAI and HOMA-IR for predicting the occurrence of NAFLD in newly diagnosed type 2 diabetic patients, and it is also an independent risk factor for advanced liver fibrosis.

Objective To observe the effect of heat needle treatment on the inflammatory reaction in the damaged tissues of rats with the third lumbar transverse process syndrome. Methods Ninety Sprague-Dawley rats were randomly divided into 6 groups, namely normal group, model group, drug group, heat needle group, conventional acupuncture group and warming acup-moxibustion group, 15 rats in each group. The rat model of the third lumbar syndrome was established by embedding gelatin sponge. Two weeks later, the rats in various groups were given corresponding treatment respectively. Before treatment and on the 7th, 14th and 28th day of treatment, the blood samples were collected from jugular vein, and then the serum inflammatory cytokines of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). On the 28th day of treatment, all of the rats were sacrificed, the expression levels of TNF-α, IL-1βand IL-6 mRNA in the muscle tissue were detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and the pathological changes in local muscle tissue were observed by HE staining method. Results The serum contents of the inflammatory cytokines of TNF-α, IL-1β and IL-6 in the model group and treatment groups after modeling were significantly higher than those in the normal group (P < 0.05). After treatment, the contents of TNF-α, IL-1β and IL-6 in various treatment groups were decreased significantly as compared with those in the model group, and the decrease was most obvious in the heat needle group. The expression levels of TNF-α, IL-1β and IL-6 mRNA in muscle tissues of drug group, heat needle group, conventional acupuncture group and warming acup-moxibustion group were lower than those of the model group (P < 0.05), and the heat needle group had the lowest levels. HE staining results showed that heat needle group had milder inflammatory reaction and better muscle fiber structure than the model group. Conclusion Heat needle treatment can improve the local inflammatory response and promote the repair of tissue damage through decreasing the expression levels of inflammatory cytokines of TNF-α, IL-1βand IL-6.

Objective To promote professional training aid on healthcare talents in western China,this paper aims to explore methods and to evaluate the effectiveness of a new training system for underprivileged medical professionals from hospitals of western China.Methods Questionnaires and interviews were conducted to 115 trainees and their employers took part in the Counterpart Support Project.A retrospective analysis was done to evaluate key indicators of the trainees' professional competence.Results More than 90％ of the trainees have seen great improvement on basic healthcare knowledge,clinical skills,and diagnosis and treatment practice.About 80％ the participating hospitals have enjoyed varied degrees of growth in outpatient visits,surgical volume,number of inpatient and operational efficiency.(Increase level from 0％ to 50％) Conclusions Within the frist 3 years of implementation,this new model has been proven effective to enhance participants' overall professional competence,and by tapping into the significant resource of first class hospitals,this model will help greatly promoting the development of healthcare talents in Western China.

Objective To quantitate coronary tortuosity and provide favorable conditions for understanding the hemodynamic effects caused by coronary tortuosity.Methods We obtained all images from 72 patients who received coronary computed tomo-graphy scanning.After image post-processing,we extracted the medial axis of three main coronary vessel and analysed its coordi-nates on three dimensions.Then we calculated the tortuosity coefficient of coronary artery.Results Tortuosity coefficient was 6.66±7.54 in anterior descending,13.43±12.85 in left circumflex,and 1 7.61 ±7.67 in right coronary artery.We had proved its validity by the changes in morphology with simulated shapes.Conclusion We proposed a new method for quantitating coronary tor-tuosity,by computed tomography coronary imaging.The measurement results would not be affected by projection plane,vessel length or other artificial factors.

Objective:To identify pathogenicity of the potential splicing variants in two Chinese Han patients with osteogenesis imperfecta.Methods:Genomic DNA was extracted using the conventional phenol-chloroform method; whole exome sequencing (WES) was used to analysis the disease-related variants in the two probands; Minigene assay was used to identify pathogenicity of the variants found in the patients' genome that possibly affect RNA splicing.Results:Two potential splicing variants, c.858+1_858+5delGTAAG in intron 12 of COL1A1 and c.1405-7C>T in intron 24 of COL1A2, were found in proband 1 and proband 2, respectively. In addition, a missense mutation, c.2972G>T (p.G991V) in exon 45 of COL1A2, was detected in proband 2. Minigene assay revealed that the variant in proband 1 caused the skipping of exon 12, while the variant in proband 2 did not lead to aberrant splicing. G199 of the COL1A2 in proband 2 was a highly conserved amino acid site, and the results suggested that c.2972G>T (p.G991V) may be the real pathogenic variant by the means of bioinformatics analysis.Conclusion:The variant c.858+1_858+5delGTAAG in COL1A1 was a causative variant that led to OI in proband 1, while the missense variant c.2972G>T (p.G991V) in COL1A2 was the cause of OI in proband 2, instead of the variant c.1405-7C>T. Minigene assay for potential splicing variants detected by WES could not only validate the pathogenicity of the candidate variants and enrich the mutation spectrum of OI, but also lay the foundation for patients' prenatal diagnosis and subsequent mechanism research.

In order to elucidate the mechanisms of p53 overexpression in nasopharyngeal carcinoma (NPC) and detect proteins associated with the function of p53 in high throughout screening, p53 which knockdown human NPC CNE2 cell line (CNE2sip53) were successfully established by using stable RNA interference (RNAi). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of CNE2sip53 and its control cell line CNE2/pSUPER, and PDQuest software was applied to analyze 2-DE images. Twenty-two differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3? etc.) , and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, the differential expression levels of the partial proteins (HSP27, 14-3-3?, GRP75) were confirmed by Western blot analysis and compared with CNE2 and CNE2 cells transfected with pcDNA3.1-FLAG, CNE2 cells transfected with pcDNA3.1-FLAG-p53 had obvious down-regulations of HSP27 and 14-3-3?, and an up-regulation of GRP75. The 22 differentially expressed proteins could be divided into five groups based on their functions: signal transduction, chaperone, transcription and translation, metabolism and cytoskeleton, which were involved in cell cycle, the transcription regulation, cell adherence,cellular metabolism and so on. The data suggest that these differential proteins may be associated with the function of p53 in NPC, and will be valuable for further to study the mechanisms of p53 overexpression and inactivation in NPC.

Objective To investigate the association between the tumour necrosis factor-α-863(TNF-α-863) polymorphism and gout in Han population from the city of Tangshan.Methods We recruited 80 gout patients and 80 healthy individuals into this study.The polymorphisms of TNF-α-863 site were analyzed by polymerase chain reaction-ligase detection reaction(PCR-LDR).The frequencies of different TNF-α-863 genotypes/alleles were analyzed in the gout group and the control subjects.Results No significant differences were observed in the genotype frequencies(x2=2.8807,P=0.0897) and allelic frequencies(x2=4.2646,P=0.1187) of TNF-α-863 site in the comparison between gout and control groups.Conclusion The result of our study suggests that the polymorphism of TNF-α-863 site may not related to gout in Han population in Tangshan.

Objective To evaluate desmopressin stimulated inferior petrosal sinus sampling in diagnosing Cushing′s disease.Methods Sixteen ACTH-dependent Cushing′s disease patients underwent bilateral desmopressin stimulated inferior petrosal sinus ( IPS ) sampling because of negative or equivocal magnetic resonance imaging.Cortisol response to high-dose dexamethasone suppression test was also evaluated.ACTH sampling was taken from a peripheral vein and bilateral IPS before and both 5 and 10 min after injection of desmopressin.Diagnosis was based on the ratio of ACTH level in between IPS to peripheral vein by desmopressin test.Diagnosis was confirmed after surgery.Results High-dose dexamethasone suppression test showed suppressible in 9 of 16 patients with Cushing′s disease.An IPS gradient ＞2 was found in 14 of the 16 cases (87.5％ )with Cushing′s disease after desmopressin injection,while before injection the respective figure was 12 of 16 (75.0％).No severe adverse effects were observed during or after the procedure.Conclusion Desmopressin test during bilateral IPS sampling is a safe and effective diagnostic procedure in Cushing′s disease.