Global quantitative proteomic analysis profiles of host protein expression in response to enterovirus A71 infection in bronchial epithelial cells based on tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS uncovers the key role of proteasome in virus replication.

Enterovirus A71 (EV-A71) is a neurotropic human pathogen which mainly caused hand, foot and mouth disease (HFMD) mostly in children under 5 years-old. Generally, EV-A71-associated HFMD is a relatively self-limiting febrile disease, but there will still be a small percentage of patients with rapid disease progression and severe neurological complications. To date, the underlying mechanism of EV-A71 inducing pathological injury of central nervous system (CNS) remains largely unclear. It has been investigated and discussed the changes of mRNA, miRNA and circRNA expression profile during infection by EV-A71 in our previous studies. However, these studies were only analyzed at the RNA level, not at the protein level. It's the protein levels that ultimately do the work in the body. Here, to address this, we performed a tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS approach to quantitatively identify cellular proteome changes at 24 h post-infection (hpi) in EV-A71-infected 16HBE cells. In total, 6615 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the EV-A71- and mock-infected groups, 210 differentially expressed proteins were found, including 86 upregulated and 124 downregulated proteins, at 24 hpi. To ensure the validity and reliability of the proteomics data, 3 randomly selected proteins were verified by Western blot and Immunofluorescence analysis, and the results were consistent with the TMT results. Subsequently, functional enrichment analysis indicated that the up-regulated and down-regulated proteins were individually involved in various biological processes and signaling pathways, including metabolic process, AMPK signaling pathway, Neurotrophin signaling pathway, Viral myocarditis, GABAergic synapse, and so on. Moreover, among these enriched functional analysis, the "Proteasome" pathway was up-regulated, which has caught our attention. Inhibition of proteasome was found to obviously suppress the EV-A71 replication. Finally, further in-depth analysis revealed that these differentially expressed proteins contained distinct domains and localized in different subcellular components. Taken together, our data provided a comprehensive view of host cell response to EV-A71 and identified host proteins may lead to better understanding of the pathogenic mechanisms and host responses to EV-A71 infection, and also to the identification of new therapeutic targets for EV-A71 infection.

Taxonomic classification and detection of potential pathogens in healthy voluntary blood donors: Based on metagenomics analysis / 中国输血杂志

【Objective】 To map a comprehensive description of microbiome presented in healthy blood donations in Liangshan Yi Autonomous Prefecture, and detect the potential pathogens and its prevalence. 【Methods】 A total of 1 299 blood samples were randomly selected from healthy blood donors in Liangshan Prefecture. Total nucleic acids were extracted and sequenced by high-throughput sequencing, and the microbiome was determined by metagenomics analysis. Quantitative real-time PCR (qPCR) and ELISA assays were used to detect antibodies against HSV DNA and HSA in each sample. The prevalence of HSV in healthy blood donors was compared in terms of gender, age, occupation, education level, and frequency of donation. 【Results】 3.49GB data were obtained from DNA pool through high-throughput sequencing. After filtering the data of human genome, the DNA sequences annotated as bacteria, parasites / fungi and viruses were 213 057, 10 623 and 15 reads, respectively. A total of 2.79GB data were obtained from cDNA pool, after filtering the data of human genome, the fragments annotated as bacteria, parasites / fungi and viruses were 4 105 600, 18 446 and 0 reads, respectively. The prevalence of IgG and / or IgM antibodies against herpes simplex virus type 1 and 2 were 8.62% (112 / 1 299) and 18.32% (238/1 299), and that of nucleic acid was 0.77 ‰ (1/1 299). 【Conclusion】 The microbiome of healthy blood donors in Liangshan Prefecture and the potential pathogens were identified in this study. Regional specificity of HSV infections emerged in Liangshan Prefecture. Considering the collaboration between HSV-2 and HIV infection, epidemiological investigation of HSV-2 infection should be conducted preferentially among different populations in Liangshan Prefecture and other HIV high prevalence areas in order to benefit the prevention and control of HIV.

Course construction and practice of clinical pathophysiology for postgraduate student / 中华医学教育探索杂志

Qinghai University lies in western China. There are some gaps between western and eastern universities at student level, education fund, experiment condition and technique supply for graduate students. To improve educational quality for graduate students, Department of Pathophysiology of Qinghai University focused on teaching methods, evaluation approaches and capacity of teachers and constructed a course named Clinical Pathophysiology for scientific research graduate students. The course adopted a series of measures such as combining lectures with students' self-learning (PBL, paper discussion, lecture for students), introducing experts from other schools to give lectures, establishing formative evaluation, optimizing teacher's group and so on, which have gained some achievements and improved the training quality for graduate students.

The expression and significance of cysteine-rich 61, connective tissue growth factor, vascular endothelial growth factor and microvascular density marked by CD105 in muscle tissue of patients with dermatomyosits and polymyosits / 中华风湿病学杂志

Objective By detecting vascular cysteine-rich 61(Cyr61) related factor,connective tissue growth factor (CTGF),vascular endothelial growth factor (VEGF) and CD105 markers of microvascular density (MVD) of muscle tissue in patients with PM/DM,the role and significance of the expression of Cyr61,CTGF,VEGF and CD105 in the process of vascular lesions of dermatomyosits (DM) and polymyosits (PM) were discussed.Methods The expression of Cyr61,CTGF,VEGF and CD105 markers of micro vascular density (MVD) were detected in 10 cases of DM,10 cases of PM and 20 controls by using immunohistochemical Envision two step method.Data were analyzed using Statistical Product and Service Solutions (SPSS) statistical software.Fisher's exact probability analysis and Spearman correlation analysis were conducted.Results Compared with the control group,Cyr61,CTGF,VEGF positive expression rate in muscle tissue of patients with DM and PM group were significantly different (P＜0.01),the positive expression rates of Cyr61,CTGF,VEGF in DM group and PM group were 90％,70％,90％,80％,80％,70％,and the control group (5％,10％,5％) respectively.In the muscle tissue of patients with DM and PM group,CD105 markers of capillaries could be seen,and MVD in DM and PM group were higher than that in the control group,the difference was statistically significant (F=8.103,P=0.001).Cyr61,CTGF and VEGF protein expression levels in muscle tissueof patients with DM and PM were positively correlated with MVD.Conclusion The muscle tissue of PM/DMpatients may have new blood vessels formation.Cyr61,CTGF,VEGF may be involved in the formation of newblood vessels in the PM/DM muscle tissue.The results of this study suggest that microvascular lesion plays animportant role in the immune pathogenesis of inflammatory myopathy such as PM/DM.

Clinicopathologic features of collagen III glomerulopathy / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features of collagen III glomerulopathy and its cause, pathogenesis and prognosis.</p><p><b>METHODS</b>Five cases of collagen III glomerulopathy that collected from 2005 to 2014 were observed by renal biopsy. The morphologic characteristics were studied by light microscopy, immunofluorescence, immunohistochemical and electron microscopy.</p><p><b>RESULTS</b>The glomerular mesangium became expansion but no hypercellularity, basement membrane appeared thickened. The glomeruli showed collagen type III deposit by immunohistochemistry method, and collagen fibers increased by electron microscopy. The patients often show serious proteinuria, nephrotic syndrome and renal function damage.</p><p><b>CONCLUSIONS</b>Collagen III glomerulopathy is an idiopathic glomerular disease, characterized by massive accumulation of collagen type III within the glomerular mesangial areas and basement membrane. Collagen III glomerulopathy is extremely rare. The etiology and pathogenesis may relate to the abnormality of collagen III gene. There is no specific treatment for it and its prognosis is poor.</p>

Innovation and practice of pathophysiological experiment examination system / 中华医学教育探索杂志

In order to adapt to the demand of culturing high quality talents and to strengthen student's comprehensive quality,pathophysiology department of medical college of Qinghai University carried on the reform on the pathophysiological experiment examination system.After researches and practices in recent years,we established a set of relatively sound examination system including experiment operating and experiment report in normal study,report for designable experiment and experiment skill and theory exam.Investigation results demonstrated that examination system after reform functioned well in training the students' operation ability,observation ability and ability to analyze and solve questions.

IL-18 IFN-γ expression in labial glands and blood of patients with primary Sj(o)ren's syndrome / 中华风湿病学杂志

ObjectiveThis study explored the change of IL-18,and IFN-γ and their role in the pathogenesis of primary Sj(o)gren's syndrome (pSS) by determining the expression of IL-18,IFN-γ in labial glands and blood of patients with pSS.MethodsThe levels of IL-18,IFN-γ in labial glands of patients with pSS were determined by immunohistochemical method.The level of serum IL-18 was determined with enzyme linked immunosorbent assay ELISA.The level of γ globulin was measured by the cellulose acetate membrane method.SPSS 17.0 statistical software was used for data analysis.The t test,Fisher's exact probability test and index correlation analysis were performed with linear correlation analysis.Spearman's test was used to examine the association between the expression of IL-18 and IFN.ResultsThe positive rate of IL-18 and IFN-γ in patients with pSS was much higher than normal control group(P＜0.05).And the IL-18 and IFN-γ levels were positively correlated withpatients with pSS (P＜0.05).The level of serum IL-18 in patients with pSS was (216±85) pg/ml,which was much higher than normal control group(39±8) pg/ml.The IL-18 level and γ globulin(23.2±2.6)％ was positively correlated in patients with pSS (r=0.538,P＜0.05).ConclusionThe expression of IL-18 and IFN-γ in labial glands of patients with pSS are much higher in patients with pSS than those of the normal controls.Theresults have shown that IL-18 and IFN-γ may be involved inthe pathogenesis of pSS.

Effects of bone morrow stromal cells transplantation on neurological behavior and Bax expression in rats with traumatic brain injury / 中华创伤杂志

ObjectiveTo explore the effects of bone morrow stromal cells (BMSCs) on the neurological behavior of rats with traumatic brain injury (TBI).MethodsTwenty-four SD rats were randomly and equally divided into control group, TBI group and BMSC group. The weight-drop device was adapted to establish the TBI model. The injury severity and its outcome were evaluated by a set of criteria termed neurological severity score (NSS). Brain tissues were harvested at day 14 to observe the survival and migration of the transplanted cells.Bax expression was detected by RT-PCR. Results NSS was (12 ±3 ) points in the TBI group, significantly higher than (7 ± 1 ) points in the BMSC group (P ＜0.05). The transplanted BMSCs could survive and migrate. Moreover, BAX, a crucail apopotosis gene, was down-regulated to 0.9 ±0.1 in the BMSC group, compared with 1.1 ±0.2 in the TBI group (P ＜0.05). ConclusionsBMSC transplantation is available to improve the neurological function, as may be associated with the Bax.

Structural basis for prokaryotic calcium-mediated regulation by a Streptomyces coelicolor calcium binding protein

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.

Construction and evaluation of DnaB split intein high expression vector and a six amino acids cyclic peptide library / 生物工程学报

A library with potential to produce six amino acids cyclic peptides was prepared using pET-28a as the starting plasmid. pVmut was used to amplify the Int(C)-dnaB-N-Int(N) fragment that was inserted into pET28a to give pEV. On pEV, DnaB split intein was expressed under the strong T7 promoter. Analyses of Escherichia coli transformed with pEV showed that DnaB split intein was produced in large quantity and the fusion protein self-spliced efficiently to produce cyclized DnaB-N. A synthesized 115 bp fragment mixture encoding 5 random amino acids was inserted into pEV to generate pEV-IS. The ligation mixture was transformed into E. coli. A library of 10(3) clones was obtained, 20 randomly picked clones were sequenced. All of them contain different sequences. Nine clones were chosen for further analysis. Split-intein-ISs were expressed in large quantity, and 90% of them self-spliced under 16 degrees C in 20 hours. After induction at 30 degrees C for 3 hours, the expressed DnaB split intein was purified using His-column, and then a molecular weight of target cyclic peptide was detected by MALDI-TOF-MS.

Homocysteine-mediated expression of SAHH, DNMTs, MBD2, and DNA hypomethylation potential pathogenic mechanism in VSMCs.

Homocysteine (Hcy) is a well-established risk factor for atherosclerosis and may cause dysregulation of gene expression, but the characteristics and the key links involved in its pathogenic mechanisms are still poorly understood. The aim of this study was to explore (i) the effects of Hcy on DNA methylation in vascular smooth muscle cells (VSMCs) and (ii) the underlying mechanism of Hcy-induced changes in DNA methylation patterns in relation to atherosclerosis. We examined the levels of gDNA methylation, namely, the Alu and line-1 element sequences, which can serve as a surrogate marker for gDNA methylation, and also investigated the effects of Hcy on the intracellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations as well as the expressions of SAH hydrolase (SAHH), DNA methyltransferase3a (DNMT3a), DNMT3b, and methyl-CpG-binding domain 2 (MBD2). We found that clinically relevant levels of Hcy (0-500 microM) induced elevation of SAH, declination of SAM and SAM/SAH ratio, and reduction in expression of SAHH and MBD2, but increased the activity of DNMT3a and DNMT3b compared to the control group (p < 0.05). We found also that the genome-wide hypomethylation is a common feature of gDNA in the VSMCs cultured with Hcy. In conclusion, these results suggest that Hcy-induced DNA methylation may be an important potential pathogenic mechanism in the development of atherosclerosis, and may become a therapeutic target for preventing Hcy-induced atherosclerosis.

Voltage-gated K+ channels are associated with cell proliferation and cell cycle of ovarian cancer cell.

OBJECTIVES: To study the role of Voltage-gated potassium ion channels (Kv) in cell proliferation and cell cycle of ovarian cancer cell. METHODS: The effects of voltage-gate K+ channels on human ovarian cancer cell line (A2780 cell) proliferation and cell cycle were observed by means of MTT and flow cytometry. A variety of K+ channel blockers were used in order to differentiate the critical subtype of K+ channels involved. Mechanism of K+ channel in cell proliferation was explored by studying the relationship between the K+ channel and Ca2+ entry. Kv and L-type Ca2+ channel gene expressions were determined by RT-PCR. RESULTS: 4-Aminopyridine, an inhibitor of voltage-gated K+ channels, significantly inhibited the proliferation of A2780 cells as measured by MTT. 4-Aminopyridine also significantly affected the cell cycle, which increased the percentage of cells in G0/G1, and reduced the percentage of cells in S phase and G2/M phase. Non-selective K+ channel blockers tetrapentylammonium (TPeA) and verapamil had similar inhibitory effects on A2780 cell proliferation and cell cycle. In contrast, iberiotoxin, a selective inhibitor of KCa channels, and glibenclamide, a potent inhibitor of KATP channels, had no effect on the cell proliferation and cell cycle of A2780. Moreover K+ channels inhibitor, TPeA and verapamil, can blocked Ca2+ influx in these cells. CONCLUSION: Voltage-gated K+ channels play an important role in the proliferation and cell cycle of ovarian cancer cell. It is likely that the influence of Kv channels on A2780 cell proliferation and cell cycle is mediated by a Ca2+-dependent mechanism.

Axonal regulation of Schwann cell differentiation and integrin α6β4 expression / 北京大学学报(医学版)

Objective: To study the axonal effect and the expression of integrin α6β4 during Schwann cell(SC) differentiation and myelination. Methods: Schwann cells were dissociated from the sciatic nerve of neonatal Waster rats and neurons dissociated from spinal cord. Singal cultures and purified populations of SC were cocultured with NC. Four methods (contrast microscope, scanning electron microscopy(SEM), immunocytochemistry method and in situ hybridization ) were used. Results: The separately cultured Schwann cells showed MBP negetive by immunocytochemistry method. But cocultured SC were shown positive. SEM showed that Schwann cells' membrane loop progressively circumnavigated around the axon during myelination, which suggested that the non-myelinating SC(nMSC) transformed to myelinating SC (MSC). In situ hybridization showed integrin α6β4 positive signals only on the outer surface of the Schwann cell-axon unit in SC coculture with NC. Conclusion: The differentiation and maturation of SC depend on axon, and the activity of integrins is expressed by axon. Axonal contact induces the expression of α6β4 during SC myelination, which suggests that integrin α6β4 is an important mediator of interactions of myelinating SC with the basal limina.

The retrospective analysis of glucose metabolism disorders in patients with hepatic cirrhosis / 中国实用内科杂志

Objective To study glucose metabolism in patients with liver cirrhosis and to explore the relationship between glucose and liver function.Methods 164 liver cirrhosis patients with abnormal glucose metabolism were divided into A,B and C groups according to Child-Turcotte-Pugh(CTP)classification system.Glucose metabolic disorder and relation between blood sugar level and liver function were observed and analyzed.Results We divided the patients into three subgroups according to their blood sugar levels:hypoglycemia group,impaired fasting glucose group and diabetes mellitus group.Patients of Grade C were with the highest incidence of hypoglycemia,with a P value P

Maternal serum tumor necrosis factor-? concentration and correlation with insulin resistance in gestational diabetes / 中华妇产科杂志

Objective To investigate se rum concentration of tumor necrosis factor(TNF)-? and its correlation with insulin resistance in pregnant women with gestational diabetes mellitus (GDM). Methods Enzyme-linked immunosorbent assay was used to measure th e fasting serum TNF-? levels of 42 women with GDM (28～39 gestational weeks), a n d 40 cases of normal pregnant women in the third trimester. Fasting plasma gluc ose, insulin, C-peptide and glycosylated hemoglobin (HbA1c) were also measured at the same time. Insulin sensitive index (ISI) was calculated. Res u lts (1)Significantly elevated serum TNF-? was found in the women w i th GDM(5.2?1.6) ng/L as compared with the healthy pregnant women in third t rimester (4.5?0.5)ng/L(P

Homocysteine promotes rat VSMCs proliferation and phenotypic transformation / 中国病理生理杂志

AIM:To provide evidence for the molecular mechanism of homocysteine (Hcy) as an independent risk factor in atherosclerosis (AS) by investigating the effect of Hcy on phenotype transformation and proliferation of vascular smooth muscle cells(VSMCs) in rats.METHODS:After treated with different concentrations of Hcy for 24 h,the cultured VSMCs were assayed for cell proliferation rate by MTT method,cell cycle by flow cytometry,the expression of SM22-? mRNA by semi-quantitative RT-PCR and the observation of morphological characteristics and the phenotype transformation by transmission electron microscopy.RESULTS:Hcy increased the cell proliferation rate and gradually reduced the proportion of the cells in G0 /G1 phase.Hcy down-regulated the expression of SM22? mRNA and the most significant effect was observed at concentration of 1 000 ?mol/L.The observations of transmission electron microscopy revealed an abundant endoplasmic reticulum,Golgi's complex,loose nucleus and puffy chromatin in VSMCs treated with high concentration of Hcy.CONCLUSION:Hcy promotes the proliferation and phenotype transformation of VSMCs simultaneously.

Influence of pathologic factors on phenotypic transformation of renal tubular epithelial cells in vitro / 中华肾脏病杂志

Objective To study the effects of high albumin, high glucose and low bovine serum on the phenotypic transformation of renal tubular epithelial cells. Methods The normal human kidney proximal tubular eel] line (HKC) was cultured for 30 days in the presence of high albumin (1.5 g/L), high glucose(25 mmol/L) and low bovine serum(2% ) . Morphological changes were observed by electronic microscopy. Immunohistochemistry stain was used to examine the expression of cytokeratin, vimentin, a-SMA, collagen Ⅰ and TGF-pl protein. Western blot was applied to further detect the process of collagen I protein expression, and in situ hybridization was used to examined the expression of collagen Ⅰ gene. Results Renal tubular epithelial cells cultured in high albumin, high glucose and low bovine serum showed obvious morphologic changes, including elongated shape, decrease of microvilli and mitochondria, and increase of rough endoplasmic reticulum under electronic microscopy. Immunohistochemistry stain revealed the reduction of cytokeratin, and enhancement of vimentin, ?-SMA, TGF-?1 and collagen Ⅰ. Western blot demonstrated that the expression of collagen Ⅰincreased in a time-dependent manner, and in situ hybridization showed that collagen type Ⅰ mRNA increased as well. Conclusion High albumin, high glucose and low bovine serum induce phenotypic transformation of renal tubular epithelial cells into mesanchymal cells.

Study of renal tubular epithelial cells transformations in rat tubulointerstitial fibrosis / 中华肾脏病杂志

Objective To observe the phenotypic changes between renal tubular epithelial cells and mesenchymal cells in rat tubulointerstitial fibrosis(TIF) .Methods The renal tubulointerstitial fibrosis models of Wistar rats were established by gavage with excessive adenine. The morphological changes of model kidney were observed by light microscopy, electron microscopy, polarizing microscopy. The?-smooth muscle actin(a-SMA), vimentin, and keratin were examined by immunohistochemistry staining. While the proliferating cell nuclear antigen (PCNA) was also assessed. Results There were many 2, 8-dioxyadenine crystals derived from the metabolite of adenine to deposit in the renal tubules, which induced the injury. Tubular epithelial cells degeneration and regeneration, loss of renal tubules, mononuclear cells infiltration in renal interstitium and the accumulation of extracellular matrix protein were observed. Both ?-SMA and vimentin were positive in some of renal tubules. And consecutive sections of immunohistochemical staining showed that the area of distribution of a-SMA positive cells was almost the same area as that of vimentin positive cells. Polarizing microscopy results showed that there was a mass of interstitial collagen ( Ⅲ and Ⅰ) accumulation. And electron microscopy examination proved that epithelial cells invaded into the renal interstitium. Conclusion There is tubular epithelial cells-myofibroblasts transdifferentiation in the process of TIF, and which plays a key role in the accumulation of extracellular matrix.