Objective To explore the sensitive blood plasma molecular markers in diabetic nephropathy (DN) .Methods Two-di-mensional fluorescence difference gel electrophoresis (2D-DIGE) was used to analyze early DN patients plasma (n=8) and normal plasma ,proteins that showed differential expression of a 1 .5 fold change were analyzed by MALDI-TOF/TOF mass spectrometry . Results 2D-DIGE maps of plasma proteins in patients with DN and normal plasma were established successfully .We validated 13 differentially expressed proteins detected by 2D-DIGE ,including complement component C3、complement component C4、apolipopro-tein E ,etc .Conclusion Proteomic analysis can objectively revealed the differences of protein expression between the two kinds of blood plasma .The 13 proteins might be the potential plasma molecular markers for the clinical diagnosis of diabetic nephropathy .

Objective To screen the sensitive plasma molecular markers in diabetic nephropathy ( DN) patients with kidney yang deficiency. Methods Two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was used to analyze the plasma which was sampled from 4 early DN patients with kidney yang deficiency and 4 healthy adult volunteers. Proteins that showed differential expression with an over 1.5-fold change were analyzed by MALDI-TOF/TOF mass spectrometry. Results Two-dimensional electrophoresis (2-DE) maps of plasma proteins in DN patients with kidney yang deficiency and healthy adults were established successfully. We validated 9 differentially expressed proteins, including complements C3 and C4, apolipoprotein E and ubiquitination factor. Conclusion Proteomic analysis by 2-D DIGE can objectively reveal the differences of plasma protein expression in DN patents with kidney yang deficiency and healthy adult volunteers. The obtained 9 proteins have potential to provide plasma molecular markers for the early clinical diagnosis and for the research of traditional Chinese medical patterns of DN.

Objective To investigate the value of hs-CRP in the assessment of risk stratification,coronary artery disease severity and prognosis for patients undergoing PCI with coronary heart disease.Methods A total of 150 coronary heart disease patients were recruited in the study,who had undergone PCI,and before that all the patients had undergone coronary angiography,and the levels of hs-CRP,TG,LDL-C,HDL-C,NT-proBNP and hs-cTnT were measured.Patients were divided into three groups according to the serum concentration of hs-CRP.Gemini score was used to determine the degree of stenosis.After PCI,patients were followed up for 6 months,the major cardiovascular events(MACE)were recorded.The relationships between hs-CRP and other coronary risk factors,Gemini score,vasculopathy count,MACE events were analyzed.Results The serum concentrations of hs-CRP and HDL-C, hs-cTnT were significantly correlated(r=-0.17,0.42,P <0.05).The level of hs-CRP had increasing tendency with the increase of Gensini score,and the serum concentration of hs-CRP in severe vascular lesions group was significantly higher than both mild vascular lesions group and moderate vascular lesions group(P <0.05).The serum concentration of hs-CRP in single-vessel disease patients,double-vessel disease patients and multi-vessel disease patients were (4.64±5.39),(9.86±8.75),(14.93±10.34)mg/L, there was significant difference among them(P <0.05 ).The correlation coefficients of hs-CRP and Gemini score was 0.21 (P <0.05),while hs-CRP and vasculopathy count was 0.18(P <0.05).The incidence of MACE in three groups were 18.52%,43.75%and 58.66%,which were statistically significantly difference(χ2 =13.42,P =0.001).Logistic regression showed that hs-CRP was an independent risk factor for MACE(OR =2.05,95%CI :1.24 -3.39,P =0.005).Conclusion Hs-CRP is an independent risk factor for coronary heart disease in patients with MACE after PCI,and hs-CRP was correlated with PCI patient′s vascular disease. Hs-CRP can be used as an indicator in the assessment of patient′s condition,PCI risk stratification and prognosis.

Objective The purpose of the study was to investigate clinical manifestation and the characters of diagnosis and treatment among children with primary angiitis of the central nervous system (cPACNS) in order to improve awareness of the disease.Methods Clinical data of 5 children with cPACNS in the First People's Hospital of Yunnan Province from January 2009 to December 2013 were collected,and the clinical manifestations and laboratory test results were analyzed and summarized.Results Five cases of children with cPACNS were misdiagnosed at the first clinic visit,and were confirmed a clear diagnosis on the average of (4±6) months; clinical manifestations of five cases of varying degrees of headache,one case with severe headache,2 patients with decreased visual acuity,a cases with hearing were loss,two cases with secondarily generalized seizures; five cases with mild abnormal cerebrospinal fluid examination; 1 case with elevated ESR and CRP level,1 case with elevated immunoglobulin IgG level; 5 cases with abnomal MRI examinations,which showed multiple bilateral lesions,diffuse,lesions,involving the cortex and deep white matter; 4 cases had vascular abnormalities on MRA,treated with corticosteroids alone or in combination with cyclophosphamide and achieved good results.Conclusion Children of primary central nervous system vasculitis is ar are autoimmmune disease primarily involving the central nervous system.It is difficult for the clinical diagnosis.Children need to be wary of the major manifestation of headache associated with vision loss,hearing loss,seizures and other focal neurological system damage.

AIM:To identify the serum proteins that might serve as biomarkers for predicting mucosal healing ( MH) in the patients with Crohn’ s disease ( CD) treated with infliximab ( IFX) .METHODS:We collected serum sam-ples before treatment (0 week, group A) and 14 weeks after treatment (group B) from 7 CD patients with IFX treatment who had achieved MH, as well as the serum samples from 7 CD patients who had not achieved MH (0 week, group C;14 weeks, group D) .Two-dimensional fluorescence difference gel electrophoresis was applied to analyze and compare the re-sults of serum profiles between groups A and B, C and D, A and C, B and D.Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and bioinformatics tools were utilized to preliminarily identify and figure out the dif-ferentially expressed proteins.RESULTS:(1) In total, there were 44 differentially expressed spots, 36, 3, 10 and 31 differentially expressed spots were detected while comparing A with B, C with D, A with C and B with D, respectively. (2) Among those spots, 17, 2, 2 and 15 proteins were identified, respectively.In total, there were 19 differentially ex-pressed proteins, including apolipoprotein E, apolipoprotein A-I, complement factor H, and so on.(3) Protein functional association networks were carried out based on STRING database.CONCLUSION: The serum protein profiles obviously change after IFX treatment in MH CD patients, and the serum protein profiles of MH patients are different from that of non-MH patients after IFX treatment.The 19 proteins we identified may serve as potential biomarkers for predicting MH in CD patients with IFX treatment.

[Objective]To apply an acetonitrile-pretreatment method for proteomic analysis of serum of patient with hepatocellular carcinoma(HCC),searching for the proteins that might be related to occurrence and development of HCC.[Methods]Serum samples were recruited from 12 patients with primary HCC and 12 health adult.Firsdy,the sera were pretreated with acetonitrile (ACN),and the high-abundance proteins were removed,then two-dimensional electrophoreaia(2-DE)was performed to screen the differentially expressed proteins between the patient with HCC and health control.Finally,the differentially expressed proteins in the 2-DE gel were identified by a MALDI-TOF/TOF mass spectrometry.[Results]After the sera were pretreated with 0%,20% and 30% ACN,2-DE analysis showed that the number of the protein spots in the gels were 532±96,623±102,and 674±123,respectively.By the proteomic analysis of the ACN-pretreatment sera of patient with HCC and health control,we found that tetraiodo-L-thyronine and proapolipoproteinin were up-regulated,vitamin D-binding protein precurser and transferrin were downregulated in the HCC serum.[Conclusion]Proteomic analysis of serum using the ACN-pretreatment method can improve the detection of proteins which were non-specifically binding to the albumin.Abnormal expression of tetraiodo-L-thyronine,proapolipoproteinin,vitamin D-binding protein precursor and transferrin in the serum are related to HCC.

OBJECTIVETo characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.

METHODSThe full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.

RESULTSThe recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.

CONCLUSIONThe pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; immunology ; Calmodulin ; immunology ; Clonorchiasis ; immunology ; Clonorchis sinensis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Library ; Immunoglobulin G ; blood ; Inflammation ; Liver Cirrhosis ; parasitology ; Male ; Mice ; Rats ; Recombinant Proteins ; immunology

Objective To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis. Methods The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein. Results The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1:51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis. Conclusion The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis-associated hepatic fibrosis.

Background and Objectives: Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction ofarticular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. Methods: and Results: Real-time reverse transcription–polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3’-untranscribed region. Conclusions These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.

Objective To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis. Methods The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein. Results The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1:51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis. Conclusion The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis-associated hepatic fibrosis.