Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A growing body of evidence has linked the gut microbiota to liver metabolism. The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health. However, the effects of Lactobacillus gasseri LA39, a potential probiotic, on liver metabolism remain unclear. Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes, and used the germ-free (GF) mouse model to evaluate host-microbe interaction. Here, we explored the effects of L. gasseri LA39 gavage on the protein expression profiles of the liver of GF mice. Our results showed that a total of 128 proteins were upregulated, whereas a total of 123 proteins were downregulated by treatment with L. gasseri LA39. Further bioinformatics analyses suggested that the primary bile acid (BA) biosynthesis pathway in the liver was activated by L. gasseri LA39. Three differentially expressed proteins (cytochrome P450 family 27 subfamily A member 1 (CYP27A1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), and cytochrome P450 family 8 subfamily B member 1 (CYP8B1)) involved in the primary BA biosynthesis pathway were further validated by western blot assay. In addition, targeted metabolomic analyses demonstrated that serum and fecal β‍-muricholic acid (a primary BA), dehydrolithocholic acid (a secondary BA), and glycolithocholic acid-3-sulfate (a secondary BA) were significantly increased by L. gasseri LA39. Thus, our data revealed that L. gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation. Based on these findings, we suggest that L. gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues

Context: Colorectal cancer (CRC) is one of the most common malignancies and one of the leading causes of cancer death worldwide. Establishing early detection methods or markers of CRC is central to improve the survival rate of CRC patients. Nowadays, new molecular tools have been developed to acquire further knowledge on tumor progression. Aims: Comparative proteomics analysis of Vietnamese colorectal carcinoma in different stages was performed to gain an insight into the molecular events taking place in CRC and metastasis. Subjects and Methods: In this study, the comparative protein expression analysis of ten paired CRC and its corresponding noncancerous tissue samples was performed using the combination of isobaric tags for relative and absolute quantitation labeling and mass spectrometry (MS). The data obtained were further analyzed with Ingenuity Pathways Analysis (IPA) system. Results: Based on the MS/MS spectra analyzed by ProteinPilot software, 684 proteins were identified, out of which 215 were observed to be differentially expressed in at least 1 sample pair. Individual protein expression and variation have been identified for each patient. IPA system demonstrated cytoskeletal signaling as the top-ranked functional pathway network associated with the oncogenic function. Conclusions: Our study supplemented the understanding about proteome of Vietnamese CRC patients and identified statistically protein expression differences among samples assisting in finding effective biomarkers for CRC diagnostics

Reproduction-related proteins differentially expressed in the testes of the mice with kidney-yang or kidney-yin deficiency / 中华男科学杂志

Objective@#To compare the differentially expressed proteins in mice with kidney-yang deficiency and those with kidney-yin deficiency induced by hydrocortisone, and explore the similar and different material bases of male infertility caused by the two types of kidney deficiency.@*METHODS@#Thirty Kunming mice were equally randomized into a normal control, a kidney-yang deficiency and a kidney-yin deficiency group. The animals of the normal control group were injected intraperitoneally with normal saline at 0.2 ml qd for 7 days, while those of the latter two groups with hydrocortisone at 25 mg/kg/d for 10 days and 50 mg/kg/d for 7 days, respectively, for establishment of kidney-yang deficiency and kidney-yin deficiency models. Then the pathological changes in the testicular tissue of the mice were observed by HE staining and the differentially expressed proteins were compared among different groups using isobaric tags for relative and absolute quantitation (iTRAQ) and the bioinformatics method.@*RESULTS@#Sod1 was found to be a reproduction-related node protein differentially expressed in the testis tissues of the two types of kidney-deficiency mice, more highly expressed in the kidney-yin than in the kidney-yang deficiency group (P < 0.05). Five reproduction-associated node proteins were co-expressed in the testes of the two groups of kidney-deficiency mice, with significantly up-regulated expression of Rps28 and down-regulated expressions of Rpl11, Rplp2, Svs2 and Svs3a (P < 0.01).@*CONCLUSIONS@#Sod1 may be one of the key material bases for the differentiation of male infertility caused by kidney-yang deficiency from that induced by kidney-yin deficiency, while Rps28, Rpl11, Rplp2, Svs2 and Svs3a may be the common material bases of male infertility caused by the two types of kidney deficiency.

Cellular stress and redox activity proteins are involved in gastric carcinogenesis associated with Helicobacter pylori infection expressing high levels of thioredoxin-1 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Helicobacter pylori infection is related to the development of gastric diseases. Our previous studies showed that high thioredoxin-1 (Trx1) expression in H. pylori can promote gastric carcinogenesis. To explore the underlying molecular mechanisms, we performed an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis of stomach tissues from Mongolian gerbil infected with H. pylori expressing high and low Trx1. Differences in the profiles of the expressed proteins were analyzed by bioinformatics and verified using Western blot analysis. We found three candidate proteins, 14-3-3α/β, glutathione-S-transferase (GST), and heat shock protein 70 (HSP70), in high Trx1 tissues compared with low Trx1 tissues and concluded that cellular stress and redox activity-related proteins were involved in the pathogenesis of gastric cancer associated with H. pylori Trx1.

Aerobic exercise improves spermatogenesis of male rats: Results of iTRAQ-based proteomic analysis of the testis tissue / 中华男科学杂志

Objective@#To investigate the effect of aerobic exercise on the spermatogenic function of male rats and screen out differentially expressed proteins related to spermatonesis-regulation by proteomic analysis.@*METHODS@#We randomly divided 24 SD male rats into groups A (non-exercise control), B (exercise), and C (weight-bearing exercise), those in the latter two groups made to swim for 60 minutes a day and those in group C bearing a load 3% of the body weight, both 6 times a week for 9 weeks. At 24 hours after the last exercise, we obtained the sperm count, measured the levels of such serum reproductive hormones as testosterone (T), luteotrophic hormone (LH), follicle-stimulating hormone (FSH), and gonadotrophin-releasing hormone (GnRH), and employed isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of the testicular tissue.@*RESULTS@#Compared with group A, group C showed significant increases in sperm concentration (［2.12 ± 0.43］ vs ［3.54 ± 0.52］ ×10⁶/ml, P 0.05) and FSH (［20.49 ± 2.44］ vs ［22.29 ± 2.31］ IU/L, P >0.05). No significant changes were observed in sperm concentration or reproductive hormone levels in group B as compared with A. Group B exhibited obviously more mature sperm and cell layers in the seminiferous epithelium than group A. A total of 47 differentially expressed proteins were identified, of which 37 were up-regulated and the other 10 down-regulated. In addition, another 5 significantly differentially expressed proteins closely related to reproductive function were identified, including up-regulated Anx A1, GPX3, Rimbp3, and Dpy19l2 and down-regulated CYP17. Enrichment analysis showed that the differentially expressed proteins were mainly involved in extracellular matrix-receptor interaction, protein digestion and absorption, and focal adhesion pathways.@*CONCLUSIONS@#Proper-intensity exercise can improve the spermatogenic function of rats. Aerobic exercise promotes spermatogenesis mainly by up-regulating the expressions of the proteins related to the production and differentiation of spermatozoa.

Analysis of differential protein expression profile of HeLa cells stably transfected with Chlamydia trachomatis pORF5 gene / 中华微生物学和免疫学杂志

Objective To analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis. Methods A lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation ( iTRAQ) approach combined with nano-liquid chromatography-tandem mass spec-trometry ( NanoLC-MS/MS) analysis was performed to understand protein expression profiles and to iden-tify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells ( pORF5-Hela) and the control HeLa cells. Quantitative real-time PCR ( qRT-PCR ) and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively. Results HeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successful-ly. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C(histone H1. 2C), HBA1(hemoglobin subunit alpha), PARK7(parkinson disease protein 7), HMGB1(high mobility group protein B1) and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1 ( chloride intracellular channel protein 1 ) , KRT7 ( typeⅡ cy-toskeletal 7), SFN(14-3-3 protein sigma) and CDKN2A(cyclin-dependent kinase inhibitor 2A) were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at pro-tein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data. Con-clusion Expression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.

Isobaric tags for relative and absolute quantitation analysis on serum markers of pancreatic cancer based on mass spectrometry / 肿瘤研究与临床

Objective To determine the expression of serum proteins in pancreatic cancer patients based on mass spectrometry to screen differential proteins and to find potential molecular biomarkers to prediagnosis of pancreatic cancer. Methods The technique of isobaric tags for relative and absolute quantitation (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) were used to analyze the serum proteins in 15 pancreatic cancer patients and 10 healthy controls. Screening for serum differential proteins was performed via retrieving Panther, Mascot and Scaffold databases. Ingenuity Pathway Analysis was used to detect the correlation between proteins. Results The expression level of 442 proteins was quantified, and 76 proteins were found to be differentially expressed which changed corresponding biological process. The up-regulation of DNA repair protein 50 (RAD50) and down-regulation of transforming growth factor β1 (TGFβ1) and apoptosis protease-activating factor 1 (APAF1) which were correlated with cell growth and apoptosis were found in pancreatic cancer. A closely interacted network was formed between these three differential proteins and other molecules, which could effect metabolism in pancreatic cancer patients. Conclusion Differential expression of serum proteins screened by iTRAQ in pancreatic cancer patients may be the potential markers of pancreatic cancer, which can provide molecular basis for early diagnosis of pancreatic cancer.

Differential proteomic analysis of the zebrafish neuron protective effect of Cerebralcare granules using iTRAQ labeling coupled with 2DLC-MS/MS / 中国药学杂志

OBJECTIVE: To screen the differentially expressed proteins before and after administration of Cerebralcare granules to zebrafish central nerve injury (CNI) models and search practical markers and explore the molecular mechanism of the treatment. METHODS: Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with nano liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) were used to analyze and identify differentially expressed serum proteins in the two groups. Bioinformatics was used to analyze the identified differentially expressed proteins, and the expression of representative differential proteins was verified by Western blotting. RESULTS: With the high throughput proteomic technology of iTRAQ coupled with Nano-LC-MS/MS, 1 933 unique proteins were identified, and 130 proteins showed ≥ 1.50 or ≤ 0.70 folds of changes during differentiation. The proteins detected in the zebrafish neuroendocrine brain had roles in the biological processes of translation, metabolic process and neuronal ion channel clustering. Ef1-α (elongation factor 1-alpha) and α-6-F (Na+/K+ transporting ATPase alpha 1 polypeptide) were validated by Western blotting. The two sets of data showed a statistically significant difference (P<0.05). These RESULTS were consistent with those from quantitative mass spectrometry. CONCLUSION: A high-throughput screen for zebrafish central nerve injury proteins can be performed by a combined use of iTRAQ and Nano-LC-MS/MS, and the molecular mechanism of Cerebralcare granules treatment can thus be preliminarily explored.

Analysis of inferior colliculus region typical proteins in auditory pathway / 中国耳鼻咽喉头颈外科

OBJECTIVE To study the proteome of inferior colliculus and determinate the region-typical proteins which may be the candidate cause of the Central Auditory Processing Disorders. METHODS The telencephalon was taken as reference, and then identified and quantified the proteome of IC of adult rats with iTRAQ. Those with higher abundance in inferior colliculus than the other three regions were considered as IC-Region typical proteins,which may lead to functional specializations. RESULTS We identified 1937 cytomembrane proteins in total, among which there are 53 IC-Region typical proteins, which may lead to functional specializations of inferior colliculus.We used GO and KEGG pathway to analyze these proteins and then found that these proteins mainly take part in the regulation of neurons development and information integrations. CONCLUSION Our quantitative comparison of inferior colliculus has revealed two candidate proteins, including CaMKII and SV2A, which may play important roles in maintaining the balance of excitatory and inhibitory transmitters release. These proteins may be the candidate proteins for Central Auditory Processing Disorders.

Proteomics analysis of rat liver fibrosis caused by sodium arsenite / 中华地方病学杂志

Objective To study the mechanism of liver fibrosis in rats caused by chronic exposure through drinking water containing sodium arsenite,to identify the differential proteins via proteomics technique.Methods Totally 40 healthy 8-week-old male Sprague Dawley (SD) rats of specific pathogen free (SPF) grade were randomly divided into 4 groups,which were control group (deionized water),0.68,1.36 and 2.73 mg/kg sodium arsenite (iAs3+) treated groups,respectively.The rats were fed with iAs-treated drinking water freely for 24 consecutive weeks.Twenty-four hour urine sample,blood and liver samples were collected.Hepatic fibrosis indices,specifically,type Ⅲ precollagen (PC Ⅲ),type Ⅳ collagen (Ⅳ-C),hyaluronic acid (HA) and laminin (LN) were detected by enzymelinked immunoassay (ELISA).Based on the isobaric tags for relative and absolute quantitation (iTRAQ) reagent 8-plex experiment,combined with 2DLC-MS/MS,the proteins in rats liver tissue of the medium dose group and the high dose group were compared with the those of control groups.Results ①The serum HA contents in the C (control) group,the L (low dose) group,the M (medium dose) group and the H (high dose) group were (198.51 ± 16.64),(218.39 ± 34.98),(261.72 ± 30.56) and (297.31 ± 35.72) ng/L;the serum PCⅢ contents in C,L,M and H groups were (15.32 ± 2.15),(16.78 ± 2.64),(19.51 ± 0.85) and (21.42 ± 1.63) μg/L;the serum LN contents in C,L,M and H groups were (734.57 ± 86.00),(792.65 ± 94.15),(916.83 ± 84.40) and (1 008.09 ± 64.17) μg/L;the serum Ⅳ-C contents in C,L,M and H groups were (52.34 ± 14.65),(59.72 ± 12.84),(74.38 ± 4.83) and (78.46 ± 4.30) μ.g/L,respectively.The differences in serological indices of liver fibrosis between-groups were statistically significant (F =21.136,19.957,22.007,14.288,all P ＜ 0.05).In multiple comparison of serum HA,PCⅢ and LN,there were no statistical significant differences between L group and C group.M and H groups were higher than L group and C group,significant statistical difference was found between H group and M group (all P ＜ 0.05).②Combining iTRAQ with 2DLC-MS/MS,based on the confidence threshold of protein (unused protScore) ＞ 1.3 and at least 1 matched peptides within the 95％ confidence interval,2 948 proteins were identified.Totally 2 162 proteins were detected in three groups compared with Venn diagram,after removing significant different proteins in C group,687 up-regulated proteins and 548 down-regulated proteins were identified in M group;633 up-regulated proteins and 519 downregulated were found in H group;the differences of protein expression between M and H groups were not statistically significant (P ＞ 0.05).③Up-regulated proteins related to the metabolism including AS3MT,MAT,SHMT,CHDH,CTH,CSAD and BHMT in M and H groups;of the two kinds of proteins of MTR,METK1 was up-regulated and F1LRB8 was down-regulated.Proteins associated with GSH including Gsta1,Gsta4,Gsta5,Gstt1,Gstt2,Gstk1,Gstp1,Gstm1,Gstm2,Gstm3,Gss,Gpx1,Gpx4,Esd,Hagh,Glo1,Mgst1 and B6DYQ5 which were all up-regulated.Proteins associated with liver fibrosis were Hic-5,Gss and six kinds of Tpm,and six kinds of Tpm subunits including two kinds of Tpm1,three kinds of Tpm2 and one kind of Tpm3 which were all up-regulated.Conclusions There is liver accumulation of arsenic after chronic arsenic exposure and resulting in liver fibrosis and decline of liver function.Expressions of AS3MT,MTR,MAT,SHMT,BHMT,CHDH,CTH and CSAD are up-regulated;arsenic meta bolism methionine cycle,folic acid cycle and sulfur transfer pathways are closely related.GSH plays an important role in arsenic metabolism and liver fibrosis,Hic-5,GSS and TPM may be associated with the occurrence of liver fibrosis.

Proteomics research on antifibrotic mechanism of combination therapy based on isobaric tags for relative and absolute quantitation technique / 中草药

Objective: To investigate the protein basis and molecular mechanism of combination therapy with epigallocatechin gallate (EGCG), taurine, and genistein on CCl4-induced liver fibrosis in rats using isobaric tags for relative and absolute quantitation (iTRAQ). Methods: The SD rats were randomly divided into normal control, model, and combination therapy (taurine 200 mg/kg + EGCG 30 mg/kg + genistein 20 mg/kg) groups. The rats in the model and combination therapy groups were induced by ig administration of 50% CCl4 for 14 weeks, and the rats in the normal control group were given peanut oil. The combination therapy was started at week 9 for 6 weeks. HE staining of liver tissue was performed to evaluate histopathologic changes. The high-abundance proteins in the serum of model and combination therapy groups were depleted by ProteoMiner Protein Enrichment Kit. Proteins differentially expressed in the serum of those two groups were identified with iTRAQ coupled with LC-ESI-MS/MS technology and analyzed with bioinformatics tools. The expression of representative differential protein (Txn1) was validated by ELISA. Results: A total of 359 proteins with the confidence coefficient above 95% were identified, of which 78 were differential expressed proteins, including 51 up-regulated ones and 27 down-regulated ones. The serum level of Txn1 increased significantly in the combination therapy group compared with the model group (P < 0.05). Conclusion: The combination therapy can alleviate the progression of liver fibrosis effectively by affecting multiple biological processes. The regulation of anti-oxidant defense system and coagulation cascade pathway may be the molecular mechanisms of the combination therapy against liver fibrosis.

Screening for membrane proteins differentially expressed between the lung adenocarcinoma cell lines with high- and low-metastatic potential using iTRAQ technology / 肿瘤

Objective: To screen for the membrane proteins differentially expressed between the lung adenocarcinoma cell lines with high- and low- metastatic potential, and to explore the potential targets for biomarkers and biological therapy. Methods: The membrane proteins were extracted from lung adenocarcinoma SPC-A-1 and SPC-A-1 sci cells which were generated from the same parental cell type and had low- and high- metastatic potential, respectively. The membrane proteins were labeled and the peptides were separated and analyzed by iTRAQ (isobaric tags for relative and absolute quantitation) technology combined with Nano-LC-MS/MS (nano liquid chromatography-tandem mass spectrometry). The identification and quantitation of the proteins were analyzed by Proteinpilot 4.0 solfware. The membrane proteins differentially expressed were analyzed and verified by GO (Gene Ontology) terms and real-time fluorescence quantitative-PCR in combination with Western blotting, respectively. Results: The identified numbers of proteins with FDRs (false discovery rates) < 1% were 1 413, 1 374, 1 297, and 1 351 in the experiment which were repeated four times by Nano LC-MS/MS, and the rate of labelling was above 95%. Among the 27 proteins up-regulated in total four experiments, 20 proteins were identified as membrane protein. Among the 32 proteins down-regulated in total four experiments, 25 proteins were identified as membrane protein. The GO analysis demonstrated the major molecular functions of the proteins differentially expressed including cytoskeletal protein binding, identical protein binding and enzyme binding as well as the catabolic process and cellular localization in biological processes. The expression levels of ITGA3 (integrin alpha-3), MYH9 (myosin, heavy chain 9), PLEC1 (plectin 1), HADHA (3-hydroxyacyl- CoA dehydrogenase), HK1 (hexokinase-1), KTN1 (kinectin 1), ESYT1 (extended synaptotagmin-like protein 1), ALDH18A1 (aldehyde dehydrogenase 18 family, member A1), ATP5A1 (ATP synthase alpha-subunit), LMNB2 (lamin-B2), CAV1 (caveolin-1) and CK-19 (keratin, type I cytoskeletal 19) mRNAs in SPC-A-1 sci cells were higher than those in the SPC-A-1 cells. The expression levels of CLTC (clathrin, heavy chain), HK1, LMNB2 and CK-19 in SPC-A-1 sci cells were also higher than those in SPC-A-1 cells. These results were consistent with those from quantitative mass spectrometry. Conclusion: A high-throughput screen for metastasis-related membrane proteins can be performed by a combined use of iTRAQ and Nano LCMS/ MS and may provide the potential metastasis-related biomarkers and therapeutic targets in diagnosis, prognostic prediction and treatment for patients with lung adenocarcinoma. Copyright © 2013 by TUMOR.

Study on serum proteomics in rats after accumulated irradiation with 137Cs γ-rays / 中华放射医学与防护杂志

Objective To investigate the changes of proteomics in serum of Sprague-Dawley(SD) rats after accumulated irradiation with 137Cs γ-rays.Methods A total of thirty mature SD rats were randomly divided into three groups:0.2 Gy group,2 Gy group and healthy control group.Rats were irradiated at a dose rate of 0.336 mGy/min for 10 d and 20 d continuously.Isobaric tags for relative and absolute quantitation (iTRAQ) was used to analyze the different protein expression in serum of irradiated rats.Gene Ontology,KEGG pathway and protein-protein interaction network analysis were conducted using softwares.Results In total,363 protein spots were identified.Twenty nine proteins were differentially expressed in both groups compared with control,of which 10 proteins were up-regulated and 19 proteins were down-regulated.Based on the information of GO categories,these differentially expressed proteins were mainly located in the cytoplasm and membrane concerning the function of binding and catalytic activity.Analysis with the PAJEK software demonstrated that 16 differentially expressed proteins could form a complicated interaction network where glutathione S-transferase P1 (GSTP1),phosphoglycerate kinase 1 (PGK1) and protein disulfide-isomerase (PDI) might be key nodes.Conclusions Accumulated irradiation can induce differentially expressed proteins in serum of irradiated rats.Analysis on functional roles of the screened proteins GSTP1,PGK1 and PDI may provide insight into further mechanistic investigations and underlying molecular biomarkers induced by accumulated irradiation.

Application of isobaric tags labeling in proteomic research of human renal proximal tubular epithelial cells / 上海第二医科大学学报

0.05). Conclusion The labeling of iTRAQ in HK-2 cells is successful with favourable reproducibi-lity,which lays a foundation for the further research of proteomics in renal diseases.