Separation and Identification of Differentially Expressed Proteins in Pistillate Flowers between Different Mulberry Cultivars

[Objective] This study aimed to explore the proteins related to pistillate flower development in different mulberry cultivars. [Method] The total proteins of the pistillate flowers of two mulberry cultivars Dal0 （Morus atropurpurea Roxb.） and SG01 （Morus muIticaulis Perr.） were extracted, separated and detected through two- dimensional electrophoresis （2-DE） and mass spectrometry. [Result] There was sig- nificant difference in the expression of proteins from the pistillate flowers of different mulberry cultivars. From the 2-DE images of Dal0 and SG01, 445_＋17 and 425_＋12 protein spots were respectively detected. The expression levels of 75 protein spots differed significantly. Thirteen spots those were expressed at high levels and well separated were analyzed by mass spectrometry, and nine of them were identified successfully. The nine proteins are involved in the glycometabolism, protein and amino acid metabolism and defense responses during the development of mulberry pistillate flower after they were pollinated. [Conclusion] The findings will provide reference for further study on the molecular mechanism of mulberry pistillate flower de- velopment.

Differentially Expressed Proteins in Rat Hippocampus after Chronic Immobilization Stress and Intervention Using Xiao Yao San Decoction

Objective To identify differentially expressed proteins in the hippocampus of rats after chronic immobilization stress(CIS)using a proteomics approach,and to study the effect of the Xiao Yao San(XYS)decoction on differentially expressed proteins.Methods Twenty-four Sprague Dawley rats were randomly assigned to one of four groups of equal body weight:control(non-stress),7-day stress,21-day stress and21-day stress+XYS treatment groups.Two-dimensional gel electrophoresis(2-DE)was used to detect differences in protein expression in rat hippocampus.One differentially expressed protein was measured and verified by western blotting.Results Seventeen proteins showed differential expression.Among these,eight could be identified:glial fibrillary acidic protein-2(GFAP-2),tubulin alpha-1c,cytoplasmic muscle actin2,14-3-3protein,β-2a tubulin,phosphatidylethanolamine binding protein,synucleinαsyn3,and a low molecular weight(18kD)protein.Six of these proteins exhibited increased expression,one showed decreased expression,and the other protein,which comprised five subtypes,were either increased or decreased.These proteins are known to be involved in immunity,signal transduction,cell cycle control,apoptosis,regulation of enzyme activity,cytoskeleton structure,and synaptic plasticity.GFAP-2was further analyzed,and its differential expression confirmed by western blotting.Conclusion Some proteins are differentially expressed in the hippocampus of rats under chronic stress.The biological functions of these differentially expressed proteins are varied.Finally,the XYS decoction can significantly up-or down-regulate these protein expression levels.

Characteristics of Atmospheric Fine Particulate Matter(PM2.5)Induced Differentially Expressed Proteins Determined by Proteomics and Bioinformatics Analyses

Objective To screen the differentially expressed proteins(DEPs)in human bronchial epithelial cells(HBE)treated with atmospheric fine particulate matter(PM2.5).Methods HBE cells were treated with PM2.5 samples from Shenzhen and Taiyuan for 24 h.To detect overall protein expression,the Q Exactive mass spectrometer was used.Gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG),and Perseus software were used to screen DEPs.Results Overall,67 DEPs were screened in the Shenzhen sample-treated group,of which 46 were upregulated and 21 were downregulated.In total,252 DEPs were screened in the Taiyuan sampletreated group,of which 134 were upregulated and 118 were downregulated.KEGG analysis demonstrated that DEPs were mainly enriched in ubiquitin-mediated proteolysis and HIF-1 signal pathways in Shenzhen PM2.5 samples-treated group.The GO analysis demonstrated that Shenzhen sample-induced DEPs were mainly involved in the biological process for absorption of various metal ions and cell components.The Taiyuan PM2.5-induced DEPs were mainly involved in biological processes of protein aggregation regulation and molecular function of oxidase activity.Additionally,three important DEPs,including ANXA2,DIABLO,and AIMP1,were screened.Conclusion Our findings provide a valuable basis for further evaluation of PM2.5-associated carcinogenesis.

Astrocytic endothelin-1 overexpression impairs learning and memory ability in ischemic stroke via altered hippocampal neurogenesis and lipid metabolism

Vascular etiology is the second most prevalent cause of cognitive impairment globally.Endothelin-1,which is produced and secreted by endothelial cells and astrocytes,is implicated in the pathogenesis of stroke.However,the way in which changes in astrocytic endothelin-1 lead to poststroke cognitive deficits following transient middle cerebral artery occlusion is not well understood.Here,using mice in which astrocytic endothelin-1 was overexpressed,we found that the selective overexpression of endothelin-1 by astrocytic cells led to ischemic stroke-related dementia(1 hour of ischemia;7 days,28 days,or 3 months of reperfusion).We also revealed that astrocytic endothelin-1 overexpression contributed to the role of neural stem cell proliferation but impaired neurogenesis in the dentate gyrus of the hippocampus after middle cerebral artery occlusion.Comprehensive proteome profiles and western blot analysis confirmed that levels of glial fibrillary acidic protein and peroxiredoxin 6,which were differentially expressed in the brain,were significantly increased in mice with astrocytic endothelin-1 overexpression in comparison with wild-type mice 28 days after ischemic stroke.Moreover,the levels of the enriched differentially expressed proteins were closely related to lipid metabolism,as indicated by Kyoto Encyclopedia of Genes and Genomes pathway analysis.Liquid chromatography-mass spectrometry nontargeted metabolite profiling of brain tissues showed that astrocytic endothelin-1 overexpression altered lipid metabolism products such as glycerol phosphatidylcholine,sphingomyelin,and phosphatidic acid.Overall,this study demonstrates that astrocytic endothelin-1 overexpression can impair hippocampal neurogenesis and that it is correlated with lipid metabolism in poststroke cognitive dysfunction.

iTRAQ-Based Proteomics Investigation of Critical Response Proteins in Embryo and Coleoptile During Rice Anaerobic Germination

Direct-seeding of rice has become popular in recent years due to its low cost and convenience,however,hypoxic condition limits seedling establishment.In this study,weedy rice WR04-6 with high germination ability under anaerobic conditions was used as a gene donor,and we successfully improved the seedling establishment rate of rice cultivar Qishanzhan(QSZ)based on selection of a new rice line R42 from the recombinant inbred line population.R42 inherited high anaerobic germination(AG)ability,and was used for isobaric tags for relative and absolute quantitation(iTRAQ)-based comparative proteomic studies with QSZ to further explore the molecular mechanism of AG.A total of 719 differentially abundant proteins(DAPs)were shared by R42 and QSZ responded to AG,and thus defined as common response DAPs.A total of 300 DAPs that responded to AG were only identified from R42,which were defined as tolerance-specific DAPs.The common response and tolerance-specific DAPs had similar biochemical reaction processes and metabolic pathways in response to anoxic stress,however,they involved different proteins.The tolerance-specific DAPs were involved in amino acid metabolism,starch and sucrose metabolism,tricarboxylic acid cycle pathway,ethylene synthesis pathway,cell wall-associated proteins and activity of active oxygen scavenging enzyme.The in silico protein-protein interactions for the top 60 DAPs indicated that tolerance-specific DAPs had relatively independent protein interaction networks in response to an anoxic environment compared with common response DAPs.The results of physiological indicators showed thatα-amylase and superoxide dismutase activities of R42 were significantly increased under anoxic conditions compared with aerobic conditions.Multiple lines of evidence from western blot,physiological analysis and quantitatDirect seeding of rice has become popular in recent years due to its low cost and convenience,however,hypoxic conditions can limit seedling establishment.In the present study,weedy rice WR04-6 with high germination ability in anaerobic conditions was used as the gene donor and successfully improved the seedling establishment rate of rice cultivar Qishanzhan(QSZ)based on selection of a new rice line R42 from the recombinant inbred line(RIL)population.R42 inherited the had high anaerobic germination(AG)ability,which was used for the isobaric tags for relative and absolute quantitation(iTRAQ)based comparative proteomic studies with QSZ to further explore the molecular mechanism of AG.A total of 719 differentially abundant proteins(DAPs)shared by R42 and QSZ responded to AG and were thus defined as common response DAPs.A total of 300 DAPs that responded to AG were only identified from R42,which were defined as tolerance-specific DAPs.The common response and tolerance-specific DAPs had similar biochemical reaction processes and metabolic pathways in response to anoxic stress,however they involved different proteins.The 300 tolerance-specific DAPs were involved in amino acid metabolism,starch and sucrose metabolism,TCA cycle pathways,ethylene synthesis pathway,cell wall-associated proteins and activity of active oxygen scavenging enzyme.The in silico protein-protein interactions for the top 60 DAPs indicated that tolerance-specific DAPs had relatively independent protein interaction networks in response to an anoxic environment compared with common response DAPs.The results of physiological indicators showed thatα-amylase and SOD activities of R42 were significantly increased under anoxic conditions compared with aerobic conditions.Multiple lines of evidence from western blot,physiological analyses and real-time PCR jointly supported the reliability of proteomics data.In summary,our findings deepened the understanding of the molecular mechanism for the rice response to AG.ive real-time PCR jointly supported the reliability of proteomics data.In summary,our findings deepened the understanding of the molecular mechanism for the rice response to AG.

Proteomic Analysis of High Temperature Stress-Responsive Proteins in Chrysanthemum Leaves

Chrysanthemum is one of the most important ornamental flowers in the world,and temperature has a significant influence on its field production.In the present study,differentially expressed proteins were investigated in the leaves of Dendranthema grandiflorum‘Jinba’under high temperature stress using label-free quantitative proteomics techniques.The expressed proteins were comparatively identified and analyzed.A total of 1,463 heat-related,differentially expressed proteins were successfully identified by Liquid Chromatography-tandem Mass Spectrometry(LC-MS/MS),and 1,463 heat-related,differentially expressed proteins were successfully identified by mass spectrometry after a high temperature treatment.Among these,701 proteins were upregulated and 762 proteins were downregulated.The in-depth bioinformatics analysis of these differentially expressed proteins revealed that these were involved in energy metabolism pathways,protein metabolism,and heat shock.In the present study,the investigators determined the changes in the levels of some proteins,and their expression at the protein and molecular levels in chrysanthemum to help reveal the mechanism of heat resistance in chrysanthemum.Furthermore,the present study elucidated some of the proteins correlated to heat resistance in chrysanthemum,and their expression changes at the protein and molecular levels to help reveal the mechanism of heat resistance in this flower species.These results provide a theoretical basis for the selection of new heat resistant varieties of chrysanthemum in the field.

Effect of cluster needling at scalp acupoints on differential protein expression in rat brain tissue after acute focal cerebral ischemia

Objective:To explore the function of cluster needling at scalp points therapy on regulating differential protein's expression at different time points in middle cerebral artery occlusion(MCAO)model rats.Methods:Fifty-four rats were divided into three groups randomly and 18 rats in each group.The groups respectively were the model group(group M,n=18),cluster needling at scalp points group(group C,n=18),false operation group(group F,n=18).Each group was then assigned in three subgroups,including 24-h,7-day,and 14-day subgroups.Six rats in each subgroup.Acupuncture at Baihui(GV20)and 2 points beside Baihui,which was 3 e4 mm away from the midline.Longa score was used to evaluated neurological effects.Proteomics methods were used to identify differentially expression proteins with a standard of fold change greater than 1.5 and P<.05 at different times.Results:1.Nerve function scoring:The nerve function scores at 7 and 14 days decreased in group C,which showed better neural function than group M(P<.05).2.Fold change in proteins:Group M showed932 differentially expressed proteins compared with group F,and among them,414 proteins showed significant changes in expression after acupuncture.The expression levels of Cdc42 and GFAP were increased,and Mag,Shank2,and MBP levels were decreased.In the Gene Ontology analysis,the cellular component consisted of the terms cytoplasm,cytoskeleton,lysosome,and plasma membrane.The main related biological processes were cellecell signaling,protein transport,aging,and cell adhesion.Many synaptic and metabolic pathways were found by KEGG analysis.Conclusion:Cluster needling at scalp acupoints can improve the nerve function score and improve dyskinesia in MCAO model rats.Cluster needling at scalp acupoints can regulate the expression of 414 proteins,including Cdc42,GFAP,Mag,Shank2,and MBP,which are related to cerebral ischemia.The differential proteins are major concentration in cytoplasm,cytoskeleton,lysosomes,and plasma membrane,participate in cellecell signaling,protein transport,aging,and cell adhesion,and act through multiple synaptic and metabolic pathways to exert their biological functions.

Differential protein expression in rat cortical astrocytes following fluid percussion injury Two-dimensional gel electrophoresis and mass-spectrum detection

BACKGROUND： The Glasgow Coma Scale, computer tomography, and nuclear magnetic resonance imaging have been frequently used to diagnose brain injury. However, these methods do not accurately and quantitatively evaluate injury degree. However, proteomics displays some advantages. To date, there are few proteomics studies based on primary astrocyte cultures from a fluid percussion injury model. OBJECTIVE： To detect differential protein expression in rat cerebral cortical astrocytes following fluid percussion injury using two-dimensional gel electrophoresis and mass spectrum and to determine specific biological markers of brain injury. DESIGN, TIME AND SETTING： Complete, randomized grouping and proteomics experiments were performed at the Molecular Pathological Laboratory, Central Laboratory and Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard of Medical College of Chinese People＇s Armed Police Force from October 2007 to May 2008. MATERIALS： Inverted phase-contrast microscope was purchased from Olympus, Japan. PROTEAN IEF Cell isoelectric focusing electrophoresis system and PROTEAN II Xi-Cell vertical electrophoresis system were purchased from Bio-Rad, USA. Autofiex MALDI-TOF mass spectrometer was purchased from Bruker, Germany. METHODS： A total of 90 culture dishes, fully coated with Sprague Dawley rat cortical astrocytes, were randomly divided into control （n = 30） and injury （n = 60） groups. Astrocytes in the injury group were subjected to fluid percussion and subdivided into 4-hour （n = 30） and 48-hour injury （n = 30） groups. MAIN OUTCOME MEASURES： Cell morphology was observed using inverted phase-contrast microscopy. Cell total protein was extracted from each group, followed by two-dimensional gel electrophoresis and silver staining, and the differential protein expression was analyzed using PDQuest 7.0 software. Protein peptide mass fingerprinting of differential protein spots was obtained by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The National Center for Biotechnology Information （NCBI） protein database was retrieved by Mascot to primarily identify protein type, Finally, differential protein expression was detected by Western blot analysis. RESULTS： Following fluid percussion injury, astrocytes displayed obvious swelling and increased intercellular space, with some cell detachment; the number of dead cells was significantly greater than the control group （P 〈 0.05）. Expression intensity of 114 protein spots was significantly greater in the injury group compared with the control group （P〈 0.05）; 9 of the 114 protein spots were identified and peptJde matching scores of 8 spots were 〉 61 （P 〈 0.05）. Protein types were identified and included cellular retinol binding protein, brain fatty acid binding protein 7, $100 calcium binding protein All, 60S acidic ribosomal protein P2, calponin 3, breast carcinoma amplified sequence 2 homolog, eukaryotic translation initiation factor 1A, and hypothetical protein LOC685814. Western blot detection revealed brain fatty acid binding protein 7 expression in cortical astrocytes, which increased with injury time compared with the control group （P 〈 0.05）. CONCLUSION： Results from this study showed morphological and proteomic changes in cortical astrocytes following fluid percussion injury. Brain fatty acid binding protein 7 was expressed in astrocytes and possibly played an important role in injury repair. Mass-spectrum identified differentially expressed proteins that correlated with cell metabolism regulation, signal transduction, and translation initiation, and could serve as specific biological markers of brain injury.

Proteomic Analysis of Proteins in the Salivary Glands of the Fed and Unfed Female Tick Rhipicephalus haemaphysaloides

Identification of differentially expressed salivary gland proteins between the fed and unfed female Rhipicephalus haemaphysaloides may obtain valuable functional molecules. In this study, comparative two-dimensional gel electrophoresis （2-DE） and mass spectrometry were used to separate and identify differentially expressed salivary gland proteins between the fed and unfed female R. haemaphysaloides. The soluble proteins from the salivary glands of fed and unfed female R. haemaphysaloides were separated by a sequential extraction method followed by 2-DE and 2-DE images. Image analysis of the gels revealed 1 096 ± 87 protein spots from the fed female ticks and 991 ±64 protein spots from the unfed female ticks. Among those protein spots, about 724 ±34 were present both in the fed and unfed female ticks. Fourteen spots from the fed ticks and six spots from the unfed ticks were selected for peptide mass fingerprinting （PMF） and sequencing assay by mass spectrometry （MS）. Bioinformatic analysis showed that a majority of the differentially expressed proteins were involved in signal transduction, metabolism, and transcriptional regulation. These differentially expressed proteins might be antigen candidates for the development of vaccines against the tick.

Effects of Space Flight on Expression of Key Proteins in Rice Leaves

As a unique form of abiotic stress, the environmental conditions of outer space are expected to induce changes in plant genomes, proteomes and metabolic pathways. However, the effect of outer space conditions on the overall physiology of plants at the protein level has yet to be reported. To investigate the effects of outer space conditions on the growth-and development-related physiological processes and metabolic pathways of rice different stages, the seeds of rice variety DN423 were sent into orbit for 12.5 d aboard the SJ-10 Returning Satellite, and then the seedlings of both treated and control rice were compared at the three-leaf stage(TLS) and tillering stage(TS). In addition to comparing plant growth and reactive oxygen species(ROS) levels, seedling proteomes were also compared using isobaric tags for relative and absolute quantitation(i TRAQ). Space flight increased TLS plant height by 20%, reduced and increased ROS levels of the TLS and TS seedlings, respectively, and affected the expression of 36 and 323 proteins in TLS and TS leaves, respectively. Furthermore, the functions of the differentially abundant proteins were mainly associated with metabolism, energy, and protein synthesis and degradation. These results suggested that the exposure of seeds to outer space conditions affects the subsequent abundance of key signaling proteins, gene expression, and the processes of protein synthesis and degradation, thereby affecting metabolic processes and promoting adaptation to the abiotic stress of outer space. As such, the present study sheds light on the effects of space flight on plants and contributes to a more comprehensive understanding of extraterrestrial biology.

1-DE/MS Analysis of Proteins Related to the Spathe Color Variation in an Anthurium andraeanum Mutant

[Objectives] This study was conducted to explore the color variation mechanism of Anthurium andraeanum spathe at the protein level. [Methods]The differential proteins of wild type and its white mutant were separated and identified by using one-dimensional gel electrophoresis and mass spectrometry( 1-DE/MS). [Results] Compared with leaves and spadices,the 1-DE patterns of two kinds of spathe proteins were significantly different,and two different bands were detected in wild type spathes and mutant spathes respectively. The four significantly differential bands were selected and analyzed by mass spectrometry,and 138,111,70 and 427 proteins were identified respectively. The results of GO functional annotation analysis showed that the molecular functions of the proteins were mainly catalytic activity and binding,and the main biological processes involved were cellular process and metabolic process. Many proteins involved in the synthesis of anthocyanins and flavonoids,sugar metabolism and some resistance proteins were screened,indicating that the spathe color difference of A. andraeanum‘Pink champion'is not only related to anthocyanin anabolism,but also regulated by various metabolic pathways. [Conclusions]The study provides a new experimental basis for elucidating the molecular mechanism of the regulation of A. andraeanum flower color.

Differential Expression of Bt Protein in Transgenic Bt Cotton

[Objective] The paper was to study the temporal and spatial dynamics of Bt protein expression in transgenic Bt cotton and to determine the inner relationship of Bt protein expression and transgenic Bt cotton. [Method] With transgenic cotton cultivar（ GK19） as the test material,Bt protein contents in different organs,main stem functional leaves at different growth stages and different positions of main stem leaves at different growth stages were studied by enzyme-linked immunosorbent assay. [Result] There were differences in Bt protein content among different organs of transgenic Bt cotton; the Bt protein content of leaves at seedling stage was the highest,followed by flowers,bubs and bolls,and those of roots and stems were relatively low. The Bt protein content of main stem function leaves gradually decreased with the progressing development. There were great differences in Bt protein content among different positions of main stem leaves at different growth stages; the Bt protein content of the 1^（st）-7^（th） top leaves at seedling stage and full budding stage gradually decreased,while those at full flowering stage and full bolling stage first slowly increased then gradually stabilized. [Conclusion] Bt protein expression was found in all organs of transgenic cotton at all growth stages,and the expression level presented temporal and spatial dynamics.

Analysis of Proteotranscriptomics Landscape Reveals Differentially Regulated Pathways in Toxoplasma gondii Infected Mouse Liver

Toxoplasma gondii (T. gondii) an intracellular protozoan parasite, infects mammals including human population world-wide. Upon primary infection, the parasite contributes to mild flu like symptoms in immune competent host, but life threatening complication is seen in immune compromised patients and in pregnant women. Understanding the host-parasite interaction is critical for understanding the pathogenesis and biology parasite reactivation in the host. In this study, we used proteotrasncriptomics analyses by integrating the transcriptomics and proteomics data of T. gondii infected mouse liver to uncover the effector molecules responsible for disease pathogenesis that can be used as candidate markers for diagnosis and drug target. With this aim, we systematically integrated transcriptomicand proteomic data, representing the parasite infected mouse liver. Out of 2758 differentially expressed genes (DEGs) and 301 differentially expressed proteins (DEPs), 159 overlapping genes were identified. Among them, 86 genes were upregulated and 72 were downregulated in their respective mRNA and protein levels in the infected condition. Gene Ontology (GO) analysis revealed that the upregulated genes were mostly associated with immune system processes whereas the downregulated genes were involved in oxidation-reduction process and metabolism of lipid, and fatty acids. Protein-protein interaction (PPI) network analysis uncovered an interaction-hub including, Psmb8, Psmb9 and Tap1 for upregulated proteins and Cyp1A2, Cyp4A10 and Cyp3A11 for down-regulated proteins. Further studies are needed to validating these effector molecules. These molecules are likely to play a vital role in disease pathogenesis, as well as can be used as potential diagnostic marker and drug target candidates.

Differentially Expressed Serum Protein Profiling in Esophageal Squamous Cell Carcinoma Patients with Different Chinese Medicine Syndromes

Objective:Esophageal carcinoma is the eighth most common malignant tumor and ranked the sixth cause of death in the worldwide careinoma.To find out effective serum markers for early diagnosis and targeted treatment is important to improve esophageal carcinoma survival rate.The aim of this study was to screen differ-entially expressed serum protein profiling in esophageal squamous cell careinoma(ESCC)patients with differ-ent Chinese medicine syndrome and establish ESCC syndrome diagnosis model,to explore the signifcance of ESCC serum differential protein in traditional Chinese medicine(TCM)syndrome diagnosis and provide exper-imental and theoretical basis for the diagnosis,clinical treatment of ESCC.Methods:Serum specimens of 80 ES-CC patients with different syndrome and 20 healthy people were selected according to distribution of their sex and age.Magnetic beads based weak cation exchange combined w ith matrix-assist ed laser desorption/ionization time-of-flight mass spectrometry prot eomic technology and Autoflex Analysis and ClinProTools software were applied to screen differentially expressed serum protein profiling.Then establishing each syndrome group diag-nosis model and verifying with new samples to evaluate the diagnosis value of each syndrome group diagnosis model.Results:18 significantly differential expression protein peaks were screened between the whole ESCC patients sera and the healthy control group.Compared with normal control group,among which,ten were signif-cant down-regulation with m/z values of4210,4267,4054,4248,2106,4091,2991,5337,3952,822,and eight were significant up-regulation with m/z values of 2900,2883,1467,1617,2862,2600,895,811.There were 16,12,11,15 differentially expressed protein peaks in phlegm and qi stagnat ion syndrome(Group P),fluid insufficiency and heat agglomeration syndrome(Group F),blood stasis syndrome(Group B),qi and yang deficiency syndrome(Group Q),respectively.Protein with m/z of4210,4091,4054 and 5337 presen-ted obviously low expression in all syndrome groups,while protein with m/z of2883,1617 presented obvious-y high expression.Protein with m/z of 4644 is an obvious and significative biomarker in the comparison of dif-ferent syndrome of ESCC.Establishing ESCC each syndrome serum different ial protein profiling diagnosis mod-el showed that the specificity and veracity in four syndrome groups were all≥80.00%.Conclusion:Our study achieved differentially expressed protein profiling in ESCC patients with different TCM syndromes,established relatively high sensitivity and specificity of different syndrome of ESCC serum differential protein profiling diag-nosis model,which implied the screened syndrome related serum differential expression protein in TCM syn-drome differentiation and diagnosis had great clinical meaning,could be the assistant diagnosis biomarker of ESCC.

Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model:A study based on proteomics

BACKGROUND Ulcerative colitis(UC)is a chronic,nonspecific intestinal inflammatory disease.Acupuncture and moxibustion is proved effective in treating UC,but the mechanism has not been clarified.Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC,which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC.AIM To investigate the mechanism of electroacupuncture(EA)and herb-partitioned moxibustion(HM)on UC rats by using proteomics technology.METHODS Male Sprague-Dawley rats were randomly divided into the normal(N)group,the dextran sulfate sodium(DSS)-induced UC model(M)group,the HM group,and the EA group.UC rat model was prepared with 3%DSS,and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group.Haematoxylin and eosin staining was used for morphological evaluation of colon tissues.Isotope-labeled relative and absolute quantification(iTRAQ)and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues,followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins(DEPs)between groups.Then western blot was used for verification of selected DEPs.RESULTS The macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group(P<0.01).Compared with the N group,a total of 202 DEPs were identified in the M group,including 111 up-regulated proteins and 91 down-regulated proteins,of which 25 and 15 proteins were reversed after HM and EA interventions,respectively.The DEPs were involved in various biological processes such as biological regulation,immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity,intestinal immune network for immunoglobulin A(IgA)production,and FcγR-mediated phagocytosis.The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups,EA and M groups both included immuneassociated and oxidative phosphorylation.Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions,and the expression of oxidative phosphorylation pathway-related proteins,including ATP synthase subunit g(ATP5L),ATP synthase beta subunit precursor(Atp5f),cytochrome c oxidase subunit 4 isoform 1(Cox4i1)were down-regulated after HM and EA interventions.Subsequent verification of selected DEPs(Synaptic vesicle glycoprotein 2A;nuclear cap binding protein subunit 1;carbamoyl phosphate synthetase 1;Cox4i1;ATP synthase subunit b,Atp5f1;doublecortin like kinase 3)by western blot confirmed the reliability of the iTRAQ data,HM and EA interventions can significantly downregulate the expression of oxidative phosphorylation-associated proteins(Cox4i1,Atp5f1)(P<0.01).CONCLUSION EA and HM could regulate the expression of ATP5L,Atp5f1,Cox4i1 that associated with oxidative phosphorylation,then might regulate immune-related pathways of intestinal immune network for IgA production,FcγR-mediated phagocytosis,thereby alleviating colonic inflammation of DSSinduced UC rats.

Proteomic study of three component interactions: plant, pathogens and antagonistic fungi

The molecular factors involved in the three-way interaction between plant, pathogenic fungi and antagonistic/biocontrol fungi, such as Trichoderma, are still poorly understood, even if they represent a matter of interest for improving crop management and developing new strategies for plant diseases control. The aim of this work is to investigate the components involved in this interaction and, for this purpose, a proteomic approach was used. 2-D maps of the protein extracts from the single components in various interactions between plants (potato, bean, tobacco or tomato), pathogens (Botrytis cinerea, Rhizoctonia solani or Pythium ultimum) and biocontrol fungi (Trichoderma atroviride strain P1 or Trichoderma harzianum strain T22) were obtained. The proteome of each partner was collected separately and extracted by acetone precipitation in presence of trichloroacetic acid and a reducing agent (DTT). The extracted proteins were separated by isoelectrofocusing (IEF), using IPG (Immobilized pH gradient) strips, followed by SDS-PAGE. In order to improve resolution the separations were performed both on wide than narrow pH range and on different gel lengths. Differential spots were noted in the proteome of the three-way interaction when compared to each single component. These were further characterized by mass spectrometry and in silico analysis with the aim of identifying and cloning the relative genes. During the in vitro interaction of T. harzianum strain T22 with tomato and the culture filtrate or cell walls of pathogens, the spot number was higher than in the presence of pathogen biomass. In terms of Trichoderma differential proteins displayed on 2D gels, the most important changes were obtained in the presence of P. ultimum . During the in vivo interaction with tomato, the antagonist proteome changed much more in presence of soilborne fungi R. solani and P. ultimum than with the foliar fungus B. cinerea, both in terms of total and increased or novel spots. In silico analysis of some of those spots revealed homology with intracellular enzymes (GTPases, hydrolases) and with stress-related proteins (heat shock proteins HSP70, bacteriocin cloacin). Specific proteins in the plant proteome, i.e. pathogenesis-related proteins, have been identified during the in vivo interaction of bean with R. solani and T. atroviride strain P1. This is in agreement with the demonstrated ability of these beneficial fungi to induce plant systemic disease resistance by activating expression of defence-related genes. Proteins extracted from T. atrovride strain P1 which were analysed by mass spectrometry, revealed some interesting homologies with a fungal hydrophobin of Pleurotus ostreatus and an ABC transporter of Ralstonia metallidurans. These could represent molecular factors involved in the antagonistic mechanisms of Trichoderma and play a role in the three-way interaction with the plant and other microbes.

Proteomic analysis reveals molecular biological details in varioliform gastritis without Helicobacter pylori infection

AIM:To investigate and elucidate the molecular mechanism underlying varioliform gastritis for early detection,prevention and intervention of gastric cancer.METHODS:A combination of two-dimensional gel electrophoresis and mass spectrometry was used to detect the differentially expressed proteins between varioliform gastritis and matched normal mucosa.The selected proteins were confirmed by Western blotting and reverse transcription polymerase chain reaction(RT-PCR) in additional samples and the function of some proteins in varioliform gastritis was analyzed by bio-method preliminarily.RESULTS:We identified 21 differentially expressed proteins in varioliform gastritis,and compared them with matched normal mucosa.Eleven proteins were upregulated and ten downregulated in varioliform gastritis when compared with the same proteins in individualmatched normal gastric mucosa.These proteins are related to metabolism,oxidation,cytoskeleton,apoptosis,signal transduction and other aspects of cells.Two novel proteins,thioredoxin domain-containing protein 5(TXNDC5) upregulated in varioliform gastritis,and neuropolypeptide h3 [phosphatidylethanolamine-binding protein 1(PEBP1)] downregulated in varioliform gastritis,were further investigated.Their expressions were validated by Western blotting and RT-PCR in 12 cases of varioliform gastritis which was matched with normal mucosa.The expression level of PEBP1 in varioliform gastritis was significantly lower(P<0.05) while that of TXNDC5 was significantly higher than that in matched normal gastric mucosa(P<0.05).CONCLUSION:There are some changes of protein expression in varioliform gastritis.Downregulation of PEBP1 and upregulation of TXNDC5 are involved in the development of varioliform gastritis.

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Comparative Proteome Analysis of Human Lung Squamous Carcinoma Tissue

Objective： To establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression tumor-associated proteins by using proteome analysis. Methods： Comparative proteome analysis with 20 human lung squamous carcinoma tissues and the paired normal bronchial epithelial tissues adjacent to tumors was carried out. The total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis （2-DE） and silver staining. The differential expression proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Results： （1） Well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained. For tumor tissue, average spots of 3 gels were 1567±46, and 1436±54 spots were matched with an average matching rate of 91.6%. For control, average spots of 3 gels were 1349±58, and 1228±35 spots were matched with an average matching rate of 91.03%. The average position deviation of matched spots was 0.924±0.128 mm in IEF direction, and 1.022±0.205 mm in SDS-PAGE direction; （2） A total of 1178±56 spots were matched between the eleetrophoretie maps of 20 human lung squamous carcinoma tissues and paired normal tumor-adjacent bronchial epithelial tissues. Seventy-six differentially expressed proteins were screened; （3） Sixty-eight differential proteins were identified by PMF, some proteins were the products of oneogenes, and others involved in the regulation of cell cycle and signal transduetion; （4） In order to validate the reliability of the identified results, the expression of 3 proteins mdm2, c-jun and EGFR, which was correlated with lung squamous carcinoma, was detected by immunohistoehemieal staining and Western blot analysis. The results revealed that mdm2, c-jun and EGFR were up-regulated in lung squamous carcinomas, whereas they were down-regulated in adjacent normal bronchial epithelial tissues, normal lung tissues and inflammatory pseudotumor, which was consistent with our proteome analysis results. Conclusion： The well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were established and 68 differential proteins were characterized by applying comparative proteome analysis successfully. These results will provide scientific foundation for screening the molecular biomarker used to diagnose and treat lung squamous carcinoma, as well as to improve the patient＇s prognosis and provide new clue for the research of lung squamous carcinogenic mechanism.

Electroacupuncture pretreatment exhibits antidepressive effects by regulating hippocampal proteomics in rats with chronic restraint stress

The clinical effect of electroacupuncture on depression is widely recognized. However, the signal transduction pathways and target proteins involved remain unclear. In the present study, rat models of chronic restraint stress were used to explore the mechanism by which electroacupuncture alleviates depression. Rats were randomly divided into control, model, and electroacupuncture groups. Chronic restraint stress was induced in the model and electroacupuncture groups by restraining rats for 28 days. In the electroacupuncture group, electroacupuncture pretreatment at Baihui（GV20） and Yintang（GV29） acupoints was performed daily（1 m A, 2 Hz, discontinuous wave, 20 minutes） prior to restraint for 28 days. Open field tests and body weight measurements were carried out to evaluate the depressive symptoms at specific time points. On day 28, the crossing number, rearing number, and body weights of the model group were significantly lower than those in the control group. Behavior test results indicated that rat models of depressive-like symptoms were successfully established by chronic restraint stress combined with solitary raising. On day 28, an isobaric tag for a relative and absolute quantitation-based quantitative proteomic approach was performed to identify differentially expressed proteins in hippocampal samples obtained from the model and electroacupuncture groups. The potential function of these differential proteins was predicted through the use of the Cluster of Orthologous Groups of proteins（COG） database. Twenty-seven differential proteins（uncharacteristic proteins expected） were selected from the model and electroacupuncture groups. In addition to unknown protein functions, COG are mainly concentrated in general prediction function, mechanism of signal transduction, amino acid transport and metabolism groups. This suggests that electroacupuncture improved depressive-like symptoms by regulating differential proteins, and most of these related proteins exist in nerve cells.