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.

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.

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.

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.

Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry

The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product （gil55628）, gfyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylgfutathione lyase, adenyfate kinase isozyme 1, two unnamed proteins products （gil55628 and gi11334163）, and poly（rC）-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

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.

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.

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.

Transforming growth factor β1 is a differentially expressed candidate protein of congestive heart failure with Qi-deficiency-blood-stasis syndrome

OBJECTIVE: To investigate the role of tongue coating fluid protein in regulation of congestive heart failure(CHF) in Qi-deficiency-blood-stasis syndrome.METHODS: We studied patients with CHF(3 patients with Qi-deficiency-blood-stasis syndrome and 3 without Qi-deficiency-blood-stasis syndrome) to investigate differentially expressed proteins. We also included a control group. A biotin label-based antibody array was used for testing tongue coating fluid samples from patients. Net-work analysis of these differentially expressed proteins was conducted using the STRING database,which can predict the relations between differentially expressed proteins and CHF with Qi-deficiency-blood-stasis syndrome.RESULTS: A total of seven differentially expressed proteins were identified, and among these, transforming growth factor β1(TGF-β1) gets a particular attention for us has drawn specific attention.Network analysis showed a homologous relationship of TGF-β1 with bone morphogenetic protein15, which is associated with myocardial fibrosis.CONCLUSION: Occurrence and development of CHF may result from certain DE-proteins and associated signaling pathways. TGF-β1 protein may be a candidate marker for assessing the risk of CHF in Qideficiency-blood-stasis syndrome.

Sodium acetate can promote the growth and astaxanthin accumulation in the unicellular green alga Haematococcus pluvialis as revealed by a proteomics approach

Haematococcus pluvialis is an ideal natural source of strong antioxidant astaxanthin.Sodium acetate(NaAc)was proven an effective organic carbon source for improving algal growth and astaxanthin production;however,the underlying mechanism remains obscure.To reveal the mechanism of NaAc at the green vegetative stage of H.pluvialis,the physiochemical characteristics and the global protein expression profiles obtained using a tandem mass tag labeling approach were compared between the control(CK)and two NaAc-addition groups.Results show that after NaAc addition,the biomass,nitrate consumption rate,and activities of three carbohydrate metabolism enzymes of H.pluvialis were significantly increased,and the net photosynthetic rate and chlorophyll content decreased.In addition,astaxanthin,total carbohydrates,and total lipids were accumulated,and some red cells appeared in the NaAc5 group.Moreover,317 differentially expressed proteins(DEPs)with the most altered expression patterns were screened out in the CK vs.NaAc5 comparison in our proteomics study.All the DEPs involved in carbohydrate metabolism and lipid metabolism were significantly increased,while most of the photosynthesis-related proteins were depressed in the two NaAc-treated groups.The proteomics results were verified and supported by parallel reaction monitoring approach and physiochemical data.Our findings demonstrate that NaAc promoted the tricarboxylic acid cycle,glyoxylate cycle,and amino acid and lipid synthesis,and inhibited the photo synthe sis-related activities,which consequently speeded up the growth and astaxanthin accumulation in this alga.

Comparative Proteomic Analysis of Wheat (Triticum aestivum L.) Hybrid Necrosis

Hybrid necrosis is the gradual premature death of leaves or plants in certain Fj hybrids of wheat （Triticum aestivum L.）. Comparison of protein expression in necrotic and normal wheat leaves showed that the abundance of 33 proteins was changed significantly, and 24 of these proteins were identified. These proteins were involved in plant growth and development, antioxidation, photosynthesis and carbon assimilation, amino acid and protein biosynthesis, cytological signal transduction, DNA and RNA modification, protein transport, folding and assembly according to their functions. The down-regulation of uroporphyrinogen decarboxylase and the up-regulation of lipoxygenases in necrotic leaves may be related to the oxidative stress in the necrotic cells. The heat shock proteins may play the cytoprotective role. The differential expression of photosynthesis and carbon assimilation related proteins indicated chlorophyll biosynthesis and chloroplast development were inhibited and might finally cause the gradual chlorosis and cell death in necrotic leaves. The results of this study give a comprehensive picture of the post-transcriptional response to necrosis in hybrid wheat leaves and serve as a platform for further characterization of gene function and regulation in wheat hybrid necrosis.

Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558

Spike number is one of three yield-related factors and is closely related to wheat yield. In the present study, we found that the inhibited and normal tillers of the 3558 line presented phenotypic differences at the elongation stage by morphological and anatomical analysis. We then initiated a proteomic study using two-dimensional electrophoresis （2-DE） and nano- scale liquid chromatography-high-definition tandem mass spectroscopy, to isolate and identify the key proteins and metabolic pathways related to spike-development inhibition. A total of 31 differentially expressed proteins （DEPs）, which were mainly involved in cell cycle regulation, photosynthesis, glycolysis, stress response, and oxidation-reduction reactions, were isolated and identified. 14-3-3-1ike proteins and proliferating cell nuclear antigen （PCNA）, involved in cell-cycle regulation, were dramatically down-regulated in inhibited tillers compared to normal tillers. Six spots corresponding to degraded Rubisco large subunits, involved in photosynthesis, were detected in different locations of the 2-DE gels and were up-regulated in inhibited tillers. In addition, the relative levels of DEPs involved in glycolysis and oxidation- reduction reactions changed dramatically. Development was blocked or delayed at the elongation stage in the inhibited tillers of 3558. Weakened energy metabolism might be one reason that the inhibited tillers could not joint and develop into spikes. These DEPs and related metabolic pathways are significant for understanding the mechanism of spike-development inhibition and studying the spike-development process in wheat.

Protein expression changes in cornea after collagen crosslinking

Riboflavin/UV-mediated corneal collagen cross-linking can increase the mechanical strength of the cornea and prevent or delay corneal expansion and keratoconus progression.We performed quantitative analysis of protein iTRAQ in rabbit eye white matter after cross-linking to explore the changes of protein expression in cornea at different times after cross-linking and to understand the process of corneal stroma remodeling after cross-linking.The screening conditions are fold Change
1.2 and P-value<0.05,we identified 713 and 38 differentially expressed proteins in cornea at 1 week and 1 month after cross-linking.There were 16 differentially expressed proteins at two time points after corneal cross-linking.By annotating the functions of these proteins,we identified some proteins that affect the mechanical properties of the cornea,and these proteins are involved in cell growth,oxidative stress response,and signal transduction in the cornea.It has a guiding role in studying the corneal stroma remodeling process after collagen crosslinking.