Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

PubMed

Wan, Cen; Lees, Jonathan G; Minneci, Federico; Orengo, Christine A; Jones, David T

2017-10-01

Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

Recombinant Human Lysyl Oxidase-like 2 Secreted from Human Embryonic Kidney Cells Displays Complex and Acidic Glycans at All Three N-Linked Glycosylation Sites.

PubMed

Go, Eden P; Moon, Hee-Jung; Mure, Minae; Desaire, Heather

2018-05-04

Human lysyl oxidase-like 2 (hLOXL2), a glycoprotein implicated in tumor progression and organ fibrosis, is a molecular target for anticancer and antifibrosis treatment. This glycoprotein contains three predicted N-linked glycosylation sites; one is near the protein's active site, and at least one more is known to facilitate the protein's secretion. Because the glycosylation impacts the protein's biology, we sought to characterize the native, mammalian glycosylation profile and to determine how closely this profile is recapitulated when the protein is expressed in insect cells. All three glycosylation sites on the protein, expressed in human embryonic kidney (HEK) cells, were characterized individually using a mass spectrometry-based glycopeptide analysis workflow. These data were compared to the glycosylation profile of the same protein expressed in insect cells. We found that the producer cell type imparts a substantial influence on the glycosylation of this important protein. The more-relevant version, expressed in HEK cells, contains large, acidic glycoforms; these glycans are not generated in insect cells. The glycosylation differences likely have structural and functional consequences, and these data should be considered when generating protein for functional studies or for high-throughput screening campaigns.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2) is a novel biomarker of myxofibrosarcoma invasion identified by global protein expression profiling.

PubMed

Kikuta, Kazutaka; Kubota, Daisuke; Yoshida, Akihiko; Qiao, Zhiwei; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Chuman, Hirokazu; Kawai, Akira; Kondo, Tadashi

2017-09-01

Myxofibrosarcoma (MFS) is a mesenchymal malignancy characterized by frequent recurrence even after radical wide resection. To optimize therapy for MFS patients, we aimed to identify candidate tissue biomarkers of MFS invasion potential. Invasion characteristics of MFS were evaluated by magnetic resonance imaging and protein expression profiling of primary tumor tissues performed using two-dimensional difference gel electrophoresis (2D-DIGE). Protein expression profiles were compared between invasive and non-invasive tumors surgically resected from 11 patients. Among the 3453 protein spots observed, 59 demonstrated statistically significant difference in intensity (≥2-fold) between invasive and non-invasive tumors (p<0.01 by Wilkoxon test), and were identified by mass spectrometry as 47 individual proteins. Among them, we further focused on discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2), a receptor tyrosine kinase with aberrant expression in malignant tumors. Immunohistochemistry analysis of 21 additional MFS cases revealed that higher DCBLD2 expression was significantly associated with invasive properties of tumor cells. DCBLD2 sensitivity and specificity, and positive and negative predictive values for MFS invasion were 69.2%, 87.5%, 90%, and 63.6%, respectively. The expression level of DCBLD2 was consistent in different portions of tumor tissues. Thus, DCBLD2 expression can be a useful biomarker to evaluate invasive properties of MFS. Further validation studies based on multi-institutional collaboration and comprehensive analysis of DCBLD2 biological functions in MFS are required to confirm its prognostic utility for clinical application. Copyright © 2017 Elsevier B.V. All rights reserved.

Alterations in the lenticular protein profile in experimental selenite-induced cataractogenesis and prevention by ellagic acid.

PubMed

Sakthivel, Muniyan; Geraldine, Pitchairaj; Thomas, Philip A

2011-08-01

Accumulating evidence suggests that oxidative stress underlies age-related formation of cataract, and that antioxidants retard cataractogenesis. This study aimed to evaluate whether ellagic acid, a natural polyphenol with antioxidant properties, prevents alterations in the lenticular protein profile in an experimental model of selenite cataract. Alterations in lenticular protein were determined by two-dimensional electrophoresis (2DE) and image analysis. Eluted αA-crystallin spots were analyzed by mass spectrometry. Western blot analysis was also performed to confirm the differential expression of certain crystallins and cytoskeletal proteins. In cataractous lenses, 2DE and image analysis revealed approximately 45 and 60 prominent spots in soluble and insoluble protein fractions respectively. Analysis of the pI and molecular weight of protein spots revealed differences in the expression of crystallin proteins in soluble and insoluble fractions. Western blot analysis confirmed changes in the expression of αA- and βB1- crystallins in both soluble and insoluble protein fractions, while mass spectrometry confirmed the degradation of αA-crystallin in selenite cataractous lenses. Western blot analysis also confirmed the occurrence of altered expression of certain cytoskeletal proteins in insoluble fractions. However, the lenticular protein profile in lenses from selenite-challenged, ellagic acid-treated rats was essentially similar to that noted in lenses from normal rats. The present study confirms the importance of structural and cytoskeletal proteins in the maintenance of lenticular transparency; the results also suggest that ellagic acid prevents lenticular protein alterations induced by selenite in an experimental setting.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

PubMed Central

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history. PMID:26560065

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

PubMed

Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

2016-01-01

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrated analysis of long noncoding RNA and mRNA expression profile in children with obesity by microarray analysis.

PubMed

Liu, Yuesheng; Ji, Yuqiang; Li, Min; Wang, Min; Yi, Xiaoqing; Yin, Chunyan; Wang, Sisi; Zhang, Meizhen; Zhao, Zhao; Xiao, Yanfeng

2018-06-08

Long noncoding RNAs (lncRNAs) have an important role in adipose tissue function and energy metabolism homeostasis, and abnormalities may lead to obesity. To investigate whether lncRNAs are involved in childhood obesity, we investigated the differential expression profile of lncRNAs in obese children compared with non-obese children. A total number of 1268 differentially expressed lncRNAs and 1085 differentially expressed mRNAs were identified. Gene Ontology (GO) and pathway analysis revealed that these lncRNAs were involved in varied biological processes, including the inflammatory response, lipid metabolic process, osteoclast differentiation and fatty acid metabolism. In addition, the lncRNA-mRNA co-expression network and the protein-protein interaction (PPI) network were constructed to identify hub regulatory lncRNAs and genes based on the microarray expression profiles. This study for the first time identifies an expression profile of differentially expressed lncRNAs in obese children and indicated hub lncRNA RP11-20G13.3 attenuated adipogenesis of preadipocytes, which is conducive to the search for new diagnostic and therapeutic strategies of childhood obesity.

Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

PubMed

Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

2016-04-01

Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; <34 weeks), and late-onset (n = 8; >36 weeks) preeclampsia and their controls who delivered preterm (n = 5; <34 weeks) or at term (n = 5; >36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value < 0.05. Quantitative real-time reverse transcriptase PCR was used to verify the results. Western blotting was performed to verify the expressions of secreted genes at the protein level. Six hundred twenty-seven genes were differentially expressed in early-compared with late-onset preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague-Dawley rats.

PubMed

Heden, Timothy D; Morris, E Matthew; Kearney, Monica L; Liu, Tzu-Wen; Park, Young-Min; Kanaley, Jill A; Thyfault, John P

2014-04-01

The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague-Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ∼27% lower in HF- than in LF-fed animals (p < 0.05); the protein expression of all other molecules was similar in the 2 diets. The fed-state hepatic TAG secretion rate was ∼39% lower (p < 0.05) in HF- (4.62 ± 0.18 mmol·h(-1)) than in LF- (7.60 ± 0.57 mmol·h(-1)) fed animals. Hepatic TAG content was ∼2-fold higher (p < 0.05) in HF- (1.07 ± 0.15 nmol·g(-1) tissue) than in LF- (0.50 ± 0.16 nmol·g(-1) tissue) fed animals. In addition, the fatty acid profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression.

Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague–Dawley rats

PubMed Central

Heden, Timothy D.; Morris, E. Matthew; Kearney, Monica L.; Liu, Tzu-Wen; Park, Young-min; Kanaley, Jill A.; Thyfault, John P.

2015-01-01

The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague–Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ~27% lower in HF- than in LF-fed animals (p < 0.05); the protein expression of all other molecules was similar in the 2 diets. The fed-state hepatic TAG secretion rate was ~39% lower (p < 0.05) in HF- (4.62 ± 0.18 mmol·h−1) than in LF- (7.60 ± 0.57 mmol·h−1) fed animals. Hepatic TAG content was ~2-fold higher (p < 0.05) in HF- (1.07 ± 0.15 nmol·g−1 tissue) than in LF- (0.50 ± 0.16 nmol·g−1 tissue) fed animals. In addition, the fatty acid profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression. PMID:24669989

Long non-coding RNA expression patterns in lung tissues of chronic cigarette smoke induced COPD mouse model.

PubMed

Zhang, Haiyun; Sun, Dejun; Li, Defu; Zheng, Zeguang; Xu, Jingyi; Liang, Xue; Zhang, Chenting; Wang, Sheng; Wang, Jian; Lu, Wenju

2018-05-15

Long non-coding RNAs (lncRNAs) have critical regulatory roles in protein-coding gene expression. Aberrant expression profiles of lncRNAs have been observed in various human diseases. In this study, we investigated transcriptome profiles in lung tissues of chronic cigarette smoke (CS)-induced COPD mouse model. We found that 109 lncRNAs and 260 mRNAs were significantly differential expressed in lungs of chronic CS-induced COPD mouse model compared with control animals. GO and KEGG analyses indicated that differentially expressed lncRNAs associated protein-coding genes were mainly involved in protein processing of endoplasmic reticulum pathway, and taurine and hypotaurine metabolism pathway. The combination of high throughput data analysis and the results of qRT-PCR validation in lungs of chronic CS-induced COPD mouse model, 16HBE cells with CSE treatment and PBMC from patients with COPD revealed that NR_102714 and its associated protein-coding gene UCHL1 might be involved in the development of COPD both in mouse and human. In conclusion, our study demonstrated that aberrant expression profiles of lncRNAs and mRNAs existed in lungs of chronic CS-induced COPD mouse model. From animal models perspective, these results might provide further clues to investigate biological functions of lncRNAs and their potential target protein-coding genes in the pathogenesis of COPD.

Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development.

PubMed

Merlaen, Britt; De Keyser, Ellen; Van Labeke, Marie-Christine

2018-01-01

The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant-water relations in the commercial strawberry ( Fragaria x ananassa ). Aquaporins are water channel proteins that allow water to cross (intra)cellular membranes. In Fragaria x ananassa , few of them have been identified hitherto, hampering the exploration of the water transport regulation at cellular level. Here, we present new aquaporin coding sequences belonging to different subclasses: plasma membrane intrinsic proteins subtype 1 and subtype 2 (PIP1 and PIP2) and tonoplast intrinsic proteins (TIP). The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues. Substrate-specific signature sequences (SSSSs) and specificity-determining positions (SDPs) predict the substrate specificity of each new aquaporin. Expression profiling in leaves, petioles and developing fruits reveals distinct patterns, even within the same (sub)class. Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression. Both upregulation and downregulation during fruit ripening occur. Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same (sub)class.

The Human Pancreas Proteome Defined by Transcriptomics and Antibody-Based Profiling

PubMed Central

Fagerberg, Linn; Hallström, Björn M.; Schwenk, Jochen M.; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia

2014-01-01

The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects. PMID:25546435

Distinct profiles of expressed sequence tags during intestinal regeneration in the sea cucumber Holothuria glaberrima

PubMed Central

Rojas-Cartagena, Carmencita; Ortíz-Pineda, Pablo; Ramírez-Gómez, Francisco; Suárez-Castillo, Edna C.; Matos-Cruz, Vanessa; Rodríguez, Carlos; Ortíz-Zuazaga, Humberto; García-Arrarás, José E.

2010-01-01

Repair and regeneration are key processes for tissue maintenance, and their disruption may lead to disease states. Little is known about the molecular mechanisms that underline the repair and regeneration of the digestive tract. The sea cucumber Holothuria glaberrima represents an excellent model to dissect and characterize the molecular events during intestinal regeneration. To study the gene expression profile, cDNA libraries were constructed from normal, 3-day, and 7-day regenerating intestines of H. glaberrima. Clones were randomly sequenced and queried against the nonredundant protein database at the National Center for Biotechnology Information. RT-PCR analyses were made of several genes to determine their expression profile during intestinal regeneration. A total of 5,173 sequences from three cDNA libraries were obtained. About 46.2, 35.6, and 26.2% of the sequences for the normal, 3-days, and 7-days cDNA libraries, respectively, shared significant similarity with known sequences in the protein database of GenBank but only present 10% of similarity among them. Analysis of the libraries in terms of functional processes, protein domains, and most common sequences suggests that a differential expression profile is taking place during the regeneration process. Further examination of the expressed sequence tag dataset revealed that 12 putative genes are differentially expressed at significant level (R > 6). Experimental validation by RT-PCR analysis reveals that at least three genes (unknown C-4677-1, melanotransferrin, and centaurin) present a differential expression during regeneration. These findings strongly suggest that the gene expression profile varies among regeneration stages and provide evidence for the existence of differential gene expression. PMID:17579180

Protein expression profile changes in human fibroblasts induced by low dose energetic protons

NASA Astrophysics Data System (ADS)

Zhang, Ye; Clement, Jade Q.; Gridley, Daila S.; Rodhe, Larry H.; Wu, Honglu

2009-12-01

Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

PubMed Central

Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí;aEsther; Sánchez del Pino, Manuel M.; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

2015-01-01

Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article “Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry” (Calderón-González et al. [1] in press). PMID:26217805

Aureolib — A Proteome Signature Library: Towards an Understanding of Staphylococcus aureus Pathophysiology

PubMed Central

Pané-Farré, Jan; Kusch, Harald; Wolf, Carmen; Reiß, Swantje; Binh, Le Thi Nguyen; Albrecht, Dirk; Riedel, Katharina; Hecker, Michael; Engelmann, Susanne

2013-01-01

Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H2O2, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σB regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides an essential tool to decipher more complex adaptation processes in S. aureus during host pathogen interaction. PMID:23967085

Dietary Lactobacillus acidophilus modulated skin mucus protein profile, immune and appetite genes expression in gold fish (Carassius auratus gibelio).

PubMed

Hosseini, Marjan; Kolangi Miandare, Hamed; Shabani, Ali; Hoseinifar, Seyed Hossein; Yarahmadi, Peyman

2016-12-01

The objective of the present study was to investigate the effect of dietary Lactobacillus acidophilus on skin mucus protein pattern, immune and appetite related genes expression as well as growth performance in gold fish (Carassius auratus gibelio). Three hundred healthy gold fish (2.5 ± 0.05) juveniles were randomly distributed in 12 glass aquariums (400-L; 25 fish per aquaria) and fed experimental diets contain different levels of L. acidophilus (0, 1.5 × 10 8 , 3 × 10 8 and 6 × 10 8 ) for 8 weeks. SDS-PAGE analysis of skin mucus protein profile at the end of the feeding trial revealed differences in protein profile of probiotic fed fish and control group; even three new bands were observed in L. acidophilus treated groups. Furthermore, fish fed 6 × 10 8  CFU g -1 supplemented diet showed up-regulation of both TNF-1α and TNF-2α gene expression (P < 0.05). Evaluation of appetite related gene expression showed down-regulation of ghrelin in probiotic fed fish compared those of control treatment (P < 0.05). However, administration of different levels of L. acidophilus had no significant effects on growth performance (P > 0.05). These results demonstrated that while no beneficial effects on growth performance, dietary L. acidophilus affects immune and appetite related genes expression as well as skin mucus protein profile. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein Profiles Reveal Diverse Responsive Signaling Pathways in Kernels of Two Maize Inbred Lines with Contrasting Drought Sensitivity

PubMed Central

Yang, Liming; Jiang, Tingbo; Fountain, Jake C.; Scully, Brian T.; Lee, Robert D.; Kemerait, Robert C.; Chen, Sixue; Guo, Baozhu

2014-01-01

Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964) with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP), and protein profiles were investigated in developing kernels (35 DAP) using iTRAQ (isobaric tags for relative and absolute quantitation). Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels. PMID:25334062

Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

PubMed Central

Yang, Wen-Kai; Kang, Chao-Kai; Chang, Chia-Hao; Hsu, An-Di; Lee, Tsung-Han; Hwang, Pung-Pung

2013-01-01

FXYD proteins are novel regulators of Na+-K+-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies. PMID:23383199

Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients

PubMed Central

Bae, Su-Mi; Min, Hyun-Jin; Ding, Guo Hua; Kwak, Sun-Young; Cho, Young-Lae; Nam, Kye-Hyun; Park, Choong Hak; Kim, Yong-Wan; Kim, Chong-Kook; Han, Byoung-Don; Lee, Young-Joo; Kim, Do Kang

2006-01-01

Purpose Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two- dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectrometer. Materials and Methods Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software™. The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. Results A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. Conclusion 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC. PMID:19771267

The plant energy-dissipating mitochondrial systems: depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots.

PubMed

Borecky, Jirí; Nogueira, Fábio T S; de Oliveira, Kívia A P; Maia, Ivan G; Vercesi, Aníbal E; Arruda, Paulo

2006-01-01

The simultaneous existence of alternative oxidases and uncoupling proteins in plants has raised the question as to why plants need two energy-dissipating systems with apparently similar physiological functions. A probably complete plant uncoupling protein gene family is described and the expression profiles of this family compared with the multigene family of alternative oxidases in Arabidopsis thaliana and sugarcane (Saccharum sp.) employed as dicot and monocot models, respectively. In total, six uncoupling protein genes, AtPUMP1-6, were recognized within the Arabidopsis genome and five (SsPUMP1-5) in a sugarcane EST database. The recombinant AtPUMP5 protein displayed similar biochemical properties as AtPUMP1. Sugarcane possessed four Arabidopsis AOx1-type orthologues (SsAOx1a-1d); no sugarcane orthologue corresponding to Arabidopsis AOx2-type genes was identified. Phylogenetic and expression analyses suggested that AtAOx1d does not belong to the AOx1-type family but forms a new (AOx3-type) family. Tissue-enriched expression profiling revealed that uncoupling protein genes were expressed more ubiquitously than the alternative oxidase genes. Distinct expression patterns among gene family members were observed between monocots and dicots and during chilling stress. These findings suggest that the members of each energy-dissipating system are subject to different cell or tissue/organ transcriptional regulation. As a result, plants may respond more flexibly to adverse biotic and abiotic conditions, in which oxidative stress is involved.

Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development.

PubMed

Guo, D; Li, H L; Tang, X; Peng, S Q

2014-12-18

In plants, homeodomain proteins play a critical role in regulating various aspects of plant growth and development. KNOX proteins are members of the homeodomain protein family. The KNOX transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of cassava (Manihot esculenta Krantz) has allowed a genome-wide search for M. esculenta KNOX (MeKNOX) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. In the present study, we identified 12 MeKNOX genes in the cassava genome and grouped them into two distinct subfamilies based on their domain composition and phylogenetic analysis. Furthermore, semi-quantitative reverse transcription polymerase chain reaction analysis was performed to elucidate the expression profiles of these genes in different tissues and during various stages of root development. The analysis of MeKNOX expression profiles of indicated that 12 MeKNOX genes display differential expressions either in their transcript abundance or expression patterns.

Identification of human cell responses to benzene and benzene metabolites.

PubMed

Gillis, Bruce; Gavin, Igor M; Arbieva, Zarema; King, Stephen T; Jayaraman, Sundararajan; Prabhakar, Bellur S

2007-09-01

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

PubMed Central

Zhang, Xianghui; Fu, Jianming; Hiromasa, Yasuaki; Pan, Hongyu; Bai, Guihua

2013-01-01

Background Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1. Methods The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins. Results Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification. Conclusions Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance. PMID:24376514

Chronic hypoxia-induced alteration of presynaptic protein profiles and neurobehavioral dysfunction are averted by supplemental oxygen in Lymnaea stagnalis.

PubMed

Fei, G-H; Feng, Z-P

2008-04-22

Chronic hypoxia causes neural dysfunction. Oxygen (O(2)) supplements have been commonly used to increase the O(2) supply, yet the therapeutic benefit of this treatment remains controversial due to a lack of cellular and molecular evidence. In this study, we examined the effects of short-burst O(2) supplementation on neural behavior and presynaptic protein expression profiles in a simple chronic hypoxia model of snail Lymnaea stagnalis. We reported that hypoxia delayed the animal response to light stimuli, suppressed locomotory activity, induced expression of stress-response proteins, hypoxia inducible factor-1alpha (HIF-1alpha) and heat shock protein 70 (HSP70), repressed syntaxin-1 (a membrane-bound presynaptic protein) and elevated vesicle-associated membrane protein-1 (VAMP-1) (a vesicle-bound presynaptic protein) level. O(2) supplements relieved suppression of neural behaviors, and corrected hypoxia-induced protein alterations in a dose-dependent manner. The effectiveness of supplemental O(2) was further evaluated by determining time courses for recovery of neural behaviors and expression of stress response proteins and presynaptic proteins after relief from hypoxia conditions. Our findings suggest that O(2) supplement improves hypoxia-induced adverse alterations of presynaptic protein expression and neurobehaviors, however, the optimal level of O(2) required for improvement is protein specific and system specific.

The protein expression landscape of mitosis and meiosis in diploid budding yeast.

PubMed

Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

2017-03-06

Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We have integrated protein profiling data using mass spectrometry with tiling array RNA profiling data and information on double-stranded RNAs (dsRNAs) by overlapping sense/antisense transcripts from an RNA-Sequencing experiment. This work therefore provides quantitative insight into protein expression during cell division and development and associates changing protein levels with developmental stage specific induction of antisense transcripts and the formation of dsRNAs. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses

PubMed Central

2010-01-01

Background Nontypeable Haemophilus influenzae colonizes and infects the airways of adults with chronic obstructive pulmonary disease, the fourth most common cause of death worldwide.Thus, H. influenzae, an exclusively human pathogen, has adapted to survive in the hostile environment of the human airways.To characterize proteins expressed by H. influenzae in the airways, a prototype strain was grown in pooled human sputum to simulate conditions in the human respiratory tract.The proteins from whole bacterial cell lysates were solubilized with a strong buffer and then quantitatively cleaned with an optimized precipitation/on-pellet enzymatic digestion procedure.Proteomic profiling was accomplished by Nano-flow liquid chromatography/mass spectroscopy with low void volume and high separation efficiency with a shallow, long gradient. Results A total of 1402 proteins were identified with high confidence, including 170 proteins that were encoded by genes that are annotated as conserved hypothetical proteins.Thirty-one proteins were present in greater abundance in sputum-grown conditions at a ratio of > 1.5 compared to chemically defined media.These included 8 anti-oxidant and 5 stress-related proteins, suggesting that expression of antioxidant activity and stress responses is important for survival in the airways.Four proteins involved in uptake of divalent anions and 9 proteins that function in uptake of various molecules were present in greater abundance in sputum-grown conditions. Conclusions Proteomic expression profiling of H. influenzae grown in pooled human sputum revealed increased expression of antioxidant, stress-response proteins and cofactor and nutrient uptake systems compared to media grown cells.These observations suggest that H. influenzae adapts to the oxidative and nutritionally limited conditions of the airways in adults with chronic obstructive pulmonary disease by increasing expression of molecules necessary for survival in these conditions. PMID:20515494

Human liver proteome project: plan, progress, and perspectives.

PubMed

He, Fuchu

2005-12-01

The Human Liver Proteome Project is the first initiative of the human proteome project for human organs/tissues and aims at writing a modern Prometheus myth. Its global scientific objectives are to reveal the "solar system" of the human liver proteome, expression profiles, modification profiles, a protein linkage (protein-protein interaction) map, and a proteome localization map, and to define an ORFeome, physiome, and pathome. Since it was first proposed in April 2002, the Human Liver Proteome Project has attracted more than 100 laboratories from all over the world. In the ensuing 3 years, we set up a management infrastructure, identified reference laboratories, confirmed standard operating procedures, initiated international research collaborations, and finally achieved the first set of expression profile data.

Gene expression profiling of Listeria monocytogenes strain F2365 during growth in ultrahigh-temperature-processed skim milk.

PubMed

Liu, Yanhong; Ream, Amy

2008-11-01

To study how Listeria monocytogenes survives and grows in ultrahigh-temperature-processed (UHT) skim milk, microarray technology was used to monitor the gene expression profiles of strain F2365 in UHT skim milk. Total RNA was isolated from strain F2365 in UHT skim milk after 24 h of growth at 4 degrees C, labeled with fluorescent dyes, and hybridized to "custom-made" commercial oligonucleotide (35-mers) microarray chips containing the whole genome of L. monocytogenes strain F2365. Compared to L. monocytogenes grown in brain heart infusion (BHI) broth for 24 h at 4 degrees C, 26 genes were upregulated (more-than-twofold increase) in UHT skim milk, whereas 14 genes were downregulated (less-than-twofold decrease). The upregulated genes included genes encoding transport and binding proteins, transcriptional regulators, proteins in amino acid biosynthesis and energy metabolism, protein synthesis, cell division, and hypothetical proteins. The downregulated genes included genes that encode transport and binding proteins, protein synthesis, cellular processes, cell envelope, energy metabolism, a transcriptional regulator, and an unknown protein. The gene expression changes determined by microarray assays were confirmed by real-time reverse transcriptase PCR analyses. Furthermore, cells grown in UHT skim milk displayed the same sensitivity to hydrogen peroxide as cells grown in BHI, demonstrating that the elevated levels of expression of genes encoding manganese transporter complexes in UHT skim milk did not result in changes in the oxidative stress sensitivity. To our knowledge, this report represents a novel study of global transcriptional gene expression profiling of L. monocytogenes in a liquid food.

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

DOE PAGES

Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo; ...

2017-10-07

We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured)more » longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.« less

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo

We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured)more » longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.« less

Uncovering Hidden Layers of Cell Cycle Regulation through Integrative Multi-omic Analysis

PubMed Central

Aviner, Ranen; Shenoy, Anjana; Elroy-Stein, Orna; Geiger, Tamar

2015-01-01

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression. PMID:26439921

An Inducible DamID System for Profiling Interactions of Nuclear Lamina Protein Component Lamin B1 with Chromosomes in Mouse Cells.

PubMed

Kozhevnikova, E N; Leshchenko, A E; Pindyurin, A V

2018-05-01

At the level of DNA organization into chromatin, there are mechanisms that define gene expression profiles in specialized cell types. Genes within chromatin regions that are located at the nuclear periphery are generally expressed at lower levels; however, the nature of this phenomenon remains unclear. These parts of chromatin interact with nuclear lamina proteins like Lamin B1 and, therefore, can be identified in a given cell type by chromatin profiling of these proteins. In this study, we created and tested a Dam Identification (DamID) system induced by Cre recombinase using Lamin B1 and mouse embryonic fibroblasts. This inducible system will help to generate genome-wide profiles of chromatin proteins in given cell types and tissues with no need to dissect tissues from organs or separate cells from tissues, which is achieved by using specific regulatory DNA elements and due to the high sensitivity of the method.

Gene Expression Profiling in Pachyonychia Congenita Skin

PubMed Central

Cao, Yu-An; Hickerson, Robyn P.; Seegmiller, Brandon L.; Grapov, Dmitry; Gross, Maren M.; Bessette, Marc R.; Phinney, Brett S.; Flores, Manuel A.; Speaker, Tycho J.; Vermeulen, Annaleen; Bravo, Albert A.; Bruckner, Anna L.; Milstone, Leonard M.; Schwartz, Mary E.; Rice, Robert H.; Kaspar, Roger L.

2015-01-01

Background Pachyonychia congenita (PC) is a skin disorder resulting from mutations in keratin (K) proteins including K6a, K6b, K16, and K17. One of the major symptoms is painful plantar keratoderma. The pathogenic sequelae resulting from the keratin mutations remain unclear. Objective To better understand PC pathogenesis. Methods RNA profiling was performed on biopsies taken from PC-involved and uninvolved plantar skin of seven genotyped PC patients (two K6a, one K6b, three K16, and one K17) as well as from control volunteers. Protein profiling was generated from tape-stripping samples. Results A comparison of PC-involved skin biopsies to adjacent uninvolved plantar skin identified 112 differentially-expressed mRNAs common to patient groups harboring K6 (i.e., both K6a and K6b) and K16 mutations. Among these mRNAs, 25 encode structural proteins including keratins, small proline-rich and late cornified envelope proteins, 20 are related to metabolism and 16 encode proteases, peptidases, and their inhibitors including kallikrein-related peptidases (KLKs), and serine protease inhibitors (SERPINs). mRNAs were also identified to be differentially expressed only in K6 (81) or K16 (141) patient samples. Furthermore, 13 mRNAs were identified that may be involved in pain including nociception and neuropathy. Protein profiling, comparing three K6a plantar tape-stripping samples to non-PC controls, showed changes in the PC corneocytes similar, but not identical, to the mRNA analysis. Conclusion Many differentially-expressed genes identified in PC-involved skin encode components critical for skin barrier homeostasis including keratinocyte proliferation, differentiation, cornification, and desquamation. The profiling data provide a foundation for unraveling the pathogenesis of PC and identifying targets for developing effective PC therapeutics. PMID:25656049

Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

PubMed

Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

2009-12-01

The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

Validation of the Swine Protein-Annotated Oligonucleotide Microarray

USDA-ARS?s Scientific Manuscript database

The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation

PubMed Central

Lin, Zhaomiao; Wang, Zunxin; Zhang, Xincheng; Li, Ganghua; Wang, Shaohua; Ding, Yanfeng

2017-01-01

Rice grain chalkiness is a highly complex trait involved in multiple metabolic pathways and controlled by polygenes and growth conditions. To uncover novel aspects of chalkiness formation, we performed an integrated profiling of gene activity in the developing grains of a notched-belly rice mutant. Using exhaustive tandem mass spectrometry-based shotgun proteomics and whole-genome RNA sequencing to generate a nearly complete catalog of expressed mRNAs and proteins, we reliably identified 38,476 transcripts and 3,840 proteins. Comparison between the translucent part and chalky part of the notched-belly grains resulted in only a few differently express genes (240) and differently express proteins (363), thus making it possible to focus on ‘core’ genes or common pathways. Several novel key pathways were identified as of relevance to chalkiness formation, in particular the shift of C and N metabolism, the down-regulation of ribosomal proteins and the resulting low abundance of storage proteins especially the 13 kDa prolamin subunit, and the suppressed photosynthetic capacity in the pericarp of the chalky part. Further, genes and proteins as transporters for carbohydrates, amino acid/peptides, proteins, lipids and inorganic ions showed an increasing expression pattern in the chalky part of the notched-belly grains. Similarly, transcripts and proteins of receptors for auxin, ABA, ethylene and brassinosteroid were also up-regulated. In summary, this joint analysis of transcript and protein profiles provides a comprehensive reference map of gene activity regarding the physiological state in the chalky endosperm. PMID:28158863

Proteomic Analysis of Whole Human Saliva Detects Enhanced Expression of Interleukin-1 Receptor Antagonist, Thioredoxin and Lipocalin-1 in Cigarette Smokers Compared to Non-Smokers

PubMed Central

Jessie, Kala; Pang, Wei Wei; Haji, Zubaidah; Rahim, Abdul; Hashim, Onn Haji

2010-01-01

A gel-based proteomics approach was used to screen for proteins of differential abundance between the saliva of smokers and those who had never smoked. Subjecting precipitated proteins from whole human saliva of healthy non-smokers to two-dimensional electrophoresis (2-DE) generated typical profiles comprising more than 50 proteins. While 35 of the proteins were previously established by other researchers, an additional 22 proteins were detected in the 2-DE saliva protein profiles generated in the present study. When the 2-DE profiles were compared to those obtained from subjects considered to be heavy cigarette smokers, three saliva proteins, including interleukin-1 receptor antagonist, thioredoxin and lipocalin-1, showed significant enhanced expression. The distribution patterns of lipocalin-1 isoforms were also different between cigarette smokers and non-smokers. The three saliva proteins have good potential to be used as biomarkers for the adverse effects of smoking and the risk for inflammatory and chronic diseases that are associated with it. PMID:21151451

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling.

PubMed

Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

2009-12-24

Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

Proteomic and transcriptomic analyses of rigid and membranous cuticles and epidermis from the elytra and hindwings of the red flour beetle, Tribolium castaneum.

PubMed

Dittmer, Neal T; Hiromasa, Yasuaki; Tomich, John M; Lu, Nanyan; Beeman, Richard W; Kramer, Karl J; Kanost, Michael R

2012-01-01

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles.

Cell and tissue microarray technologies for protein and nucleic acid expression profiling.

PubMed

Cardano, Marina; Diaferia, Giuseppe R; Falavigna, Maurizio; Spinelli, Chiara C; Sessa, Fausto; DeBlasio, Pasquale; Biunno, Ida

2013-02-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

[Plasma proteomic analysis in children with infectious mononucleosis].

PubMed

Ran, Zhi-Ling; Xiao, Bin; Liu, Hong-Rui; Liu, You-Ping; Sheng, Qiao-Ni

2015-03-01

To explore the abnormal expression of plasma proteins by analysis of proteomic expression profile in children with infectious mononucleosis (IM). Two dimensional gel electrophoresis (2-DE) followed by the mass spectrometry was used to examine important protein spots with different expression levels between children with IM and normal controls. Seven differential proteins were obtained: hemopexin, vitamin D binding protein, fetuin A, C-reactive protein, apolipoprotein A, haptoglobin and transthyretin. Compared with the control group, haptoglobin showed a higher expression level in children with IM, and the expression levels of the other proteins were obviously down-regulated. The expression changes of differential proteins identified in this study are all related with the liver acute injury, suggesting that children with IM are associated with acute liver injury. Further studies on the characteristics of above proteins will contribute to the diagnosis and treatment of pediatric IM.

Comparative Proteomic Analysis of Yak Follicular Fluid during Estrus

PubMed Central

Guo, Xian; Pei, Jie; Ding, Xuezhi; Chu, Min; Bao, Pengjia; Wu, Xiaoyun; Liang, Chunnian; Yan, Ping

2016-01-01

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development. PMID:26954118

Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles

EPA Science Inventory

We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing twodimensional...

Distinct effects of calorie restriction on adipose tissue cytokine and angiogenesis profiles in obese and lean mice

PubMed Central

2012-01-01

Background Obesity associates with low-grade inflammation and adipose tissue remodeling. Using sensitive high-throughput protein arrays we here investigated adipose tissue cytokine and angiogenesis-related protein profiles from obese and lean mice, and in particular, the influence of calorie restriction (CR). Methods Tissue samples from visceral fat were harvested from obese mice fed with a high-fat diet (60% of energy), lean controls receiving low-fat control diet as well as from obese and lean mice kept under CR (energy intake 70% of ad libitum intake) for 50 days. Protein profiles were analyzed using mouse cytokine and angiogenesis protein array kits. Results In obese and lean mice, CR was associated with 11.3% and 15.6% reductions in body weight, as well as with 4.0% and 4.6% reductions in body fat percentage, respectively. Obesity induced adipose tissue cytokine expressions, the most highly upregulated cytokines being IL-1ra, IL-2, IL-16, MCP-1, MIG, RANTES, C5a, sICAM-1 and TIMP-1. CR increased sICAM-1 and TIMP-1 expression both in obese and lean mice. Overall, CR showed distinct effects on cytokine expressions; in obese mice CR largely decreased but in lean mice increased adipose tissue cytokine expressions. Obesity was also associated with increased expressions of angiogenesis-related proteins, in particular, angiogenin, endoglin, endostatin, endothelin-1, IGFBP-3, leptin, MMP-3, PAI-1, TIMP-4, CXCL16, platelet factor 4, DPPIV and coagulation factor III. CR increased endoglin, endostatin and platelet factor 4 expressions, and decreased IGFBP-3, NOV, MMP-9, CXCL16 and osteopontin expressions both in obese and lean mice. Interestingly, in obese mice, CR decreased leptin and TIMP-4 expressions, whereas in lean mice their expressions were increased. CR decreased MMP-3 and PAI-1 only in obese mice, whereas CR decreased FGF acidic, FGF basic and coagulation factor III, and increased angiogenin and DPPIV expression only in lean mice. Conclusions CR exerts distinct effects on adipocyte cytokine and angiogenesis profiles in obese and lean mice. Our study also underscores the importance of angiogenesis-related proteins and cytokines in adipose tissue remodeling and development of obesity. PMID:22748184

Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

NASA Astrophysics Data System (ADS)

Zhou, Qian; Wu, Changgong; Dong, Bo; Li, Fuhua; Liu, Fengqi; Xiang, Jianhai

2010-03-01

Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica. Fourteen differentially displayed protein spots were detected and seven of them were identified. Three spots were up-expressed and identified: actin, heat shock protein 70, and chaperone subunit 1; three down-regulated proteins were identified: arginine kinase, elongation factor-2, and glycine-rich protein; and a newly expressed protein was identified as peroxiredoxin. The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression, and in the survival of A. sinica in the presence of copper and other heavy metals; the findings improve understanding of the organism’s adaptive responses and resistance.

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Proteomic Profiling of Paraffin-Embedded Samples Identifies Metaplasia-Specific and Early-Stage Gastric Cancer Biomarkers

PubMed Central

Sousa, Josane F.; Ham, Amy-Joan L.; Whitwell, Corbin; Nam, Ki Taek; Lee, Hyuk-Joon; Yang, Han-Kwang; Kim, Woo Ho; Zhang, Bing; Li, Ming; LaFleur, Bonnie; Liebler, Daniel C.; Goldenring, James R.

2013-01-01

Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis. PMID:22944598

Comparative proteomic profiling and possible toxicological mechanism of acute injury induced by carbon ion radiation in pubertal mice testes

NASA Astrophysics Data System (ADS)

Zhang, Hong

2016-07-01

We investigated potential mechanisms of acute injury in pubertal mice testes after exposure to carbon ion radiation (CIR). Serum testosterone was measured following whole-body irradiation with a 2Gy carbon ion beam. Comparative proteomic profiling and Western blotting were applied to identify potential biomarkers and measure protein expression, and terminal dUTP nick end-labeling (TUNEL) was performed to detect apoptotic cells. Immunohistochemistry and immunofluorescence were used to investigate protein localization. Serum testosterone was lowest at 24h after CIR, and 10 differentially expressed proteins were identified at this time point that included eIF4E, an important regulator of initiation that combines with mTOR and 4EBP1 to control protein synthesis via the mTOR signalling pathway during proliferation and apoptosis. Protein expression and localization studies confirmed their association with acute injury following exposure to CIR. These three proteins may be useful molecular markers for detecting abnormal spermatogenesis following exposure to environmental and cosmic radiation

Proteomic analysis associated with coronary artery dilatation caused by Kawasaki disease using serum exosomes.

PubMed

Zhang, Li; Wang, Wei; Bai, Jun; Xu, Yu-Fen; Li, Lai-Qing; Hua, Liang; Deng, Li; Jia, Hong-Ling

2016-05-01

The aim of this study was to investigate the serum exosome proteome profile of coronary artery dilatation (CAD) caused by Kawasaki disease (KD). Two-dimensional electrophoresis was implemented on proteins of serum exosomes obtained from children with CAD caused by KD and from healthy controls. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis. We identified 38 differentially expressed proteins (13 up-regulated and 25 down-regulated) from serum exosomes of patients with CAD caused by KD compared with healthy controls. Expression levels of three differentially expressed proteins (leucine-rich alpha-2-glycoprotein, sex hormone-binding globulin, and serotransferrin) were validated using western blot analysis. Classification and protein-protein network analysis showed that they are associated with multiple functional groups involved in the acute inflammatory response, defense response, complement activation, humoral immune response, and response to wounding. The majority of the proteins are involved in the inflammation and coagulation cascades. These findings establish a comprehensive proteome profile of CAD caused by KD and increase our knowledge of scientific insight into its mechanisms. Copyright © 2016 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG).

PubMed

Pentheroudakis, George; Kotoula, Vassiliki; Eleftheraki, Anastasia G; Tsolaki, Eleftheria; Wirtz, Ralph M; Kalogeras, Konstantine T; Batistatou, Anna; Bobos, Mattheos; Dimopoulos, Meletios A; Timotheadou, Eleni; Gogas, Helen; Christodoulou, Christos; Papadopoulou, Kyriaki; Efstratiou, Ioannis; Scopa, Chrisoula D; Papaspyrou, Irene; Vlachodimitropoulos, Dimitrios; Linardou, Helena; Samantas, Epaminontas; Pectasides, Dimitrios; Pavlidis, Nicholas; Fountzilas, George

2013-01-01

Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes. In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029). ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.

Impact of pyrrolidine-bispyrrole DNA minor groove binding agents and chirality on global proteomic profile in Escherichia Coli.

PubMed

Yang, Ya-Ting; Lin, Chun-Yu; Jeng, Jingyueh; Ong, Chi-Wi

2013-05-23

There is great interest in the design of small molecules that selectively target minor grooves of duplex DNA for controlling specific gene expression implicated in a disease. The design of chiral small molecules for rational drug design has attracted increasing attention due to the chirality of DNA. Yet, there is limited research on the chirality effect of minor groove binders on DNA interaction, especially at the protein expression level. This paper is an attempt to illustrate that DNA binding affinity might not provide a full picture on the biological activities. Drug interacting at the genomic level can be translated to the proteomic level. Here we have illustrated that although the chiral bispyrrole-pyrrolidine-oligoamides, PySSPy and PyRSPy, showed low binding affinity to DNA, their influence at the proteomic level is significant. More importantly, the chirality also plays a role. Two-dimensional proteomic profile to identify the differentially expressed protein in Escherichia coli DH5α (E coli DH5α) were investigated. E coli DH5α incubated with the chiral PySSPy and PyRSPy, diastereomeric at the pyrrolidine ring, showed differential expression of eighteen proteins as observed through two dimensional proteomic profiling. These eighteen proteins identified by MALDI_TOF/TOF MS include antioxidant defense, DNA protection, protein synthesis, chaperone, and stress response proteins. No statistically significant toxicity was observed at the tested drug concentrations as measured via MTT assay. The current results showed that the chiral PySSPy and PyRSPy impact on the proteomic profiling of E coli DH5α, implicating the importance of drug chirality on biological activities at the molecular level.

Proteomic Analysis of Propiconazole Responses in Mouse Liver-Comparison of Genomic and Proteomic Profiles

EPA Science Inventory

We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this commonly used fungicide. Utilizing t...

Usefulness of DIGE for the detection of protein profile in retained and released bovine placental tissues.

PubMed

Kankofer, M; Wawrzykowski, J; Miller, I; Hoedemaker, M

2015-02-01

Regardless intensive research, the etiology and mechanisms of retention of fetal membranes in cows, still require elucidation. In our research approach, difference in gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization (MALDI) identification were used to obtain first results on protein profile of bovine placental membranes which were properly released or retained for more than 12 h after parturition. Placentomes from 6 cows that released placenta and from 6 cows that retained fetal membranes were homogenized, fluorescence labeled and subjected to DIGE. Selected spots that significantly differed between retained and released placenta as well as spots with constant appearance were identified by MALDI. This allowed identification of the following proteins with high statistical reliability: Transforming growth factor beta 2 - high expression in maternal and fetal part of retained fetal membranes, Short transient receptor potential channel 5 -high expression in maternal part of retained and not retained fetal membranes, Rab GDP dissociation inhibitor beta - high expression in fetal part of retained and not retained fetal membranes, Proline dehydrogenase 2 - similar expression in all examined samples, Ras-related protein Rab-7b -high expression only in maternal part of not retained fetal membranes. Up to now, these proteins have not been considered as possibly important molecules for the separation/retention of fetal membranes, but their biological roles may suggest it. Further studies are necessary to establish a full profile of bovine placental proteins and define target molecules that may be involved in separation/retention of fetal membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

PubMed

Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

2012-01-01

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

Profiling of acyl-CoA oxidase-deficient and peroxisome proliferator Wy14,643-treated mouse liver protein by surface-enhanced laser desorption/ionization ProteinChip Biology System.

PubMed

Chu, Ruiyin; Zhang, Weihua; Lim, Hanjo; Yeldandi, Anjana V; Herring, Chris; Brumfield, Laura; Reddy, Janardan K; Davison, Matthew

2002-01-01

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including fatty acids. Mice lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation. To investigate proteins involved in peroxisome proliferation, we adopted a novel surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology to compare the protein profiles of control (wild-type), AOX-/-, and wild-type mice treated with peroxisome proliferator, Wy-14,643. The results indicated that the protein profiles of AOX-/- mice were similar to the wild-type mice treated with Wy14,643, but significantly different from the nontreated wild-type mice. Using four different ProteinChip Arrays, a total of 40 protein peaks showed more than twofold changes. Among these differentially expressed peaks, a downregulated peak was identified as the major urinary protein in both AOX-/- and Wyl4,643-treated mice by SELDI. The identification of MUP was further confirmed by two-dimensional electrophoresis and liquid chromatography coupled tandem mass spectrometry (LC-MS-MS). This SELDI method offers several technical advantages for detection of differentially expressed proteins, including ease and speed of screening, no need for chromatographic processing, and small sample size.

Differentiating disease subtypes by using pathway patterns constructed from gene expressions and protein networks.

PubMed

Hung, Fei-Hung; Chiu, Hung-Wen

2015-01-01

Gene expression profiles differ in different diseases. Even if diseases are at the same stage, such diseases exhibit different gene expressions, not to mention the different subtypes at a single lesion site. Distinguishing different disease subtypes at a single lesion site is difficult. In early cases, subtypes were initially distinguished by doctors. Subsequently, further differences were found through pathological experiments. For example, a brain tumor can be classified according to its origin, its cell-type origin, or the tumor site. Because of the advancements in bioinformatics and the techniques for accumulating gene expressions, researchers can use gene expression data to classify disease subtypes. Because the operation of a biopathway is closely related to the disease mechanism, the application of gene expression profiles for clustering disease subtypes is insufficient. In this study, we collected gene expression data of healthy and four myelodysplastic syndrome subtypes and applied a method that integrated protein-protein interaction and gene expression data to identify different patterns of disease subtypes. We hope it is efficient for the classification of disease subtypes in adventure.

Application of Protein Expression Profiling to Screen Chemicals for Androgenic Activity.

EPA Science Inventory

Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) coupled with a s...

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

Proteomic changes in brain tissues of marine medaka (Oryzias melastigma) after chronic exposure to two antifouling compounds: butenolide and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT).

PubMed

Chen, Lianguo; Zhang, Huoming; Sun, Jin; Wong, Yue-Him; Han, Zhuang; Au, Doris W T; Bajic, Vladimir B; Qian, Pei-Yuan

2014-12-01

SeaNine 211 with active ingredient of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) has been used as a "green" antifouling agent worldwide but has raised serious biosafety concerns in coastal environments. DCOIT has the potential to disrupt the neurotransmission in nervous system, but the underlying mechanism has not been clarified. In the present study, we used TMT six-plex labeling coupled with two-dimensional LC-MS/MS analysis to investigate the protein expression profiles in brain tissues of the marine medaka (Oryzias melastigma) after a 28-day exposure to environmentally-realistic concentration of DCOIT at 2.55 μg/L (0.009 μM) or butenolide, one promising antifouling compound, at 2.31 μg/L (0.012 μM). DCOIT and butenolide induced differential expression of 26 and 18 proteins in male brains and of 27 and 23 proteins in female brains, respectively. Distinct mechanisms of toxicity were initiated by DCOIT and butenolide in males, whereas the protein expression profiles were largely similar in females treated by these two compounds. In males, DCOIT exposure mainly led to disruption of mitogen-activated protein kinase (MAPK) signaling pathway, while butenolide affected proteins related to the cytoskeletal disorganization that is considered as a general response to toxicant stress. Furthermore, a sex-dependent protein expression profile was also noted between male and female fish, as evident by the inverse changes in the expressions of common proteins (5 proteins for butenolide- and 2 proteins for DCOIT-exposed fish). Overall, this study provided insight into the molecular mechanisms underlying the toxicity of DCOIT and butenolide. The extremely low concentrations used in this study highlighted the ecological relevance, arguing for thorough assessments of their ecological risks before the commercialization of any new antifouling compound. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular profiles of Quadriceps muscle in myostatin-null mice reveal PI3K and apoptotic pathways as myostatin targets

PubMed Central

Chelh, Ilham; Meunier, Bruno; Picard, Brigitte; Reecy, Mark James; Chevalier, Catherine; Hocquette, Jean-François; Cassar-Malek, Isabelle

2009-01-01

Background Myostatin (MSTN), a member of the TGF-β superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass. Results Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed. Conclusion All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3ε protein, TCTP/GSK-3β). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo. PMID:19397818

Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Mingqun ..; Kikuchi, Takane; Brewer, Heather M.

2011-02-17

Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular {alpha}-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins ({approx}99%) with known functions were expressed, whereas only approximately 80% of hypothetical proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in bothmore » bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.« less

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

PubMed Central

2012-01-01

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

PubMed

Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

2012-03-02

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

Identifying Dynamic Protein Complexes Based on Gene Expression Profiles and PPI Networks

PubMed Central

Li, Min; Chen, Weijie; Wang, Jianxin; Pan, Yi

2014-01-01

Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of “closeness” and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling

PubMed Central

Cardano, Marina; Diaferia, Giuseppe R.; Falavigna, Maurizio; Spinelli, Chiara C.; Sessa, Fausto; DeBlasio, Pasquale

2013-01-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform. PMID:23172795

HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma.

PubMed

Thuring, Camilla; Follin, Elna; Geironson, Linda; Freyhult, Eva; Junghans, Victoria; Harndahl, Mikkel; Buus, Søren; Paulsson, Kajsa M

2015-09-15

Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with β2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols.

Analysis of the Human Prostate-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling Identifies TMEM79 and ACOXL as Two Putative, Diagnostic Markers in Prostate Cancer

PubMed Central

O'Hurley, Gillian; Busch, Christer; Fagerberg, Linn; Hallström, Björn M.; Stadler, Charlotte; Tolf, Anna; Lundberg, Emma; Schwenk, Jochen M.; Jirström, Karin; Bjartell, Anders; Gallagher, William M.; Uhlén, Mathias; Pontén, Fredrik

2015-01-01

To better understand prostate function and disease, it is important to define and explore the molecular constituents that signify the prostate gland. The aim of this study was to define the prostate specific transcriptome and proteome, in comparison to 26 other human tissues. Deep sequencing of mRNA (RNA-seq) and immunohistochemistry-based protein profiling were combined to identify prostate specific gene expression patterns and to explore tissue biomarkers for potential clinical use in prostate cancer diagnostics. We identified 203 genes with elevated expression in the prostate, 22 of which showed more than five-fold higher expression levels compared to all other tissue types. In addition to previously well-known proteins we identified two poorly characterized proteins, TMEM79 and ACOXL, with potential to differentiate between benign and cancerous prostatic glands in tissue biopsies. In conclusion, we have applied a genome-wide analysis to identify the prostate specific proteome using transcriptomics and antibody-based protein profiling to identify genes with elevated expression in the prostate. Our data provides a starting point for further functional studies to explore the molecular repertoire of normal and diseased prostate including potential prostate cancer markers such as TMEM79 and ACOXL. PMID:26237329

Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

PubMed

Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

2015-05-01

Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

Skin transcriptome profiles associated with coat color in sheep

PubMed Central

2013-01-01

Background Previous molecular genetic studies of physiology and pigmentation of sheep skin have focused primarily on a limited number of genes and proteins. To identify additional genes that may play important roles in coat color regulation, Illumina sequencing technology was used to catalog global gene expression profiles in skin of sheep with white versus black coat color. Results There were 90,006 and 74,533 unigenes assembled from the reads obtained from white and black sheep skin, respectively. Genes encoding for the ribosomal proteins and keratin associated proteins were most highly expressed. A total of 2,235 known genes were differentially expressed in black versus white sheep skin, with 479 genes up-regulated and 1,756 genes down-regulated. A total of 845 novel genes were differentially expressed in black versus white sheep skin, consisting of 107 genes which were up-regulated (including 2 highly expressed genes exclusively expressed in black sheep skin) and 738 genes that were down-regulated. There was also a total of 49 known coat color genes expressed in sheep skin, from which 13 genes showed higher expression in black sheep skin. Many of these up-regulated genes, such as DCT, MATP, TYR and TYRP1, are members of the components of melanosomes and their precursor ontology category. Conclusion The white and black sheep skin transcriptome profiles obtained provide a valuable resource for future research to understand the network of gene expression controlling skin physiology and melanogenesis in sheep. PMID:23758853

Neuronal proteins are novel components of podocyte major processes and their expression in glomerular crescents supports their role in crescent formation.

PubMed

Sistani, Laleh; Rodriguez, Patricia Q; Hultenby, Kjell; Uhlen, Mathias; Betsholtz, Christer; Jalanko, Hannu; Tryggvason, Karl; Wernerson, Annika; Patrakka, Jaakko

2013-01-01

The podocyte has a central role in the glomerular filtration barrier typified by a sophisticated morphology of highly organized primary (major) and secondary (foot) processes. The molecular makeup of foot processes is well characterized, but that of major processes is poorly known. Previously, we profiled the glomerular transcriptome through large-scale sequencing and microarray profiling. Unexpectedly, the survey found expression of three neuronal proteins (Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a)), all enriched in the glomerulus. These proteins were expressed exclusively by podocytes, wherein they localized to major processes as verified by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. During podocyte development, these proteins colocalized with vimentin, confirming their association with major processes. Using immunohistochemistry, we found coexpression of Hip1 and Olfml2a along with the recognized podocyte markers synaptopodin and Pdlim2 in glomerular crescents of human kidneys, indicating the presence of podocytes in these lesions. Thus, three neuronal proteins are highly expressed in podocyte major process. Using these new markers we found that podocytes contribute to the formation of glomerular crescents.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

PubMed Central

2009-01-01

Background Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion. PMID:20034395

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

PubMed

Baraúna, Rafael A; Santos, Agenor V; Graças, Diego A; Santos, Daniel M; Ghilardi, Rubens; Pimenta, Adriano M C; Carepo, Marta S P; Schneider, Maria P C; Silva, Artur

2015-05-01

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

Advanced colorectal adenoma related gene expression signature may predict prognostic for colorectal cancer patients with adenoma-carcinoma sequence.

PubMed

Li, Bing; Shi, Xiao-Yu; Liao, Dai-Xiang; Cao, Bang-Rong; Luo, Cheng-Hua; Cheng, Shu-Jun

2015-01-01

There are still no absolute parameters predicting progression of adenoma into cancer. The present study aimed to characterize functional differences on the multistep carcinogenetic process from the adenoma-carcinoma sequence. All samples were collected and mRNA expression profiling was performed by using Agilent Microarray high-throughput gene-chip technology. Then, the characteristics of mRNA expression profiles of adenoma-carcinoma sequence were described with bioinformatics software, and we analyzed the relationship between gene expression profiles of adenoma-adenocarcinoma sequence and clinical prognosis of colorectal cancer. The mRNA expressions of adenoma-carcinoma sequence were significantly different between high-grade intraepithelial neoplasia group and adenocarcinoma group. The biological process of gene ontology function enrichment analysis on differentially expressed genes between high-grade intraepithelial neoplasia group and adenocarcinoma group showed that genes enriched in the extracellular structure organization, skeletal system development, biological adhesion and itself regulated growth regulation, with the P value after FDR correction of less than 0.05. In addition, IPR-related protein mainly focused on the insulin-like growth factor binding proteins. The variable trends of gene expression profiles for adenoma-carcinoma sequence were mainly concentrated in high-grade intraepithelial neoplasia and adenocarcinoma. The differentially expressed genes are significantly correlated between high-grade intraepithelial neoplasia group and adenocarcinoma group. Bioinformatics analysis is an effective way to study the gene expression profiles in the adenoma-carcinoma sequence, and may provide an effective tool to involve colorectal cancer research strategy into colorectal adenoma or advanced adenoma.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

PubMed

de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

2017-08-10

Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open reading frames were predicted for 7947 transcripts. This class represented 17% of the differentially expressed transcripts, suggesting a potential transcriptional regulatory function of long non-coding RNA in tick blood-feeding. The assembled sialotranscriptome greatly expands the sequence availability of R. zambeziensis, assists in our understanding of the transcription of secretory proteins during blood-feeding and will be a valuable resource for future vaccine candidate selection.

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data.

PubMed

Raju, Hemalatha B; Tsinoremas, Nicholas F; Capobianco, Enrico

2016-01-01

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein-protein interaction networks, other connectivity paths were identified between proteins already investigated in studies on disorders, such as Parkinson, Down syndrome, Huntington disease, and Alzheimer. Our findings suggest the importance of reusing gene expression data by meta-analysis approaches.

Analyzing gene expression time-courses based on multi-resolution shape mixture model.

PubMed

Li, Ying; He, Ye; Zhang, Yu

2016-11-01

Biological processes actually are a dynamic molecular process over time. Time course gene expression experiments provide opportunities to explore patterns of gene expression change over a time and understand the dynamic behavior of gene expression, which is crucial for study on development and progression of biology and disease. Analysis of the gene expression time-course profiles has not been fully exploited so far. It is still a challenge problem. We propose a novel shape-based mixture model clustering method for gene expression time-course profiles to explore the significant gene groups. Based on multi-resolution fractal features and mixture clustering model, we proposed a multi-resolution shape mixture model algorithm. Multi-resolution fractal features is computed by wavelet decomposition, which explore patterns of change over time of gene expression at different resolution. Our proposed multi-resolution shape mixture model algorithm is a probabilistic framework which offers a more natural and robust way of clustering time-course gene expression. We assessed the performance of our proposed algorithm using yeast time-course gene expression profiles compared with several popular clustering methods for gene expression profiles. The grouped genes identified by different methods are evaluated by enrichment analysis of biological pathways and known protein-protein interactions from experiment evidence. The grouped genes identified by our proposed algorithm have more strong biological significance. A novel multi-resolution shape mixture model algorithm based on multi-resolution fractal features is proposed. Our proposed model provides a novel horizons and an alternative tool for visualization and analysis of time-course gene expression profiles. The R and Matlab program is available upon the request. Copyright © 2016 Elsevier Inc. All rights reserved.

Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging*

PubMed Central

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers. PMID:24217021

Label-free quantitative protein profiling of vastus lateralis muscle during human aging.

PubMed

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.

Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

PubMed

Parmar, Mehtab S; Mishra, Smruti Ranjan; Somal, Anjali; Pandey, Sriti; Kumar, G Sai; Sarkar, Mihir; Chandra, Vikash; Sharma, G Taru

2017-05-01

The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs. Copyright © 2017 Elsevier B.V. All rights reserved.

Nano-LC-ESI MS/MS analysis of proteins in dried sea dragon Solenognathus hardwickii and bioinformatic analysis of its protein expression profiling.

PubMed

Zhang, Dong-Mei; Feng, Li-Xing; Li, Lu; Liu, Miao; Jiang, Bao-Hong; Yang, Min; Li, Guo-Qiang; Wu, Wan-Ying; Guo, De-An; Liu, Xuan

2016-09-01

The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Expression profiling analysis: Uncoupling protein 2 deficiency improves hepatic glucose, lipid profiles and insulin sensitivity in high-fat diet-fed mice by modulating expression of genes in peroxisome proliferator-activated receptor signaling pathway.

PubMed

Zhou, Mei-Cen; Yu, Ping; Sun, Qi; Li, Yu-Xiu

2016-03-01

Uncoupling protein 2 (UCP2), which was an important mitochondrial inner membrane protein associated with glucose and lipid metabolism, widely expresses in all kinds of tissues including hepatocytes. The present study aimed to explore the impact of UCP2 deficiency on glucose and lipid metabolism, insulin sensitivity and its effect on the liver-associated signaling pathway by expression profiling analysis. Four-week-old male UCP2-/- mice and UCP2+/+ mice were randomly assigned to four groups: UCP2-/- on a high-fat diet, UCP2-/- on a normal chow diet, UCP2+/+ on a high-fat diet and UCP2+/+ on a normal chow diet. The differentially expressed genes in the four groups on the 16th week were identified by Affymetrix gene array. The results of intraperitoneal glucose tolerance test and insulin tolerance showed that blood glucose and β-cell function were improved in the UCP2-/- group on high-fat diet. Enhanced insulin sensitivity was observed in the UCP2-/- group. The differentially expressed genes were mapped to 23 pathways (P < 0.05). We concentrated on the 'peroxisome proliferator-activated receptor (PPAR) signaling pathway' (P = 3.19 × 10(-11)), because it is closely associated with the regulation of glucose and lipid profiles. In the PPAR signaling pathway, seven genes (PPARγ, Dbi, Acsl3, Lpl, Me1, Scd1, Fads2) in the UCP2-/- mice were significantly upregulated. The present study used gene arrays to show that activity of the PPAR signaling pathway involved in the improvement of glucose and lipid metabolism in the liver of UCP2-deficient mice on a long-term high-fat diet. The upregulation of genes in the PPAR signaling pathway could explain our finding that UCP2 deficiency ameliorated insulin sensitivity. The manipulation of UCP2 protein expression could represent a new strategy for the prevention and treatment of diabetes.

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

PubMed Central

Schäpe, Paul; Müller-Hagen, Dirk; Ouedraogo, Jean-Paul; Heiderich, Caroline; Jedamzick, Johanna; van den Hondel, Cees A.; Ram, Arthur F.; Meyer, Vera

2016-01-01

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes. PMID:27835655

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger.

PubMed

Paege, Norman; Jung, Sascha; Schäpe, Paul; Müller-Hagen, Dirk; Ouedraogo, Jean-Paul; Heiderich, Caroline; Jedamzick, Johanna; Nitsche, Benjamin M; van den Hondel, Cees A; Ram, Arthur F; Meyer, Vera

2016-01-01

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes.

Application of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry technology for the diagnosis of colorectal adenoma.

PubMed

Zhou, Zhong-Yin; Tao, DI-DI; Cao, Ji-Wang; Luo, He-Sheng

2013-06-01

The aim of the present study was to identify a specific biological marker for the diagnosis of colorectal adenomas through the analysis of variations in serum protein profiling in colorectal adenoma patients. The study was conducted at the Renmin Hospital of Wuhan University (Wuhan, China) between September 2011 and May 2012. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was performed to compare the serum protein profiles of 50 patients with colorectal adenoma and 50 healthy individuals. The obtained protein profiles were analyzed using Biomarker Wizard software. Twenty protein peaks were identified to exhibit differences in average intensity between colorectal adenomas compared with normal controls, including peaks 8,565.84, 8,694.51 and 5,910.50 Da, in which the intensity between the patients and control individuals was significantly different. Two peaks, 8,565.84 and 8,694.51 Da, were observed to be highly expressed in the colorectal adenomas, however, expression was low in the control samples. By contrast, 5,910.50 Da expression was low in the colorectal adenomas and high in the controls. The results of the current study indicate that the three protein peaks may represent specific biomarkers for colorectal adenomas.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula.

PubMed

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling; Wang, Yun

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components.

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula

PubMed Central

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components. PMID:29692857

Nasal protein profiles in work-related asthma caused by different exposures.

PubMed

Suojalehto, H; Lindström, I; Wolff, H; Puustinen, A

2018-03-01

The mechanisms of work-related asthma (WRA) are incompletely delineated. Nasal cell samples may be informative about processes in the lower airways. Our aim was to determine the nasal protein expression profiles of WRA caused by different kind of exposures. We collected nasal brush samples from 82 nonsmoking participants, including healthy controls and WRA patients exposed to (i) protein allergens, (ii) isocyanates and (iii) welding fumes the day after relevant exposure. The proteome changes in samples were analysed by two-dimensional difference gel electrophoresis, and the differentially regulated proteins found were identified by mass spectrometry. Immunological comparison was carried out using Western blot. We detected an average of 2500 spots per protein gel. Altogether, 228 protein spots were chosen for identification, yielding 77 different proteins. Compared to the controls, exposure to protein allergens had the largest effects on the proteome. Hierarchical clustering revealed that protein allergen- and isocyanate-related asthma had similar profiles, whereas asthma related to welding fumes differed. The highly overrepresented functional categories in the asthma groups were defence response, protease inhibitor activity, inflammatory and calcium signalling, complement activation and cellular response to oxidative stress. Immunological analysis confirmed the found abundance differences in galectin 10 and protein S100-A9 between the groups. Work-related asthma patients exposed to protein allergens and isocyanates elicit similar nasal proteome responses and the profiles of welders and healthy controls were alike. Revealed biological activities of the protein expression changes are associated with allergic inflammation and asthma. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Differences in shotgun protein expression profile between superficial bladder transitional cell carcinoma and normal urothelium.

PubMed

Niu, Hai Tao; Zhang, Yi Bing; Jiang, Hai Ping; Cheng, Bo; Sun, Guang; Wang, Yi; E, Ya Jun; Pang, De Quan; Chang, Ji Wu

2009-01-01

This study was undertaken to identify differences in protein expression profiles between superficial bladder transitional cell carcinoma (BTCC) and normal urothelial cells. We used laser capture microdissection (LCM) to harvest purified cells, and used two-dimensional liquid chromatography (2D-LC) followed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to separate and identify the peptide mixture. A total of 440/438 proteins commonly appeared in 4 paired specimens. Multi-step bioinformatic procedures were used for the analysis of identified proteins; 175/179 of the 293/287 proteins that were specific expressed in tumor/normal cells own gene ontology (GO) biological process annotation. Compared with the entire list of the international protein index (IPI), there are 52/46 GO terms exhibited as enriched and 6/10 exhibited as depleted, respectively. Significantly altered pathways between tumor and normal cells mainly include oxidative phosphorylation, focal adhesion, etc. Finally, descriptive statistics show that the shotgun proteomics strategy has practice directive significance for biomarker discovery by two-dimensional electrophoresis (2-DE) technology.

Apoptosis induced by low-intensity ultrasound in vitro: Alteration of protein profile and potential molecular mechanism

NASA Astrophysics Data System (ADS)

Feng, Yi; Wan, Mingxi

2017-03-01

To analyze the potential mechanism related to the apoptosis induced by low intensity focused ultrasound, comparative proteomic method was introduced in the study. After ultrasound irradiation (3.0 W/cm2, 1 minute, 6 hours incubation post-irradiation), the human SMMC-7721 hepatocarcinoma cells were stained by trypan blue to detect the morphologic changes, and then the percentage of early apoptosis were tested by the flow cytometry with double staining of FITC-labelled Annexin V/Propidium iodide. Two-dimensional SDS polyacrylamide gel electrophoresis was used to get the protein profile and some proteins differently expressed after ultrasound irradiation were identified by MALDI-TOF mass spectrometry. It's proved early apoptosis of cells were induced by low intentisy focused ultrasound. After ultrasound irradiation, the expressing characteristics of several proteins changed, in which protein p53 and heat shock proteins are associated with apoptosis initiation. It is suggested that the low-intensity ultrasound-induced apoptotic cancer therapy has the potential application via understanding its relevant molecular signaling and key proteins. Moreover, the comparative proteomic method is proved to be useful to supply information about the protein expression to analyze the metabolic processes related to bio-effects of biomedical ultrasound.

Mono-allelic expression of variegating transgene locus in the mouse.

PubMed

Opsahl, Margaret L; Springbett, Anthea; Lathe, Richard; Colman, Alan; McClenaghan, Margaret; Whitelaw, C Bruce A

2003-12-01

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Global PROTOMAP profiling to search for biomarkers of early-recurrent hepatocellular carcinoma.

PubMed

Taoka, Masato; Morofuji, Noriaki; Yamauchi, Yoshio; Ojima, Hidenori; Kubota, Daisuke; Terukina, Goro; Nobe, Yuko; Nakayama, Hiroshi; Takahashi, Nobuhiro; Kosuge, Tomoo; Isobe, Toshiaki; Kondo, Tadashi

2014-11-07

This study used global protein expression profiling to search for biomarkers to predict early recurrent hepatocellular carcinoma (HCC). HCC tissues surgically resected from patients with or without recurrence within 2 years (early recurrent) after surgery were compared with adjacent nontumor tissue and with normal liver tissue. We used the PROTOMAP strategy for comparative profiling, which integrates denaturing polyacrylamide gel electrophoresis migratory rates and high-resolution, semiquantitative mass-spectrometry-based identification of in-gel-digested tryptic peptides. PROTOMAP allows examination of global changes in the size, topography, and abundance of proteins in complex tissue samples. This approach identified 8438 unique proteins from 45 708 nonredundant peptides and generated a proteome-wide map of changes in expression and proteolytic events potentially induced by intrinsic apoptotic/necrotic pathways. In the early recurrent HCC tissue, 87 proteins were differentially expressed (≥20-fold) relative to the other tissues, 46 of which were up-regulated or specifically proteolyzed and 41 of which were down-regulated. This data set consisted of proteins that fell into various functional categories, including signal transduction and cell organization and, notably, the major catalytic pathways responsible for liver function, such as the urea cycle and detoxification metabolism. We found that aberrant proteolysis appeared to occur frequently during recurrence of HCC in several key signal transducers, including STAT1 and δ-catenin. Further investigation of these proteins will facilitate the development of novel clinical applications.

A focused microarray approach to functional glycomics: transcriptional regulation of the glycome.

PubMed

Comelli, Elena M; Head, Steven R; Gilmartin, Tim; Whisenant, Thomas; Haslam, Stuart M; North, Simon J; Wong, Nyet-Kui; Kudo, Takashi; Narimatsu, Hisashi; Esko, Jeffrey D; Drickamer, Kurt; Dell, Anne; Paulson, James C

2006-02-01

Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes, we employed the Affymetrix technology to develop a focused and highly annotated glycogene-chip representing human and murine glycogenes, including glycosyltransferases, nucleotide sugar transporters, glycosidases, proteoglycans, and glycan-binding proteins. In this report, the array has been used to generate glycogene-expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm reveals that expression profiles in immune tissues (thymus [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell-type-specific glycosylation. Comparison of gene expression profiles with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and Siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org).

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

PubMed

Proszkowiec-Weglarz, M; Richards, M P

2009-01-01

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

PubMed

Cui, Hao-Ran; Zhang, Zheng-Rong; Lv, Wei; Xu, Jia-Ning; Wang, Xiao-Yun

2015-08-01

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

An emerging cyberinfrastructure for biodefense pathogen and pathogen-host data.

PubMed

Zhang, C; Crasta, O; Cammer, S; Will, R; Kenyon, R; Sullivan, D; Yu, Q; Sun, W; Jha, R; Liu, D; Xue, T; Zhang, Y; Moore, M; McGarvey, P; Huang, H; Chen, Y; Zhang, J; Mazumder, R; Wu, C; Sobral, B

2008-01-01

The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host-pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at http://www.proteomicsresource.org/.

Characterization and expression analysis of two cDNAs encoding Xa1 and oxysterol binding proteins in sorghum (Sorghum bicolor)

USDA-ARS?s Scientific Manuscript database

Using suppression subtractive hybridization (SSH) and subsequent microarray analysis, expression profiles of sorghum genes responsive to greenbug phloem-feeding were obtained and identified. Among the profiles, two cDNAs designated to MM73 and MM95 were identified to encode Xa1 (Xa1) and oxysterol ...

Acute myeloid leukaemia: expression of MYC protein and its association with cytogenetic risk profile and overall survival.

PubMed

Mughal, Muhammad Kashif; Akhter, Ariz; Street, Lesley; Pournazari, Payam; Shabani-Rad, Meer-Taher; Mansoor, Adnan

2017-09-01

Acute myeloid leukaemia (AML) is a clinically aggressive disease with marked genetic heterogeneity. Cytogenetic abnormalities provide the basis for risk stratification into clinically favourable, intermediate, and unfavourable groups. There are additional genetic mutations, which further influence the prognosis of patients with AML. Most of these result in molecular aberrations whose downstream target is MYC. It is therefore logical to study the relationship between MYC protein expression and cytogenetic risk groups. We studied MYC expression by immunohistochemistry in a large cohort (n = 199) of AML patients and correlated these results with cytogenetic risk profile and overall survival (OS). We illustrated differential expression of MYC protein across various cytogenetic risk groups (p = 0.03). Highest expression of MYC was noted in AML patients with favourable cytogenetic risk group. In univariate analysis, MYC expression showed significant negative influence of OS in favourable and intermediate cytogenetic risk group (p = 0.001). Interestingly, MYC expression had a protective effect in the unfavourable cytogenetic risk group. In multivariate analysis, while age and cytogenetic risk group were significant factors influencing survival, MYC expression by immunohistochemistry methods also showed some marginal impact (p = 0.069). In conclusion, we have identified differential expression of MYC protein in relation to cytogenetic risk groups in AML patients and documented its possible impact on OS in favourable and intermediate cytogenetic risk groups. These preliminary observations mandate additional studies to further investigate the routine clinical use of MYC protein expression in AML risk stratification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma

PubMed Central

Smith, Ashlee L.; Sun, Mai; Bhargava, Rohit; Stewart, Nicolas A.; Flint, Melanie S.; Bigbee, William L.; Krivak, Thomas C.; Strange, Mary A.; Cooper, Kristine L.; Zorn, Kristin K.

2013-01-01

Objective: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. Results: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. Conclusion: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute. PMID:28250404

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing; Ma, Zihao; Carr, Steven A.

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC).more » Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.060301, 121–134, 2017.« less

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472

PubMed Central

Baraúna, Rafael A.; Santos, Agenor V.; Graças, Diego A.; Santos, Daniel M.; Ghilardi, Rubens; Pimenta, Adriano M. C.; Carepo, Marta S. P.; Schneider, Maria P.C.; Silva, Artur

2015-01-01

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field. PMID:26273227

Comprehensive RNA-Seq Expression Analysis of Sensory Ganglia with a Focus on Ion Channels and GPCRs in Trigeminal Ganglia

PubMed Central

Manteniotis, Stavros; Lehmann, Ramona; Flegel, Caroline; Vogel, Felix; Hofreuter, Adrian; Schreiner, Benjamin S. P.; Altmüller, Janine; Becker, Christian; Schöbel, Nicole; Hatt, Hanns; Gisselmann, Günter

2013-01-01

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain. PMID:24260241

Protein Expression Profiles of Permissive, Semi-Permissive and Non-Permissive Cells Infected by Baculovirus

USDA-ARS?s Scientific Manuscript database

Amassing information on the in vitro protein expression of an insect host challenged by an entomopathogenic agent, such as a baculovirus, is paramount to an enhanced understanding of how host-pathogen interactions determine the success or failure of a pathogen. In this study, 2D-gel electrophoresis...

Hessian fly larval attack triggers elevated expression of disease resistance dirigent-like protein-encoding gene, HfrDrd, in resistant wheat.

USDA-ARS?s Scientific Manuscript database

Dirigent proteins regulate coupling of monolignol plant phenols to generate the structural cell wall polymers lignins and lignans that are involved in structural fortification of cell wall and defense against pathogens and pests. Microarray expression profiling of resistant wheat (Triticum aestivum)...

ABC gene expression profiles have clinical importance and possibly form a new hallmark of cancer.

PubMed

Dvorak, Pavel; Pesta, Martin; Soucek, Pavel

2017-05-01

Adenosine triphosphate-binding cassette proteins constitute a large family of active transporters through extracellular and intracellular membranes. Increased drug efflux based on adenosine triphosphate-binding cassette protein activity is related to the development of cancer cell chemoresistance. Several articles have focused on adenosine triphosphate-binding cassette gene expression profiles (signatures), based on the expression of all 49 human adenosine triphosphate-binding cassette genes, in individual tumor types and reported connections to established clinicopathological features. The aim of this study was to test our theory about the existence of adenosine triphosphate-binding cassette gene expression profiles common to multiple types of tumors, which may modify tumor progression and provide clinically relevant information. Such general adenosine triphosphate-binding cassette profiles could constitute a new attribute of carcinogenesis. Our combined cohort consisted of tissues from 151 cancer patients-breast, colorectal, and pancreatic carcinomas. Standard protocols for RNA isolation and quantitative real-time polymerase chain reaction were followed. Gene expression data from individual tumor types as well as a merged tumor dataset were analyzed by bioinformatics tools. Several general adenosine triphosphate-binding cassette profiles, with differences in gene functions, were established and shown to have significant relations to clinicopathological features such as tumor size, histological grade, or clinical stage. Genes ABCC7, A3, A8, A12, and C8 prevailed among the most upregulated or downregulated ones. In conclusion, the results supported our theory about general adenosine triphosphate-binding cassette gene expression profiles and their importance for cancer on clinical as well as research levels. The presence of ABCC7 (official symbol CFTR) among the genes with key roles in the profiles supports the emerging evidence about its crucial role in various cancers. Graphical abstract.

Protein-protein interaction network of gene expression in the hydrocortisone-treated keloid.

PubMed

Chen, Rui; Zhang, Zhiliang; Xue, Zhujia; Wang, Lin; Fu, Mingang; Lu, Yi; Bai, Ling; Zhang, Ping; Fan, Zhihong

2015-01-01

In order to explore the molecular mechanism of hydrocortisone in keloid tissue, the gene expression profiles of keloid samples treated with hydrocortisone were subjected to bioinformatics analysis. Firstly, the gene expression profiles (GSE7890) of five samples of keloid treated with hydrocortisone and five untreated keloid samples were downloaded from the Gene Expression Omnibus (GEO) database. Secondly, data were preprocessed using packages in R language and differentially expressed genes (DEGs) were screened using a significance analysis of microarrays (SAM) protocol. Thirdly, the DEGs were subjected to gene ontology (GO) function and KEGG pathway enrichment analysis. Finally, the interactions of DEGs in samples of keloid treated with hydrocortisone were explored in a human protein-protein interaction (PPI) network, and sub-modules of the DEGs interaction network were analyzed using Cytoscape software. Based on the analysis, 572 DEGs in the hydrocortisone-treated samples were screened; most of these were involved in the signal transduction and cell cycle. Furthermore, three critical genes in the module, including COL1A1, NID1, and PRELP, were screened in the PPI network analysis. These findings enhance understanding of the pathogenesis of the keloid and provide references for keloid therapy. © 2015 The International Society of Dermatology.

Differential expression profile of membrane proteins in L-02 cells exposed to trichloroethylene.

PubMed

Hong, Wen-Xu; Huang, Aibo; Lin, Sheng; Yang, Xifei; Yang, Linqing; Zhou, Li; Huang, Haiyan; Wu, Desheng; Huang, Xinfeng; Xu, Hua; Liu, Jianjun

2016-10-01

Trichloroethylene (TCE), a halogenated organic solvent widely used in industries, is known to cause severe hepatotoxicity. However, the mechanisms underlying TCE hepatotoxicity are still not well understood. It is predicted that membrane proteins are responsible for key biological functions, and recent studies have revealed that TCE exposure can induce abnormal levels of membrane proteins in body fluids and cultured cells. The aim of this study is to investigate the TCE-induced alterations of membrane proteins profiles in human hepatic L-02 liver cells. A comparative membrane proteomics analysis was performed in combination with two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 15 proteins were identified as differentially expressed (4 upregulated and 11 downregulated) between TCE-treated cells and normal controls. Among this, 14 of them are suggested as membrane-associated proteins by their transmembrane domain and/or subcellular location. Furthermore, the differential expression of β subunit of adenosine triphosphate synthase (ATP5B) and prolyl 4-hydroxylase, β polypeptide (P4HB) were verified by Western blot analysis in TCE-treated L-02 cells. Our work not only reveals the association between TCE exposure and altered expression of membrane proteins but also provides a novel strategy to discover membrane biomarkers and elucidate the potential mechanisms involving with membrane proteins response to chemical-induced toxic effect. © The Author(s) 2015.

Overexpression of the S100A2 protein as a prognostic marker for patients with stage II and III colorectal cancer

PubMed Central

MASUDA, TAIKI; ISHIKAWA, TOSHIAKI; MOGUSHI, KAORU; OKAZAKI, SATOSHI; ISHIGURO, MEGUMI; IIDA, SATORU; MIZUSHIMA, HIROSHI; TANAKA, HIROSHI; UETAKE, HIROYUKI; SUGIHARA, KENICHI

2016-01-01

We aimed to identify a novel prognostic biomarker related to recurrence in stage II and III colorectal cancer (CRC) patients. Stage II and III CRC tissue mRNA expression was profiled using an Affymetrix Gene Chip, and copy number profiles of 125 patients were generated using an Affymetrix 250K Sty array. Genes showing both upregulated expression and copy number gains in cases involving recurrence were extracted as candidate biomarkers. The protein expression of the candidate gene was assessed using immunohistochemical staining of tissue from 161 patients. The relationship between protein expression and clinicopathological features was also examined. We identified 9 candidate genes related to recurrence of stage II and III CRC, whose mRNA expression was significantly higher in CRC than in normal tissue. Of these proteins, the S100 calcium-binding protein A2 (S100A2) has been observed in several human cancers. S100A2 protein overexpression in CRC cells was associated with significantly worse overall survival and relapse-free survival, indicating that S100A2 is an independent risk factor for stage II and III CRC recurrence. S100A2 overexpression in cancer cells could be a biomarker of poor prognosis in stage II and III CRC recurrence and a target for treatment of this disease. PMID:26783118

Identification of GRB2 and GAB1 Coexpression as an Unfavorable Prognostic Factor for Hepatocellular Carcinoma by a Combination of Expression Profile and Network Analysis

PubMed Central

Yang, Mei; Wang, Danhua; Yu, Lingxiang; Guo, Chaonan; Guo, Xiaodong; Lin, Na

2013-01-01

Aim To screen novel markers for hepatocellular carcinoma (HCC) by a combination of expression profile, interaction network analysis and clinical validation. Methods HCC significant molecules which are differentially expressed or had genetic variations in HCC tissues were obtained from five existing HCC related databases (OncoDB.HCC, HCC.net, dbHCCvar, EHCO and Liverome). Then, the protein-protein interaction (PPI) network of these molecules was constructed. Three topological features of the network ('Degree', 'Betweenness', and 'Closeness') and the k-core algorithm were used to screen candidate HCC markers which play crucial roles in tumorigenesis of HCC. Furthermore, the clinical significance of two candidate HCC markers growth factor receptor-bound 2 (GRB2) and GRB2-associated-binding protein 1 (GAB1) was validated. Results In total, 6179 HCC significant genes and 977 HCC significant proteins were collected from existing HCC related databases. After network analysis, 331 candidate HCC markers were identified. Especially, GAB1 has the highest k-coreness suggesting its central localization in HCC related network, and the interaction between GRB2 and GAB1 has the largest edge-betweenness implying it may be biologically important to the function of HCC related network. As the results of clinical validation, the expression levels of both GRB2 and GAB1 proteins were significantly higher in HCC tissues than those in their adjacent nonneoplastic tissues. More importantly, the combined GRB2 and GAB1 protein expression was significantly associated with aggressive tumor progression and poor prognosis in patients with HCC. Conclusion This study provided an integrative analysis by combining expression profile and interaction network analysis to identify a list of biologically significant HCC related markers and pathways. Further experimental validation indicated that the aberrant expression of GRB2 and GAB1 proteins may be strongly related to tumor progression and prognosis in patients with HCC. The overexpression of GRB2 in combination with upregulation of GAB1 may be an unfavorable prognostic factor for HCC. PMID:24391994

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data

PubMed Central

Raju, Hemalatha B.; Tsinoremas, Nicholas F.; Capobianco, Enrico

2016-01-01

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein–protein interaction networks, other connectivity paths were identified between proteins already investigated in studies on disorders, such as Parkinson, Down syndrome, Huntington disease, and Alzheimer. Our findings suggest the importance of reusing gene expression data by meta-analysis approaches. PMID:27803687

Computational Prediction and Validation of BAHD1 as a Novel Molecule for Ulcerative Colitis

NASA Astrophysics Data System (ADS)

Zhu, Huatuo; Wan, Xingyong; Li, Jing; Han, Lu; Bo, Xiaochen; Chen, Wenguo; Lu, Chao; Shen, Zhe; Xu, Chenfu; Chen, Lihua; Yu, Chaohui; Xu, Guoqiang

2015-07-01

Ulcerative colitis (UC) is a common inflammatory bowel disease (IBD) producing intestinal inflammation and tissue damage. The precise aetiology of UC remains unknown. In this study, we applied a rank-based expression profile comparative algorithm, gene set enrichment analysis (GSEA), to evaluate the expression profiles of UC patients and small interfering RNA (siRNA)-perturbed cells to predict proteins that might be essential in UC from publicly available expression profiles. We used quantitative PCR (qPCR) to characterize the expression levels of those genes predicted to be the most important for UC in dextran sodium sulphate (DSS)-induced colitic mice. We found that bromo-adjacent homology domain (BAHD1), a novel heterochromatinization factor in vertebrates, was the most downregulated gene. We further validated a potential role of BAHD1 as a regulatory factor for inflammation through the TNF signalling pathway in vitro. Our findings indicate that computational approaches leveraging public gene expression data can be used to infer potential genes or proteins for diseases, and BAHD1 might act as an indispensable factor in regulating the cellular inflammatory response in UC.

Functional discovery via a compendium of expression profiles.

PubMed

Hughes, T R; Marton, M J; Jones, A R; Roberts, C J; Stoughton, R; Armour, C D; Bennett, H A; Coffey, E; Dai, H; He, Y D; Kidd, M J; King, A M; Meyer, M R; Slade, D; Lum, P Y; Stepaniants, S B; Shoemaker, D D; Gachotte, D; Chakraburtty, K; Simon, J; Bard, M; Friend, S H

2000-07-07

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

PubMed

Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

2016-02-18

Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease in early xylem zones. We observed upregulation of two forms of xylem cysteine protease (Potri.002G005700.1 and Potri.005G256000.2; Pt-XCP2.1) in early xylem and their downregulation in late maturing xylem. Our data also show that Pt-KOR1.3 (Potri.003G151700.2) exhibits an expression pattern that supports the hypothesis put forward in previous studies that this is a key xyloglucanase involved in cellulose biosynthesis in primary cell walls and reduction of cellulose crystallinity in secondary walls. Our novel multivariate approach highlights important processes and provides confirmatory insights into the molecular foundations of wood development.

Protein expression profiling in head fragments during planarian regeneration after amputation.

PubMed

Chen, Xiaoguang; Xu, Cunshuan

2015-04-01

Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various cell types. Taken together, our study demonstrated that proteomic analysis approach used by us is a powerful tool in understanding molecular process related to head regeneration of planarian.

Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pena-Castillo, Lourdes; Mercer, Ryan; Gurinovich, Anastasia

2014-08-28

The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigatedmore » preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. We identified R. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in R. sphaeroides whereas modules related to RcGTA production and signalling showed lack of preservation in R. sphaeroides. In addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mRNA expression and protein abundance data for which simple correlation measurements have previously had mixed results.« less

Changes of protein expression profiles in the amygdala during the process of morphine-induced conditioned place preference in rats.

PubMed

Lin, XiaoJing; Wang, QingSong; Cheng, Yong; Ji, JianGuo; Yu, Long-Chuan

2011-08-01

Repeated exposures to addictive drugs result in persistent or even permanent expression changes of proteins in addiction-related brain regions, such as nucleus accumbens, hippocampus and prefrontal cortex while the changes of protein content in amygdala were seldom studied. Here we aimed to find the proteins involved in the process of morphine-induced conditioned place preference (CPP). The model of morphine-induced CPP was established in rats and the rat amygdala tissues were obtained in different stages of morphine-induced CPP: establishment group, extinction group, reinstatement group and saline group as a control. Two-dimensional electrophoresis (2-DE) was performed to analyze and compare the changes of protein expression profiles in the amygdala of rats during the process of morphine-induced CPP. There were eighty proteins with 1.3-fold changes in amygdala relative to saline group, most of which were down-regulated. These differentially expressed proteins were mainly involved in metabolism, structure, cell signaling pathway and ubiquitin-proteasome pathway. And we further used methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting to confirm the results of proteomics. Mitosis activated protein kinase1 (MAPK1) was increased in the stages of extinction and reinstatement of morphine-induced CPP, while glial fibrillary acidic protein (GFAP) was decreased in the stage of extinction. Our results provide some proteins and cellular signaling pathways involved in the molecular mechanisms of opioid addiction in amygdala. Copyright © 2011 Elsevier B.V. All rights reserved.

Gene Expression Profiles of Chlamydophila pneumoniae during the Developmental Cycle and Iron Depletion–Mediated Persistence

PubMed Central

Mäurer, André P; Mehlitz, Adrian; Mollenkopf, Hans J; Meyer, Thomas F

2007-01-01

The obligate intracellular, gram-negative bacterium Chlamydophila pneumoniae (Cpn) has impact as a human pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence. The latter stage has been linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 h post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using self-organizing map clustering and manual sorting into the “early”, “mid”, “late”, and “tardy” cluster classes. The latter two were differentiated because the “tardy” class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between “late” genes and genes coding for EB proteins, whereas “tardy” genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion–mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion–mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Here, we describe the Cpn transcriptome during the acute infection, differentiating “late” genes, which correlate to EB proteins, and “tardy” genes, which lead to EB mRNA. Expression profiles during iron mediated–persistence led us to propose the hypothesis that the transcriptomic “clock” is arrested during acute mid-cycle. PMID:17590080

Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae.

PubMed

Buck, Teresa M; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L; Needham, Patrick G; Kleyman, Thomas R; Brodsky, Jeffrey L

2015-06-01

Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. Copyright © 2015 the American Physiological Society.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

PubMed

Amjadi, Fatemehsadat; Mehdizadeh, Mehdi; Ashrafi, Mahnaz; Nasrabadi, Davood; Taleahmad, Sara; Mirzaei, Mehdi; Gupta, Vivek; Salekdeh, Ghasem Hosseini; Aflatoonian, Reza

2018-04-21

What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Protein profiles associated with survival in lung adenocarcinoma

PubMed Central

Chen, Guoan; Gharib, Tarek G; Wang, Hong; Huang, Chiang-Ching; Kuick, Rork; Thomas, Dafydd G.; Shedden, Kerby A.; Misek, David E.; Taylor, Jeremy M. G.; Giordano, Thomas J.; Kardia, Sharon L. R.; Iannettoni, Mark D.; Yee, John; Hogg, Philip J.; Orringer, Mark B.; Hanash, Samir M.; Beer, David G.

2003-01-01

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer. PMID:14573703

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers

PubMed Central

Li, Yanjie; Lu, Yue; Lin, Kevin; Hauser, Lauren A.; Lynch, David R.

2017-01-01

ABSTRACT Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers. PMID:29125828

White adipose tissue genome wide-expression profiling and adipocyte metabolic functions after soy protein consumption in rats.

PubMed

Frigolet, Maria E; Torres, Nimbe; Uribe-Figueroa, Laura; Rangel, Claudia; Jimenez-Sanchez, Gerardo; Tovar, Armando R

2011-02-01

Obesity is associated with an increase in adipose tissue mass due to an imbalance between high dietary energy intake and low physical activity; however, the type of dietary protein may contribute to its development. The aim of the present work was to study the effect of soy protein versus casein on white adipose tissue genome profiling, and the metabolic functions of adipocytes in rats with diet-induced obesity. The results showed that rats fed a Soy Protein High-Fat (Soy HF) diet gained less weight and had lower serum leptin concentration than rats fed a Casein High-Fat (Cas HF) diet, despite similar energy intake. Histological studies indicated that rats fed the Soy HF diet had significantly smaller adipocytes than those fed the Cas HF diet, and this was associated with a lower triglyceride/DNA content. Fatty acid synthesis in isolated adipocytes was reduced by the amount of fat consumed but not by the type of protein ingested. Expression of genes of fatty acid oxidation increased in adipose tissue of rats fed Soy diets; microarray analysis revealed that Soy protein consumption modified the expression of 90 genes involved in metabolic functions and inflammatory response in adipose tissue. Network analysis showed that the expression of leptin was regulated by the type of dietary protein and it was identified as a central regulator of the expression of lipid metabolism genes in adipose tissue. Thus, soy maintains the size and metabolic functions of adipose tissue through biochemical adaptations, adipokine secretion, and global changes in gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Innate immune parameters and haemolymph protein expression profile to evaluate the immunotoxicity of tributyltin on abalone (Haliotis diversicolor supertexta).

PubMed

Zhou, Jin; Cai, Zhong-Hua; Zhu, Xiao-Shan; Li, Lei; Gao, Yun-Feng

2010-10-01

The immunotoxicity of tributyltin (TBT) on marine gastropods has been comparatively little studied although risks to wildlife associated with this compound are well known. In this study, a 30-day trial was conducted to evaluate the immunotoxic effects on abalone (Haliotis diversicolor supertexta) by exposing a range of doses of TBT (0, 2, 10, and 50 ng/L). Innate immune parameters, including phagocytic ability (PA), lysozyme activity, phenoloxidase (PO) level and superoxide dismutase (SOD) activity were monitored at intervals of 5, 15 and 30 days. Haemolymph protein expression profile was also examined at the end of the experiment. The results showed that PA value, lysozyme activity and PO level significantly decreased compared with the controls (P < 0.05), which indicated that TBT exposure markedly suppressed non-specific immune competence. Exposure to TBT also caused variation in protein expression patterns of haemolymph. Among the protein spots of differential expressions, seven proteins from the haemolymph of TBT-treated abalone were successfully identified by MALDI-TOF-MS analysis. Three protein spots increased and were identified as carrier-like peptide, peroxidase 21 precursor and creatine phosphokinase. These proteins are believed to up-regulate in expression as a response to detoxification and antioxidative stress mechanisms. The other four protein spots that down-regulated in TBT-treated groups were identified as aromatase-like protein, protein kinase C, ceruloplasmin and microtubule-actin crosslinking factor 1, and these proteins play an important role in endocrine regulation and immune defense. Taken together, the results demonstrate that TBT impair abalone immunological ability and is a potential immune disruptor. 2010 Elsevier Ltd. All rights reserved.

Proteomic Profiling of Tissue-Engineered Blood Vessel Walls Constructed by Adipose-Derived Stem Cells

PubMed Central

Wang, Chen; Guo, Fangfang; Zhou, Heng; Zhang, Yun; Xiao, Zhigang

2013-01-01

Adipose-derived stem cells (ASCs) can differentiate into smooth muscle cells and have been engineered into elastic small diameter blood vessel walls in vitro. However, the mechanisms involved in the development of three-dimensional (3D) vascular tissue remain poorly understood. The present study analyzed protein expression profiles of engineered blood vessel walls constructed by human ASCs using methods of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). These results were compared to normal arterial walls. A total of 1701±15 and 1265±26 protein spots from normal and engineered blood vessel wall extractions were detected by 2DE, respectively. A total of 20 spots with at least 2.0-fold changes in expression were identified, and 38 differently expressed proteins were identified by 2D electrophoresis and ion trap MS. These proteins were classified into seven functional categories: cellular organization, energy, signaling pathway, enzyme, anchored protein, cell apoptosis/defense, and others. These results demonstrated that 2DE, followed by ion trap MS, could be successfully utilized to characterize the proteome of vascular tissue, including tissue-engineered vessels. The method could also be employed to achieve a better understanding of differentiated smooth muscle protein expression in vitro. These results provide a basis for comparative studies of protein expression in vascular smooth muscles of different origin and could provide a better understanding of the mechanisms of action needed for constructing blood vessels that exhibit properties consistent with normal blood vessels. PMID:22963350

Proteomic profiling of tissue-engineered blood vessel walls constructed by adipose-derived stem cells.

PubMed

Wang, Chen; Guo, Fangfang; Zhou, Heng; Zhang, Yun; Xiao, Zhigang; Cui, Lei

2013-02-01

Adipose-derived stem cells (ASCs) can differentiate into smooth muscle cells and have been engineered into elastic small diameter blood vessel walls in vitro. However, the mechanisms involved in the development of three-dimensional (3D) vascular tissue remain poorly understood. The present study analyzed protein expression profiles of engineered blood vessel walls constructed by human ASCs using methods of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). These results were compared to normal arterial walls. A total of 1701±15 and 1265±26 protein spots from normal and engineered blood vessel wall extractions were detected by 2DE, respectively. A total of 20 spots with at least 2.0-fold changes in expression were identified, and 38 differently expressed proteins were identified by 2D electrophoresis and ion trap MS. These proteins were classified into seven functional categories: cellular organization, energy, signaling pathway, enzyme, anchored protein, cell apoptosis/defense, and others. These results demonstrated that 2DE, followed by ion trap MS, could be successfully utilized to characterize the proteome of vascular tissue, including tissue-engineered vessels. The method could also be employed to achieve a better understanding of differentiated smooth muscle protein expression in vitro. These results provide a basis for comparative studies of protein expression in vascular smooth muscles of different origin and could provide a better understanding of the mechanisms of action needed for constructing blood vessels that exhibit properties consistent with normal blood vessels.

Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

PubMed Central

Wang, Huaishan; Yang, Jia; Yang, Qi; Fu, Yi; Hu, Yu; Liu, Fang; Wang, Weiqing; Cui, Lianxian; Chen, Hui; Zhang, Jianmin; He, Wei

2016-01-01

Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel’s Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj’s findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis. PMID:27907096

Diet-Induced Over-Expression of Flightless-I Protein and Its Relation to Flightlessness in Mediterranean Fruit Fly, Ceratitis capitata

PubMed Central

Cho, Il Kyu; Chang, Chiou Ling; Li, Qing X.

2013-01-01

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation. PMID:24312525

Differential Protein Expression Profiles in Glaucomatous Trabecular Meshwork: An Evaluation Study on a Small Primary Open Angle Glaucoma Population.

PubMed

Micera, Alessandra; Quaranta, Luciano; Esposito, Graziana; Floriani, Irene; Pocobelli, Augusto; Saccà, Sergio Claudio; Riva, Ivano; Manni, Gianluca; Oddone, Francesco

2016-02-01

Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the "National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation".

Molecular evolution and expression profile of the chemerine encoding gene RARRES2 in baboon and chimpanzee.

PubMed

González-Alvarez, Rafael; Garza-Rodríguez, María de Lourdes; Delgado-Enciso, Iván; Treviño-Alvarado, Víctor Manuel; Canales-Del-Castillo, Ricardo; Martínez-De-Villarreal, Laura Elia; Lugo-Trampe, Ángel; Tejero, María Elizabeth; Schlabritz-Loutsevitch, Natalia E; Rocha-Pizaña, María Del Refugio; Cole, Shelley A; Reséndez-Pérez, Diana; Moises-Alvarez, Mario; Comuzzie, Anthony G; Barrera-Saldaña, Hugo Alberto; Garza-Guajardo, Raquel; Barboza-Quintana, Oralia; Rodríguez-Sánchez, Irám Pablo

2015-06-12

Chemerin, encoded by the retinoic acid receptor responder 2 (RARRES2) gene is an adipocytesecreted protein with autocrine/paracrine functions in adipose tissue, metabolism and inflammation with a recently described function in vascular tone regulation, liver, steatosis, etc. This molecule is believed to represent a critical endocrine signal linking obesity to diabetes. There are no data available regarding evolution of RARRES2 in non-human primates and great apes. Expression profile and orthology in RARRES2 genes are unknown aspects in the biology of this multigene family in primates. Thus; we attempt to describe expression profile and phylogenetic relationship as complementary knowledge in the function of this gene in primates. To do that, we performed A RT-PCR from different tissues obtained during necropsies. Also we tested the hypotheses of positive evolution, purifying selection, and neutrality. And finally a phylogenetic analysis was made between primates RARRES2 protein. RARRES2 transcripts were present in liver, lung, adipose tissue, ovary, pancreas, heart, hypothalamus and pituitary tissues. Expression in kidney and leukocytes were not detectable in either species. It was determined that the studied genes are orthologous. RARRES2 evolution fits the hypothesis of purifying selection. Expression profiles of the RARRES2 gene are similar in baboons and chimpanzees and are also phylogenetically related.

Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.

2008-02-01

WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 tomore » 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.« less

Identification of Potential Biomarkers for Rhegmatogenous Retinal Detachment Associated with Choroidal Detachment by Vitreous iTRAQ-Based Proteomic Profiling

PubMed Central

Wu, Zhifeng; Ding, Nannan; Yu, Mengxi; Wang, Ke; Luo, Shasha; Zou, Wenjun; Zhou, Ying; Yan, Biao; Jiang, Qin

2016-01-01

Rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) is a complicated and serious type of rhegmatogenous retinal detachment (RRD). In this study, we identified differentially expressed proteins in the vitreous humors of RRDCD and RRD using isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography-electrospray ion trap-mass spectrometry-mass spectrometry (nano-LC-ESI-MS/MS) and bioinformatic analysis. Our result shows that 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins were identified in RRDCD. Gene ontology (GO) analysis suggested that most of the differentially expressed proteins were extracellular．The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis suggested that proteins related to complement and coagulation cascades were significantly enriched. iTRAQ-based proteomic profiling reveals that complement and coagulation cascades and inflammation may play important roles in the pathogenesis of RRDCD. This study may provide novel insights into the pathogenesis of RRDCD and offer potential opportunities for the diagnosis and treatment of RRDCD. PMID:27941623

A transcriptome-based assessment of the astrocytic dystrophin-associated complex in the developing human brain.

PubMed

Simon, Matthew J; Murchison, Charles; Iliff, Jeffrey J

2018-02-01

Astrocytes play a critical role in regulating the interface between the cerebral vasculature and the central nervous system. Contributing to this is the astrocytic endfoot domain, a specialized structure that ensheathes the entirety of the vasculature and mediates signaling between endothelial cells, pericytes, and neurons. The astrocytic endfoot has been implicated as a critical element of the glymphatic pathway, and changes in protein expression profiles in this cellular domain are linked to Alzheimer's disease pathology. Despite this, basic physiological properties of this structure remain poorly understood including the developmental timing of its formation, and the protein components that localize there to mediate its functions. Here we use human transcriptome data from male and female subjects across several developmental stages and brain regions to characterize the gene expression profile of the dystrophin-associated complex (DAC), a known structural component of the astrocytic endfoot that supports perivascular localization of the astroglial water channel aquaporin-4. Transcriptomic profiling is also used to define genes exhibiting parallel expression profiles to DAC elements, generating a pool of candidate genes that encode gene products that may contribute to the physiological function of the perivascular astrocytic endfoot domain. We found that several genes encoding transporter proteins are transcriptionally associated with DAC genes. © 2017 Wiley Periodicals, Inc.

Different HER2 protein expression profiles aid in the histologic differential diagnosis between urothelial carcinoma in situ (CIS) and non-CIS conditions (dysplasia and reactive atypia) of the urinary bladder mucosa.

PubMed

Gunia, Sven; Koch, Stefan; Hakenberg, Oliver W; May, Matthias; Kakies, Christoph; Erbersdobler, Andreas

2011-12-01

We evaluated HER2 expression profiles in 32 carcinoma in situ (CIS) and 31 non-CIS conditions (5 dysplasia and 26 reactive atypia) of the urinary bladder mucosa by applying breast cancer scoring rules. In situ hybridization was performed on tissue microarrays to assess HER2 gene amplification status. Our immunoprofiling data disclosed moderate to strong HER2 expression in CIS, including the basal layer of the urothelium, and absent to weak HER2 expression in non-CIS conditions. From the histologic differential diagnostic standpoint, immunostaining for HER2 protein represents a useful adjunct to aid in the delineation between CIS and non-CIS conditions of the bladder mucosa. Pathogenically, aberrant HER2 protein expression in CIS seems to be more commonly associated with polysomy than with gene amplification. From a therapeutic viewpoint, prospective clinical studies should investigate the potential benefit of HER2-targeted therapies in CIS, particularly in cases unresponsive to conventional therapeutic regimens.

Cassava root membrane proteome reveals activities during storage root maturation.

PubMed

Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

2016-01-01

Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

Expression profiling of chickpea genes differentially regulated during a resistance response to Ascochyta rabiei.

PubMed

Coram, Tristan E; Pang, Edwin C K

2006-11-01

Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) expressed sequence tags (ESTs) and lentil (Lens culinaris Med.) resistance gene analogues, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, in which samples from mock-inoculated controls acted as reference against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye swap), the inclusion of negative controls and strict selection criteria for differentially expressed genes, including a fold change cut-off determined by self-self hybridizations, Student's t-test and multiple testing correction (P < 0.05). Microarray observations were also validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The time course expression patterns of 756 microarray features resulted in the differential expression of 97 genes in at least one genotype at one time point. k-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and A. rabiei-susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, Ca-binding protein and several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on the functional validation of the genes of interest.

Characterization of two distinct dual specificity phosphatases encoded in alternative open reading frames of a single gene located on human chromosome 10q22.2.

PubMed

Chen, Hsu-Hsin; Luche, Ralf; Wei, Bo; Tonks, Nicholas K

2004-10-01

Dual specificity phosphatases (DSPs) are members of the protein-tyrosine phosphatase superfamily that dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Many DSPs have been found to play important roles in various aspects of cellular function and to be involved in human disease. We have identified a gene located on human chromosome 10q22.2, which utilizes alternative open reading frames (ORFs) to encode the following two distinct DSPs: the previously described testis and skeletal muscle-specific dual specificity phosphatase (TMDP) and a novel DSP, muscle-restricted dual specificity phosphatase (MDSP). Use of alternative ORFs encoding distinct proteins from a single gene is extremely rare in eukaryotes, and in all previously reported cases the two proteins produced from one gene are unrelated. To our knowledge this is the first example of a gene from which two distinct proteins of the same family are expressed using alternative ORFs. Here we provide evidence that both MDSP and TMDP proteins are expressed in vivo and are restricted to specific tissues, skeletal muscle and testis, respectively. Most interestingly, the protein expression profiles of both MDSP and TMDP during mouse postnatal development are strikingly similar. MDSP is expressed at very low levels in myotubes and early postnatal muscle. TMDP is not detectable in testis lysate in the first 3 weeks of life. The expression of both MDSP and TMDP proteins was markedly increased at approximately the 3rd week after birth and continued to increase gradually into adulthood, implying that the physiological functions of both DSPs are specific to the mature/late-developing organs. The conserved gene structure and the similarity in postnatal expression profile of these two proteins suggest biological significance of the unusual gene arrangement.

Effects of nitric oxide on expressions of nitrosocysteine and calcium-activated potassium channels in the supraoptic nuclei and neural lobe of dehydrated rats

PubMed Central

Kadekaro, Massako; Su, Guangxiao; Chu, Rong; Lei, Yongzhong; Li, Junfa; Fang, Li

2007-01-01

Nitric oxide (NO) is an important gas mediator in the signal transduction cascade regulating osmotic function in the hypothalamo-neurohypophysial system. We previously found that increased nitric oxide synthase (NOS) activity in the supraoptic nuclei (SON) and neural lobe following osmotic stimulation and NO could regulate the expression of Ca2+-activated K+ channel (BK channels) protein in the magnocellular system during dehydration. The aim of the current study is to examine the role of NO in the regulation of nitrosocysteine and BK channel protein in the magnocellular system in dehydrated animals. Using Western blot analysis and quantitative immunofluorescent staining study, we found that water deprivation in rats significantly enhanced the expression of nitrosocysteine protein in SON and neural lobes. Immunohistochemistry study indicated that dehydration significantly increased the profiles of SON neurons co-expressing nitrosocysteine with BK-channel protein. Intracerebroventricular administration of L-NAME (an inhibitor of NO synthase) significantly reduced the neuronal profiles of nitrosocysteine, as well as their co-expression with BK-channel in SON of dehydrated rats. However, treatment of sodium nitroprusside (a donor of NO) increased this co-expression. Our results indicate that NO signaling cascade may control the expression of BK channels through the regulation of nitrosocysteine in SON and neural lobe of rats during osmotic regulation. PMID:17098363

Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

PubMed

Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

2011-12-01

Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

PubMed

Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

2017-03-14

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

NASA Astrophysics Data System (ADS)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

PubMed Central

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

Transcriptional profiling of Haemophilus parasuis SH0165 response to tilmicosin.

PubMed

Liu, Yingyu; Chen, Pin; Wang, Yang; Li, Wentao; Cheng, Shuang; Wang, Chunmei; Zhang, Anding; He, Qigai

2012-12-01

The Haemophilus parasuis respiratory tract pathogen poses a severe threat to the swine industry despite available antimicrobial therapies. To gain a more detailed understanding of the molecular mechanisms underlying H. parasuis response to tilmicosin treatment, microarray technology was applied to analyze the variation in gene expression of isolated H. parasuis SH0165 treated in vitro with subinhibitory (0.25 μg/ml) and inhibitory (8 μg/ml) concentrations. Tilmicosin treatment induced differential expression of 405 genes, the encoded products of which are mainly involved in the heat shock response, protein synthesis, and intracellular transportation. The subinhibitory and inhibitory concentrations of tilmicosin induced distinctive gene expression profiles of shared and unique changes, respectively. These changes included 302 genes mainly involved in protein export and the phosphotransferase system to sustain cell growth, and 198 genes mainly related to RNA polymerase, recombination, and repair to inhibit cell growth. In silico analysis of functions related to the differentially expressed genes suggested that adaptation of H. parasuis SH0165 to tilmicosin involves modulation of protein synthesis and membrane transport. Collectively, the genes comprising each transcriptional profile of H. parasuis response to tilmicosin provide novel insights into the physiological functions of this economically significant bacterium and may represent targets of future molecular therapeutic strategies.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

PubMed

Wu, Pei-Wen; Mason, Katelyn E; Durbin-Johnson, Blythe P; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Parker, Glendon J; Rice, Robert H

2017-07-01

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spleen-dependent regulation of antigenic variation in malaria parasites: Plasmodium knowlesi SICAvar expression profiles in splenic and asplenic hosts.

PubMed

Lapp, Stacey A; Korir-Morrison, Cindy; Jiang, Jianlin; Bai, Yaohui; Corredor, Vladimir; Galinski, Mary R

2013-01-01

Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera. We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry. SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment.

Genome-wide organization and expression profiling of the NAC transcription factor family in potato (Solanum tuberosum L.).

PubMed

Singh, Anil Kumar; Sharma, Vishal; Pal, Awadhesh Kumar; Acharya, Vishal; Ahuja, Paramvir Singh

2013-08-01

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

Whole blood genome-wide expression profiling and network analysis suggest MELAS master regulators.

PubMed

Mende, Susanne; Royer, Loic; Herr, Alexander; Schmiedel, Janet; Deschauer, Marcus; Klopstock, Thomas; Kostic, Vladimir S; Schroeder, Michael; Reichmann, Heinz; Storch, Alexander

2011-07-01

The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation. To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome. Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1 complex, nuclear factor Y and cAMP responsive element-binding protein-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction. Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.

An emerging cyberinfrastructure for biodefense pathogen and pathogen–host data

PubMed Central

Zhang, C.; Crasta, O.; Cammer, S.; Will, R.; Kenyon, R.; Sullivan, D.; Yu, Q.; Sun, W.; Jha, R.; Liu, D.; Xue, T.; Zhang, Y.; Moore, M.; McGarvey, P.; Huang, H.; Chen, Y.; Zhang, J.; Mazumder, R.; Wu, C.; Sobral, B.

2008-01-01

The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host–pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at http://www.proteomicsresource.org/. PMID:17984082

Honeybee (Apis mellifera ligustica) drone embryo proteomes.

PubMed

Li, Jianke; Fang, Yu; Zhang, Lan; Begna, Desalegn

2011-03-01

Little attention has been paid to the drone honeybee (Apis mellifera ligustica) which is a haploid individual carrying only the set of alleles that it inherits from its mother. Molecular mechanisms underlying drone embryogenesis are poorly understood. This study evaluated protein expression profiles of drone embryogenesis at embryonic ages of 24, 48 and 72h. More than 100 reproducible proteins were analyzed by mass spectrometry on 2D electrophoresis gels. Sixty-two proteins were significantly changed at the selected three experimental age points. Expression of the metabolic energy requirement-related protein peaked at the embryonic age of 48h, whereas development and metabolizing amino acid-related proteins expressed optimally at 72h. Cytoskeleton, protein folding and antioxidant-related proteins were highly expressed at 48 and 72h. Protein networks of the identified proteins were constructed and protein expressions were validated at the transcription level. This first proteomic study of drone embryogenesis in the honeybee may provide geneticists an exact timetable and candidate protein outline for further manipulations of drone stem cells. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

PubMed Central

2012-01-01

Background The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation. Results We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified. The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways. Conclusions The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation. PMID:22537182

Alteration of gene expression profiling including GPR174 and GNG2 is associated with vasovagal syncope.

PubMed

Huang, Yu-Juan; Zhou, Zai-wei; Xu, Miao; Ma, Qing-wen; Yan, Jing-bin; Wang, Jian-yi; Zhang, Quo-qin; Huang, Min; Bao, Liming

2015-03-01

Vasovagal syncope (VVS) causes accidental harm for susceptible patients. However, pathophysiology of this disorder remains largely unknown. In an effort to understanding of molecular mechanism for VVS, genome-wide gene expression profiling analyses were performed on VVS patients at syncope state. A total of 66 Type 1 VVS child patients and the same number healthy controls were enrolled in this study. Peripheral blood RNAs were isolated from all subjects, of which 10 RNA samples were randomly selected from each groups for gene expression profile analysis using Gene ST 1.0 arrays (Affymetrix). The results revealed that 103 genes were differently expressed between the patients and controls. Significantly, two G-proteins related genes, GPR174 and GNG2 that have not been related to VVS were among the differently expressed genes. The microarray results were confirmed by qRT-PCR in all the tested individuals. Ingenuity pathway analysis and gene ontology annotation study showed that the differently expressed genes are associated with stress response and apoptosis, suggesting that the alteration of some gene expression including G-proteins related genes is associated with VVS. This study provides new insight into the molecular mechanism of VVS and would be helpful to further identify new molecular biomarkers for the disease.

Proteomic plasma profile of psoriatic patients.

PubMed

Gęgotek, Agnieszka; Domingues, Pedro; Wroński, Adam; Wójcik, Piotr; Skrzydlewska, Elżbieta

2018-06-05

Psoriasis is a chronic, immune-mediated inflammatory skin disease with severe consequences for the whole organism. The lack of complete knowledge of the main factors predisposing an individual to the appearance of psoriatic lesions, has recently led to the search for modifications in biochemical pathways participating in the development of this disease. We therefore aimed to investigate changes in the plasma proteomic profile of patients with psoriasis. A proteomics approach was used to analyze the expression of proteins in plasma from psoriatic patients and healthy controls (sex- and age-matched individuals). The analysis was performed using gel electrophoresis, followed by nanoflow LC-MS/MS using a Q-Exactive OrbiTrap mass spectrometer. Proteomic data indicated a significant decrease in the level of proteins involved in lipid metabolism, such as apolipoprotein M, and proteins involved in the management of vitamin D levels in psoriatic patients' plasma. These changes were accompanied by the expression of proteins involved in immune response and signal transduction. This was particularly evident by the level of transcriptional factors, including AT motif binding factor 1, which regulates excessive cellular proliferation and differentiation. It was also suggested that psoriasis development was associated with increased expression of proteins directly involved in signaling molecule secretion [biotinidase and BAI1-associated protein 3]. In addition, the lipid peroxidation product - 4-hydroxynonenal (4-HNE) generates higher level of adducts with proteins in the plasma of psoriatic patients. Moreover, plasma proteins from healthy subjects creating with 4-HNE adducts were mainly characterized as structural, while in the plasma of psoriatic patients, increased levels of 4-HNE-protein adducts with catalytic activity were observed. The results presented herein confirm the current knowledge about the profile of proteins responsible for the immune response and management of vitamin D in the plasma of psoriatic patients. However, several new proteins were also identified, which are involved in signal transduction and lipid metabolism as well as catalytic activity. The expression or structure of these proteins was shown to change through the course of the development of psoriasis. This knowledge may help contribute to the design of more specific pharmacotherapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

PubMed

Perkins, Lydia A; Yan, Qi; Schmidt, Brigitte F; Kolodieznyi, Dmytro; Saurabh, Saumya; Larsen, Mads Breum; Watkins, Simon C; Kremer, Laura; Bruchez, Marcel P

2018-02-06

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

The AlternativeTranslational Profile That Underlies the Immune-Evasive State of Persistence in Chlamydiaceae Exploits Differential Tryptophan Contents of the Protein Repertoire

PubMed Central

Lo, Chien-Chi; Bonner, Carol A.

2012-01-01

Summary: One form of immune evasion is a developmental state called “persistence” whereby chlamydial pathogens respond to the host-mediated withdrawal of l-tryptophan (Trp). A sophisticated survival mode of reversible quiescence is implemented. A mechanism has evolved which suppresses gene products necessary for rapid pathogen proliferation but allows expression of gene products that underlie the morphological and developmental characteristics of persistence. This switch from one translational profile to an alternative translational profile of newly synthesized proteins is proposed to be accomplished by maximizing the Trp content of some proteins needed for rapid proliferation (e.g., ADP/ATP translocase, hexose-phosphate transporter, phosphoenolpyruvate [PEP] carboxykinase, the Trp transporter, the Pmp protein superfamily for cell adhesion and antigenic variation, and components of the cell division pathway) while minimizing the Trp content of other proteins supporting the state of persistence. The Trp starvation mechanism is best understood in the human-Chlamydia trachomatis relationship, but the similarity of up-Trp and down-Trp proteomic profiles in all of the pathogenic Chlamydiaceae suggests that Trp availability is an underlying cue relied upon by this family of pathogens to trigger developmental transitions. The biochemically expensive pathogen strategy of selectively increased Trp usage to guide the translational profile can be leveraged significantly with minimal overall Trp usage by (i) regional concentration of Trp residue placements, (ii) amplified Trp content of a single protein that is required for expression or maturation of multiple proteins with low Trp content, and (iii) Achilles'-heel vulnerabilities of complex pathways to high Trp content of one or a few enzymes. PMID:22688818

Evaluation of Beauveria bassiana infection in the hemolymph serum proteins of the housefly, Musca domestica L. (Diptera: Muscidae).

PubMed

Mishra, Sapna; Kumar, Peeyush; Malik, Anushree

2017-11-01

Beauveria bassiana plays a prominent role in biocontrol of houseflies, Musca domestica (L.). Thus, a deeper insight into immune response of M. domestica during B. bassiana infection was warranted to assist the production of more efficient mycoinsecticides. The present study investigates changes in protein profile of M. domestica hemolymph serum post B. bassiana infection using two-dimensional difference gel electrophoresis (2D-DIGE) followed by identification of selected proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The non-infected or control group of flies showed an expression of 54 proteins, while M. domestica infected with B. bassiana expressed a total of 68 hemolymph serum proteins. Thirty three proteins were expressed in both groups of houseflies, whereas 35 proteins were exclusively expressed in infected flies and 21 proteins were exclusively expressed in control flies. Among the 33 proteins which were expressed in both groups of houseflies, 17 proteins showed downregulation, while16 proteins were upregulated in the infected flies compared to the non-infected ones. The results from this study are expected to facilitate better understanding of insect's immune response mechanism.

Genome-wide survey of B-box proteins in potato (Solanum tuberosum)-Identification, characterization and expression patterns during diurnal cycle, etiolation and de-etiolation.

PubMed

Talar, Urszula; Kiełbowicz-Matuk, Agnieszka; Czarnecka, Jagoda; Rorat, Tadeusz

2017-01-01

Plant B-box domain proteins (BBX) mediate many light-influenced developmental processes including seedling photomorphogenesis, seed germination, shade avoidance and photoperiodic regulation of flowering. Despite the wide range of potential functions, the current knowledge regarding BBX proteins in major crop plants is scarce. In this study, we identify and characterize the StBBX gene family in potato, which is composed of 30 members, with regard to structural properties and expression profiles under diurnal cycle, etiolation and de-etiolations. Based on domain organization and phylogenetic relationships, StBBX genes have been classified into five groups. Using real-time quantitative PCR, we found that expression of most of them oscillates following a 24-h rhythm; however, large differences in expression profiles were observed between the genes regarding amplitude and position of the maximal and minimal expression levels in the day/night cycle. On the basis of the time-of-day/time-of-night, we distinguished three expression groups specifically expressed during the light and two during the dark phase. In addition, we showed that the expression of several StBBX genes is under the control of the circadian clock and that some others are specifically associated with the etiolation and de-etiolation conditions. Thus, we concluded that StBBX proteins are likely key players involved in the complex diurnal and circadian networks regulating plant development as a function of light conditions and day duration.

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

PubMed

Judelson, Howard S; Ah-Fong, Audrey M V; Aux, George; Avrova, Anna O; Bruce, Catherine; Cakir, Cahid; da Cunha, Luis; Grenville-Briggs, Laura; Latijnhouwers, Maita; Ligterink, Wilco; Meijer, Harold J G; Roberts, Samuel; Thurber, Carrie S; Whisson, Stephen C; Birch, Paul R J; Govers, Francine; Kamoun, Sophien; van West, Pieter; Windass, John

2008-04-01

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

Ribosome profiling reveals the what, when, where and how of protein synthesis.

PubMed

Brar, Gloria A; Weissman, Jonathan S

2015-11-01

Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products.

Identification and Comparative Expression Profiles of Chemoreception Genes Revealed from Major Chemoreception Organs of the Rice Leaf Folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

PubMed Central

Zeng, Fang-Fang; Zhao, Zhen-Fei; Yan, Miao-Jun; Zhou, Wen; Zhang, Zan; Zhang, Aijun; Lu, Zhong-Xian; Wang, Man-Qun

2015-01-01

To better understand the olfactory mechanisms in the rice leaf folder, Cnaphalocrocis medinalis (Guenée), a serious pest of rice in Asia, we established six partial transcriptomes from antennae, protarsus, and reproductive organs of male and female adults. A total of 102 transcripts were identified, including 29 odorant receptors (ORs), 15 ionotropic receptors (IRs), 30 odorant-binding proteins (OBPs), 26 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs). The expression patterns of these genes were calculated by fragments per kilobase of exon per million fragments mapped (FPKM) and validated by real-time quantitative PCR (RT-qPCR). Some transcripts were exclusively expressed in specific organs, such as female protarsus, whereas others were universally expressed, this varied expression profile may provide insights into the specific functions mediated by chemoreception proteins in insects. To the best of our knowledge, among the 102 identified transcripts, 81 are novel and have never been reported before. In addition, it also is the first time that ORs and IRs are identified in C. medinalis. Our findings significantly enhance the currently limited understanding olfactory mechanisms of the olfactory mechanisms underlying the chemoreception system in C. medinalis. PMID:26657286

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

NASA Astrophysics Data System (ADS)

Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

2009-09-01

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Expression of EGFP and NPTII protein is not associated with organ abnormalities in deceased transgenic cloned cattle.

PubMed

Liu, Yan; Wu, Qian; Cui, Huiting; Li, Qinghe; Zhao, Yiqiang; Luo, Juan; Liu, Qiuyue; Sun, Xiuzhu; Tang, Bo; Zhang, Lei; Dai, Yunping; Li, Ning

2008-12-01

Both enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase type II enzyme (NPTII) are widely used in transgenic studies, but their side effects have not been extensively investigated. In this study, we evaluated the expression profiles of the two marker genes and the relationship between their expression and organ abnormalities. Eight transgenic cloned cattle were studied, four harboring both EGFP and NPTII, and four harboring only the NPTII gene. Four age-matched cloned cattle were used as controls. EGFP and NPTII expression were measured and detected by Q-PCR, Western blot, ELISA, and RIA in heart, liver, and lungs, and the values ranged from 0.3 to 5 microg/g. The expression profiles exhibited differential or mosaic pattern between the organs, the pathologic symptoms of which were identified, but were similar to those of age-matched cloned cattle. All data indicated that the expression of EGFP and NPTII is not associated with organ abnormalities in transgenic cloned cattle.

Gene expression profile change and growth inhibition in Drosophila larvae treated with azadirachtin.

PubMed

Lai, Duo; Jin, Xiaoyong; Wang, Hao; Yuan, Mei; Xu, Hanhong

2014-09-20

Azadirachtin is a botanical insecticide that affects various biological processes. The effects of azadirachtin on the digital gene expression profile and growth inhibition in Drosophila larvae have not been investigated. In this study, we applied high-throughput sequencing technology to detect the differentially expressed genes of Drosophila larvae regulated by azadirachtin. A total of 15,322 genes were detected, and 28 genes were found to be significantly regulated by azadirachtin. Biological process and pathway analysis showed that azadirachtin affected starch and sucrose metabolism, defense response, signal transduction, instar larval or pupal development, and chemosensory behavior processes. The genes regulated by azadirachtin were mainly enriched in starch and sucrose metabolism. This study provided a general digital gene expression profile of dysregulated genes in response to azadirachtin and showed that azadirachtin provoked potent growth inhibitory effects in Drosophila larvae by regulating the genes of cuticular protein, amylase, and odorant-binding protein. Finally, we propose a potential mechanism underlying the dysregulation of the insulin/insulin-like growth factor signaling pathway by azadirachtin. Copyright © 2014 Elsevier B.V. All rights reserved.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts.

PubMed

Rozenchan, Patricia Bortman; Carraro, Dirce Maria; Brentani, Helena; de Carvalho Mota, Louise Danielle; Bastos, Elen Pereira; e Ferreira, Elisa Napolitano; Torres, Cesar H; Katayama, Maria Lúcia Hirata; Roela, Rosimeire Aparecida; Lyra, Eduardo C; Soares, Fernando Augusto; Folgueira, Maria Aparecida Azevedo Koike; Góes, João Carlos Guedes Sampaio; Brentani, Maria Mitzi

2009-12-15

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGFbeta1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. Copyright (c) 2009 UICC.

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression ofmore » surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.« less

Expression Profiles, Characterization and Function of HbTCTP in Rubber Tree (Hevea brasiliensis)

PubMed Central

Deng, Zhi; Chen, Jiangshu; Leclercq, Julie; Zhou, Zhuangzhi; Liu, Changren; Liu, Hui; Yang, Hong; Montoro, Pascal; Xia, Zhihui; Li, Dejun

2016-01-01

As a highly conserved protein, the translationally controlled tumor protein (TCTP) carries out vital roles in various life processes. In rubber tree, two TCTP genes, HbTCTP and HbTCTP1, were cloned, but only HbTCTP1 was studied in details. In this study, cis-acting regulatory elements, expression patterns, subcellular localization, interacting proteins, and antioxidant activity of HbTCTP were systematically analyzed. Besides the common cis-acting regulatory elements, HbTCTP promoter also harbored various known cis-elements that respond to hormone/stresses. Being consistent with the aforementioned results, HbTCTP was regulated by drought, low temperature, high salt, ethylene (ET), wounding, H2O2, and methyl jasmonate (MeJA) treatments. HbTCTP was expressed throughout different tissues and developmental stages of leaves. In addition, HbTCTP was associated with tapping panel dryness (TPD). HbTCTP was localized in the membrane, cytoplasm and the nucleus, and interacted with four proteins rubber elongation factor (REF), 17.5 kDa heat shock family protein, annexin, and REF-like stress related protein 1. Being similar to HbTCTP1, HbTCTP also indicated antioxidant activity in metal-catalyzed oxidation (MCO) system. Our results are useful for further understanding the molecular characterization and expression profiles of HbTCTP, but also lay a solid foundation for elucidating the function of HbTCTP in rubber tree. PMID:27375647

Multiple biomarkers in molecular oncology. II. Molecular diagnostics applications in breast cancer management.

PubMed

Malinowski, Douglas P

2007-05-01

In recent years, the application of genomic and proteomic technologies to the problem of breast cancer prognosis and the prediction of therapy response have begun to yield encouraging results. Independent studies employing transcriptional profiling of primary breast cancer specimens using DNA microarrays have identified gene expression profiles that correlate with clinical outcome in primary breast biopsy specimens. Recent advances in microarray technology have demonstrated reproducibility, making clinical applications more achievable. In this regard, one such DNA microarray device based upon a 70-gene expression signature was recently cleared by the US FDA for application to breast cancer prognosis. These DNA microarrays often employ at least 70 gene targets for transcriptional profiling and prognostic assessment in breast cancer. The use of PCR-based methods utilizing a small subset of genes has recently demonstrated the ability to predict the clinical outcome in early-stage breast cancer. Furthermore, protein-based immunohistochemistry methods have progressed from using gene clusters and gene expression profiling to smaller subsets of expressed proteins to predict prognosis in early-stage breast cancer. Beyond prognostic applications, DNA microarray-based transcriptional profiling has demonstrated the ability to predict response to chemotherapy in early-stage breast cancer patients. In this review, recent advances in the use of multiple markers for prognosis of disease recurrence in early-stage breast cancer and the prediction of therapy response will be discussed.

Protein Expression Profile of Twenty-Week-Old Diabetic db/db and Non-Diabetic Mice Livers: A Proteomic and Bioinformatic Analysis.

PubMed

Guzmán-Flores, Juan Manuel; Flores-Pérez, Elsa Cristina; Hernández-Ortiz, Magdalena; Vargas-Ortiz, Katya; Ramírez-Emiliano, Joel; Encarnación-Guevara, Sergio; Pérez-Vázquez, Victoriano

2018-06-01

Type 2 diabetes mellitus is characterized by insulin resistance in the liver. Insulin is not only involved in carbohydrate metabolism, it also regulates protein synthesis. This work describes the expression of proteins in the liver of a diabetic mouse and identifies the metabolic pathways involved. Twenty-week-old diabetic db/db mice were hepatectomized, after which proteins were separated by 2D-Polyacrylamide Gel Electrophoresis (2D-PAGE). Spots varying in intensity were analyzed using mass spectrometry, and biological function was assigned by the Database for Annotation, Visualization and Integrated Discovery (DAVID) software. A differential expression of 26 proteins was identified; among these were arginase-1, pyruvate carboxylase, peroxiredoxin-1, regucalcin, and sorbitol dehydrogenase. Bioinformatics analysis indicated that many of these proteins are mitochondrial and participate in metabolic pathways, such as the citrate cycle, the fructose and mannose metabolism, and glycolysis or gluconeogenesis. In addition, these proteins are related to oxidation⁻reduction reactions and molecular function of vitamin binding and amino acid metabolism. In conclusion, the proteomic profile of the liver of diabetic mouse db/db exhibited mainly alterations in the metabolism of carbohydrates and nitrogen. These differences illustrate the heterogeneity of diabetes in its different stages and under different conditions and highlights the need to improve treatments for this disease.

A combined analysis of genome-wide expression profiling of bipolar disorder in human prefrontal cortex.

PubMed

Wang, Jinglu; Qu, Susu; Wang, Weixiao; Guo, Liyuan; Zhang, Kunlin; Chang, Suhua; Wang, Jing

2016-11-01

Numbers of gene expression profiling studies of bipolar disorder have been published. Besides different array chips and tissues, variety of the data processes in different cohorts aggravated the inconsistency of results of these genome-wide gene expression profiling studies. By searching the gene expression databases, we obtained six data sets for prefrontal cortex (PFC) of bipolar disorder with raw data and combinable platforms. We used standardized pre-processing and quality control procedures to analyze each data set separately and then combined them into a large gene expression matrix with 101 bipolar disorder subjects and 106 controls. A standard linear mixed-effects model was used to calculate the differentially expressed genes (DEGs). Multiple levels of sensitivity analyses and cross validation with genetic data were conducted. Functional and network analyses were carried out on basis of the DEGs. In the result, we identified 198 unique differentially expressed genes in the PFC of bipolar disorder and control. Among them, 115 DEGs were robust to at least three leave-one-out tests or different pre-processing methods; 51 DEGs were validated with genetic association signals. Pathway enrichment analysis showed these DEGs were related with regulation of neurological system, cell death and apoptosis, and several basic binding processes. Protein-protein interaction network further identified one key hub gene. We have contributed the most comprehensive integrated analysis of bipolar disorder expression profiling studies in PFC to date. The DEGs, especially those with multiple validations, may denote a common signature of bipolar disorder and contribute to the pathogenesis of disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein-expression profiles in mouse blood-plasma following acute whole-body exposure to (137)Cs gamma rays.

PubMed

Rithidech, Kanokporn Noy; Honikel, Louise; Rieger, Robert; Xie, Weiping; Fischer, Thomas; Simon, Sanford R

2009-05-01

To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of (137)Cs gamma rays. Two-dimensional electrophoresis gel coupled with mass spectrometry was used to analyze blood-samples collected at days 2 and 7 post-irradiation. At each sacrifice-time, alterations in expression-level of protein spots between control- and exposed-groups were analyzed statistically by the PDQuest software using Student's t-test (at the significance level of p < 0.05). Mass spectrometry was used to identify the identity of protein-spots with significantly altered expression-level. At day 2, 18 proteins were significantly up-regulated in exposed-mice. These included: alpha-2-Heremans-Schmid (HS)-glycoprotein, apolipoprotein (Apo)-AII-precursor, Apo-E, beta-2-glycoprotein-I, clusterin, fibrinogen-alpha-chain, fibrinogen-gamma-polypeptide, fetuin-B, haptoglobin, high-molecular-weight (HMW)-kininogen (Kng), low-MW-Kng, Kng1-precursor, liver-carboxylesterase-I-precursor, major-urinary-protein-6-precursor, mannose-binding-protein-C-precursor, mannose-binding-lectin-C, and prothrombin-precursor. Gelsolin was detected in control-mice only. At day 7, high expression-levels of 14 proteins were detected in control-mice (i.e., alpha-1-antitrypsin-precursor, carboxylesterase-N, cholesterol-7-alpha-hydroxylase, contraspin, coagulation-factor-II, coagulation-factor-XIII, gelsolin, immunoglobulin-G-heavy-chain, neurexin, prothrombin-precursor, protein-phosphatase, putative-calcium-influx-channel, vitamin-D-binding-protein, and 1110018G07Rik); while 15 proteins were highly expressed in exposed-mice. These included: alpha-1-acid-glycoprotein, alpha-2-HS-glycoprotein, alpha-1-protease-inhibitor-2, ApoA-IV, ApoC-I, ApoH, beta-1-globin, clusterin, complement-component-3, fibrinogen-beta-chain, HMW-Kng, major-histocompatibility-complex-class-Ia-H2-K, serine-(cysteine)-proteinase-inhibitor, retinoblastoma-associated-protein-140, and vascular-cell-adhesion-molecule-1. Although different proteins (mostly involved in inflammatory responses) were detected in exposed-mice, alterations in expression-levels of clusterin, gelsolin, kininogen, and alpha-2-HS-glycoproteins were found at both times. Despite the need for validation, the results suggested that alterations in expression-levels of specific proteins may be indicative of radiation-exposure. The results also provided the important step in an eventual establishment of blood-based biomarkers of radiation-exposure in vivo.

Structure-related clustering of gene expression fingerprints of thp-1 cells exposed to smaller polycyclic aromatic hydrocarbons.

PubMed

Wan, B; Yarbrough, J W; Schultz, T W

2008-01-01

This study was undertaken to test the hypothesis that structurally similar PAHs induce similar gene expression profiles. THP-1 cells were exposed to a series of 12 selected PAHs at 50 microM for 24 hours and gene expressions profiles were analyzed using both unsupervised and supervised methods. Clustering analysis of gene expression profiles revealed that the 12 tested chemicals were grouped into five clusters. Within each cluster, the gene expression profiles are more similar to each other than to the ones outside the cluster. One-methylanthracene and 1-methylfluorene were found to have the most similar profiles; dibenzothiophene and dibenzofuran were found to share common profiles with fluorine. As expression pattern comparisons were expanded, similarity in genomic fingerprint dropped off dramatically. Prediction analysis of microarrays (PAM) based on the clustering pattern generated 49 predictor genes that can be used for sample discrimination. Moreover, a significant analysis of Microarrays (SAM) identified 598 genes being modulated by tested chemicals with a variety of biological processes, such as cell cycle, metabolism, and protein binding and KEGG pathways being significantly (p < 0.05) affected. It is feasible to distinguish structurally different PAHs based on their genomic fingerprints, which are mechanism based.

Modeling T-cell activation using gene expression profiling and state-space models.

PubMed

Rangel, Claudia; Angus, John; Ghahramani, Zoubin; Lioumi, Maria; Sotheran, Elizabeth; Gaiba, Alessia; Wild, David L; Falciani, Francesco

2004-06-12

We have used state-space models to reverse engineer transcriptional networks from highly replicated gene expression profiling time series data obtained from a well-established model of T-cell activation. State space models are a class of dynamic Bayesian networks that assume that the observed measurements depend on some hidden state variables that evolve according to Markovian dynamics. These hidden variables can capture effects that cannot be measured in a gene expression profiling experiment, e.g. genes that have not been included in the microarray, levels of regulatory proteins, the effects of messenger RNA and protein degradation, etc. Bootstrap confidence intervals are developed for parameters representing 'gene-gene' interactions over time. Our models represent the dynamics of T-cell activation and provide a methodology for the development of rational and experimentally testable hypotheses. Supplementary data and Matlab computer source code will be made available on the web at the URL given below. http://public.kgi.edu/~wild/LDS/index.htm

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients

PubMed Central

Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

2016-01-01

Objectives To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Methods Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). Results DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. Conclusions The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions. PMID:27846214

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients.

PubMed

Li, Li; Zhang, Jiangyu; Deng, Qingshan; Li, Jieming; Li, Zhengfen; Xiao, Yao; Hu, Shuiwang; Li, Tiantian; Tan, Qiuxiao; Li, Xiaofang; Luo, Bingshu; Mo, Hui

2016-01-01

To identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS). Twenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC). DIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC. The present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

PubMed

Santana, Flávia A; Nunes, Francis M F; Vieira, Carlos U; Machado, Maria Alice M S; Kerr, Warwick E; Silva, Wilson A; Bonetti, Ana Maria

2006-03-01

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

Identification and Analysis of Mitogen-Activated Protein Kinase (MAPK) Cascades in Fragaria vesca.

PubMed

Zhou, Heying; Ren, Suyue; Han, Yuanfang; Zhang, Qing; Qin, Ling; Xing, Yu

2017-08-13

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes, including yeasts, plants and animals. MAPK cascades are responsible for protein phosphorylation during signal transduction events, and typically consist of three protein kinases: MAPK, MAPK kinase, and MAPK kinase kinase. In this current study, we identified a total of 12 FvMAPK , 7 FvMAPKK , 73 FvMAPKKK , and one FvMAPKKKK genes in the recently published Fragaria vesca genome sequence. This work reported the classification, annotation and phylogenetic evaluation of these genes and an assessment of conserved motifs and the expression profiling of members of the gene family were also analyzed here. The expression profiles of the MAPK and MAPKK genes in different organs and fruit developmental stages were further investigated using quantitative real-time reverse transcription PCR (qRT-PCR). Finally, the MAPK and MAPKK expression patterns in response to hormone and abiotic stresses (salt, drought, and high and low temperature) were investigated in fruit and leaves of F. vesca . The results provide a platform for further characterization of the physiological and biochemical functions of MAPK cascades in strawberry.

Identification and expression profiles of the WRKY transcription factor family in Ricinus communis.

PubMed

Li, Hui-Liang; Zhang, Liang-Bo; Guo, Dong; Li, Chang-Zhu; Peng, Shi-Qing

2012-07-25

In plants, WRKY proteins constitute a large family of transcription factors. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. A large number of WRKY transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of castor bean (Ricinus communis) has allowed a genome-wide search for R. communis WRKY (RcWRKY) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. A total of 47 WRKY genes were identified in the castor bean genome. According to the structural features of the WRKY domain, the RcWRKY are classified into seven main phylogenetic groups. Furthermore, putative orthologs of RcWRKY proteins in Arabidopsis and rice could now be assigned. An analysis of expression profiles of RcWRKY genes indicates that 47 WRKY genes display differential expressions either in their transcript abundance or expression patterns under normal growth conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into Regulated and Coordinated Gene Expression in Lotus japonicus and Rhizophagus irregularis.

PubMed

Handa, Yoshihiro; Nishide, Hiroyo; Takeda, Naoya; Suzuki, Yutaka; Kawaguchi, Masayoshi; Saito, Katsuharu

2015-08-01

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

PubMed

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Plant uncoupling mitochondrial proteins.

PubMed

Vercesi, Aníbal Eugênio; Borecký, Jiri; Maia, Ivan de Godoy; Arruda, Paulo; Cuccovia, Iolanda Midea; Chaimovich, Hernan

2006-01-01

Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide-sensitive free fatty acid-activated H(+) flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (Deltamu(H)+) and production of reactive oxygen species.

Proteomic Analysis of Hair Follicles

NASA Astrophysics Data System (ADS)

Ishioka, Noriaki; Terada, Masahiro; Yamada, Shin; Seki, Masaya; Takahashi, Rika; Majima, Hideyuki J.; Higashibata, Akira; Mukai, Chiaki

2013-02-01

Hair root cells actively divide in a hair follicle, and they sensitively reflect physical conditions. By analyzing the human hair, we can know stress levels on the human body and metabolic conditions caused by microgravity environment and cosmic radiation. The Japan Aerospace Exploration Agency (JAXA) has initiated a human research study to investigate the effects of long-term space flight on gene expression and mineral metabolism by analyzing hair samples of astronauts who stayed in the International Space Station (ISS) for 6 months. During long-term flights, the physiological effects on astronauts include muscle atrophy and bone calcium loss. Furthermore, radiation and psychological effects are important issue to consider. Therefore, an understanding of the effects of the space environment is important for developing countermeasures against the effects experienced by astronauts. In this experiment, we identify functionally important target proteins that integrate transcriptome, mineral metabolism and proteome profiles from human hair. To compare the protein expression data with the gene expression data from hair roots, we developed the protein processing method. We extracted the protein from five strands of hair using ISOGEN reagents. Then, these extracted proteins were analyzed by LC-MS/MS. These collected profiles will give us useful physiological information to examine the effect of space flight.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

PubMed

Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

2016-01-01

Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa ( Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and contributes to the understanding of the basic molecular mechanisms during the alfalfa flowering process. These results may offer insight into potential strategies for improving seed yield, quality, and stress tolerance in alfalfa.

Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development

PubMed Central

Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

2016-01-01

Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and contributes to the understanding of the basic molecular mechanisms during the alfalfa flowering process. These results may offer insight into potential strategies for improving seed yield, quality, and stress tolerance in alfalfa. PMID:27757120

Protein Profiles for Muscle Development and Intramuscular Fat Accumulation at Different Post-Hatching Ages in Chickens.

PubMed

Liu, Jie; Fu, Ruiqi; Liu, Ranran; Zhao, Guiping; Zheng, Maiqing; Cui, Huanxian; Li, Qinghe; Song, Jiao; Wang, Jie; Wen, Jie

2016-01-01

Muscle development and growth influences the efficiency of poultry meat production, and is closely related to deposition of intramuscular fat (IMF), which is crucial in meat quality. To clarify the molecular mechanisms underlying muscle development and IMF deposition in chickens, protein expression profiles were examined in the breast muscle of Beijing-You chickens at ages 1, 56, 98 and 140 days, using isobaric tags for relative and absolute quantification (iTRAQ). Two hundred and four of 494 proteins were expressed differentially. The expression profile at day 1 differed greatly from those at day 56, 98 and 140. KEGG pathway analysis of differential protein expression from pair-wise comparisons (day 1 vs. 56; 56 vs. 98; 98 vs. 140), showed that the fatty acid degradation pathway was more active during the stage from day 1 to 56 than at other periods. This was consistent with the change in IMF content, which was highest at day 1 and declined dramatically thereafter. When muscle growth was most rapid (days 56-98), pathways involved in muscle development were dominant, including hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, tight junctions and focal adhesion. In contrast with hatchlings, the fatty acid degradation pathway was downregulated from day 98 to 140, which was consistent with the period for IMF deposition following rapid muscle growth. Changes in some key specific proteins, including fast skeletal muscle troponin T isoform, aldehyde dehydrogenase 1A1 and apolipoprotein A1, were verified by Western blotting, and could be potential biomarkers for IMF deposition in chickens. Protein-protein interaction networks showed that ribosome-related functional modules were clustered in all three stages. However, the functional module involved in the metabolic pathway was only clustered in the first stage (day 1 vs. 56). This study improves our understanding of the molecular mechanisms underlying muscle development and IMF deposition in chickens.

Expression profiles and functional associations of endogenous androgen receptor and caveolin-1 in prostate cancer cell lines.

PubMed

Bennett, Nigel C; Hooper, John D; Johnson, David W; Gobe, Glenda C

2014-05-01

In prostate cancer (PCa) patients, the protein target for androgen deprivation and blockade therapies is androgen receptor (AR). AR interacts with many proteins that function to either co-activate or co-repress its activity. Caveolin-1 (Cav-1) is not found in normal prostatic epithelium, but is found in PCa, and may be an AR co-regulator protein. We investigated cell line-specific signatures and associations of endogenous AR and Cav-1 in six PCa cell lines of known androgen sensitivity: LNCaP (androgen sensitive); 22Rv1 (androgen responsive); PC3, DU145, and ALVA41 (androgen non-reliant); and RWPE1 (non-malignant). Protein and mRNA expression profiles were compared and electron microscopy used to identify cells with caveolar structures. For cell lines expressing both AR and Cav-1, knockdown techniques using small interfering RNA against AR or Cav-1 were used to test whether diminished expression of one affected the other. Co-sedimentation of AR and Cav-1 was used to test their association. A reporter assay for AR genomic activity was utilized following Cav-1 knockdown. AR-expressing LNCaP and 22Rv1 cells had low endogenous Cav-1 mRNA and protein. Cell lines that expressed little or no AR (DU145, PC3, ALVA41, and RWPE1) expressed high endogenous levels of Cav-1. AR knockdown in LNCaP cells had little effect on Cav-1, but Cav-1 knockdown inhibited AR expression and genomic activity. These data show endogenous AR and Cav-1 mRNA and protein expression is inversely related in PCa cells, with Cav-1 acting on the androgen/AR signaling axis possibly as an AR co-activator, demonstrated by diminished AR genomic activity following Cav-1 knockdown. © 2013 Wiley Periodicals, Inc.

Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

PubMed

M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2016-11-01

The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers. Copyright © 2016 Elsevier GmbH. All rights reserved.

Integrated and comparative proteomics of high-oil and high-protein soybean seeds.

PubMed

Xu, Xiu Ping; Liu, Hui; Tian, Lihong; Dong, Xiang Bai; Shen, Shi Hua; Qu, Le Qing

2015-04-01

We analysed the global protein expression in seeds of a high-oil soybean cultivar (Jiyu 73, JY73) by proteomics. More than 700 protein spots were detected and 363 protein spots were successfully identified. Comparison of the protein profile of JY73 with that of a high-protein cultivar (Zhonghuang 13, ZH13) revealed 40 differentially expressed proteins, including oil synthesis, redox/stress, hydrolysis and storage-related proteins. All redox/stress proteins were less or not expressed in JY73, whereas the expression of the major storage proteins, nitrogen and carbon metabolism-related proteins was higher in ZH13. Biochemical analysis of JY73 revealed that it was in a low oxidation state, with a high content of polyunsaturated fatty acids and vitamin E. Vitamin E was more active than antioxidant enzymes and protected the soybean seed in a lower oxidation state. The characteristics of high oil and high protein in soybean, we revealed, might provide a reference for soybean nutrition and soybean breeding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative proteomic expression profile in all-trans retinoic acid differentiated neuroblastoma cell line.

PubMed

Cimmino, Flora; Spano, Daniela; Capasso, Mario; Zambrano, Nicola; Russo, Roberta; Zollo, Massimo; Iolascon, Achille

2007-07-01

Neuroblastoma (NB) is an infant tumor which frequently differentiates into neurons. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to analyze the cytosolic and nuclear protein expression patterns of LAN-5 cells following neuronal differentiating agent all-trans-retinoic acid treatment. We identified several candidate proteins, from which G beta2 and Prefoldin 3 may have a role on NB development. These results strength the use of proteomics to discover new putative protein targets in cancer.

Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats

PubMed Central

2012-01-01

Background Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR). Results Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril. Conclusion Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension. PMID:22416803

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.

PubMed

Ghaffari, Abdi; Li, Yunyaun; Karami, Ali; Ghaffari, Mazyar; Tredget, Edward E; Ghahary, Aziz

2006-05-15

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions. Copyright 2006 Wiley-Liss, Inc.

Heterogeneous expression pattern of tandem duplicated sHsps genes during fruit ripening in two tomato species

NASA Astrophysics Data System (ADS)

Arce, DP; Krsticevic, FJ; Ezpeleta, J.; Ponce, SD; Pratta, GR; Tapia, E.

2016-04-01

The small heat shock proteins (sHSPs) have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the gene expression profile of four sHsps with a tandem gene structure arrangement in the domesticated Solanum lycopersicum (Heinz 1706) genome and its wild close relative Solanum pimpinellifolium (LA1589), differential gene expression analysis using RNA-Seq was conducted in three ripening stages in both cultivars fruits. Gene promoter analysis was performed to explain the heterogeneous pattern of gene expression found for these tandem duplicated sHsps. In silico analysis results contribute to refocus wet experiment analysis in tomato sHsp family proteins.

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu

2013-02-15

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomicmore » approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more diverse spectrum of CYP isozymes than male. ► First data showing expression of 2F1, 2S1, 2W1, 4A22, 4X1, 26A1 on a protein level.« less

Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

PubMed Central

Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

2013-01-01

Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

Polysome Profiling in Leishmania, Human Cells and Mouse Testis.

PubMed

Karamysheva, Zemfira N; Tikhonova, Elena B; Grozdanov, Petar N; Huffman, James C; Baca, Kristen R; Karamyshev, Alexander; Denison, R Brian; MacDonald, Clinton C; Zhang, Kai; Karamyshev, Andrey L

2018-04-08

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

CsGOGAT Is Important in Dynamic Changes of Theanine Content in Postharvest Tea Plant Leaves under Different Temperature and Shading Spreadings.

PubMed

Liu, Zhi-Wei; Li, Hui; Wang, Wen-Li; Wu, Zhi-Jun; Cui, Xin; Zhuang, Jing

2017-11-08

We analyzed the changes of theanine content in postharvest tea leaves under high temperature (38 °C), low temperature (4 °C), and shading spreadings by using ultrahigh-performance liquid chromatography. The differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT, which are involved in theanine biosynthesis pathway, were identified from the corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology. The expression profiles of 17 genes in theanine metabolism, including CsFd-GOGAT and CsNADH-GOGAT, were analyzed by quantitative real-time polymerase chain reaction. The correlations between the dynamic changes of theanine content and expression profiles of related genes and DEPs were analyzed. This study preliminarily proved the importance of CsGOGAT in dynamic changes of theanine content in postharvest tea leaves during spreading.

Genome-Wide Investigation and Expression Profiling of HD-Zip Transcription Factors in Foxtail Millet (Setaria italica L.).

PubMed

Chai, Wenbo; Si, Weina; Ji, Wei; Qin, Qianqian; Zhao, Manli; Jiang, Haiyang

2018-01-01

HD-Zip proteins represent the major transcription factors in higher plants, playing essential roles in plant development and stress responses. Foxtail millet is a crop to investigate the systems biology of millet and biofuel grasses and the HD-Zip gene family has not been studied in foxtail millet. For further investigation of the expression profile of the HD-Zip gene family in foxtail millet, a comprehensive genome-wide expression analysis was conducted in this study. We found 47 protein-encoding genes in foxtail millet using BLAST search tools; the putative proteins were classified into four subfamilies, namely, subfamilies I, II, III, and IV. Gene structure and motif analysis indicate that the genes in one subfamily were conserved. Promotor analysis showed that HD-Zip gene was involved in abiotic stress. Duplication analysis revealed that 8 (~17%) hdz genes were tandemly duplicated and 28 (58%) were segmentally duplicated; purifying duplication plays important roles in gene expansion. Microsynteny analysis revealed the maximum relationship in foxtail millet-sorghum and foxtail millet-rice. Expression profiling upon the abiotic stresses of drought and high salinity and the biotic stress of ABA revealed that some genes regulated responses to drought and salinity stresses via an ABA-dependent process, especially sihdz29 and sihdz45. Our study provides new insight into evolutionary and functional analyses of HD-Zip genes involved in environmental stress responses in foxtail millet.

Genome-wide analysis of WRKY gene family in Cucumis sativus

PubMed Central

2011-01-01

Background WRKY proteins are a large family of transcriptional regulators in higher plant. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. Prior to the present study, only one full-length cucumber WRKY protein had been reported. The recent publication of the draft genome sequence of cucumber allowed us to conduct a genome-wide search for cucumber WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such as Arabidopsis. Results We identified a total of 55 WRKY genes in the cucumber genome. According to structural features of their encoded proteins, the cucumber WRKY (CsWRKY) genes were classified into three groups (group 1-3). Analysis of expression profiles of CsWRKY genes indicated that 48 WRKY genes display differential expression either in their transcript abundance or in their expression patterns under normal growth conditions, and 23 WRKY genes were differentially expressed in response to at least one abiotic stresses (cold, drought or salinity). The expression profile of stress-inducible CsWRKY genes were correlated with those of their putative Arabidopsis WRKY (AtWRKY) orthologs, except for the group 3 WRKY genes. Interestingly, duplicated group 3 AtWRKY genes appear to have been under positive selection pressure during evolution. In contrast, there was no evidence of recent gene duplication or positive selection pressure among CsWRKY group 3 genes, which may have led to the expressional divergence of group 3 orthologs. Conclusions Fifty-five WRKY genes were identified in cucumber and the structure of their encoded proteins, their expression, and their evolution were examined. Considering that there has been extensive expansion of group 3 WRKY genes in angiosperms, the occurrence of different evolutionary events could explain the functional divergence of these genes. PMID:21955985

Genome-wide analysis of WRKY gene family in Cucumis sativus.

PubMed

Ling, Jian; Jiang, Weijie; Zhang, Ying; Yu, Hongjun; Mao, Zhenchuan; Gu, Xingfang; Huang, Sanwen; Xie, Bingyan

2011-09-28

WRKY proteins are a large family of transcriptional regulators in higher plant. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. Prior to the present study, only one full-length cucumber WRKY protein had been reported. The recent publication of the draft genome sequence of cucumber allowed us to conduct a genome-wide search for cucumber WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such as Arabidopsis. We identified a total of 55 WRKY genes in the cucumber genome. According to structural features of their encoded proteins, the cucumber WRKY (CsWRKY) genes were classified into three groups (group 1-3). Analysis of expression profiles of CsWRKY genes indicated that 48 WRKY genes display differential expression either in their transcript abundance or in their expression patterns under normal growth conditions, and 23 WRKY genes were differentially expressed in response to at least one abiotic stresses (cold, drought or salinity). The expression profile of stress-inducible CsWRKY genes were correlated with those of their putative Arabidopsis WRKY (AtWRKY) orthologs, except for the group 3 WRKY genes. Interestingly, duplicated group 3 AtWRKY genes appear to have been under positive selection pressure during evolution. In contrast, there was no evidence of recent gene duplication or positive selection pressure among CsWRKY group 3 genes, which may have led to the expressional divergence of group 3 orthologs. Fifty-five WRKY genes were identified in cucumber and the structure of their encoded proteins, their expression, and their evolution were examined. Considering that there has been extensive expansion of group 3 WRKY genes in angiosperms, the occurrence of different evolutionary events could explain the functional divergence of these genes.

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

PubMed Central

Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

2013-01-01

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

Network-Induced Classification Kernels for Gene Expression Profile Analysis

PubMed Central

Dror, Gideon; Shamir, Ron

2012-01-01

Abstract Computational classification of gene expression profiles into distinct disease phenotypes has been highly successful to date. Still, robustness, accuracy, and biological interpretation of the results have been limited, and it was suggested that use of protein interaction information jointly with the expression profiles can improve the results. Here, we study three aspects of this problem. First, we show that interactions are indeed relevant by showing that co-expressed genes tend to be closer in the network of interactions. Second, we show that the improved performance of one extant method utilizing expression and interactions is not really due to the biological information in the network, while in another method this is not the case. Finally, we develop a new kernel method—called NICK—that integrates network and expression data for SVM classification, and demonstrate that overall it achieves better results than extant methods while running two orders of magnitude faster. PMID:22697242

Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells.

PubMed

Mlakar, Vid; Todorovic, Vesna; Cemazar, Maja; Glavac, Damjan; Sersa, Gregor

2009-08-26

Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses on human malignant melanoma cells in vitro in order to understand and predict the possible effect of electric pulses on gene expression and their possible effect on cell behaviour. We used microarrays with 2698 different oligonucleotides to obtain the expression profile of genes involved in apoptosis and cancer development in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Cells exposed to ECT pulses showed a 68.8% average survival rate, while cells exposed to EGT pulses showed a 31.4% average survival rate. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce an HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Our results show that electroporation does not significantly change the expression profile of major tumour suppressor genes or oncogenes of the cell cycle. Moreover, electroporation also does not changes the expression of genes involved in the stability of DNA, supporting current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method, it does to some extent induce stress, which resulted in the expression of the environmental stress response mechanism, HSP70.

Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

PubMed

Gamboa, Maribet; Tsuchiya, Maria Claret; Matsumoto, Suguru; Iwata, Hisato; Watanabe, Kozo

2017-11-01

Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies. © 2017 Wiley Periodicals, Inc.

Proteomic analysis on the alteration of protein expression in the early-stage placental villous tissue of electromagnetic fields associated with cell phone exposure.

PubMed

Luo, Qiong; Jiang, Ying; Jin, Min; Xu, Jian; Huang, He-Feng

2013-09-01

To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified- procollagen-proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects.

A Comparison of the Costs and Benefits of Bacterial Gene Expression

DOE PAGES

Price, Morgan N.; Wetmore, Kelly M.; Deutschbauer, Adam M.; ...

2016-10-06

In order to study how a bacterium allocates its resources, we compared the costs and benefits of most (86%) of the proteins in Escherichia coli K-12 during growth in minimal glucose medium. The cost or investment in each protein was estimated from ribosomal profiling data, and the benefit of each protein was measured by assaying a library of transposon mutants. We found that proteins that are important for fitness are usually highly expressed, and 95% of these proteins are expressed at above 13 parts per million (ppm). Conversely, proteins that do not measurably benefit the host (with a benefit ofmore » less than 5% per generation) tend to be weakly expressed, with a median expression of 13 ppm. In aggregate, genes with no detectable benefit account for 31% of protein production, or about 22% if we correct for genetic redundancy. Though some of the apparently unnecessary expression could have subtle benefits in minimal glucose medium, the majority of the burden is due to genes that are important in other conditions. We propose that at least 13% of the cell's protein is on standby in case conditions change.« less

A Comparison of the Costs and Benefits of Bacterial Gene Expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Wetmore, Kelly M.; Deutschbauer, Adam M.

In order to study how a bacterium allocates its resources, we compared the costs and benefits of most (86%) of the proteins in Escherichia coli K-12 during growth in minimal glucose medium. The cost or investment in each protein was estimated from ribosomal profiling data, and the benefit of each protein was measured by assaying a library of transposon mutants. We found that proteins that are important for fitness are usually highly expressed, and 95% of these proteins are expressed at above 13 parts per million (ppm). Conversely, proteins that do not measurably benefit the host (with a benefit ofmore » less than 5% per generation) tend to be weakly expressed, with a median expression of 13 ppm. In aggregate, genes with no detectable benefit account for 31% of protein production, or about 22% if we correct for genetic redundancy. Though some of the apparently unnecessary expression could have subtle benefits in minimal glucose medium, the majority of the burden is due to genes that are important in other conditions. We propose that at least 13% of the cell's protein is on standby in case conditions change.« less

The transcriptional response of Escherichia coli to recombinant protein insolubility.

PubMed

Smith, Harold E

2007-03-01

Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

PubMed

Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

2014-04-01

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

The protein expression landscape of the Arabidopsis root

PubMed Central

Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

2012-01-01

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*

PubMed Central

Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

2015-01-01

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues. PMID:26106084

The effect of disease on human cardiac protein expression profiles in paired samples from right and left ventricles

PubMed Central

2014-01-01

Background Cardiac diseases (e.g. coronary and valve) are associated with ventricular cellular remodeling. However, ventricular biopsies from left and right ventricles from patients with different pathologies are rare and thus little is known about disease-induced cellular remodeling in both sides of the heart and between different diseases. We hypothesized that the protein expression profiles between right and left ventricles of patients with aortic valve stenosis (AVS) and patients with coronary artery disease (CAD) are different and that the protein profile is different between the two diseases. Left and right ventricular biopsies were collected from patients with either CAD or AVS. The biopsies were processed for proteomic analysis using isobaric tandem mass tagging and analyzed by reverse phase nano-LC-MS/MS. Western blot for selected proteins showed strong correlation with proteomic analysis. Results Proteomic analysis between ventricles of the same disease (intra-disease) and between ventricles of different diseases (inter-disease) identified more than 500 proteins detected in all relevant ventricular biopsies. Comparison between ventricles and disease state was focused on proteins with relatively high fold (±1.2 fold difference) and significant (P < 0.05) differences. Intra-disease protein expression differences between left and right ventricles were largely structural for AVS patients and largely signaling/metabolism for CAD. Proteins commonly associated with hypertrophy were also different in the AVS group but with lower fold difference. Inter-disease differences between left ventricles of AVS and CAD were detected in 9 proteins. However, inter-disease differences between the right ventricles of CAD and AVS patients were associated with differences in 73 proteins. The majority of proteins which had a significant difference in one ventricle compared to the other pathology also had a similar trend in the adjacent ventricle. Conclusions This work demonstrates for the first time that left and right ventricles have a different proteome and that the difference is dependent on the type of disease. Inter-disease differential expression was more prominent for right ventricles. The finding that a protein change in one ventricle was often associated with a similar trend in the adjacent ventricle for a large number of proteins suggests cross-talk proteome remodeling between adjacent ventricles. PMID:25249829

Proteomic Analysis of Interactions between a Deep-Sea Thermophilic Bacteriophage and Its Host at High Temperature ▿ †

PubMed Central

Wei, Dahai; Zhang, Xiaobo

2010-01-01

The virus-host interaction is essential to understanding the role that viruses play in ecological and geochemical processes in deep-sea vent ecosystems. Virus-induced changes in cellular gene expression and host physiology have been studied extensively. However, the molecular mechanism of interaction between a bacteriophage and its host at high temperature remains poorly understood. In the present study, the virus-induced gene expression profile of Geobacillus sp. E263, a thermophile isolated from a deep-sea hydrothermal ecosystem, was characterized. Based on proteomic analysis and random arbitrarily primed PCR (RAP-PCR) of Geobacillus sp. E263 cultured under non-bacteriophage GVE2 infection and GVE2 infection conditions, there were two types of protein/gene profiles in response to GVE2 infection. Twenty differentially expressed genes and proteins were revealed that could be grouped into 3 different categories based on cellular function, suggesting a coordinated response to infection. These differentially expressed genes and proteins were further confirmed by Northern blot analysis. To characterize the host proteins in response to virus infection, aspartate aminotransferase (AST) was inactivated to construct the AST mutant of Geobacillus sp. E263. The results showed that the AST protein was essential in virus infection. Thus, transcriptional and proteomic analyses and functional analysis revealed previously unknown host responses to deep-sea thermophilic virus infection. PMID:20015994

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients.

PubMed

Schlick, Bettina; Massoner, Petra; Lueking, Angelika; Charoentong, Pornpimol; Blattner, Mirjam; Schaefer, Georg; Marquart, Klaus; Theek, Carmen; Amersdorfer, Peter; Zielinski, Dirk; Kirchner, Matthias; Trajanoski, Zlatko; Rubin, Mark A; Müllner, Stefan; Schulz-Knappe, Peter; Klocker, Helmut

2016-01-01

Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients

PubMed Central

Schlick, Bettina; Massoner, Petra; Lueking, Angelika; Charoentong, Pornpimol; Blattner, Mirjam; Schaefer, Georg; Marquart, Klaus; Theek, Carmen; Amersdorfer, Peter; Zielinski, Dirk; Kirchner, Matthias; Trajanoski, Zlatko; Rubin, Mark A.; Müllner, Stefan; Schulz-Knappe, Peter; Klocker, Helmut

2016-01-01

Background Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Methods Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Results Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). Conclusions We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation. PMID:26863016

Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

PubMed Central

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest. PMID:25774904

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

PubMed

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

The Rice B-Box Zinc Finger Gene Family: Genomic Identification, Characterization, Expression Profiling and Diurnal Analysis

PubMed Central

Huang, Jianyan; Zhao, Xiaobo; Weng, Xiaoyu; Wang, Lei; Xie, Weibo

2012-01-01

Background The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now. Methodology/Principal Findings In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern. Conclusions/Significance The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes. PMID:23118960

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection

PubMed Central

De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain

2016-01-01

ABSTRACT Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete’s foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates. PMID:27822542

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

PubMed

Tran, Van Du T; De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain; Pagni, Marco; Monod, Michel

2016-01-01

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae . Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum . IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae . Comparing gene expression during infection on guinea pigs with keratin degradation in vitro , which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo , encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

Comparative prion disease gene expression profiling using the prion disease mimetic, cuprizone

PubMed Central

Moody, Laura R; Herbst, Allen J; Yoo, Han Sang; Vanderloo, Joshua P

2009-01-01

Identification of genes expressed in response to prion infection may elucidate biomarkers for disease, identify factors involved in agent replication, mechanisms of neuropathology and therapeutic targets. Although several groups have sought to identify gene expression changes specific to prion disease, expression profiles rife with cell population changes have consistently been identified. Cuprizone, a neurotoxicant, qualitatively mimics the cell population changes observed in prion disease, resulting in both spongiform change and astrocytosis. The use of cuprizone-treated animals as an experimental control during comparative expression profiling allows for the identification of transcripts whose expression increases during prion disease and remains unchanged during cuprizone-triggered neuropathology. In this study, expression profiles from the brains of mice preclinically and clinically infected with Rocky Mountain Laboratory (RML) mouse-adapted scrapie agent and age-matched controls were profiled using Affymetrix gene arrays. In total, 164 genes were differentially regulated during prion infection. Eighty-three of these transcripts have been previously undescribed as differentially regulated during prion disease. A 0.4% cuprizone diet was utilized as a control for comparative expression profiling. Cuprizone treatment induced spongiosis and astrocyte proliferation as indicated by glial fibrillary acidic protein (Gfap) transcriptional activation and immunohistochemistry. Gene expression profiles from brain tissue obtained from cuprizone-treated mice identified 307 differentially regulated transcript changes. After comparative analysis, 17 transcripts unaffected by cuprizone treatment but increasing in expression from preclinical to clinical prion infection were identified. Here we describe the novel use of the prion disease mimetic, cuprizone, to control for cell population changes in the brain during prion infection. PMID:19535908

Proteomics Analysis of Lactobacillus casei Zhang, a New Probiotic Bacterium Isolated from Traditional Home-made Koumiss in Inner Mongolia of China*

PubMed Central

Wu, Rina; Wang, Weiwei; Yu, Dongliang; Zhang, Wenyi; Li, Yan; Sun, Zhihong; Wu, Junrui; Meng, He; Zhang, Heping

2009-01-01

Lactobacillus casei Zhang, isolated from traditional home-made koumiss in Inner Mongolia of China, was considered as a new probiotic bacterium by probiotic selection tests. We carried out a proteomics study to identify and characterize proteins expressed by L. casei Zhang in the exponential phase and stationary phase. Cytosolic proteins of the strain cultivated in de Man, Rogosa, and Sharpe broth were resolved by two-dimensional gel electrophoresis using pH 4–7 linear gradients. The number of protein spots quantified from the gels was 487 ± 21 (exponential phase) and 494 ± 13 (stationary phase) among which a total of 131 spots were identified by MALDI-TOF/MS and/or MALDI-TOF/TOF according to significant growth phase-related differences or high expression intensity proteins. Accompanied by the cluster of orthologous groups (COG), codon adaptation index (CAI), and GRAVY value analysis, the study provided a very first insight into the profile of protein expression as a reference map of L. casei. Forty-seven spots were also found in the study that showed statistically significant differences between exponential phase and stationary phase. Thirty-three of the spots increased at least 2.5-fold in the stationary phase in comparison with the exponential phase, including 19 protein spots (e.g. Hsp20, DnaK, GroEL, LuxS, pyruvate kinase, and GalU) whose intensity up-shifted above 3.0-fold. Transcriptional profiles were conducted to confirm several important differentially expressed proteins by using real time quantitative PCR. The analysis suggests that the differentially expressed proteins were mainly categorized as stress response proteins and key components of central and intermediary metabolism, indicating that these proteins might play a potential important role for the adaptation to the surroundings, especially the accumulation of lactic acid in the course of growth, and the physiological processes in bacteria cell. PMID:19508964

Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx.

PubMed

Xie, Hai-Yang; Cheng, Jun; Xing, Chun-Yang; Wang, Jin-Jin; Su, Rong; Wei, Xu-Yong; Zhou, Lin; Zheng, Shu-Sen

2011-06-01

Hepatitis B virus (HBV) is one of the major pathogens of human liver disease. Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication. In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx, thereby detecting the mechanisms of action of HBx on virion replication. HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA). The replication level of HBV was evaluated by microparticle enzyme immunoassay analysis of HBsAg and HBeAg in the culture supernatant, and real-time quantitative PCR analysis of HBV DNA. Two-dimensional electrophoresis combined with MALDI-TOF/TOF was performed to analyze the changes in protein expression profile after treatment with HBx siRNA. Knockdown of HBx disturbed HBV replication in vitro. HBx target siRNA significantly inhibited the expression of HBsAg, HBeAg and the replication of HBV DNA. Twelve significantly changed proteins (7 upregulated and 5 downregulated) were successfully identified by MALDI-TOF/TOF using proteomics differential expression analysis after the knockdown of HBx. Among these identified proteins, HSP70 was validated by Western blotting. The results of the study indicated the positive effect of HBx on HBV replication, and a group of downstream target proteins of HBx may be responsible for this effect.

Light of DNA-alkylating agents in castration-resistant prostate cancer cells: a novel mixed EGFR/DNA targeting combi-molecule.

PubMed

Liang, Guan-Can; Zheng, Hao-Feng; Chen, Yan-Xiong; Li, Teng-Cheng; Liu, Wei; Fang, You-Qiang

2017-01-01

The mechanism underlying the therapeutic effects of combi-molecule JDF12 on prostate cancer (PCa) DU145 cells remains still unclear. This study aimed to investigate the proteomic profile after JDF12 treatment in DU145 cells by comparing with that in Iressa treated cells and untreated cells. MTT was used to evaluate drug cytotoxicity, DAPI staining was done to assess apoptosis of cells, and flow cytometry was used to analyze cell cycle. iTRAQ and qPCR were employed to obtain the proteomic profiles of JDF12 treated, Iressa treated, and untreated DU145 cells, and validate the expression of selected differentially expressed proteins, respectively. JDF12 could significantly inhibit the proliferation and increase the apoptosis of DU145 cells when compared with Iressa or blank group. In total, 5071 proteins were obtained, out of which, 42, including 21 up-regulated and 21 down-regulated proteins, were differentially expressed in JDF12 group when compared with Iressa and blank groups. The up-regulated proteins were mainly involved in DNA damage/repair and energy metabolism; while the down-regulated proteins were mainly associated with cell apoptosis. qPCR confirmed the expression of several biologically important proteins in DU145 cells after JDF12 treatment. The molecular mechanisms of DNA alkylating agents on PCa therapy that with the assistant of EGFR-blocker were revealed on proteomic level, which may increase the possible applications of DNA alkylating agents and JDF12 on PCa therapy.

Expression profile and ligand-binding characterization of odorant-binding protein 2 in Batocera horsfieldi (Hope)

USDA-ARS?s Scientific Manuscript database

Odorant-binding proteins (OBPs) are important components in insect olfactory systems that transport semiochemicals through the aqueous sensillum lymph to surface of olfactory receptor neurons. In this study, we cloned the cDNA of odorant-binding protein 2 (BhorOBP2) in Batocera horsfieldi (Hope) and...

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Serum proteomic profiling for autism using magnetic bead-assisted matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a pilot study.

PubMed

Chen, Yan-Ni; Du, Hui-Ying; Shi, Zhuo-Yue; He, Li; He, Yu-Ying; Wang, Duan

2018-01-24

The pathogenesis of autism spectrum disorders remains elusive and currently there are no diagnostic or predictive biomarkers in autism available. Proteomic profiling has been used in a wide range of neurodevelopmental disorder studies, which could produce deeper perceptions of the molecular bases behind certain disease and potentially becomes useful in discovering biomarkers in autism spectrum disorders. Serum samples were collected from autistic children about 3 years old in age (n = 32) and healthy controls (n = 20) in similar age and gender. The samples were identified specific proteins that are differentially expressed by magnetic bead-based pre-fractionation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Eight protein peaks were significantly different in autistic children from the healthy controls (P < 0.0001). The two peaks with the most significant differences were 6428 and 7758 Da in size. According to differences in serum protein profiles between the autistic children and healthy controls, this study identified a set of differentially expressed proteins those are significant for further evaluation and might function as biomarkers in autism.

Proteomic signatures differentiating Bacillus anthracis Sterne sporulation on soil relative to laboratory media.

PubMed

Wunschel, D S; Hutchison, J R; Deatherage Kaiser, B L; Merkley, E D; Hess, B M; Lin, A; Warner, M G

2017-12-18

The process of sporulation is vital for the stability and infectious cycle of Bacillus anthracis. The spore is the infectious form of the organism and therefore relevant to biodefense. While the morphological and molecular events occurring during sporulation have been well studied, the influence of growth medium and temperature on the proteins expressed in sporulated cultures is not well understood. Understanding the features of B. anthracis sporulation specific to natural vs. laboratory production will address an important question in microbial forensics. In an effort to bridge this knowledge gap, a system for sporulation on two types of agar-immobilized soils was used for comparison to cultures sporulated on two common types of solid laboratory media, and one liquid sporulation medium. The total number of proteins identified as well as their identity differed between samples generated in each medium and growth temperature, demonstrating that sporulation environment significantly impacts the protein content of the spore. In addition, a subset of proteins common in all of the soil-cultivated samples was distinct from the expression profiles in laboratory medium (and vice versa). These differences included proteins involved in thiamine and phosphate metabolism in the sporulated cultures produced on soils with a notable increase in expression of ATP binding cassette (ABC) transporters annotated to be for phosphate and antimicrobial peptides. A distinct set of ABC transporters for amino acids, sugars and oligopeptides were found in cultures produced on laboratory media as well as increases in carbon and amino acid metabolism-related proteins. These protein expression changes indicate that the sporulation environment impacts the protein profiles in specific ways that are reflected in the metabolic and membrane transporter proteins present in sporulated cultures.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Bo; Huang Bo; School of Public Health, University of South China, Hengyang, Hunan 421001

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe,more » such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.« less

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE.

PubMed

Michaux, Charlotte; Holmqvist, Erik; Vasicek, Erin; Sharan, Malvika; Barquist, Lars; Westermann, Alexander J; Gunn, John S; Vogel, Jörg

2017-06-27

The functions of many bacterial RNA-binding proteins remain obscure because of a lack of knowledge of their cellular ligands. Although well-studied cold-shock protein A (CspA) family members are induced and function at low temperature, others are highly expressed in infection-relevant conditions. Here, we have profiled transcripts bound in vivo by the CspA family members of Salmonella enterica serovar Typhimurium to link the constitutively expressed CspC and CspE proteins with virulence pathways. Phenotypic assays in vitro demonstrated a crucial role for these proteins in membrane stress, motility, and biofilm formation. Moreover, double deletion of cspC and cspE fully attenuates Salmonella in systemic mouse infection. In other words, the RNA ligand-centric approach taken here overcomes a problematic molecular redundancy of CspC and CspE that likely explains why these proteins have evaded selection in previous virulence factor screens in animals. Our results highlight RNA-binding proteins as regulators of pathogenicity and potential targets of antimicrobial therapy. They also suggest that globally acting RNA-binding proteins are more common in bacteria than currently appreciated.

Multiplexed quantitation of protein expression and phosphorylation based on functionalized soluble nanopolymers

PubMed Central

Pan, Li; Iliuk, Anton; Yu, Shuai; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity, and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events. PMID:23088311

FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues.

PubMed

El-Manzalawy, Yasser; Abbas, Mostafa; Malluhi, Qutaibah; Honavar, Vasant

2016-01-01

A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM)-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles). Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein and protein-DNA interfaces.

Single-cell transcriptional analysis of taste sensory neuron pair in Caenorhabditis elegans.

PubMed

Takayama, Jun; Faumont, Serge; Kunitomo, Hirofumi; Lockery, Shawn R; Iino, Yuichi

2010-01-01

The nervous system is composed of a wide variety of neurons. A description of the transcriptional profiles of each neuron would yield enormous information about the molecular mechanisms that define morphological or functional characteristics. Here we show that RNA isolation from single neurons is feasible by using an optimized mRNA tagging method. This method extracts transcripts in the target cells by co-immunoprecipitation of the complexes of RNA and epitope-tagged poly(A) binding protein expressed specifically in the cells. With this method and genome-wide microarray, we compared the transcriptional profiles of two functionally different neurons in the main C. elegans gustatory neuron class ASE. Eight of the 13 known subtype-specific genes were successfully detected. Additionally, we identified nine novel genes including a receptor guanylyl cyclase, secreted proteins, a TRPC channel and uncharacterized genes conserved among nematodes, suggesting the two neurons are substantially different than previously thought. The expression of these novel genes was controlled by the previously known regulatory network for subtype differentiation. We also describe unique motif organization within individual gene groups classified by the expression patterns in ASE. Our study paves the way to the complete catalog of the expression profiles of individual C. elegans neurons.

Progression Rate Associated Peripheral Blood Biomarkers of Parkinson's Disease.

PubMed

Fan, Yanxia; Xiao, Shuping

2018-06-23

Parkinson disease (PD) is one of the most frequent neurodegenerative disorders. The aim of this study was to identify blood biomarkers capable to discriminate PD patients with different progression rates. Differentially expressed genes (DEGs) were acquired by comparing the expression profiles of PD patients with rapid and slow progression rates, using an expression dataset from Gene Expression Omnibus (GEO) under accession code of GSE80599. Altered biological processes and pathways were revealed by functional annotation. Potential biomarkers of PD were identified by protein-protein interaction (PPI) network analysis. Critical transcription factors (TFs) and miRNAs regulating DEGs were predicted by TF analysis and miRNA analysis. A total of 225 DEGs were identified between PD patients with rapid and slow progression profiles. These genes were significantly enriched in biological processes and pathways related to fatty acid metabolism. Among these DEGs, ZFAND4, SRMS, UBL4B, PVALB, DIRAS1, PDP2, LRCH1, and MYL4 were potential progression rate associated biomarkers of PD. Additionally, these DEGs may be regulated by miRNAs of the miR-30 family and TFs STAT1 and GRHL3. Our results may contribute to our understanding of the molecular mechanisms underlying different PD progression profiles.

Expression profiles of adult T-cell leukemia–lymphoma and associations with clinical responses to zidovudine and interferon α

PubMed Central

ALIZADEH, ASH A.; BOHEN, SEAN P.; LOSSOS, CHEN; MARTINEZ-CLIMENT, JOSE A.; RAMOS, JUAN CARLOS; CUBEDO-GIL, ELENA; HARRINGTON, WILLIAM J.; LOSSOS, IZIDORE S.

2014-01-01

Adult T-cell leukemia–lymphoma (ATLL) is an HTLV-1-associated lymphoproliferative malignancy that is frequently fatal. We compared gene expression profiles (GEPs) of leukemic specimens from nine patients with ATLL at the time of diagnosis and immediately after combination therapy with zidovudine (AZT) and interferon α (IFNα). GEPs were also related to genetic aberrations determined by comparative genomic hybridization. We identified several genes anomalously over-expressed in the ATLL leukemic cells at the mRNA level, including LYN, CSPG2, and LMO2, and confirmed LMO2 expression in ATLL cells at the protein level. In vivo AZT–IFNα therapy evoked a marked induction of interferon-induced genes accompanied by repression of cell-cycle regulated genes, including those encoding ribosomal proteins. Remarkably, patients not responding to AZT–IFNα differed most from responding patients in lower expression of these same IFN-responsive genes, as well as components of the antigen processing and presentation apparatus. Demonstration of specific gene expression signatures associated with response to AZT–IFNα therapy may provide novel insights into the mechanisms of action in ATLL. PMID:20370541

Detailed expression profile of the six Glypicans and their modifying enzyme, Notum during chick limb and feather development.

PubMed

Saad, Kawakeb; Theis, Susanne; Otto, Anthony; Luke, Graham; Patel, Ketan

2017-04-30

The development of vertebrate appendages, especially the limb and feather buds are orchestrated by numerous secreted signalling molecules including Sonic Hedgehog, Bone Morphogenetic Proteins, Fibroblast Growth Factors and Wnts. These proteins coordinate the growth and patterning of ectodermal and mesenchymal cells. The influence of signalling molecules is affected over large distances by their concentration (morphogen activity) but also at local levels by the presence of proteins that either attenuate or promote their activity. Glypicans are cell surface molecules that regulate the activity of the major secreted signalling molecules expressed in the limb and feather bud. Here we investigated the expression of all Glypicans during chick limb and feather development. In addition we profiled the expression of Notum, an enzyme that regulates Glypican activity. We show that five of the six Glypicans and Notum are expressed in a dynamic manner during the development of limbs and feathers. We also investigated the expression of key Glypicans and show that they are controlled by signalling molecules highlighting the presence of feedback loops. Lastly we show that Glypicans and Notum are expressed in a tissue specific manner in adult chicken tissues. Our results strongly suggest that the Glypicans and Notum have many as yet undiscovered roles to play during the development of vertebrate appendages. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrophoretic characterisation of the outer membrane proteins of Yersinia pestis isolated in north-east Brazil.

PubMed Central

Abath, F. G.; Almeida, A. M.; Ferreira, L. C.

1989-01-01

The outer membrane proteins of 38 Yersinia pestis isolates from all known plague foci of north-east Brazil were analysed by SDS-PAGE. Approximately 20 bands were consistently found in all strains analysed and 11 were selected for comparative studies. Although qualitative differences among the electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates were not observed, quantitative alterations were clearly noted for most of these proteins. No particular quantitative alteration of the electrophoretic profile of outer membrane proteins could be associated with the period of isolation and geographic origin of the isolates. The 64 kDa outer membrane protein was significantly expressed in higher amounts among Y. pestis strains isolated from a recent plague outbreak. The possible use of electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates as a tool for epidemiological studies and for the analysis of virulence determinants is discussed. Images Fig. 2 PMID:2606164

Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation

PubMed Central

Tang, Guo-Qing; Maxwell, E. Stuart

2008-01-01

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus. PMID:18032731

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

PubMed

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

Early Lung Cancer Detection via Global Protein Modification Profiles

DTIC Science & Technology

2013-12-01

Increased DNMT1 protein expression has also been shown to play a critical role in the malignant progression of hepatocellular carcinoma (HCC) and be a...the Malignant Potential and Poor Prognosis of Human Hepatocellular Carcinomas , Int. J. Cancer, 105:527-532.

Simultaneous measurement of multiple radiation-induced protein expression profiles using the Luminex(TM) system

NASA Technical Reports Server (NTRS)

Desai, N.; Wu, H.; George, K.; Gonda, S. R.; Cucinotta, F. A.; Cucniotta, F. A. (Principal Investigator)

2004-01-01

Space flight results in the exposure of astronauts to a mixed field of radiation composed of energetic particles of varying energies, and biological indicators of space radiation exposure provides a better understanding of the associated long-term health risks. Current methods of biodosimetry have employed the use of cytogenetic analysis for biodosimetry, and more recently the advent of technological progression has led to advanced research in the use of genomic and proteomic expression profiling to simultaneously assess biomarkers of radiation exposure. We describe here the technical advantages of the Luminex(TM) 100 system relative to traditional methods and its potential as a tool to simultaneously profile multiple proteins induced by ionizing radiation. The development of such a bioassay would provide more relevant post-translational dynamics of stress response and will impart important implications in the advancement of space and other radiation contact monitoring. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

PubMed Central

Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

2016-01-01

Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

Genome-wide identification of translationally inhibited and degraded miR-155 targets using RNA-interacting protein-IP

PubMed Central

Meier, Jan; Hovestadt, Volker; Zapatka, Marc; Pscherer, Armin; Lichter, Peter; Seiffert, Martina

2013-01-01

MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity. PMID:23673373

Differential Expression Patterns in Chemosensory and Non-Chemosensory Tissues of Putative Chemosensory Genes Identified by Transcriptome Analysis of Insect Pest the Purple Stem Borer Sesamia inferens (Walker)

PubMed Central

Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

2013-01-01

Background A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. Methodology/Principal Findings We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Conclusion Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects. PMID:23894529

Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker).

PubMed

Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

2013-01-01

A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects.

Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

PubMed Central

Lichtenfels, Rudolf; Mougiakakos, Dimitrios; Johansson, C. Christian; Dressler, Sven P.; Recktenwald, Christian V.; Kiessling, Rolf; Seliger, Barbara

2012-01-01

The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA+ and their memory/effector CD45RO+ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H2O2) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA+ and CD45RO+ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress. PMID:22911781

Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

PubMed

Baev, Vesselin; Milev, Ivan; Naydenov, Mladen; Vachev, Tihomir; Apostolova, Elena; Mehterov, Nikolay; Gozmanva, Mariyana; Minkov, Georgi; Sablok, Gaurav; Yahubyan, Galina

2014-11-01

Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

PubMed

Deng, Wei-Wei; Sasamoto, Hamako; Ashihara, Hiroshi

2015-05-01

It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were β-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.

PubMed

Martínez, Cristina; Rodiño-Janeiro, Bruno K; Lobo, Beatriz; Stanifer, Megan L; Klaus, Bernd; Granzow, Martin; González-Castro, Ana M; Salvo-Romero, Eloisa; Alonso-Cotoner, Carmen; Pigrau, Marc; Roeth, Ralph; Rappold, Gudrun; Huber, Wolfgang; González-Silos, Rosa; Lorenzo, Justo; de Torres, Inés; Azpiroz, Fernando; Boulant, Steeve; Vicario, María; Niesler, Beate; Santos, Javier

2017-09-01

Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs. Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells. IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins. Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses, Hepatic Gene Expression, and Stress Protein Profiling.

PubMed

Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

2016-02-01

In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na(+) -K(+) -ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation-related genes expression and stress-related proteins profiling in fish liver. Oxidative stress indices and Na(+) -K(+) -ATPase showed tissues-specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF-I axis and ion-regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose-dependent manner under TBT stress. In short, TBT-induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long-term experiments at lower concentrations will be necessary in the future. © 2015 Wiley Periodicals, Inc.

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

NASA Astrophysics Data System (ADS)

Petersson, Linn; Berthet Duroure, Nathalie; Auger, Angèle; Dexlin-Mellby, Linda; Borrebaeck, Carl AK; Ait Ikhlef, Ali; Wingren, Christer

2014-07-01

Miniaturized (Ø 10 μm), multiplexed (>5-plex), and high-density (>100 000 spots cm-2) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, BioplumeTM—consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels—to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced—based on miniaturized spot features (78.5 um2, Ø 10 μm) at a 7-125-times increased spot density (250 000 spots cm-2), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.

High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner*

PubMed Central

Kugler, Jamie E.; Horsch, Marion; Huang, Di; Furusawa, Takashi; Rochman, Mark; Garrett, Lillian; Becker, Lore; Bohla, Alexander; Hölter, Sabine M.; Prehn, Cornelia; Rathkolb, Birgit; Racz, Ildikó; Aguilar-Pimentel, Juan Antonio; Adler, Thure; Adamski, Jerzy; Beckers, Johannes; Busch, Dirk H.; Eickelberg, Oliver; Klopstock, Thomas; Ollert, Markus; Stöger, Tobias; Wolf, Eckhard; Wurst, Wolfgang; Yildirim, Ali Önder; Zimmer, Andreas; Gailus-Durner, Valérie; Fuchs, Helmut; Hrabě de Angelis, Martin; Garfinkel, Benny; Orly, Joseph; Ovcharenko, Ivan; Bustin, Michael

2013-01-01

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1tm1/tm1, Hmgn3tm1/tm1, and Hmgn5tm1/tm1 mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgntm1/tm1 lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner. PMID:23620591

Feeding milk replacer instead of whole milk affects blood plasma proteome and lipid profile in preruminant calves.

PubMed

Lepczyński, A; Herosimczyk, A; Ożgo, M; Skrzypczak, W F

2015-01-01

The study was undertaken to determine the effect of feeding milk or milk-replacer on the blood plasma proteome and lipid profile in calves during the second week of life. Feeding milk-replacer significantly decreased the expression of plasma apoA-I. Age of calves affected apoA-I expression, which was higher on the 8th than on the 11th and 14th day of life. A significant effect of interaction between diet and age was also observed. The expression of apoA-IV, was significantly affected by diet and was lower in calves fed milk replacer. Expression of this protein was significantly lower at the 8th day of life and was up-regulated in the calves fed milk-replacer at the second week of life. Calves fed milk-replacer had greater expression of haptoglobin, which differed significantly between days of blood sampling, being higher on the 8th than on the 11th and 14th day. The interactive effect of diet and age affected haptoglobin expression, which was successively down-regulated in calves fed milk re- placer. Diet had a significant effect on the plasma lipid profile. Animals fed milk had a greater concentration of TC, HDLC and LDLC. The composition of milk-replacer, especially fat source, is probably the main factor that affects expression of proteins involved in cholesterol metabolism and level of components of lipid profile in calves fed formula. We claim that the initially increased level of haptoglobin, followed by its decrease during the second week of life in calves fed milk-replacer may indicate the presence of short-term stress induced by changes in the feeding system.

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

PubMed

Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

2016-04-01

The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-)Sca-1(+) CD49f(+) Trop2(high)-phenotype) and human (Lin(-) CD49f(+) TROP2(high)) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+)/TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+)/CD24(+)/CD29(+)/CD44(+)/CD47(+)/CD49f(+)/CD104(+)/CD147(+)/CD326(+)/Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Proteomic profiles of white sucker (Catostomus commersonii) sampled from within the Thunder Bay Area of Concern reveal up-regulation of proteins associated with tumor formation and exposure to environmental estrogens.

PubMed

Simmons, Denina B D; Bols, Niels C; Duncker, Bernard P; McMaster, Mark; Miller, Jason; Sherry, James P

2012-02-07

White sucker (Catostomus commersonii) sampled from the Thunder Bay Area of Concern were assessed for health using a shotgun approach to compile proteomic profiles. Plasma proteins were sampled from male and female fish from a reference location, an area in recovery within Thunder Bay Harbour, and a site at the mouth of the Kaministiquia River where water and sediment quality has been degraded by industrial activities. The proteins were characterized using reverse-phase liquid chromatography tandem to a quadrupole-time-of-flight (LC-Q-TOF) mass spectrometer and were identified by searching in peptide databases. In total, 1086 unique proteins were identified. The identified proteins were then examined by means of a bioinformatics pathway analysis to gain insight into the biological functions and disease pathways that were represented and to assess whether there were any significant changes in protein expression due to sampling location. Female white sucker exhibited significant (p = 0.00183) site-specific changes in the number of plasma proteins that were related to tumor formation, reproductive system disease, and neurological disease. Male fish plasma had a significantly different (p < 0.0001) number of proteins related to neurological disease and tumor formation. Plasma concentrations of vitellogenin were significantly elevated in females from the Kaministiquia River compared to the Thunder Bay Harbour and reference sites. The protein expression profiles indicate that white sucker health has benefited from the remediation of the Thunder Bay Harbour site, whereas white sucker from the Kaministiquia River site are impacted by ongoing contaminant discharges.

Investigation of protein expression profiles of erythritol-producing Candida magnoliae in response to glucose perturbation.

PubMed

Kim, Hyo Jin; Lee, Hyeong-Rho; Kim, Chang Sup; Jin, Yong-Su; Seo, Jin-Ho

2013-08-15

Protein expression patterns of an erythritol-producing yeast, Candida magnoliae, were analyzed to identify differentially expressed proteins in response to glucose perturbation. Specifically, wild type C. magnoliae was grown under high and low glucose conditions and the cells were harvested at both mid-exponential and erythritol production phases for proteomic studies. In order to analyze intracellular protein abundances from the harvested cells quantitatively, total intracellular proteins were extracted and applied to two-dimensional gel electrophoresis for separation and visualization of individual proteins. Among the proteins distributed in the range of pI 4-7 and molecular weight 29-97kDa, five osmo-responsive proteins were drastically changed in response to glucose perturbation. Hsp60 (Heat-shock protein 60), transaldolase and NADH:quinone oxidoreductase were down-regulated under the high glucose condition and Bro1 (BCK1-like Resistance to Osmotic shock) and Eno1 (enolase1) were up-regulated. These proteins are directly or indirectly related with cellular stress response. Importantly, protein expression patterns of Hsp60, Bro1 and Eno1 were strongly correlated with previous studies identifying the proteins perturbed by osmotic stress for other organisms including Saccharomyces cerevisiae. Copyright © 2013 Elsevier Inc. All rights reserved.

Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

PubMed

Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

2012-04-15

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division. Published by Elsevier GmbH.

Chitin enhances biocontrol of Rhodotorula mucilaginosa to postharvest decay of peaches.

PubMed

Zhang, Hongyin; Yang, Qiya; Ge, Lingling; Zhang, Guochao; Zhang, Xiaoli; Zhang, Xiaoyun

2016-07-01

Biological control using microbial antagonists is a promising alternative approach to synthetic fungicides. However, effective biological control requires enhancing the consistency and efficacy of the antagonists used to control postharvest diseases. This study investigated the effect of chitin on the biocontrol efficacy of Rhodotorula mucilaginosa against blue mold and Rhizopus decay of peaches and on the protein expression profiles of R. mucilaginosa. The antagonistic activity of R. mucilaginosa harvested from the nutrient yeast dextrose broth (NYDB) with 0.5% chitin added was significantly improved compared with culture in NYDB without chitin. The R. mucilaginosa population cultured in chitin-supplement NYDB and nutrient yeast chitin borth (NYCB) harvested from peach wounds was more than that of R. mucilaginosa cultured in NYDB without chitin throughout the storage period except at 1 d. The protein expression profiles findings revealed that there were several differentially expressed proteins of R. mucilaginosa in the 0.5% chitin-supplemented NYDB and NYCB compared with that of R. mucilaginosa in NYDB. Most of these were cellular proteomes relating to the primary metabolic reactions such as glycoside hydrolases, phosphoribosyl pyrophosphate, and NADH dehydrogenases. Some proteins were also related to signal transmission and stress response. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao.

PubMed

Niemenak, Nicolas; Kaiser, Edward; Maximova, Siela N; Laremore, Tatiana; Guiltinan, Mark J

2015-05-15

Two dimensional electrophoresis and nano-LC-MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L(-1)) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses. Copyright © 2015 Elsevier GmbH. All rights reserved.

Two-dimensional proteomic analysis of gonads of air-breathing catfish, Clarias batrachus after the exposure of endosulfan and malathion.

PubMed

Laldinsangi, C; Vijayaprasadarao, K; Rajakumar, A; Murugananthkumar, R; Prathibha, Y; Sudhakumari, C C; Mamta, S K; Dutta-Gupta, A; Senthilkumaran, B

2014-05-01

Endocrine disrupting chemicals have raised public concern, since their effects have been found to interfere with the physiological systems of various organisms, especially during critical stage of development and reproduction. Endosulfan and malathion, pesticides widely used for agricultural purposes, have been known to disrupt physiological functions in aquatic organisms. The current work analyzes the effects of endosulfan (2.5 parts per billion [ppb]) and malathion (10 ppb) on the reproductive physiology of catfish (Clarias batrachus) by evaluating protein expression profiles after 21 days of exposure. The proteomic profile of testis and ovary after exposure to endosulfan showed downregulation of proteins such as ubiquitin and Esco2, and upregulation in melanocortin-receptor-2 respectively. Malathion exposed ovary showed upregulated prolactin levels. Identification of proteins differentially expressed in gonads due to the exposure to these pesticides may serve as crucial indications to denote their disruptive effects at the level of proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Nodulation Characterization and Proteomic Profiling of Bradyrhizobium liaoningense CCBAU05525 in Response to Water-Soluble Humic Materials

PubMed Central

Guo Gao, Tong; Yuan Xu, Yuan; Jiang, Feng; Zhen Li, Bao; Shui Yang, Jin; Tao Wang, En; Li Yuan, Hong

2015-01-01

The lignite biodegradation procedure to produce water-soluble humic materials (WSHM) with a Penicillium stain was established by previous studies in our laboratory. This study researched the effects of WSHM on the growth of Bradyrhizobium liaoningense CCBAU05525 and its nodulation on soybean. Results showed that WSHM enhanced the cell density of CCBAU05525 in culture, and increased the nodule number, nodule fresh weight and nitrogenase activity of the inoculated soybean plants. Then the chemical compounds of WSHM were analyzed and flavonoid analogues were identified in WSHM through tetramethyl ammonium hydroxide (TMAH)-py-GC/MS analysis. Protein expression profiles and nod gene expression of CCBAU05525 in response to WSHM or genistein were compared to illustrate the working mechanism of WSHM. The differently expressed proteins in response to WSHM were involved in nitrogen and carbon metabolism, nucleic acid metabolism, signaling, energy production and some transmembrane transports. WSHM was found more effective than genistein in inducing the nod gene expression. These results demonstrated that WSHM stimulated cell metabolism and nutrient transport, which resulted in increased cell density of CCBAU05525 and prepared the bacteria for better bacteroid development. Furthermore, WSHM had similar but superior functions to flavone in inducing nod gene and nitrogen fixation related proteins expression in CCBAU05525. PMID:26054030

Methylation and expression profiles of MGMT gene in thymic epithelial tumors.

PubMed

Mokhtar, Mohamed; Kondo, Kazuya; Namura, Toshiaki; Ali, Abdellah H K; Fujita, Yui; Takai, Chikako; Takizawa, Hiromitsu; Nakagawa, Yasushi; Toba, Hiroaki; Kajiura, Koichiro; Yoshida, Mitsuteru; Kawakami, Gyokei; Sakiyama, Shoji; Tangoku, Akira

2014-02-01

A key challenge in diagnosis and treatment of thymic epithelial tumors (TET) is in improving our understanding of the genetic and epigenetic changes of these relatively rare tumors. Methylation specific PCR (MSP) and immunohistochemistry were applied to 66 TET to profile the methylation status of DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) and its protein expression in TET to clarify the association between MGMT status and clinicopathological features, response to chemotherapy and overall survival. MGMT methylation was significantly more frequent in thymic carcinoma than in thymoma (17/23, 74% versus 13/44, 29%; P<0.001). Loss of expression of MGMT protein was significantly more frequent in thymic carcinoma than in thymoma (20/23, 87% versus 10/44, 23%; P<0.0001). There is a significant correlation between of MGMT methylation and loss of its protein expression (P<0.0003). MGMT methylation and loss of expression were significantly more frequent in advanced thymic epithelial tumors (III/IV) than in early tumors (I/II). MGMT methylation plays a soul role in development of TET, especially in thymic carcinoma. Therefore, translation of our results from basic molecular research to clinical practice may have important implication for considering MGMT methylation as a marker and a target of future therapies in TET. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Simultaneous chromate and sulfate removal by Streptomyces sp. MC1. Changes in intracellular protein profile induced by Cr(VI).

PubMed

Bonilla, José Oscar; Callegari, Eduardo Alberto; Delfini, Claudio Daniel; Estevez, María Cristina; Villegas, Liliana Beatriz

2016-11-01

The purpose of this study was to investigate the influence of increasing sulfate concentrations on chromium removal, to evaluate the effect of the presence of Cr(VI) on sulfate removal by Streptomyces sp. MC1 and to analyze the differential protein expression profile in the presence of this metal for the identification of proteins repressed or overexpressed. In the presence of Cr(VI) but in the absence of sulfate ions, bacterial growth was negligible, showing the Cr(VI) toxicity for this bacterium. However, the sulfate presence stimulated bacterium growth and Cr(VI) removal, regardless of its concentrations. Streptomyces sp. MC1 showed ability to remove chromium and sulfate simultaneously. Also, the sulfate presence favored the decrease of total chromium concentration from supernatants reaching a decrease of 50% at 48 h. In presence of chromium, seven proteins were down-expressed and showed homology to proteins involved in protein biosynthesis, energy production and free radicals detoxification while two proteins involved in oxidation-reduction processes identified as dihydrolipoamide dehydrogenase and S-adenosyl-l-methionine synthase were overexpressed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Growth hormone regulation of metabolic gene expression in muscle: a microarray study in hypopituitary men.

PubMed

Sjögren, Klara; Leung, Kin-Chuen; Kaplan, Warren; Gardiner-Garden, Margaret; Gibney, James; Ho, Ken K Y

2007-07-01

Muscle is a target of growth hormone (GH) action and a major contributor to whole body metabolism. Little is known about how GH regulates metabolic processes in muscle or the extent to which muscle contributes to changes in whole body substrate metabolism during GH treatment. To identify GH-responsive genes that regulate substrate metabolism in muscle, we studied six hypopituitary men who underwent whole body metabolic measurement and skeletal muscle biopsies before and after 2 wk of GH treatment (0.5 mg/day). Transcript profiles of four subjects were analyzed using Affymetrix GeneChips. Serum insulin-like growth factor I (IGF-I) and procollagens I and III were measured by RIA. GH increased serum IGF-I and procollagens I and III, enhanced whole body lipid oxidation, reduced carbohydrate oxidation, and stimulated protein synthesis. It induced gene expression of IGF-I and collagens in muscle. GH reduced expression of several enzymes regulating lipid oxidation and energy production. It reduced calpain 3, increased ribosomal protein L38 expression, and displayed mixed effects on genes encoding myofibrillar proteins. It increased expression of circadian gene CLOCK, and reduced that of PERIOD. In summary, GH exerted concordant effects on muscle expression and blood levels of IGF-I and collagens. It induced changes in genes regulating protein metabolism in parallel with a whole body anabolic effect. The discordance between muscle gene expression profiles and metabolic responses suggests that muscle is unlikely to contribute to GH-induced stimulation of whole body energy and lipid metabolism. GH may regulate circadian function in skeletal muscle by modulating circadian gene expression with possible metabolic consequences.

Proteomic Analysis of Saliva Identifies Potential Biomarkers for Orthodontic Tooth Movement

PubMed Central

Ellias, Mohd Faiz; Zainal Ariffin, Shahrul Hisham; Karsani, Saiful Anuar; Abdul Rahman, Mariati; Senafi, Shahidan; Megat Abdul Wahab, Rohaya

2012-01-01

Orthodontic treatment has been shown to induce inflammation, followed by bone remodelling in the periodontium. These processes trigger the secretion of various proteins and enzymes into the saliva. This study aims to identify salivary proteins that change in expression during orthodontic tooth movement. These differentially expressed proteins can potentially serve as protein biomarkers for the monitoring of orthodontic treatment and tooth movement. Whole saliva from three healthy female subjects were collected before force application using fixed appliance and at 14 days after 0.014′′ Niti wire was applied. Salivary proteins were resolved using two-dimensional gel electrophoresis (2DE) over a pH range of 3–10, and the resulting proteome profiles were compared. Differentially expressed protein spots were then identified by MALDI-TOF/TOF tandem mass spectrometry. Nine proteins were found to be differentially expressed; however, only eight were identified by MALDI-TOF/TOF. Four of these proteins—Protein S100-A9, immunoglobulin J chain, Ig alpha-1 chain C region, and CRISP-3—have known roles in inflammation and bone resorption. PMID:22919344

In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole.

PubMed

Gautam, Poonam; Mushahary, Dolly; Hassan, Wazid; Upadhyay, Santosh Kumar; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Sarma, Puranam Usha

2016-07-01

Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 μg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles*

PubMed Central

Wong, Emily S. W.; Morgenstern, David; Mofiz, Ehtesham; Gombert, Sara; Morris, Katrina M.; Temple-Smith, Peter; Renfree, Marilyn B.; Whittington, Camilla M.; King, Glenn F.; Warren, Wesley C.; Papenfuss, Anthony T.; Belov, Katherine

2012-01-01

The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution. PMID:22899769

Proteomics and deep sequencing comparison of seasonally active venom glands in the platypus reveals novel venom peptides and distinct expression profiles.

PubMed

Wong, Emily S W; Morgenstern, David; Mofiz, Ehtesham; Gombert, Sara; Morris, Katrina M; Temple-Smith, Peter; Renfree, Marilyn B; Whittington, Camilla M; King, Glenn F; Warren, Wesley C; Papenfuss, Anthony T; Belov, Katherine

2012-11-01

The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.

Expression profiling of various genes during the fruit development and ripening of mango.

PubMed

Pandit, Sagar S; Kulkarni, Ram S; Giri, Ashok P; Köllner, Tobias G; Degenhardt, Jörg; Gershenzon, Jonathan; Gupta, Vidya S

2010-06-01

Mango (Mangifera indica L. cv. Alphonso) development and ripening are the programmed processes; conventional indices and volatile markers help to determine agronomically important stages of fruit life (fruit-setting, harvesting maturity and ripening climacteric). However, more and precise markers are required to understand this programming; apparently, fruit's transcriptome can be a good source of such markers. Therefore, we isolated 18 genes related to the physiology and biochemistry of the fruit and profiled their expression in developing and ripening fruits, flowers and leaves of mango using relative quantitation PCR. In most of the tissues, genes related to primary metabolism, abiotic stress, ethylene response and protein turnover showed high expression as compared to that of the genes related to flavor production. Metallothionin and/or ethylene-response transcription factor showed highest level of transcript abundance in all the tissues. Expressions of mono- and sesquiterpene synthases and 14-3-3 lowered during ripening; whereas, that of lipoxygenase, ethylene-response factor and ubiquitin-protein ligase increased during ripening. Based on these expression profiles, flower showed better positive correlation with developing and ripening fruits than leaf. Most of the genes showed their least expression on the second day of harvest, suggesting that harvesting signals significantly affect the fruit metabolism. Important stages in the fruit life were clearly indicated by the significant changes in the expression levels of various genes. These indications complemented those from the previous analyses of fruit development, ripening and volatile emission, revealing the harmony between physiological, biochemical and molecular activities of the fruit.

Identification of G-Protein-Coupled-Receptors (GPCRs) in Pulmonary Artery Smooth Muscle Cells as Novel Therapeutic Targets

DTIC Science & Technology

2016-10-01

also assessed GPCR expression in PASMCs from mice and rats and in SMCs from the human aorta and coronary artery. RESULTS: PASMCs expressed ~120...among biological replicates of non-diseased human PASMCs. GPCR expression profiles were similar, between non-IPAH PASMCs and SMCs from the aorta and

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

PubMed

Sassi, N; Laadhar, L; Allouche, M; Zandieh-Doulabi, B; Hamdoun, M; Klein-Nulend, J; Makni, S; Sellami, S

2014-01-01

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

Widespread seasonal gene expression reveals annual differences in human immunity and physiology

PubMed Central

Dopico, Xaquin Castro; Evangelou, Marina; Ferreira, Ricardo C.; Guo, Hui; Pekalski, Marcin L.; Smyth, Deborah J.; Cooper, Nicholas; Burren, Oliver S.; Fulford, Anthony J.; Hennig, Branwen J.; Prentice, Andrew M.; Ziegler, Anette-G.; Bonifacio, Ezio; Wallace, Chris; Todd, John A.

2015-01-01

Seasonal variations are rarely considered a contributing component to human tissue function or health, although many diseases and physiological process display annual periodicities. Here we find more than 4,000 protein-coding mRNAs in white blood cells and adipose tissue to have seasonal expression profiles, with inverted patterns observed between Europe and Oceania. We also find the cellular composition of blood to vary by season, and these changes, which differ between the United Kingdom and The Gambia, could explain the gene expression periodicity. With regards to tissue function, the immune system has a profound pro-inflammatory transcriptomic profile during European winter, with increased levels of soluble IL-6 receptor and C-reactive protein, risk biomarkers for cardiovascular, psychiatric and autoimmune diseases that have peak incidences in winter. Circannual rhythms thus require further exploration as contributors to various aspects of human physiology and disease. PMID:25965853

Genome-wide identification and characterisation of F-box family in maize.

PubMed

Jia, Fengjuan; Wu, Bingjiang; Li, Hui; Huang, Jinguang; Zheng, Chengchao

2013-11-01

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.

ReTrOS: a MATLAB toolbox for reconstructing transcriptional activity from gene and protein expression data.

PubMed

Minas, Giorgos; Momiji, Hiroshi; Jenkins, Dafyd J; Costa, Maria J; Rand, David A; Finkenstädt, Bärbel

2017-06-26

Given the development of high-throughput experimental techniques, an increasing number of whole genome transcription profiling time series data sets, with good temporal resolution, are becoming available to researchers. The ReTrOS toolbox (Reconstructing Transcription Open Software) provides MATLAB-based implementations of two related methods, namely ReTrOS-Smooth and ReTrOS-Switch, for reconstructing the temporal transcriptional activity profile of a gene from given mRNA expression time series or protein reporter time series. The methods are based on fitting a differential equation model incorporating the processes of transcription, translation and degradation. The toolbox provides a framework for model fitting along with statistical analyses of the model with a graphical interface and model visualisation. We highlight several applications of the toolbox, including the reconstruction of the temporal cascade of transcriptional activity inferred from mRNA expression data and protein reporter data in the core circadian clock in Arabidopsis thaliana, and how such reconstructed transcription profiles can be used to study the effects of different cell lines and conditions. The ReTrOS toolbox allows users to analyse gene and/or protein expression time series where, with appropriate formulation of prior information about a minimum of kinetic parameters, in particular rates of degradation, users are able to infer timings of changes in transcriptional activity. Data from any organism and obtained from a range of technologies can be used as input due to the flexible and generic nature of the model and implementation. The output from this software provides a useful analysis of time series data and can be incorporated into further modelling approaches or in hypothesis generation.

Different Expression Profiles Suggest Functional Differentiation Among Chemosensory Proteins in Nilaparvata lugens (Hemiptera: Delphacidae)

PubMed Central

Yang, Ke; He, Peng; Dong, Shuang-Lin

2014-01-01

Abstract Chemosensory proteins (CSPs) play various roles in insect physiology including olfaction and development. The brown planthopper, Nilaparvata lugens Stål , is one of the most notorious rice pests worldwide. The wing-from variation and annually long distance migration imply that olfaction would play a key role in N. lugens behavior. In this study, full-length cDNAs of nine CSPs were cloned by the rapid amplification of cDNA ends procedure, and their expression profiles were determined by the quantitative real-time Polymerase Chain Reaction (qPCR), with regard to developmental stage, wing-form, gender, and tissues of short-wing adult. These NlugCSP genes showed distinct expression patterns, indicating different roles they play. In particular, NlugCSP5 was long wing form biased and highly expressed in female wings among tissues; NlugCSP1 was mainly expressed in male adults and abdomen; NlugCSP7 was widely expressed in chemosensory tissues but little in the nonchemosensory abdomen. The function of NlugCSP7 in olfaction was further explored by the competitive fluorescence binding assay using the recombinant protein. However, the recombinant NlugCSP7 showed no obvious binding with all tested volatile compounds, suggesting that it may participate in physiological processes other than olfaction. Our results provide bases and some important clues for the function of NlugCSPs . PMID:25527582

Transcriptome and Gene Expression Analysis of the Rice Leaf Folder, Cnaphalocrosis medinalis

PubMed Central

Li, Shang-Wei; Yang, Hong; Liu, Yue-Feng; Liao, Qi-Rong; Du, Juan; Jin, Dao-Chao

2012-01-01

Background The rice leaf folder (RLF), Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae), is one of the most destructive pests affecting rice in Asia. Although several studies have been performed on the ecological and physiological aspects of this species, the molecular mechanisms underlying its developmental regulation, behavior, and insecticide resistance remain largely unknown. Presently, there is a lack of genomic information for RLF; therefore, studies aimed at profiling the RLF transcriptome expression would provide a better understanding of its biological function at the molecular level. Principal Findings De novo assembly of the RLF transcriptome was performed via the short read sequencing technology (Illumina). In a single run, we produced more than 23 million sequencing reads that were assembled into 44,941 unigenes (mean size = 474 bp) by Trinity. Through a similarity search, 25,281 (56.82%) unigenes matched known proteins in the NCBI Nr protein database. The transcriptome sequences were annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). Additionally, we profiled gene expression during RLF development using a tag-based digital gene expression (DGE) system. Five DGE libraries were constructed, and variations in gene expression were compared between collected samples: eggs vs. 3rd instar larvae, 3rd instar larvae vs. pupae, pupae vs. adults. The results demonstrated that thousands of genes were significantly differentially expressed during various developmental stages. A number of the differentially expressed genes were confirmed by quantitative real-time PCR (qRT-PCR). Conclusions The RLF transcriptome and DGE data provide a comprehensive and global gene expression profile that would further promote our understanding of the molecular mechanisms underlying various biological characteristics, including development, elevated fecundity, flight, sex differentiation, olfactory behavior, and insecticide resistance in RLF. Therefore, these findings could help elucidate the intrinsic factors involved in the RLF-mediated destruction of rice and offer sustainable insect pest management. PMID:23185238

Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

PubMed

Cavalla, Franco; Biguetti, Claudia; Jain, Sameer; Johnson, Cleverick; Letra, Ariadne; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

2017-09-01

Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

PubMed

Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

2013-11-01

Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

Role of 14-3-3η protein on cardiac fatty acid metabolism and macrophage polarization after high fat diet induced type 2 diabetes mellitus.

PubMed

Sreedhar, Remya; Arumugam, Somasundaram; Thandavarayan, Rajarajan A; Karuppagounder, Vengadeshprabhu; Koga, Yusuke; Nakamura, Takashi; Harima, Meilei; Watanabe, Kenichi

2017-07-01

Diabetic cardiomyopathy (DCM), a metabolic disorder, is one of the leading causes of mortality around the world and its pathogenesis involves cardiac inflammation and altered metabolic profile. Altered fatty acid metabolism during DCM can cause macrophage polarization in which inflammatory M1 phenotype dominates over the anti-inflammatory M2 phenotype. Hence, it is essential to identify a specific target, which could revert the metabolic profile and thereby reducing the M1 macrophage polarization. 14-3-3η protein has several cellular protective functions especially in the heart as plenty of reports available in various animal models of heart failure including diabetes mellitus. However, its role in the cardiac fatty acid metabolism and macrophage polarization remains unidentified. The present study has been designed to delineate the effect of cardiospecific dominant negative mutation of 14-3-3η protein (DN14-3-3) on various lipid metabolism related marker proteins expressions and cardiac macrophage phenotype in high fat diet (HFD) fed mice. Feeding HFD for 12 weeks has produced significant increase in body weight in the DN14-3-3 (TG) mice than C57BL6/J (WT) mice. Western blotting and immunohistochemical staining analysis of the heart tissue has revealed an increase in the expression of markers of cardiac fatty acid synthesis related proteins in addition to the reduced expression of fatty acid oxidation related proteins in TG mice fed HFD than WT mice fed HFD. Furthermore, the M1 macrophage marker proteins were increasingly expressed while M2 markers expressions were reduced in the hearts of TG mice fed HFD. In conclusion, our current study has identified that there is a definite role for the 14-3-3η protein against the pathogenesis of heart failure via regulation of cardiac fatty acid metabolism and macrophage polarization. Copyright © 2017. Published by Elsevier Ltd.

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients.

PubMed

Pan, Hai-Tao; Ding, Hai-Gang; Fang, Min; Yu, Bin; Cheng, Yi; Tan, Ya-Jing; Fu, Qi-Qin; Lu, Bo; Cai, Hong-Guang; Jin, Xin; Xia, Xian-Qing; Zhang, Tao

2018-01-01

Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antennal transcriptome analysis of the Asian longhorned beetle Anoplophora glabripennis

PubMed Central

Hu, Ping; Wang, Jingzhen; Cui, Mingming; Tao, Jing; Luo, Youqing

2016-01-01

Olfactory proteins form the basis of insect olfactory recognition, which is crucial for host identification, mating, and oviposition. Using transcriptome analysis of Anoplophora glabripennis antenna, we identified 42 odorant-binding proteins (OBPs), 12 chemosensory proteins (CSPs), 14 pheromone-degrading enzymes (PDEs), 1 odorant-degrading enzymes (ODE), 37 odorant receptors (ORs), 11 gustatory receptors (GRs), 2 sensory neuron membrane proteins (SNMPs), and 4 ionotropic receptor (IR). All CSPs and PBPs were expressed in antennae, confirming the authenticity of the transcriptome data. CSP expression profiles showed that AglaCSP3, AglaCSP6, and AglaCSP12 were expressed preferentially in maxillary palps and AglaCSP7 and AglaCSP9 were strongly expressed in antennae. The vast majority of CSPs were highly expressed in multiple chemosensory tissues, suggesting their participation in olfactory recognition in almost all olfactory tissues. Intriguingly, the PBP AglaPBP2 was preferentially expressed in antenna, indicating that it is the main protein involved in efficient and sensitive pheromone recognition. Phylogenetic analysis of olfactory proteins indicated AglaGR1 may detect CO2. This study establishes a foundation for determining the chemoreception molecular mechanisms of A. glabripennis, which would provide a new perspective for controlling pest populations, especially those of borers. PMID:27222053

ExAtlas: An interactive online tool for meta-analysis of gene expression data.

PubMed

Sharov, Alexei A; Schlessinger, David; Ko, Minoru S H

2015-12-01

We have developed ExAtlas, an on-line software tool for meta-analysis and visualization of gene expression data. In contrast to existing software tools, ExAtlas compares multi-component data sets and generates results for all combinations (e.g. all gene expression profiles versus all Gene Ontology annotations). ExAtlas handles both users' own data and data extracted semi-automatically from the public repository (GEO/NCBI database). ExAtlas provides a variety of tools for meta-analyses: (1) standard meta-analysis (fixed effects, random effects, z-score, and Fisher's methods); (2) analyses of global correlations between gene expression data sets; (3) gene set enrichment; (4) gene set overlap; (5) gene association by expression profile; (6) gene specificity; and (7) statistical analysis (ANOVA, pairwise comparison, and PCA). ExAtlas produces graphical outputs, including heatmaps, scatter-plots, bar-charts, and three-dimensional images. Some of the most widely used public data sets (e.g. GNF/BioGPS, Gene Ontology, KEGG, GAD phenotypes, BrainScan, ENCODE ChIP-seq, and protein-protein interaction) are pre-loaded and can be used for functional annotations.

Computational synchronization of microarray data with application to Plasmodium falciparum.

PubMed

Zhao, Wei; Dauwels, Justin; Niles, Jacquin C; Cao, Jianshu

2012-06-21

Microarrays are widely used to investigate the blood stage of Plasmodium falciparum infection. Starting with synchronized cells, gene expression levels are continually measured over the 48-hour intra-erythrocytic cycle (IDC). However, the cell population gradually loses synchrony during the experiment. As a result, the microarray measurements are blurred. In this paper, we propose a generalized deconvolution approach to reconstruct the intrinsic expression pattern, and apply it to P. falciparum IDC microarray data. We develop a statistical model for the decay of synchrony among cells, and reconstruct the expression pattern through statistical inference. The proposed method can handle microarray measurements with noise and missing data. The original gene expression patterns become more apparent in the reconstructed profiles, making it easier to analyze and interpret the data. We hypothesize that reconstructed gene expression patterns represent better temporally resolved expression profiles that can be probabilistically modeled to match changes in expression level to IDC transitions. In particular, we identify transcriptionally regulated protein kinases putatively involved in regulating the P. falciparum IDC. By analyzing publicly available microarray data sets for the P. falciparum IDC, protein kinases are ranked in terms of their likelihood to be involved in regulating transitions between the ring, trophozoite and schizont developmental stages of the P. falciparum IDC. In our theoretical framework, a few protein kinases have high probability rankings, and could potentially be involved in regulating these developmental transitions. This study proposes a new methodology for extracting intrinsic expression patterns from microarray data. By applying this method to P. falciparum microarray data, several protein kinases are predicted to play a significant role in the P. falciparum IDC. Earlier experiments have indeed confirmed that several of these kinases are involved in this process. Overall, these results indicate that further functional analysis of these additional putative protein kinases may reveal new insights into how the P. falciparum IDC is regulated.

Gene expression profile differences in left and right liver lobes from mid-gestation fetal baboons: a cautionary tale

PubMed Central

Cox, Laura A; Schlabritz-Loutsevitch, Natalia; Hubbard, Gene B; Nijland, Mark J; McDonald, Thomas J; Nathanielsz, Peter W

2006-01-01

Interpretation of gene array data presents many potential pitfalls in adult tissues. Gene array techniques applied to fetal tissues present additional confounding pitfalls. The left lobe of the fetal liver is supplied with blood containing more oxygen than the right lobe. Since synthetic activity and cell function are oxygen dependent, we hypothesized major differences in mRNA expression between the fetal right and left liver lobes. Our aim was to demonstrate the need to evaluate RNA samples from both lobes. We performed whole genome expression profiling on left and right liver lobe RNA from six 90-day gestation baboon fetuses (term 180 days). Comparing right with left, we found 875 differentially expressed genes – 312 genes were up-regulated and 563 down-regulated. Pathways for damaged DNA binding, endonuclease activity, interleukin binding and receptor activity were up-regulated in right lobe; ontological pathways related to cell signalling, cell organization, cell biogenesis, development, intracellular transport, phospholipid metabolism, protein biosynthesis, protein localization, protein metabolism, translational regulation and vesicle mediated transport were down-regulated in right lobe. Molecular pathway analysis showed down-regulation of pathways related to heat shock protein binding, ion channel and transporter activities, oxygen binding and transporter activities, translation initiation and translation regulator activities. Genes involved in amino acid biosynthesis, lipid biosynthesis and oxygen transport were also differentially expressed. This is the first demonstration of RNA differences between the two lobes of the fetal liver. The data support the argument that a complete interpretation of gene expression in the developing liver requires data from both lobes. PMID:16484296

Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

PubMed

Sá-Pereira, Paula; Diniz, Mário S; Moita, Liliana; Pinheiro, Teresa; Mendonça, Elsa; Paixão, Susana M; Picado, Ana

2018-05-01

The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO 2 -NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO 2 -NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO 2 -NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO 2 -NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO 2 -NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO 2 -NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO 2 -NP toxicity in D. magna, providing useful information for future research.

Protein profiling of preeclampsia placental tissues.

PubMed

Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y; He, Jin; Ye, Fei

2014-01-01

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

Protein Profiling of Preeclampsia Placental Tissues

PubMed Central

Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y.

2014-01-01

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia. PMID:25392996

Analysis of proteome changes in doxorubicin-treated adult rat cardiomyocyte

PubMed Central

Kumar, Suresh N.; Konorev, Eugene A.; Aggarwal, Deepika; Kalyanaraman, Balaraman

2011-01-01

Doxorubicin-induced cardiomyopathy in cancer patients is well established. The proposed mechanism of cardiac damage includes generation of reactive oxygen species, mitochondrial dysfunction and cardiomyocyte apoptosis. Exposure of adult rat cardiomyocytes to low levels of DOX for 48 h induced apoptosis. Analysis of protein expression showed a differential regulation of several key proteins including the voltage dependent anion selective channel protein 2 and methylmalonate semialdehyde dehydrogenase. In comparison, proteomic evaluation of DOX-treated rat heart showed a slightly different set of protein changes that suggests nuclear accumulation of DOX. Using a new solubilization technique, changes in low abundant protein profiles were monitored. Altered protein expression, modification and function related to oxidative stress response may play an important role in DOX cardiotoxicity. PMID:21338723

Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

PubMed

Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

2014-01-01

Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pheromone Binding Protein EhipPBP1 Is Highly Enriched in the Male Antennae of the Seabuckthorn Carpenterworm and Is Binding to Sex Pheromone Components

PubMed Central

Hu, Ping; Gao, Chenglong; Zong, Shixiang; Luo, Youqing; Tao, Jing

2018-01-01

The seabuckthorn carpenterworm moth Eogystia hippophaecolus is a major threat to seabuckthorn plantations, causing considerable ecological and economic losses in China. Transcriptomic analysis of E. hippophaecolus previously identified 137 olfactory proteins, including three pheromone-binding proteins (PBPs). We investigated the function of E. hippophaecolus PBP1 by studying its mRNA and protein expression profiles and its binding ability with different compounds. The highest levels of expression were in the antennae, particularly in males, with much lower levels of expression in the legs and external genitals. Recombinant PBP1 showed strong binding to sex-pheromone components, suggesting that antennal EhipPBP1 is involved in binding sex-pheromone components during pheromone communication. PMID:29755369

Hemostatic properties and protein expression profile of therapeutic apheresis plasma treated with amotosalen and ultraviolet A for pathogen inactivation.

PubMed

Ohlmann, Philippe; Hechler, Béatrice; Chafey, Philippe; Ravanat, Catherine; Isola, Hervé; Wiesel, Marie-Louise; Cazenave, Jean-Pierre; Gachet, Christian

2016-09-01

The INTERCEPT Blood System (IBS) using amotosalen-HCl and ultraviolet (UV)A inactivates a large spectrum of microbial pathogens and white blood cells in therapeutic plasma. Our aim was to evaluate to what extent IBS modifies the capacity of plasma to generate thrombin and induces qualitative or quantitative modifications of plasma proteins. Plasma units from four donors were collected by apheresis. Samples were taken before (control [CTRL]) and after IBS treatment and stored at -80°C until use. The activities of plasma coagulation factors and inhibitors and the thrombin generation potential were determined using assays measuring clotting times and the calibrated automated thrombogram (CAT), respectively. The proteomic profile of plasma proteins was examined using a two-dimensional differential in-gel electrophoresis (2D-DIGE) method. Nearly all of the procoagulant and antithrombotic factors tested retained at least 78% of their initial pre-IBS activity. Only FVII and FVIII displayed a lower level of conservation (67%), which nevertheless remained within the reference range for conventional plasma coagulation factors. The thrombin generation profile of plasma was conserved after IBS treatment. Among the 1331 protein spots revealed by 2D-DIGE analysis, only four were differentially expressed in IBS plasma compared to CTRL plasma and two were identified by mass spectrometric analysis as transthyretin and apolipoprotein A1. The IBS technique for plasma moderately decreases the activities of plasma coagulation factors and antithrombotic proteins, with no impact on the thrombin generation potential of plasma and very limited modifications of the proteomic profile. © 2016 AABB.

Proteomic profile of serum of pregnant women carring a fetus with Down syndrome using nano uplc Q-tof ms/ms technology.

PubMed

López Uriarte, Graciela Arelí; Burciaga Flores, Carlos Horacio; Torres de la Cruz, Víctor Manuel; Medina Aguado, María Magdalena; Gómez Puente, Viviana Maricela; Romero Gutiérrez, Liliana Nayeli; Martínez de Villarreal, Laura Elia

2018-06-01

Prenatal diagnosis of Down syndrome (DS) is based on the calculated risk of maternal age, biochemical and ultrasonographic markers and recently by cfDNA. Differences in proteomic profiles may give an opportunity to find new biomarkers. Characterize proteome of serum of mothers carrying DS fetus. Blood serum samples of three groups of women were obtained, (a) 10 non-pregnant, (b) 10 pregnant with healthy fetus by ultrasound evaluation, (c) nine pregnant with DS fetus. Sample preparation was as follows: Albumin/IgG depletion, desalting, and trypsin digestion; the process was performed in nanoUPLC MS/MS. Data analysis was made with Mass Lynx 4.1 and ProteinLynx Global Server 3.0, peptide and protein recognition by MASCOT algorithm and UNIPROT-Swissprot database. Each group showed different protein profiles. Some proteins were shared between groups. Only sera from pregnant women showed proteins related to immune and clot pathways. Mothers with DS fetus had 42 specific proteins. We found a different serum protein profile in mothers carrying DS fetuses that do not reflect expression of genes in the extra chromosome. Further studies will be necessary to establish the role of these proteins in aneuploid fetus and analyze their possible use as potential biomarkers.

Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don) Endl

PubMed Central

2010-01-01

Background Restoration of rooting competence is important for rejuvenation in Sequoia sempervirens (D. Don) Endl and is achieved by repeatedly grafting Sequoia shoots after 16 and 30 years of cultivation in vitro. Results Mass spectrometry-based proteomic analysis revealed three proteins that differentially accumulated in different rejuvenation stages, including oxygen-evolving enhancer protein 2 (OEE2), glycine-rich RNA-binding protein (RNP), and a thaumatin-like protein. OEE2 was found to be phosphorylated and a phosphopeptide (YEDNFDGNSNVSVMVpTPpTDK) was identified. Specifically, the protein levels of OEE2 increased as a result of grafting and displayed a higher abundance in plants during the juvenile and rejuvenated stages. Additionally, SsOEE2 displayed the highest expression levels in Sequoia shoots during the juvenile stage and less expression during the adult stage. The expression levels also steadily increased during grafting. Conclusion Our results indicate a positive correlation between the gene and protein expression patterns of SsOEE2 and the rejuvenation process, suggesting that this gene is involved in the rejuvenation of Sequoia sempervirens. PMID:21143964

Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don) Endl.

PubMed

Chang, Ing-Feng; Chen, Peng-Jen; Shen, Chin-Hui; Hsieh, Tsung-Ju; Hsu, Ya-Wen; Huang, Bau-Lian; Kuo, Ching-I; Chen, Yu-Ting; Chu, Hsiu-An; Yeh, Kai-Wun; Huang, Li-Chun

2010-12-10

Restoration of rooting competence is important for rejuvenation in Sequoia sempervirens (D. Don) Endl and is achieved by repeatedly grafting Sequoia shoots after 16 and 30 years of cultivation in vitro. Mass spectrometry-based proteomic analysis revealed three proteins that differentially accumulated in different rejuvenation stages, including oxygen-evolving enhancer protein 2 (OEE2), glycine-rich RNA-binding protein (RNP), and a thaumatin-like protein. OEE2 was found to be phosphorylated and a phosphopeptide (YEDNFDGNSNVSVMVpTPpTDK) was identified. Specifically, the protein levels of OEE2 increased as a result of grafting and displayed a higher abundance in plants during the juvenile and rejuvenated stages. Additionally, SsOEE2 displayed the highest expression levels in Sequoia shoots during the juvenile stage and less expression during the adult stage. The expression levels also steadily increased during grafting. Our results indicate a positive correlation between the gene and protein expression patterns of SsOEE2 and the rejuvenation process, suggesting that this gene is involved in the rejuvenation of Sequoia sempervirens.

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows.

PubMed

Tacoma, Rinske; Fields, Julia; Ebenstein, David B; Lam, Ying-Wai; Greenwood, Sabrina L

2016-01-01

Milk is a highly nutritious natural product that provides not only a rich source of amino acids to the consumer but also hundreds of bioactive peptides and proteins known to elicit health-benefitting activities. We investigated the milk protein profile produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions using proteomic approaches that optimize protein extraction and characterization of the low abundance proteins within the skim milk fraction of bovine milk. In total, 935 low abundance proteins were identified. Gene ontology classified all proteins identified into various cellular localization and function categories. A total of 43 low abundance proteins were differentially expressed between the two dairy breeds. Bioactive proteins involved in host-defense, including lactotransferrin (P=0.0026) and complement C2 protein (P=0.0001), were differentially expressed by the two breeds, whereas others such as osteopontin (P=0.1788) and lactoperoxidase (P=0.2973) were not. This work is the first to outline the protein profile produced by two important breeds of dairy cattle maintained under the same diet, environment and management conditions in order to observe likely true breed differences. This research now allows us to better understand and contrast further research examining the bovine proteome that includes these different breeds. Within the last decade, the amount of research characterizing the bovine milk proteome has increased due to growing interest in the bioactive proteins that are present in milk. Proteomic analysis of low abundance whey proteins has mainly focused on human breast milk; however, previous research has highlighted the presence of bioactive proteins in bovine milk. Recent publications outlining the cross-reactivity of bovine bioactive proteins on human biological function highlight the need for further investigation into the bovine milk proteome. The rationale behind this study is to characterize and compare the low abundance protein profile in the skim milk fraction produced from Holstein and Jersey breeds of dairy cattle, which are two major dairy cattle breeds in the USA. A combination of fractionation strategies was used to efficiently enrich the low abundance proteins from bovine skim milk for proteomic profiling. A total of 935 low abundance proteins were identified and compared between the two bovine breeds. The results from this study provide insight into breed differences and similarities in the milk proteome profile produced by two breeds of dairy cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of leaf incubation temperature profiles on Agrobacterium tumefaciens-mediated transient expression.

PubMed

Jung, Sang-Kyu; McDonald, Karen A; Dandekar, Abhaya M

2015-01-01

Agrobacterium tumefaciens-mediated transient expression is known to be highly dependent on incubation temperature. Compared with early studies that were conducted at constant temperature, we examined the effect of variable leaf incubation temperature on transient expression. As a model system, synthetic endoglucanase (E1) and endoxylanase (Xyn10A) genes were transiently expressed in detached whole sunflower leaves via vacuum infiltration for biofuel applications. We found that the kinetics of transient expression strongly depended on timing of the temperature change as well as leaf incubation temperature. Surprisingly, we found that high incubation temperature (27-30 °C) which is suboptimal for T-DNA transfer, significantly enhanced transient expression if the high temperature was applied during the late phase (Day 3-6) of leaf incubation whereas incubation temperature in a range of 20-25 °C for an early phase (Day 0-2) resulted in higher production. On the basis of these results, we propose that transient expression is governed by both T-DNA transfer and protein synthesis in plant cells that have different temperature dependent kinetics. Because the phases were separated in time and had different optimal temperatures, we were then able to develop a novel two phase optimization strategy for leaf incubation temperature. Applying the time-varying temperature profile, we were able to increase the protein accumulation by fivefold compared with the control at a constant temperature of 20 °C. From our knowledge, this is the first report illustrating the effect of variable temperature profiling for improved transient expression. © 2015 American Institute of Chemical Engineers.

Magnesium supplementation, metabolic and inflammatory markers, and global genomic and proteomic profiling: a randomized, double-blind, controlled, crossover trial in overweight individuals.

PubMed

Chacko, Sara A; Sul, James; Song, Yiqing; Li, Xinmin; LeBlanc, James; You, Yuko; Butch, Anthony; Liu, Simin

2011-02-01

Dietary magnesium intake has been favorably associated with reduced risk of metabolic outcomes in observational studies; however, few randomized trials have introduced a systems-biology approach to explore molecular mechanisms of pleiotropic metabolic actions of magnesium supplementation. We examined the effects of oral magnesium supplementation on metabolic biomarkers and global genomic and proteomic profiling in overweight individuals. We undertook this randomized, crossover, pilot trial in 14 healthy, overweight volunteers [body mass index (in kg/m(2)) ≥25] who were randomly assigned to receive magnesium citrate (500 mg elemental Mg/d) or a placebo for 4 wk with a 1-mo washout period. Fasting blood and urine specimens were collected according to standardized protocols. Biochemical assays were conducted on blood specimens. RNA was extracted and subsequently hybridized with the Human Gene ST 1.0 array (Affymetrix, Santa Clara, CA). Urine proteomic profiling was analyzed with the CM10 ProteinChip array (Bio-Rad Laboratories, Hercules, CA). We observed that magnesium treatment significantly decreased fasting C-peptide concentrations (change: -0.4 ng/mL after magnesium treatment compared with +0.05 ng/mL after placebo treatment; P = 0.004) and appeared to decrease fasting insulin concentrations (change: -2.2 μU/mL after magnesium treatment compared with 0.0 μU/mL after placebo treatment; P = 0.25). No consistent patterns were observed across inflammatory biomarkers. Gene expression profiling revealed up-regulation of 24 genes and down-regulation of 36 genes including genes related to metabolic and inflammatory pathways such as C1q and tumor necrosis factor-related protein 9 (C1QTNF9) and pro-platelet basic protein (PPBP). Urine proteomic profiling showed significant differences in the expression amounts of several peptides and proteins after treatment. Magnesium supplementation for 4 wk in overweight individuals led to distinct changes in gene expression and proteomic profiling consistent with favorable effects on several metabolic pathways. This trial was registered at clinicaltrials.gov as NCT00737815.

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds.

PubMed

Li, Runzhi; Yu, Keshun; Wu, Yongmei; Tateno, Mizuki; Hatanaka, Tomoko; Hildebrand, David F

2012-01-01

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein markers for identification of Yersinia pestis and their variation related to culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wunschel, David S.; Engelmann, Heather E.; Victry, Kristin D.

2013-12-11

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y.more » pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.« less

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Effect of hypoxia on lung gene expression and proteomic profile: insights into the pulmonary surfactant response

PubMed Central

Olmeda, Bárbara; Umstead, Todd M.; Silveyra, Patricia; Pascual, Alberto; López-Barneo, José; Phelps, David S.; Floros, Joanna; Pérez-Gil, Jesús

2014-01-01

Exposure of lung to hypoxia has been previously reported to be associated with significant alterations in the protein content of bronchoalveolar lavage (BAL) and lung tissue. In the present work we have used a proteomic approach to describe the changes in protein complement induced by moderate long-term hypoxia (rats exposed to 10% O2 for 72 hours) in BAL and lung tissue, with a special focus on the proteins associated with pulmonary surfactant, which could indicate adaptation of this system to limited oxygen availability. The analysis of the general proteomic profile indicates a hypoxia-induced increase in proteins associated with inflammation both in lavage and lung tissue. Analysis at mRNA and protein levels revealed no significant changes induced by hypoxia on the content in surfactant proteins or their apparent oligomeric state. In contrast, we detected a hypoxia-induced significant increase in the expression and accumulation of hemoglobin in lung tissue, at both mRNA and protein levels, as well as an accumulation of hemoglobin both in BAL and associated with surface-active membranes of the pulmonary surfactant complex. Evaluation of pulmonary surfactant surface activity from hypoxic rats showed no alterations in its spreading ability, ruling out inhibition by increased levels of serum or inflammatory proteins. PMID:24576641

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

PubMed

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [ P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [ P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine

PubMed Central

O’Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O’Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-01-01

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION This first study providing “tri-omics” analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression. PMID:29151691

Cloning and expression profiling of odorant-binding proteins in the tarnished plant bug, Lygus lineolaris

USDA-ARS?s Scientific Manuscript database

In insects, the perception and discrimination of odorants requires the involvement of odorant binding proteins (OBPs). To gain a better molecular understanding of olfaction in the agronomic pest, Lygus lineolaris (tarnished plant bug), we used a transcriptomics-based approach to identify potential ...

Identification and Validation of Selected Universal Stress Protein Domain Containing Drought-Responsive Genes in Pigeonpea (Cajanus cajan L.)

PubMed Central

Sinha, Pallavi; Pazhamala, Lekha T.; Singh, Vikas K.; Saxena, Rachit K.; Krishnamurthy, L.; Azam, Sarwar; Khan, Aamir W.; Varshney, Rajeev K.

2016-01-01

Pigeonpea is a resilient crop, which is relatively more drought tolerant than many other legume crops. To understand the molecular mechanisms of this unique feature of pigeonpea, 51 genes were selected using the Hidden Markov Models (HMM) those codes for proteins having close similarity to universal stress protein domain. Validation of these genes was conducted on three pigeonpea genotypes (ICPL 151, ICPL 8755, and ICPL 227) having different levels of drought tolerance. Gene expression analysis using qRT-PCR revealed 6, 8, and 18 genes to be ≥2-fold differentially expressed in ICPL 151, ICPL 8755, and ICPL 227, respectively. A total of 10 differentially expressed genes showed ≥2-fold up-regulation in the more drought tolerant genotype, which encoded four different classes of proteins. These include plant U-box protein (four genes), universal stress protein A-like protein (four genes), cation/H(+) antiporter protein (one gene) and an uncharacterized protein (one gene). Genes C.cajan_29830 and C.cajan_33874 belonging to uspA, were found significantly expressed in all the three genotypes with ≥2-fold expression variations. Expression profiling of these two genes on the four other legume crops revealed their specific role in pigeonpea. Therefore, these genes seem to be promising candidates for conferring drought tolerance specifically to pigeonpea. PMID:26779199

Transcription profile of brewery yeast under fermentation conditions.

PubMed

James, T C; Campbell, S; Donnelly, D; Bond, U

2003-01-01

Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.

Two-dimensional gel analysis of protein expression in ovarian tumors shows a low degree of intratumoral heterogeneity.

PubMed

Alaiya, A A; Franzén, B; Moberger, B; Silfverswärd, C; Linder, S; Auer, G

1999-01-01

The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Two-dimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. In the present study, tumor cells were prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression was low. In contrast, large differences were observed when the protein profiles of different tumors were compared. The differences in gene expression between pairs of malignant carcinomas were slightly larger than the differences observed between pairs of benign tumors. We conclude that 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.

MOPED 2.5—An Integrated Multi-Omics Resource: Multi-Omics Profiling Expression Database Now Includes Transcriptomics Data

PubMed Central

Montague, Elizabeth; Stanberry, Larissa; Higdon, Roger; Janko, Imre; Lee, Elaine; Anderson, Nathaniel; Choiniere, John; Stewart, Elizabeth; Yandl, Gregory; Broomall, William; Kolker, Natali

2014-01-01

Abstract Multi-omics data-driven scientific discovery crucially rests on high-throughput technologies and data sharing. Currently, data are scattered across single omics repositories, stored in varying raw and processed formats, and are often accompanied by limited or no metadata. The Multi-Omics Profiling Expression Database (MOPED, http://moped.proteinspire.org) version 2.5 is a freely accessible multi-omics expression database. Continual improvement and expansion of MOPED is driven by feedback from the Life Sciences Community. In order to meet the emergent need for an integrated multi-omics data resource, MOPED 2.5 now includes gene relative expression data in addition to protein absolute and relative expression data from over 250 large-scale experiments. To facilitate accurate integration of experiments and increase reproducibility, MOPED provides extensive metadata through the Data-Enabled Life Sciences Alliance (DELSA Global, http://delsaglobal.org) metadata checklist. MOPED 2.5 has greatly increased the number of proteomics absolute and relative expression records to over 500,000, in addition to adding more than four million transcriptomics relative expression records. MOPED has an intuitive user interface with tabs for querying different types of omics expression data and new tools for data visualization. Summary information including expression data, pathway mappings, and direct connection between proteins and genes can be viewed on Protein and Gene Details pages. These connections in MOPED provide a context for multi-omics expression data exploration. Researchers are encouraged to submit omics data which will be consistently processed into expression summaries. MOPED as a multi-omics data resource is a pivotal public database, interdisciplinary knowledge resource, and platform for multi-omics understanding. PMID:24910945

Perturbation in protein expression of the sterile salmonid hybrids between female brook trout Salvelinus fontinalis and male masu salmon Oncorhynchus masou during early spermatogenesis.

PubMed

Zheng, Liang; Senda, Yoshie; Abe, Syuiti

2013-05-01

Most males and females of intergeneric hybrid (BM) between female brook trout (Bt) Salvelinus fontinalis and male masu salmon (Ms) Oncorhynchus masou had undeveloped gonads, with abnormal germ cell development shown by histological examination. To understand the cause of this hybrid sterility, expression profiles of testicular proteins in the BM and parental species were examined with 2-DE coupled with MALDI-TOF/TOF MS. Compared with the parental species, more than 60% of differentially expressed protein spots were down-regulated in BM. A total of 16 up-regulated and 48 down-regulated proteins were identified in BM. Up-regulated were transferrin and other somatic cell-predominant proteins, whereas down-regulated were some germ cell-specific proteins such as DEAD box RNA helicase Vasa. Other pronouncedly down-regulated proteins included tubulins and heat shock proteins that are supposed to have roles in spermatogenesis. The present findings suggest direct association of the observed perturbation in protein expression with the failure of spermatogenesis and the sterility in the examined salmonid hybrids. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection

USDA-ARS?s Scientific Manuscript database

Using quantitative PCR technique, the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes [NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein (BPI), cathepsin D, hepcidin, and liver-expressed antimicrobial peptide 2 ...

Gene expression profiling in the hippocampus of learned helpless and nonhelpless rats.

PubMed

Kohen, R; Kirov, S; Navaja, G P; Happe, H Kevin; Hamblin, M W; Snoddy, J R; Neumaier, J F; Petty, F

2005-01-01

In the learned helplessness (LH) animal model of depression, failure to attempt escape from avoidable environmental stress, LH, indicates behavioral despair, whereas nonhelpless (NH) behavior reflects behavioral resilience to the effects of environmental stress. Comparing hippocampal gene expression with large-scale oligonucleotide microarrays, we found that stress-resilient (NH) rats, although behaviorally indistinguishable from controls, showed a distinct gene expression profile compared to LH, sham stressed, and naïve control animals. Genes that were confirmed as differentially expressed in the NH group by quantitative PCR strongly correlated in their levels of expression across all four animal groups. Differential expression could not be confirmed at the protein level. We identified several shared degenerate sequence motifs in the 3' untranslated region (3'UTR) of differentially expressed genes that could be a factor in this tight correlation of expression levels among differentially expressed genes.

Epigenetic modifications and chromatin loop organization explain the different expression profiles of the Tbrg4, WAP and Ramp3 genes

USDA-ARS?s Scientific Manuscript database

Whey Acidic Protein (WAP) gene expression is specific to the mammary gland and regulated by lactogenic hormones to peak during lactation. It differs markedly from the more constitutive expression of the two flanking genes, Ramp3 and Tbrg4. Our results show that the tight regulation of WAP gene expre...

Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies

PubMed Central

Ayoglu, Burcu; Chaouch, Amina; Lochmüller, Hanns; Politano, Luisa; Bertini, Enrico; Spitali, Pietro; Hiller, Monika; Niks, Eric H; Gualandi, Francesca; Pontén, Fredrik; Bushby, Kate; Aartsma-Rus, Annemieke; Schwartz, Elena; Le Priol, Yannick; Straub, Volker; Uhlén, Mathias; Cirak, Sebahattin; ‘t Hoen, Peter A C; Muntoni, Francesco; Ferlini, Alessandra; Schwenk, Jochen M; Nilsson, Peter; Al-Khalili Szigyarto, Cristina

2014-01-01

Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavoprotein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies. PMID:24920607

E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chopjitt, Peechanika; Pientong, Chamsai; Sunthamala, Nuchsupha

HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E andmore » E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant. - Highlights: • E6D25E HPV16 specifically modulates protein profile of human keratinocytes. • E6D25E HPV16 modulates protein profile which involves in TLR signalling and transformation of squamocolumnar junction cells. • E6D25E oncoprotein may correlate to impair of immune response against viral infection and cells transformation.« less

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

PubMed

Gai, Yingping; Yuan, Shuo-Shuo; Liu, Zhao-Yang; Zhao, Huai-Ning; Liu, Qi; Qin, Yong-Li; Fang, Li-Jing; Ji, Xian-Ling

2018-05-30

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signalling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000 ) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Unraveling the proteomic profile of mice testis during the initiation of meiosis.

PubMed

Shao, Binbin; Guo, Yueshuai; Wang, Lei; Zhou, Quan; Gao, Tingting; Zheng, Bo; Zheng, Haoyu; Zhou, Tao; Zhou, Zuomin; Guo, Xuejiang; Huang, Xiaoyan; Sha, Jiahao

2015-04-29

In mice, once primordial germ cells (PGCs) are generated, they continue to proliferate and migrate to eventually reach the future gonads. They initiate sexual differentiation after their colonization of the gonads. During this process, retinoic acid (RA) induces meiosis in the female germ cells, which proceeds to the diplotene stage of meiotic prophase I, whereas the male germ cells initiate growth arrest. After birth, meiosis is initiated in mice spermatogonia by their conversion to preleptotene spermatocytes. There are evidences showing the roles of RA in the regulation of spermatogonial differentiation and meiosis initiation. However, it is still not well known on what responds to RA and how RA signaling engages meiosis. Thus, we constructed a proteomic profile of proteins associated with meiosis onset during testis development in mouse and identified 104 differentially expressed proteins (≥1.5 folds). Bioinformatic analysis showed proteins functioning in specific cell processes. The expression patterns of five selected proteins were verified via Western blot, of which we found that Tfrc gene was RA responsive, with a RA responsive element, and could be up regulated by RA in spermatogonial stem cell (SSC) line. Taken together, the results provide an important reference profile for further functional study of meiosis initiation. Spermatogenesis involves mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis, in which meiosis is a unique event to germ cells, and not in the somatic cells. Till now, the detailed molecular mechanisms of the transition from mitosis to meiosis are still not elucidated. With high-throughput proteomic technology, it is now possible to systemically identify proteins possibly involved. With TMT-6plex based quantification, we identified 104 proteins differentially between testes without meiosis (day 8.5) and those that were meiosis initiated (day 10.5). And a well-known protein essential for meiosis initiation, stra8, was identified to be differentially expressed in the study. And bioinformatic analysis and functional studies revealed several proteins regulated by retinoic acid, a chemical known to regulate the meiosis initiation. Thus, this quantitative proteomic approach can identify meiosis initiation regulating proteins, and further functional studies of these proteins will help elucidate the mechanisms of meiosis initiation. Copyright © 2015. Published by Elsevier B.V.

BloodChIP: a database of comparative genome-wide transcription factor binding profiles in human blood cells.

PubMed

Chacon, Diego; Beck, Dominik; Perera, Dilmi; Wong, Jason W H; Pimanda, John E

2014-01-01

The BloodChIP database (http://www.med.unsw.edu.au/CRCWeb.nsf/page/BloodChIP) supports exploration and visualization of combinatorial transcription factor (TF) binding at a particular locus in human CD34-positive and other normal and leukaemic cells or retrieval of target gene sets for user-defined combinations of TFs across one or more cell types. Increasing numbers of genome-wide TF binding profiles are being added to public repositories, and this trend is likely to continue. For the power of these data sets to be fully harnessed by experimental scientists, there is a need for these data to be placed in context and easily accessible for downstream applications. To this end, we have built a user-friendly database that has at its core the genome-wide binding profiles of seven key haematopoietic TFs in human stem/progenitor cells. These binding profiles are compared with binding profiles in normal differentiated and leukaemic cells. We have integrated these TF binding profiles with chromatin marks and expression data in normal and leukaemic cell fractions. All queries can be exported into external sites to construct TF-gene and protein-protein networks and to evaluate the association of genes with cellular processes and tissue expression.

Global Expression Profiling of Globose Basal Cells and Neurogenic Progression Within the Olfactory Epithelium

PubMed Central

Krolewski, Richard C.; Packard, Adam; Schwob, James E.

2013-01-01

Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of enhanced green fluorescent protein (eGFP) and cell surface markers to FACS-isolate ΔSox2-eGFP(+) GBCs, Neurog1-eGFP(+) GBCs and immature neurons, and ΔOMP-eGFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2,100 genes that are upregulated compared to sustentacular cells. A further cohort of >1,200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-eGFP(+) population. In contrast, the abbreviated lifespan of ΔOMP-eGFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream ΔSox2-eGFP(+) GBCs and Neurog1-eGFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion. PMID:22847514

A High Protein Diet during Pregnancy Affects Hepatic Gene Expression of Energy Sensing Pathways along Ontogenesis in a Porcine Model

PubMed Central

Oster, Michael; Murani, Eduard; Metges, Cornelia C.; Ponsuksili, Siriluck; Wimmers, Klaus

2011-01-01

In rodent models and in humans the impact of gestational diets on the offspring's phenotype was shown experimentally and epidemiologically. The underlying programming of fetal development was shown to be associated with an increased risk of degenerative diseases in adulthood, including the metabolic syndrome. There are clues that diet-dependent modifications of the metabolism during fetal life can persist until adulthood. This leads to the hypothesis that the offspring's transcriptomes show short-term and long-term changes depending on the maternal diet. To this end pregnant German landrace gilts were fed either a high protein diet (HP, 30% CP) or an adequate protein diet (AP, 12% CP) throughout pregnancy. Hepatic transcriptome profiles of the offspring were analyzed at prenatal (94 dpc) and postnatal stages (1, 28, 188 dpn). Depending on the gestational dietary exposure, mRNA expression levels of genes related to energy metabolism, N-metabolism, growth factor signaling pathways, lipid metabolism, nucleic acid metabolism and stress/immune response were affected either in a short-term or in a long-term manner. Gene expression profiles at fetal stage 94 dpc were almost unchanged between the diets. The gestational HP diet affected the hepatic expression profiles at prenatal and postnatal stages. The effects encompassed a modulation of the genome in terms of an altered responsiveness of energy and nutrient sensing pathways. Differential expression of genes related to energy production and nutrient utilization contribute to the maintenance of development and growth performance within physiological norms, however the modulation of these pathways may be accompanied by a predisposition for metabolic disturbances up to adult stages. PMID:21789176

A novel gene expression profile in lymphatics associated with tumor growth and nodal metastasis.

PubMed

Clasper, Steven; Royston, Daniel; Baban, Dilair; Cao, Yihai; Ewers, Stephan; Butz, Stefan; Vestweber, Dietmar; Jackson, David G

2008-09-15

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., >or=2-fold up- or down-regulated, P

Gene expression profiling in respond to TBT exposure in small abalone Haliotis diversicolor.

PubMed

Jia, Xiwei; Zou, Zhihua; Wang, Guodong; Wang, Shuhong; Wang, Yilei; Zhang, Ziping

2011-10-01

In this study, we investigated the gene expression profiling of small abalone, Haliotis diversicolor by tributyltin (TBT) exposure using a cDNA microarray containing 2473 unique transcripts. Totally, 107 up-regulated genes and 41 down-regulated genes were found. For further investigation of candidate genes from microarray data and EST analysis, quantitative real-time PCR was performed at 6 h, 24 h, 48 h, 96 h and 192 h TBT exposure. 26 genes were found to be significantly differentially expressed in different time course, 3 of them were unknown. Some gene homologues like cellulose, endo-beta-1,4-glucanase, ferritin subunit 1 and thiolester containing protein II CG7052-PB might be the good biomarker candidate for TBT monitor. The identification of stress response genes and their expression profiles will permit detailed investigation of the defense responses of small abalone genes. Published by Elsevier Ltd.

The E2F3—Oncomir 1 axis is activated in Wilms Tumor

PubMed Central

Kort, Eric J.; Farber, Leslie; Tretiakova, Maria; Petillo, David; Furge, Kyle A.; Yang, Ximing J.; Cornelius, Albert; Teh, Bin T.

2008-01-01

Oncomir-1 is an oncogenic cluster of microRNAs located on chromosome 13. Previous in vitro studies demonstrated that it is transcriptionally regulated by the transcription factor E2F3. In this report we combine expression profiling of both messenger RNA (mRNA) and micro RNAs (miRNA) in Wilms tumor (WT) samples to provide the first evidence that the E2F3—Oncomir 1 axis, previously identified in cell culture, is deregulated in primary human tumors. Analysis of RNA expression signatures demonstrated that an E2F3 gene signature was activated in all Wilms tumor samples analyzed, in contrast to other kidney tumors. This finding was validated by immunohistochemistry (IHC) on the protein level. Expression of E2F3 was lowest in early stage tumors, and highest in metastatic tissue. Expression profiling of miRNAs in WT showed that expression of each measured member of the Oncomir-1 family was highest in WT relative to other kidney tumor subtypes. Quantitative polymerase chain reaction (PCR) confirmed that these microRNAs were overexpressed in Wilms tumor relative to normal kidney tissue. These results suggest that the E2F3—Oncomir-1 axis is activated in Wilms tumor. Our study also demonstrates the utility of integrated genomics combining gene signature analysis with miRNA expression profiling to identify protein-miRNA interactions that are perturbed in disease states. PMID:18519660

Evaluation of the collaborative network of highly correlating skin proteins and its change following treatment with glucocorticoids

PubMed Central

2010-01-01

Background Glucocorticoids (GC) represent the core treatment modality for many inflammatory diseases. Its mode of action is difficult to grasp, not least because it includes direct modulation of many components of the extracellular matrix as well as complex anti-inflammatory effects. Protein expression profile of skin proteins is being changed with topical application of GC, however, the knowledge about singular markers in this regard is only patchy and collaboration is ill defined. Material/Methods Scar formation was observed under different doses of GC, which were locally applied on the back skin of mice (1 to 3 weeks). After euthanasia we analyzed protein expression of collagen I and III (picrosirius) in scar tissue together with 16 additional protein markers, which are involved in wound healing, with immunhistochemistry. For assessing GC's effect on co-expression we compared our results with a model of random figures to estimate how many significant correlations should be expected by chance. Results GC altered collagen and protein expression with distinct results in different areas of investigation. Most often we observed a reduced expression after application of low dose GC. In the scar infiltrate a multivariate analysis confirmed the significant impact of both GC concentrations. Calculation of Spearman's correlation coefficient similarly resulted in a significant impact of GC, and furthermore, offered the possibility to grasp the entire interactive profile in between all variables studied. The biological markers, which were connected by significant correlations could be arranged in a highly cross-linked network that involved most of the markers measured. A marker highly cross-linked with more than 3 significant correlations was indicated by a higher variation of all its correlations to the other variables, resulting in a standard deviation of > 0.2. Conclusion In addition to immunohistochemical analysis of single protein markers multivariate analysis of co-expressions by use of correlation coefficients reveals the complexity of biological relationships and identifies complex biological effects of GC on skin scarring. Depiction of collaborative clusters will help to understand functional pathways. The functional importance of highly cross-linked proteins will have to be proven in subsequent studies. PMID:20509951

Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells

PubMed Central

2009-01-01

Background Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses on human malignant melanoma cells in vitro in order to understand and predict the possible effect of electric pulses on gene expression and their possible effect on cell behaviour. Methods We used microarrays with 2698 different oligonucleotides to obtain the expression profile of genes involved in apoptosis and cancer development in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Results Cells exposed to ECT pulses showed a 68.8% average survival rate, while cells exposed to EGT pulses showed a 31.4% average survival rate. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce an HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Conclusion Our results show that electroporation does not significantly change the expression profile of major tumour suppressor genes or oncogenes of the cell cycle. Moreover, electroporation also does not changes the expression of genes involved in the stability of DNA, supporting current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method, it does to some extent induce stress, which resulted in the expression of the environmental stress response mechanism, HSP70. PMID:19709437

Profiling Antibody Responses to Infections by Chlamydia abortus Enables Identification of Potential Virulence Factors and Candidates for Serodiagnosis

PubMed Central

Forsbach-Birk, Vera; Foddis, Corinna; Simnacher, Ulrike; Wilkat, Max; Longbottom, David; Walder, Gernot; Benesch, Christiane; Ganter, Martin; Sachse, Konrad; Essig, Andreas

2013-01-01

Enzootic abortion of ewes (EAE) due to infection with the obligate intracellular pathogen Chlamydia (C.) abortus is an important zoonosis leading to considerable economic loss to agriculture worldwide. The pathogen can be transmitted to humans and may lead to serious infection in pregnant women. Knowledge about epidemiology, clinical course and transmission to humans is hampered by the lack of reliable diagnostic tools. Immunoreactive proteins, which are expressed in infected animals and humans, may serve as novel candidates for diagnostic marker proteins and represent putative virulence factors. In order to broaden the spectrum of immunogenic C. abortus proteins we applied 2D immunoblot analysis and screening of an expression library using human and animal sera. We have identified 48 immunoreactive proteins representing potential diagnostic markers and also putative virulence factors, such as CAB080 (homologue of the “macrophage infectivity potentiator”, MIP), CAB167 (homologue of the “translocated actin recruitment protein”, TARP), CAB712 (homologue of the “chlamydial protease-like activity factor”, CPAF), CAB776 (homologue of the “Polymorphic membrane protein D”, PmpD), and the “hypothetical proteins” CAB063, CAB408 and CAB821, which are predicted to be type III secreted. We selected two putative virulence factors for further characterization, i.e. CAB080 (cMIP) and CAB063, and studied their expression profiles at transcript and protein levels. Analysis of the subcellular localization of both proteins throughout the developmental cycle revealed CAB063 being the first C. abortus protein shown to be translocated to the host cell nucleus. PMID:24260366

Genome-wide transcriptional analysis of two soybean genotypes under dehydration and rehydration conditions

PubMed Central

2013-01-01

Background Soybean is an important crop that provides valuable proteins and oils for human use. Because soybean growth and development is extremely sensitive to water deficit, quality and crop yields are severely impacted by drought stress. In the face of limited water resources, drought-responsive genes are therefore of interest. Identification and analysis of dehydration- and rehydration-inducible differentially expressed genes (DEGs) would not only aid elucidation of molecular mechanisms of stress response, but also enable improvement of crop stress tolerance via gene transfer. Using Digital Gene Expression Tag profiling (DGE), a new technique based on Illumina sequencing, we analyzed expression profiles between two soybean genotypes to identify drought-responsive genes. Results Two soybean genotypes—drought-tolerant Jindou21 and drought-sensitive Zhongdou33—were subjected to dehydration and rehydration conditions. For analysis of DEGs under dehydration conditions, 20 cDNA libraries were generated from roots and leaves at two different time points under well-watered and dehydration conditions. We also generated eight libraries for analysis under rehydration conditions. Sequencing of the 28 libraries produced 25,000–33,000 unambiguous tags, which were mapped to reference sequences for annotation of expressed genes. Many genes exhibited significant expression differences among the libraries. DEGs in the drought-tolerant genotype were identified by comparison of DEGs among treatments and genotypes. In Jindou21, 518 and 614 genes were differentially expressed under dehydration in leaves and roots, respectively, with 24 identified both in leaves and roots. The main functional categories enriched in these DEGs were metabolic process, response to stresses, plant hormone signal transduction, protein processing, and plant-pathogen interaction pathway; the associated genes primarily encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significantly expressed (|log2 ratio| ≥ 8) genes— Glyma15g03920, Glyma05g02470, Glyma15g15010, Glyma05g09070, Glyma06g35630, Glyma08g12590, and Glyma11g16000—are more likely to determine drought stress tolerance. The expression patterns of eight randomly-selected genes were confirmed by quantitative RT-PCR; the results of QRT-PCR analysis agreed with transcriptional profile data for 96 out of 128 (75%) data points. Conclusions Many soybean genes were differentially expressed between drought-tolerant and drought-sensitive genotypes. Based on GO functional annotation and pathway enrichment analysis, some of these genes encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significant DEGs are candidates for improving soybean drought tolerance. These findings will be helpful for analysis and elucidation of molecular mechanisms of drought tolerance; they also provide a basis for cultivating new varieties of drought-tolerant soybean. PMID:24093224

Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

PubMed

Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

2012-01-01

Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

A Systems Biology Approach to Link Nuclear Factor Kappa B Activation with Lethal Prostate Cancer

DTIC Science & Technology

2014-05-01

developed as a routine clinical assay. 12 Task 1B: Perform protein profiling of circulating blood proteins and determine whether a protein...or set of proteins indicative of NFκB activation are associated with lethal prostate cancer. Circulating proteins will be assessed in two cohorts of...throughput functional genomic data. Nucleic acids research 2009;37:D885-90. 3. Parkinson H, Kapushesky M, Kolesnikov N, et al. ArrayExpress update--from

Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

PubMed

Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

2016-09-01

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

Identification of inflammation-related proteins in a murine colitis model by 2D fluorescence difference gel electrophoresis and mass spectrometry.

PubMed

Naito, Yuji; Takagi, Tomohisa; Okada, Hitomi; Omatsu, Tatsushi; Mizushima, Katsura; Handa, Osamu; Kokura, Satoshi; Ichikawa, Hiroshi; Fujiwake, Hideshi; Yoshikawa, Toshikazu

2010-05-01

The aim of this study was to identify new intestinal proteins potentially associated with acute inflammation using proteomic profiling of an in vivo mice model of ulcerative colitis. 2D fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight spectrometer (MALDI-TOF) peptide mass fingerprinting were used to determine differentially expressed proteins between normal and inflamed intestinal mucosa. Acute colitis was induced by 8.0% dextran sodium sulfate (DSS) given p.o. for 7 days. Among a total of seven protein spots showing differential expression, we identified five different proteins, of which two were upregulated and three downregulated in colitis in comparison to normal mucosa, using the MASCOT search engine. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase 2 and serpin b1a were upregulated proteins, and protein disulfide-isomerase A3, peroxiredoxin-6 and vimentin were identified as downregulated proteins. These identified proteins may be responsible for the development of the intestinal inflammation. 2D-DIGE and MALDI-TOF mass spectrometry are useful in the search for the differentially expressed proteins.

Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

PubMed

Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

2013-01-14

The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

ALK Expression Is a Novel Marker for the WNT-activated Type of Pediatric Medulloblastoma and an Indicator of Good Prognosis for Patients.

PubMed

Łastowska, Maria; Trubicka, Joanna; Niemira, Magdalena; Paczkowska-Abdulsalam, Magdalena; Karkucińska-Więckowska, Agnieszka; Kaleta, Magdalena; Drogosiewicz, Monika; Tarasińska, Magdalena; Perek-Polnik, Marta; Krętowski, Adam; Dembowska-Bagińska, Bożenna; Grajkowska, Wiesława; Pronicki, Maciej; Matyja, Ewa

2017-06-01

ALK gene rearrangements were identified in a variety of cancers, including neuroblastoma, where the presence of ALK expression is associated with adverse prognosis. ALK mutations have recently been found in the pediatric brain tumor medulloblastoma, and microarray data indicate that ALK is highly expressed in a subset of these tumors. Therefore, we investigated whether ALK expression correlates with transcriptional profiles and clinical features of medulloblastoma. Tumors from 116 medulloblastoma patients were studied at diagnosis for the detection of ALK expression at the RNA level by an application of NanoString technology and at the protein level by immunohistochemistry using antibody ALK clone D5F3. The results indicate that ALK expression, at both the RNA and the protein levels, is strongly associated with the WNT-activated type of tumors and therefore may serve as a useful marker for the detection of this type of medulloblastoma. Importantly, ALK protein expression alone is also an indicator of good prognosis for medulloblastoma patients.

Anatomical and histological profiling of orphan G-protein-coupled receptor expression in gastrointestinal tract of C57BL/6J mice.

PubMed

Ito, Junko; Ito, Masahiko; Nambu, Hirohide; Fujikawa, Toru; Tanaka, Kenichi; Iwaasa, Hisashi; Tokita, Shigeru

2009-11-01

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors and regulate a variety of physiological and disease processes. Although the roles of many non-odorant GPCRs have been identified in vivo, several GPCRs remain orphans (oGPCRs). The gastrointestinal (GI) tract is the largest endocrine organ and is a promising target for drug discovery. Given their close link to physiological function, the anatomical and histological expression profiles of benchmark GI-related GPCRs, such as the cholecystokinin-1 receptor and GPR120, and 106 oGPCRs were investigated in the mucosal and muscle-myenteric nerve layers in the GI tract of C57BL/6J mice by quantitative real-time polymerase chain reaction. The mRNA expression patterns of these benchmark molecules were consistent with previous in situ hybridization and immunohistochemical studies, validating the experimental protocols in this study. Of 96 oGPCRs with significant mRNA expression in the GI tract, several oGPCRs showed unique expression patterns. GPR85, GPR37, GPR37L1, brain-specific angiogenesis inhibitor (BAI) 1, BAI2, BAI3, and GPRC5B mRNAs were preferentially expressed in the muscle-myenteric nerve layer, similar to GPCRs that are expressed in both the central and enteric nerve systems and that play multiple regulatory roles throughout the gut-brain axis. In contrast, GPR112, trace amine-associated receptor (TAAR) 1, TAAR2, and GPRC5A mRNAs were preferentially expressed in the mucosal layer, suggesting their potential roles in the regulation of secretion, immunity, and epithelial homeostasis. These anatomical and histological mRNA expression profiles of oGPCRs provide useful clues about the physiological roles of oGPCRs in the GI tract.

Gene expression profiles in the bovine corpus luteum (CL) during the estrous cycle and pregnancy: Possible roles of chemokines in regulating CL function during pregnancy

PubMed Central

SAKUMOTO, Ryosuke; HAYASHI, Ken-Go; HOSOE, Misa; IGA, Kosuke; KIZAKI, Keiichiro; OKUDA, Kiyoshi

2014-01-01

To determine functional differences between the corpus luteum (CL) of the estrous cycle and pregnancy in cows, gene expression profiles were compared using a 15 K bovine oligo DNA microarray. In the pregnant CL at days 20–25, 40–45 and 150–160, the expressions of 138, 265 and 455 genes differed by a factor of > 2-fold (P < 0.05) from their expressions in the cyclic CL (days 10–12 of the estrous cycle). Messenger RNA expressions of chemokines (eotaxin, lymphotactin and ENA-78) and their receptors (CCR3, XCR1 and CXCR2) were validated by quantitative real-time PCR. Transcripts of eotaxin were more abundant in the CL at days 40–45 and 150–160 of pregnancy than in the cyclic CL (P < 0.01). In contrast, the mRNA expressions of lymphotactin, ENA-78 and XCR1 were lower in the CL of pregnancy (P < 0.05). Messenger RNAs of CCR3 and CXCR2 were similarly detected both in the cyclic and pregnant CL. Tissue protein levels of eotaxin were significantly higher in the CL at days 150–160 of pregnancy than in the CL at other stages, whereas the lymphotactin protein levels in the CL at days 20–25 of pregnancy were lower (P < 0.05). Immunohistochemical staining showed that CCR3 was expressed in the luteal cells and that XCR1 was expressed in both the luteal cells and endothelial cells. Collectively, the different gene expression profiles may contribute to functional differences between the cyclic and pregnant CL, and chemokines including eotaxin and lymphotactin may regulate CL function during pregnancy in cows. PMID:25382605

Soybean TCP transcription factors: Evolution, classification, protein interaction and stress and hormone responsiveness.

PubMed

Feng, Zhi-Juan; Xu, Sheng-Chun; Liu, Na; Zhang, Gu-Wen; Hu, Qi-Zan; Gong, Ya-Ming

2018-06-01

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, a family of plant-specific proteins, play crucial roles in plant growth and development and stress response. However, systematical information is unknown regarding the TCP gene family in soybean. In the present study, a total of 54 GmTCPs were identified in soybean, which were grouped into 11 groups with the typical TCP conserved domains. Phylogenetic relationship, protein motif and gene structure analyses distinguished the GmTCPs into two homology classes: Class I and Class II. Class II was then differentiated into two subclasses: CIN and CYC/TB1. Unique cis-element number and composition existed in the promoter regions which might be involved in the gene transcriptional regulation of different GmTCPs. Tissue expression analysis demonstrated the diverse spatiotemporal expression profiles of GmTCPs. Furthermore, the interaction protein of one previously functionally unknown TCP protein-GmTCP8 was investigated. Yeast two-hybrid assay showed the interaction between GmTCP8 and an abscisic acid receptor (GmPYL10). QRT-PCR assays indicated the distinct expression profiles of GmTCPs in response to abiotic stresses (heat, drought and salt) and stress-related signals (abscisic acid, brassinolide, salicylicacid and methyl jasmonate). These results will facilitate to uncover the possible roles of GmTCPs under abiotic stress and hormone signal responses in soybean. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Genome-wide Expression Profiling, In Vivo DNA Binding Analysis, and Probabilistic Motif Prediction Reveal Novel Abf1 Target Genes during Fermentation, Respiration, and Sporulation in Yeast

PubMed Central

Schlecht, Ulrich; Erb, Ionas; Demougin, Philippe; Robine, Nicolas; Borde, Valérie; van Nimwegen, Erik; Nicolas, Alain

2008-01-01

The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein's target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein's variable DNA binding pattern during mitotic growth and meiotic development. PMID:18305101

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

PubMed Central

Osório, Nádia; Pereira, Carla; Simões, Sara; Delgadillo, Ivonne

2018-01-01

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells. PMID:29518018

Global Gene Expression Profiling in PAI-1 Knockout Murine Heart and Kidney: Molecular Basis of Cardiac-Selective Fibrosis

PubMed Central

Ghosh, Asish K.; Murphy, Sheila B.; Kishore, Raj; Vaughan, Douglas E.

2013-01-01

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed. PMID:23724005

A Straightforward and Highly Efficient Precipitation/On-pellet Digestion Procedure Coupled to a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-free Expression Profiling of the Swine Heart Mitochondrial Proteome

PubMed Central

Duan, Xiaotao; Young, Rebecca; Straubinger, Robert M.; Page, Brian J.; Cao, Jin; Wang, Hao; Yu, Haoying; Canty, John M.; Qu, Jun

2009-01-01

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes. PMID:19290621

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

PubMed

Cathcart, Mary-Clare; Gray, Steven G; Baird, Anne-Marie; Boyle, Elaine; Gately, Kathy; Kay, Elaine; Cummins, Robert; Pidgeon, Graham P; O'Byrne, Kenneth J

2011-11-15

Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. PGIS expression was reduced/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. Copyright © 2011 American Cancer Society.

Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development.

PubMed

Fuentealba, Jaime; Toro-Tapia, Gabriela; Rodriguez, Marion; Arriagada, Cecilia; Maureira, Alejandro; Beyer, Andrea; Villaseca, Soraya; Leal, Juan I; Hinrichs, Maria V; Olate, Juan; Caprile, Teresa; Torrejón, Marcela

2016-09-01

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development. Copyright © 2016 Elsevier B.V. All rights reserved.

G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

PubMed Central

2013-01-01

Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors. PMID:24252460

A human protein atlas for normal and cancer tissues based on antibody proteomics.

PubMed

Uhlén, Mathias; Björling, Erik; Agaton, Charlotta; Szigyarto, Cristina Al-Khalili; Amini, Bahram; Andersen, Elisabet; Andersson, Ann-Catrin; Angelidou, Pia; Asplund, Anna; Asplund, Caroline; Berglund, Lisa; Bergström, Kristina; Brumer, Harry; Cerjan, Dijana; Ekström, Marica; Elobeid, Adila; Eriksson, Cecilia; Fagerberg, Linn; Falk, Ronny; Fall, Jenny; Forsberg, Mattias; Björklund, Marcus Gry; Gumbel, Kristoffer; Halimi, Asif; Hallin, Inga; Hamsten, Carl; Hansson, Marianne; Hedhammar, My; Hercules, Görel; Kampf, Caroline; Larsson, Karin; Lindskog, Mats; Lodewyckx, Wald; Lund, Jan; Lundeberg, Joakim; Magnusson, Kristina; Malm, Erik; Nilsson, Peter; Odling, Jenny; Oksvold, Per; Olsson, Ingmarie; Oster, Emma; Ottosson, Jenny; Paavilainen, Linda; Persson, Anja; Rimini, Rebecca; Rockberg, Johan; Runeson, Marcus; Sivertsson, Asa; Sköllermo, Anna; Steen, Johanna; Stenvall, Maria; Sterky, Fredrik; Strömberg, Sara; Sundberg, Mårten; Tegel, Hanna; Tourle, Samuel; Wahlund, Eva; Waldén, Annelie; Wan, Jinghong; Wernérus, Henrik; Westberg, Joakim; Wester, Kenneth; Wrethagen, Ulla; Xu, Lan Lan; Hober, Sophia; Pontén, Fredrik

2005-12-01

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus

PubMed Central

Hu, Ping; Wang, Tao; Tao, Jing; Zong, Shixiang

2017-01-01

Seabuckthorn carpenter moth, Eogystia hippophaecolus (Lepidoptera: Cossidae), is an important pest of sea buckthorn (Hippophae rhamnoides), which is a shrub that has significant ecological and economic value in China. E. hippophaecolus is highly cold tolerant, but limited studies have been conducted to elucidate the molecular mechanisms underlying its cold resistance. Here we sequenced the E. hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads. We investigated the larval response to cold stress by comparing gene expression profiles between treatments. We obtained 118,034 unigenes, of which 22,161 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. These resulted in 57 GO terms and 193 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparing transcriptome profiles for differential gene expression, we identified many differentially expressed proteins and genes, including heat shock proteins and cuticular proteins which have previously been reported to be involved in cold resistance of insects. This study provides a global transcriptome analysis and an assessment of differential gene expression in E. hippophaecolus under cold stress. We found seven differential expressed genes in common between developmental stages, which were verified with qPCR. Our findings facilitate future genomic studies aimed at improving our understanding of the molecular mechanisms underlying the response of insects to low temperatures. PMID:29131867

Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus.

PubMed

Cui, Mingming; Hu, Ping; Wang, Tao; Tao, Jing; Zong, Shixiang

2017-01-01

Seabuckthorn carpenter moth, Eogystia hippophaecolus (Lepidoptera: Cossidae), is an important pest of sea buckthorn (Hippophae rhamnoides), which is a shrub that has significant ecological and economic value in China. E. hippophaecolus is highly cold tolerant, but limited studies have been conducted to elucidate the molecular mechanisms underlying its cold resistance. Here we sequenced the E. hippophaecolus transcriptome using RNA-Seq technology and performed de novo assembly from the short paired-end reads. We investigated the larval response to cold stress by comparing gene expression profiles between treatments. We obtained 118,034 unigenes, of which 22,161 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. These resulted in 57 GO terms and 193 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparing transcriptome profiles for differential gene expression, we identified many differentially expressed proteins and genes, including heat shock proteins and cuticular proteins which have previously been reported to be involved in cold resistance of insects. This study provides a global transcriptome analysis and an assessment of differential gene expression in E. hippophaecolus under cold stress. We found seven differential expressed genes in common between developmental stages, which were verified with qPCR. Our findings facilitate future genomic studies aimed at improving our understanding of the molecular mechanisms underlying the response of insects to low temperatures.

Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy.

PubMed

Gholamnezhadjafari, Reza; Tajik, Nader; Falak, Reza; Aflatoonian, Reza; Dehghan, Sanaz; Rezaei, Abbas

2017-07-01

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

PubMed Central

Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

2012-01-01

Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study. PMID:22272336

Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes

PubMed Central

Huang, Yong; Huang, Jinxiu; Qi, Renli; Wang, Qi; Wu, Yongjiang; Wang, Jing

2016-01-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes. PMID:27783036

Expression profile analysis of genes involved in horizontal gravitropism bending growth in the creeping shoots of ground-cover chrysanthemum by suppression subtractive hybridization.

PubMed

Xia, Shengjun; Chen, Yu; Jiang, Jiafu; Chen, Sumei; Guan, Zhiyong; Fang, Weimin; Chen, Fadi

2013-01-01

The molecular mechanisms underlying gravitropic bending of shoots are poorly understood and how genes related with this growing progress is still unclear. To identify genes related to asymmetric growth in the creeping shoots of chrysanthemum, suppression subtractive hybridization was used to visualize differential gene expression in the upper and lower halves of creeping shoots of ground-cover chrysanthemum under gravistimulation. Sequencing of 43 selected clones produced 41 unigenes (40 singletons and 1 unigenes), which were classifiable into 9 functional categories. A notable frequency of genes involve in cell wall biosynthesis up-regulated during gravistimulation in the upper side or lower side were found, such as beta tubulin (TUB), subtilisin-like protease (SBT), Glutathione S-transferase (GST), and expensing-like protein (EXP), lipid transfer proteins (LTPs), glycine-rich protein (GRP) and membrane proteins. Our findings also highlighted the function of some metal transporter during asymmetric growth, including the boron transporter (BT) and ZIP transporter (ZT), which were thought primarily for maintaining the integrity of cell walls and played important roles in cellulose biosynthesis. CmTUB (beta tubulin) was cloned, and the expression profile and phylogeny was examined, because the cytoskeleton of plant cells involved in the plant gravitropic bending growth is well known.

Proteomic profiling of developing cotton fibers from wild and domesticated Gossypium barbadense.

PubMed

Hu, Guanjing; Koh, Jin; Yoo, Mi-Jeong; Grupp, Kara; Chen, Sixue; Wendel, Jonathan F

2013-10-01

Pima cotton (Gossypium barbadense) is widely cultivated because of its long, strong seed trichomes ('fibers') used for premium textiles. These agronomically advanced fibers were derived following domestication and thousands of years of human-mediated crop improvement. To gain an insight into fiber development and evolution, we conducted comparative proteomic and transcriptomic profiling of developing fiber from an elite cultivar and a wild accession. Analyses using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology identified 1317 proteins in fiber. Of these, 205 were differentially expressed across developmental stages, and 190 showed differential expression between wild and cultivated forms, 14.4% of the proteome sampled. Human selection may have shifted the timing of developmental modules, such that some occur earlier in domesticated than in wild cotton. A novel approach was used to detect possible biased expression of homoeologous copies of proteins. Results indicate a significant partitioning of duplicate gene expression at the protein level, but an approximately equal degree of bias for each of the two constituent genomes of allopolyploid cotton. Our results demonstrate the power of complementary transcriptomic and proteomic approaches for the study of the domestication process. They also provide a rich database for mining for functional analyses of cotton improvement or evolution. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Genome-wide investigation and expression analyses of WD40 protein family in the model plant foxtail millet (Setaria italica L.).

PubMed

Mishra, Awdhesh Kumar; Muthamilarasan, Mehanathan; Khan, Yusuf; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I-V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

PubMed Central

Mishra, Awdhesh Kumar; Muthamilarasan, Mehanathan; Khan, Yusuf; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses. PMID:24466268

Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

PubMed Central

Parry, Jesse J.; Chen, Ronald; Andrews, Rebecca; Lears, Kimberly A.

2012-01-01

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling. PMID:22495673

Directed Shotgun Proteomics Guided by Saturated RNA-seq Identifies a Complete Expressed Prokaryotic Proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Omasits, U.; Quebatte, Maxime; Stekhoven, Daniel J.

2013-11-01

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, wemore » could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ~90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.« less

Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome

PubMed Central

Omasits, Ulrich; Quebatte, Maxime; Stekhoven, Daniel J.; Fortes, Claudia; Roschitzki, Bernd; Robinson, Mark D.; Dehio, Christoph; Ahrens, Christian H.

2013-01-01

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, we could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ∼90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor. PMID:23878158

Quantitative proteomics revealed partial fungistatic mechanism of ammonia against conidial germination of nematode-trapping fungus Arthrobotrys oligospora ATCC24927.

PubMed

Liu, Tong; Tian, Dong-Wei; Zou, Li-Juan; Liu, Fang-Yu; Can, Qi-Yan; Yang, Jin-Kui; Xu, Jian-Ping; Huang, Xiao-Wei; Xi, Jia-Qin; Zhu, Ming-Liang; Mo, Ming-He; Zhang, Ke-Qin

2018-05-01

Ammonia is one of the fungistatic factors in soil that can suppress conidial germination, but the molecular mechanism underlying the suppression is unknown. In this study, the proteomes of fungistatic conidia, fresh conidia and germinated conidia of Arthrobotrys oligospora ATCC24927 were determined and quantified. The protein expression profile of fungistatic conidia was significantly different from those in the other two conditions. 281 proteins were down expressed in fungistatic conidia and characterized by GO annotation. Gene transcription analysis and inhibition of puromycin (a protein translation inhibitor) on conidial germination suggested that down expression of 33 protein translation related proteins might well result in repression of protein synthesis and inhibition of conidial germination. In addition, 16 down-expressed proteins were mapped to the Ras/mitogen-activated protein (Ras/MAP) regulatory networks which regulate conidial DNA synthesis. The conidial DNA synthesis was found to be definitely inhibited under by ammonia, and function studies of two Ras/MAP proteins by using knock-out strains provided partial evidence that Ras/MAP pathway regulate the conidial germination. These results suggested that down-expression of Ras/MAP related proteins might result in inhibition of DNA synthesis and finally result in inhibition conidial germination. This study revealed partial fungistatic mechanism of ammonia against conidial germination. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

PubMed Central

Michel, Audrey M; Baranov, Pavel V

2013-01-01

Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

Expression of multidrug resistance-related protein (MRP-1), lung resistance-related protein (LRP) and topoisomerase-II (TOPO-II) in Wilms' tumor: immunohistochemical study using TMA methodology.

PubMed

Fridman, Eduard; Skarda, Jozef; Pinthus, Jonatan H; Ramon, Jonathan; Mor, Yoran

2008-06-01

MRP-1, LRP and TOPO-II are all associated with protection of the cells from the adverse effects of various chemotherapeutics. The aim of this study was to measure the expression of these proteins in Wilms' tumor (WT). TMA block was constructed from 14 samples of WT's and from xenografts derived from them. Sections of the TMA were used for immunostaining against MRP-1, LRP and TOPO-IIa. All normal kidneys expressed MRP-1 but were either weakly or negatively stained for LRP and TOPO-IIa. In WT samples, MRP-1 was universally expressed, exclusively in the tubular component, while there was no expression of LRP and TOPO-IIa showed heterogeneous distribution. The xenografts varied in their MRP-1 and TOPO-IIa expression and exhibited weak/negative staining of LRP. This study shows that although all the proteins evaluated, had different expression patterns in the tumor samples, the most prominent changes in expression were found for MRP-1. The exact clinical implications of these changes in expression and their relevance to the resistance of these tumors to chemotherapy requires further investigation. The finding of different expression profiles for the multidrug resistance proteins in the original WT's and their xenografts suggests that the results of animal cancer models may be difficult to interpret.

Divergence between motoneurons: gene expression profiling provides a molecular characterization of functionally discrete somatic and autonomic motoneurons

PubMed Central

Cui, Dapeng; Dougherty, Kimberly J.; Machacek, David W.; Sawchuk, Michael; Hochman, Shawn; Baro, Deborah J.

2009-01-01

Studies in the developing spinal cord suggest that different motoneuron (MN) cell types express very different genetic programs, but the degree to which adult programs differ is unknown. To compare genetic programs between adult MN columnar cell types, we used laser capture micro-dissection (LCM) and Affymetrix microarrays to create expression profiles for three columnar cell types: lateral and medial MNs from lumbar segments and sympathetic preganglionic motoneurons located in the thoracic intermediolateral nucleus. A comparison of the three expression profiles indicated that ~7% (813/11,552) of the genes showed significant differences in their expression levels. The largest differences were observed between sympathetic preganglionic MNs and the lateral motor column, with 6% (706/11,552) of the genes being differentially expressed. Significant differences in expression were observed for 1.8% (207/11,552) of the genes when comparing sympathetic preganglionic MNs with the medial motor column. Lateral and medial MNs showed the least divergence, with 1.3% (150/11,552) of the genes being differentially expressed. These data indicate that the amount of divergence in expression profiles between identified columnar MNs does not strictly correlate with divergence of function as defined by innervation patterns (somatic/muscle vs. autonomic/viscera). Classification of the differentially expressed genes with regard to function showed that they underpin all fundamental cell systems and processes, although most differentially expressed genes encode proteins involved in signal transduction. Mining the expression profiles to examine transcription factors essential for MN development suggested that many of the same transcription factors participatein combinatorial codes in embryonic and adult neurons, but patterns of expression change significantly. PMID:16317082

Expression profile of osteoprotegerin, RANK and RANKL genes in the femoral head of patients with avascular necrosis.

PubMed

Samara, Stavroula; Dailiana, Zoe; Chassanidis, Christos; Koromila, Theodora; Papatheodorou, Loukia; Malizos, Konstantinos N; Kollia, Panagoula

2014-02-01

Femoral head avascular necrosis (AVN) is a recalcitrant disease of the hip that leads to joint destruction. Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B (RANK) and RANK ligand (RANKL) regulate the balance between osteoclasts-osteoblasts. The expression of these genes affects the maturation and function of osteoblasts-osteoclasts and bone remodeling. In this study, we investigated the molecular pathways leading to AVN by studying the expression profile of OPG, RANK and RANKL genes. Quantitative Real Time-PCR was performed for evaluation of OPG, RANK and RANKL expression. Analysis was based on parallel evaluation of mRNA and protein levels in normal/necrotic sites of 42 osteonecrotic femoral heads (FHs). OPG and RANKL protein levels were estimated by western blotting. The OPG mRNA levels were higher (insignificantly) in the necrotic than the normal site (p > 0.05). Although the expression of RANK and RANKL was significantly lower than OPG in both sites, RANK and RANKL mRNA levels were higher in the necrotic part than the normal (p < 0.05). Protein levels of OPG and RANKL showed no remarkable divergence. Our results indicate that differential expression mechanisms for OPG, RANK and RANKL that could play an important role in the progress of bone remodeling in the necrotic area, disturbing bone homeostasis. This finding may have an effect on the resulting bone destruction and the subsequent collapse of the hip joint. Copyright © 2013. Published by Elsevier Inc.

Protein profile in HBx transfected cells: a comparative iTRAQ-coupled 2D LC-MS/MS analysis.

PubMed

Feng, Huixing; Li, Xi; Niu, Dandan; Chen, Wei Ning

2010-06-16

The x protein of HBV (HBx) has been involved in the development of hepatocellular carcinoma (HCC), with a possible link to individual genotypes. Nevertheless, the underlying mechanism remains obscure. In this study, we aim to identify the HBx-induced protein profile in HepG2 cells by LC-MS/MS proteomics analysis. Our results indicated that proteins were differentially expressed in HepG2 cells transfected by HBx of various genotypes. Proteins associated with cytoskeleton were found to be either up-regulated (MACF1, HMGB1, Annexin A2) or down-regulated (Lamin A/C). These may in turn result in the decrease of focal adhesion and increase of cell migration in response to HBx. Levels of other cellular proteins with reported impact on the function of extracellular matrix (ECM) proteins and cell migration, including Ca(2+)-binding proteins (S100A11, S100A6, and S100A4) and proteasome protein (PSMA3), were affected by HBx. The differential protein profile identified in this study was also supported by our functional assay which indicated that cell migration was enhanced by HBx. Our preliminary study provided a new platform to establish a comprehensive cellular protein profile by LC-MS/MS proteomics analysis. Further downstream functional assays, including our reported cell migration assay, should provide new insights in the association between HCC and HBx. Copyright 2009 Elsevier B.V. All rights reserved.

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

MALDI-TOF Mass Spectrometry Enables a Comprehensive and Fast Analysis of Dynamics and Qualities of Stress Responses of Lactobacillus paracasei subsp. paracasei F19

PubMed Central

Schott, Ann-Sophie; Behr, Jürgen; Quinn, Jennifer; Vogel, Rudi F.

2016-01-01

Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected Lactobacillus (Lb.) paracasei subsp. paracasei TMW 1.1434 (F19) to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress). We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS), which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC), it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses. PMID:27783652

Renal Proteome in Mice with Different Susceptibilities to Fluorosis

PubMed Central

Peres-Buzalaf, Camila; Salvato, Fernanda; Labate, Carlos Alberto; Everett, Eric T.; Whitford, Gary Milton; Buzalaf, Marília Afonso Rabelo

2013-01-01

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies. PMID:23308176

Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

PubMed

Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

2018-04-11

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

In vivo Assessment and Potential Diagnosis of Xenobiotics that Perturb the Thyroid Pathway: Proteomic Analysis of Xenopus laevis Brain Tissue following Exposure to Model T4 Inhibitors

EPA Science Inventory

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression profiles in the brain of an amphibian model (Xenopus laevis) following in vivo exposur...

Global-scale profiling of differential expressed lysine acetylated proteins in colorectal cancer tumors and paired liver metastases.

PubMed

Shen, Zhanlong; Wang, Bo; Luo, Jianyuan; Jiang, Kewei; Zhang, Hui; Mustonen, Harri; Puolakkainen, Pauli; Zhu, Jun; Ye, Yingjiang; Wang, Shan

2016-06-16

Lysine acetylated modification was indicated to impact colorectal cancer (CRC)'s distant metastasis. However, the global acetylated proteins in CRC and the differential expressed acetylated proteins and acetylated sites between CRC primary and distant metastatic tumor remains unclear. Our aim was to construct a complete atlas of acetylome in CRC and paired liver metastases. Combining high affinity enrichment of acetylated peptides with high sensitive mass spectrometry, we identified 603 acetylation sites from 316 proteins, among which 462 acetylation sites corresponding to 243 proteins were quantified. We further classified them into groups according to cell component, molecular function and biological process and analyzed the metabolic pathways, domain structures and protein interaction networks. Finally, we evaluated the differentially expressed lysine acetylation sites and revealed that 31 acetylated sites of 22 proteins were downregulated in CRC liver metastases compared to that in primary CRC while 40 acetylated sites of 32 proteins were upregulated, of which HIST2H3AK19Ac and H2BLK121Ac were the acetylated histones most changed, while TPM2 K152Ac and ADH1B K331Ac were the acetylated non-histones most altered. These results provide an expanded understanding of acetylome in CRC and its distant metastasis, and might prove applicable in the molecular targeted therapy of metastatic CRC. This study described provides, for the first time, that full-scale profiling of lysine acetylated proteins were identified and quantified in colorectal cancer (CRC) and paired liver metastases. The novelty of the study is that we constructed a complete atlas of acetylome in CRC and paired liver metastases. Moreover, we analyzed these differentially expressed acetylated proteins in cell component, molecular function and biological process. In addition, metabolic pathways, domain structures and protein interaction networks of acetylated proteins were also investigated. Our approaches shows that of the differentially expressed proteins, HIST2H3AK19Ac and H2BLK121Ac were the acetylated histones most changed, while TPM2 K152Ac and ADH1B K331Ac were the acetylated non-histones most altered. Our findings provide an expanded understanding of acetylome in CRC and its distant metastasis, and might prove applicable in the molecular targeted therapy of metastatic CRC. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

NASA Astrophysics Data System (ADS)

Bhargava, Maneesh

Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

Altered protein expression profile associated with phenotypic changes in lung fibroblasts co-cultured with gold nanoparticle-treated small airway epithelial cells.

PubMed

Ng, Cheng-Teng; Yung, Lin-Yue Lanry; Swa, Hannah Lee-Foon; Poh, Rebecca Wan-Yan; Gunaratne, Jayantha; Bay, Boon-Huat

2015-01-01

Despite the availability of toxicity studies on cellular exposure to gold nanoparticles (AuNPs), there is scarcity of information with regard to the bystander effects induced by AuNPs on neighboring cells not exposed to the NPs. In this study, we showed that exposure of small airway epithelial cells (SAECs) to AuNPs induced changes in protein expression associated with functional effects in neighboring MRC5 lung fibroblasts in a co-culture system. Uptake of 20 nm size AuNPs by SAECs was first verified by focused ion beam scanning electron microscopy. Subsequently, pretreated SAECs were co-cultured with unexposed MRC5 lung fibroblasts, which then underwent proteome profiling using a quantitative proteomic approach. Stable-isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry identified 109 proteins (which included 47 up-regulated and 62 down-regulated proteins) that were differentially expressed in the lung fibroblasts co-cultured with AuNP pretreated SAECs. There was altered expression of proteins such as Paxillin, breast cancer anti-estrogen resistance 1 and Caveolin-1, which are known to be involved in the cell adhesion process. Morphological studies revealed that there was a concomitant increase in cell adhesion and altered F-actin stress fiber arrangement involving vinculin in the lung fibroblasts. It is likely that phenotypic changes observed in the underlying lung fibroblasts were mediated by AuNP-induced downstream signals in the pretreated SAECs and cell-cell cross talk. Copyright © 2014 Elsevier Ltd. All rights reserved.

EFFECTS OF TRENBOLONE ON EXPRESSION OF ESTROGEN-RESPONSIVE PLASMA PROTEINS IN ADULT SHEEPSHEAD MINNOW, (CYPRINODON VARIEGATUS)

EPA Science Inventory

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies using sheepshead minnows (SHM), mass spectral analysis detected four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/z...

Stress amplifies sex differences in primate prefrontal profiles of gene expression.

PubMed

Lee, Alex G; Hagenauer, Megan; Absher, Devin; Morrison, Kathleen E; Bale, Tracy L; Myers, Richard M; Watson, Stanley J; Akil, Huda; Schatzberg, Alan F; Lyons, David M

2017-11-02

Stress is a recognized risk factor for mood and anxiety disorders that occur more often in women than men. Prefrontal brain regions mediate stress coping, cognitive control, and emotion. Here, we investigate sex differences and stress effects on prefrontal cortical profiles of gene expression in squirrel monkey adults. Dorsolateral, ventrolateral, and ventromedial prefrontal cortical regions from 18 females and 12 males were collected after stress or no-stress treatment conditions. Gene expression profiles were acquired using HumanHT-12v4.0 Expression BeadChip arrays adapted for squirrel monkeys. Extensive variation between prefrontal cortical regions was discerned in the expression of numerous autosomal and sex chromosome genes. Robust sex differences were also identified across prefrontal cortical regions in the expression of mostly autosomal genes. Genes with increased expression in females compared to males were overrepresented in mitogen-activated protein kinase and neurotrophin signaling pathways. Many fewer genes with increased expression in males compared to females were discerned, and no molecular pathways were identified. Effect sizes for sex differences were greater in stress compared to no-stress conditions for ventromedial and ventrolateral prefrontal cortical regions but not dorsolateral prefrontal cortex. Stress amplifies sex differences in gene expression profiles for prefrontal cortical regions involved in stress coping and emotion regulation. Results suggest molecular targets for new treatments of stress disorders in human mental health.

Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: Comparison of genomic and proteomic responses and anchoring to histopathological parameters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberemm, A., E-mail: axel.oberemm@bfr.bund.d; Ahr, H.-J.; Bannasch, P.

2009-12-01

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplasticmore » and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.« less

Molecular Mechanisms of Increased Heart Rate in Shenxianshengmai-treated Bradycardia Rabbits.

PubMed

Liu, Zhou-Ying; Huang, Jian; Liu, Na-Na; Zheng, Min; Zhao, Tao; Zhao, Bu-Chang; Wang, Yi-Min; Pu, Jie-Lin

2017-01-20

The molecular mechanisms of Shenxianshengmai (SXSM), a traditional Chinese medicine, on bradycardia have been incompletely understood. The study tried to investigate the gene expression profile and proteomics of bradycardia rabbits' hearts after SXSM treatment. Twenty-four adult rabbits were randomly assigned in four groups: sham, model, model plus SXSM treatment, and sham plus SXSM treatment groups. Heart rate was recorded in all rabbits. Then, total RNA of atria and proteins of ventricle were isolated and quantified, respectively. Gene expression profiling was conducted by gene expression chip, and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the results of gene expression chip. We used isobaric tags for elative and absolute quantitation and Western blotting to identify altered proteins after SXSM treatment. There was a constant decrease in the mean heart rate (32%, from 238 ± 6 beats/min to 149 ± 12 beats/min) after six weeks in model compared with that in sham group. This effect was partially reversed by 4-week SXSM treatment. Complementary DNA microarray demonstrated that the increased acetylcholinesterase and reduced nicotinic receptor were take responsibility for the increased heart rate. In addition, proteins involved in calcium handling and signaling were affected by SXSM treatment. Real-time RT-PCR verified the results from gene chip. Results from proteomics demonstrated that SXSM enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle in ventricular myocardium to improve ATP generation. Long-term SXSM stimulates sympathetic transmission by increasing the expression of acetylcholinesterase and reduces the expression of nicotinic receptor to increase heart rate. SXSM also restored the calcium handling genes and altered genes involved in signaling. In addition, SXSM improves the ATP supply of ventricular myocardium by increasing proteins involved in TCA cycle and oxidation-respiratory chain.

Molecular Mechanisms of Increased Heart Rate in Shenxianshengmai-treated Bradycardia Rabbits

PubMed Central

Liu, Zhou-Ying; Huang, Jian; Liu, Na-Na; Zheng, Min; Zhao, Tao; Zhao, Bu-Chang; Wang, Yi-Min; Pu, Jie-Lin

2017-01-01

Background: The molecular mechanisms of Shenxianshengmai (SXSM), a traditional Chinese medicine, on bradycardia have been incompletely understood. The study tried to investigate the gene expression profile and proteomics of bradycardia rabbits’ hearts after SXSM treatment. Methods: Twenty-four adult rabbits were randomly assigned in four groups: sham, model, model plus SXSM treatment, and sham plus SXSM treatment groups. Heart rate was recorded in all rabbits. Then, total RNA of atria and proteins of ventricle were isolated and quantified, respectively. Gene expression profiling was conducted by gene expression chip, and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the results of gene expression chip. We used isobaric tags for elative and absolute quantitation and Western blotting to identify altered proteins after SXSM treatment. Results: There was a constant decrease in the mean heart rate (32%, from 238 ± 6 beats/min to 149 ± 12 beats/min) after six weeks in model compared with that in sham group. This effect was partially reversed by 4-week SXSM treatment. Complementary DNA microarray demonstrated that the increased acetylcholinesterase and reduced nicotinic receptor were take responsibility for the increased heart rate. In addition, proteins involved in calcium handling and signaling were affected by SXSM treatment. Real-time RT-PCR verified the results from gene chip. Results from proteomics demonstrated that SXSM enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle in ventricular myocardium to improve ATP generation. Conclusions: Long-term SXSM stimulates sympathetic transmission by increasing the expression of acetylcholinesterase and reduces the expression of nicotinic receptor to increase heart rate. SXSM also restored the calcium handling genes and altered genes involved in signaling. In addition, SXSM improves the ATP supply of ventricular myocardium by increasing proteins involved in TCA cycle and oxidation-respiratory chain. PMID:28091410

Lung tumor diagnosis and subtype discovery by gene expression profiling.

PubMed

Wang, Lu-yong; Tu, Zhuowen

2006-01-01

The optimal treatment of patients with complex diseases, such as cancers, depends on the accurate diagnosis by using a combination of clinical and histopathological data. In many scenarios, it becomes tremendously difficult because of the limitations in clinical presentation and histopathology. To accurate diagnose complex diseases, the molecular classification based on gene or protein expression profiles are indispensable for modern medicine. Moreover, many heterogeneous diseases consist of various potential subtypes in molecular basis and differ remarkably in their response to therapies. It is critical to accurate predict subgroup on disease gene expression profiles. More fundamental knowledge of the molecular basis and classification of disease could aid in the prediction of patient outcome, the informed selection of therapies, and identification of novel molecular targets for therapy. In this paper, we propose a new disease diagnostic method, probabilistic boosting tree (PB tree) method, on gene expression profiles of lung tumors. It enables accurate disease classification and subtype discovery in disease. It automatically constructs a tree in which each node combines a number of weak classifiers into a strong classifier. Also, subtype discovery is naturally embedded in the learning process. Our algorithm achieves excellent diagnostic performance, and meanwhile it is capable of detecting the disease subtype based on gene expression profile.

A Systems Biology Approach to Link Nuclear Factor Kappa B Activation with Lethal Prostate Cancer

DTIC Science & Technology

2013-05-01

developed as a routine clinical assay. Task 1B: Perform protein profiling of circulating blood proteins and determine whether a protein or set of...proteins indicative of NFκB activation are associated with lethal prostate cancer. Circulating proteins will be assessed in two cohorts of 312...functional genomic data. Nucleic Acids Res 2009;37:D885-90. 3. Parkinson H, Kapushesky M, Kolesnikov N, et al. ArrayExpress update--from an archive of

Gene expression profiling of a pressure-tolerant Listeria monocytogenes Scott A CtsR deletion mutant

USDA-ARS?s Scientific Manuscript database

Listeria monocytogenes is a food-borne pathogen of significant threat to public health. High hydrostatic pressure (HPP) treatment can be used to control Listeria monocytogenes in food. The CtsR (class three stress gene repressor) protein negatively regulates the expression of class III heat shock ...

Wounding induces expression of genes involved in tuber closing layer and wound-periderm development

USDA-ARS?s Scientific Manuscript database

Little is known about the coordinate induction of genes that may be involved in important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using tuber...

Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients

USDA-ARS?s Scientific Manuscript database

More than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the...

Fatty acid profile of maternal and fetal erythrocytes and placental expression of fatty acid transport proteins in normal and intrauterine growth restriction pregnancies.

PubMed

Assumpção, Renata P; Mucci, Daniela B; Fonseca, Fernanda C P; Marcondes, Henrique; Sardinha, Fátima L C; Citelli, Marta; Tavares do Carmo, Maria G

2017-10-01

Long-chain polyunsaturated fatty acids (LC-PUFA), mainly docosahexaenoic (DHA) and arachidonic acids (AA), are critical for adequate fetal growth and development. We investigated mRNA expression of proteins involved in hydrolysis, uptake and/or transport of fatty acids in placenta of fifteen full term normal pregnancies and eleven pregnancies complicated by intrauterine growth restriction (IUGR) with normal umbilical blood flows. The mRNA expression of LPL, FATPs (-1, -2 and -4) and FABPs (-1 and -3) was increased in IUGR placentas, however, tissue profile of LC-PUFA was not different between groups. Erythrocytes from both mothers and fetuses of the IUGR group showed lower concentrations of AA and DHA and inferior DHA/ALA ratio compared to normal pregnancies (P < 0.05). We hypothesize that reduced circulating levels of AA and DHA could up-regulate mRNA expression of placental fatty acids transporters, as a compensatory mechanism, however this failed to sustain normal LC-PUFA supply to the fetus in IUGR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-Wide Identification and Expression Profiling Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family in Tobacco (Nicotiana tabacum L.).

PubMed

Wang, Meng; Xu, Zongchang; Ding, Anming; Kong, Yingzhen

2018-05-24

Xyloglucan endotransglucosylase/hydrolase genes ( XTHs ) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum , Nicotiana sylvestrisis , Nicotiana tomentosiformis , Arabidopsis , and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.

Comparative proteomics of umbilical vein blood plasma from normal and gestational diabetes mellitus patients reveals differentially expressed proteins associated with childhood obesity.

PubMed

Miao, Zhijing; Wang, Jianqing; Wang, Fuqiang; Liu, Lan; Ding, Hongjuan; Shi, Zhonghua

2016-11-01

Offspring obesity is one of long-term complications of gestational diabetes mellitus (GDM). The aim of this study is to identify proteins differentially expressed in the umbilical vein blood plasma, which could become markers for early diagnosis of childhood obesity. Umbilical vein plasma samples were collected from 30 control and 30 GDM patients in 2007-2008 whose offspring were suffering from obesity at 6-7 years old. Multiplexed isobaric tandem mass tag labeling combined with LC-MS/MS was used to identify differentially expressed proteins. Ingenuity pathway analysis was performed to identify canonical pathways, biological functions, and networks of interacting proteins. Western blotting was used to verify the expression of three selected proteins. A total of 318 proteins were identified, of which 12 proteins were upregulated in GDM group while 24 downregulated. Lipid metabolism was the top category identified by ingenuity pathway analysis. Three randomly chosen proteins were validated by Western blotting, which were consistent with LC-MS. There are significant differences of protein profile in the umbilical vein blood plasma between normal and GDM patients with obese offspring. The results indicate that a variety of proteins and biological mechanisms may contribute to childhood obesity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

UV laser radiation alters the embryonic protein profile of adrenal-kidney-gonadal complex and gonadal differentiation in the lizard, Calotes Versicolor.

PubMed

Khodnapur, Bharati S; Inamdar, Laxmi S; Nindi, Robertraj S; Math, Shivkumar A; Mulimani, B G; Inamdar, Sanjeev R

2015-02-01

To examine the impact of ultraviolet (UV) laser radiation on the embryos of Calotes versicolor in terms of its effects on the protein profile of the adrenal-kidney-gonadal complex (AKG), sex determination and differentiation, embryonic development and hatching synchrony. The eggs of C. versicolor, during thermo-sensitive period (TSP), were exposed to third harmonic laser pulses at 355 nm from a Q-switched Nd:YAG laser for 180 sec. Subsequent to the exposure they were incubated at the male-producing temperature (MPT) of 25.5 ± 0.5°C. The AKG of hatchlings was subjected to protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and to histology. The UV laser radiation altered the expression of the protein banding pattern in the AKG complex of hatchlings and it also affected the gonadal sex differentiation. SDS-PAGE of AKG of one-day-old hatchlings revealed a total of nine protein bands in the control group whereas UV laser irradiated hatchlings expressed a total of seven protein bands only one of which had the same Rf as a control band. The UV laser treated hatchlings have an ovotestes kind of gonad exhibiting a tendency towards femaleness instead of the typical testes. It is inferred that 355 nm UV laser radiation during TSP induces changes in the expression of proteins as well as their secretions. UV laser radiation had an impact on the gonadal differentiation pathway but no morphological anomalies were noticed.

Expression characteristics of BMP2, BMPR-IA and Noggin in different stages of hair follicle in yak skin.

PubMed

Song, Liang-Li; Cui, Yan; Yu, Si-Jiu; Liu, Peng-Gang; Liu, Jun; Yang, Xue; He, Jun-Feng; Zhang, Qian

2018-05-01

Bone morphogenetic protein 2 (BMP2), BMP receptor-IA (BMPR-IA), and the BMP2 antagonist Noggin are important proteins involved in regulating the hair follicle (HF) cycle in skin. In order to explore the expression profiles of BMP2, BMPR-IA, and Noggin in the HF cycle of yak skin, we collected adult yak skin in the telogen, proanagen, and midanagen phases of HFs and evaluated gene and protein expression by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. qRT-PCR and western blotting results showed that BMP2 and BMPR-IA expression levels were highest in the telogen of HFs and higher than that of Noggin in the same phase. The expression of Noggin was significantly higher in proanagen and midanagen phases of HFs than in the telogen phase, with the highest expression observed in the proanagen phase. Moreover, the expression of Noggin in the proanagen phase was significantly higher than those of BMP2 and BMPR-IA during the same phase. Immunohistochemistry results showed that BMP2, BMPR-IA, and Noggin were expressed in the skin epidermis, sweat glands, sebaceous glands, HF outer root sheath, and hair matrix. In summary, the characteristic expression profiles of BMP2, BMPR-IA, and Noggin suggested that BMP2 and BMPR-IA had inhibitory effects on the growth of HFs in yaks, whereas Noggin promoted the growth of yak HFs, mainly by affecting skin epithelial cell activity. These results provide a basis for further studies of HF development and cycle transition in yak skin. Copyright © 2017. Published by Elsevier Inc.

Genes associated with thermosensitive genic male sterility in rice identified by comparative expression profiling.

PubMed

Pan, Yufang; Li, Qiaofeng; Wang, Zhizheng; Wang, Yang; Ma, Rui; Zhu, Lili; He, Guangcun; Chen, Rongzhi

2014-12-16

Thermosensitive genic male sterile (TGMS) lines and photoperiod-sensitive genic male sterile (PGMS) lines have been successfully used in hybridization to improve rice yields. However, the molecular mechanisms underlying male sterility transitions in most PGMS/TGMS rice lines are unclear. In the recently developed TGMS-Co27 line, the male sterility is based on co-suppression of a UDP-glucose pyrophosphorylase gene (Ugp1), but further study is needed to fully elucidate the molecular mechanisms involved. Microarray-based transcriptome profiling of TGMS-Co27 and wild-type Hejiang 19 (H1493) plants grown at high and low temperatures revealed that 15462 probe sets representing 8303 genes were differentially expressed in the two lines, under the two conditions, or both. Environmental factors strongly affected global gene expression. Some genes important for pollen development were strongly repressed in TGMS-Co27 at high temperature. More significantly, series-cluster analysis of differentially expressed genes (DEGs) between TGMS-Co27 plants grown under the two conditions showed that low temperature induced the expression of a gene cluster. This cluster was found to be essential for sterility transition. It includes many meiosis stage-related genes that are probably important for thermosensitive male sterility in TGMS-Co27, inter alia: Arg/Ser-rich domain (RS)-containing zinc finger proteins, polypyrimidine tract-binding proteins (PTBs), DEAD/DEAH box RNA helicases, ZOS (C2H2 zinc finger proteins of Oryza sativa), at least one polyadenylate-binding protein and some other RNA recognition motif (RRM) domain-containing proteins involved in post-transcriptional processes, eukaryotic initiation factor 5B (eIF5B), ribosomal proteins (L37, L1p/L10e, L27 and L24), aminoacyl-tRNA synthetases (ARSs), eukaryotic elongation factor Tu (eEF-Tu) and a peptide chain release factor protein involved in translation. The differential expression of 12 DEGs that are important for pollen development, low temperature responses or TGMS was validated by quantitative RT-PCR (qRT-PCR). Temperature strongly affects global gene expression and may be the common regulator of fertility in PGMS/TGMS rice lines. The identified expression changes reflect perturbations in the transcriptomic regulation of pollen development networks in TGMS-Co27. Findings from this and previous studies indicate that sets of genes involved in post-transcriptional and translation processes are involved in thermosensitive male sterility transitions in TGMS-Co27.

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer

PubMed Central

2011-01-01

Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series. PMID:21266046

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

PubMed

Peraldo-Neia, Caterina; Migliardi, Giorgia; Mello-Grand, Maurizia; Montemurro, Filippo; Segir, Raffaella; Pignochino, Ymera; Cavalloni, Giuliana; Torchio, Bruno; Mosso, Luciano; Chiorino, Giovanna; Aglietta, Massimo

2011-01-25

Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

Coculture of Bifidobacterium longum and Bifidobacterium breve alters their protein expression profiles and enzymatic activities.

PubMed

Ruiz, Lorena; Sánchez, Borja; de Los Reyes-Gavilán, Clara G; Gueimonde, Miguel; Margolles, Abelardo

2009-07-31

Some strains of the genus Bifidobacterium are probiotic bacteria commonly added to functional dairy products. The influence of coculturing Bifidobacterium longum NCIMB8809 and Bifidobacterium breve NCIMB8807 on their physiology was studied. 2DE separation of protein extracts, coupled to MS protein analysis allowed the identification of 16 proteins whose expression drastically changed when cells were grown in compartmentalized coculture, compared to monoculture. These included ribosomal proteins and proteins involved in carbohydrate metabolism, gene regulation, cell envelope biogenesis and transport processes. Significant changes in some glycoside-hydrolysing activities (beta-d-xylopyranosidase, alpha-l-arabinofuranosidase and beta-d-glucopyranosidase) were also detected. Furthermore, qRT-PCR experiments using as targets the B. breve genes clgR (transcriptional regulator) clpP1, clpP2 and clpC (chaperone- and protease-encoding genes positively regulated by clgR) supported the proteomic results, the four genes displaying a higher expression level in coculture. This study provides new insights to understand the communication among Bifidobacterium species.

Identification, Expression Profiling and Fluorescence-Based Binding Assays of a Chemosensory Protein Gene from the Western Flower Thrips, Frankliniella occidentalis

PubMed Central

Zhang, Zhi-Ke; Lei, Zhong-Ren

2015-01-01

Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four—cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating the development of the first instar nymph and mediate F. occidentalis host recognition. PMID:25635391

Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella occidentalis.

PubMed

Zhang, Zhi-Ke; Lei, Zhong-Ren

2015-01-01

Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four-cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating the development of the first instar nymph and mediate F. occidentalis host recognition.

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

PubMed

Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

2010-12-01

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

Ixodes ricinus Tick Lipocalins: Identification, Cloning, Phylogenetic Analysis and Biochemical Characterization

PubMed Central

Beaufays, Jérôme; Adam, Benoît; Decrem, Yves; Prévôt, Pierre-Paul; Santini, Sébastien; Brasseur, Robert; Brossard, Michel; Lins, Laurence

2008-01-01

Background During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. Methodology/Principal Findings Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for “Lipocalin from I. ricinus” and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50–70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4. Conclusions/Significance This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4. PMID:19096708

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

PubMed Central

2012-01-01

Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. PMID:22703293

Cerebrospinal Fluid Cytokine Expression Profile in Multiple Sclerosis and Chronic Inflammatory Demyelinating Polyneuropathy.

PubMed

Bonin, Serena; Zanotta, Nunzia; Sartori, Arianna; Bratina, Alessio; Manganotti, Paolo; Trevisan, Giusto; Comar, Manola

2018-02-01

Cerebrospinal fluid (CSF) analysis in patients with particular neurologic disorders is a powerful tool to evaluate specific central nervous system inflammatory markers for diagnostic needs, because CSF represents the specific immune micro-environment to the central nervous system. CSF samples from 49 patients with multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), and non-inflammatory neurologic disorders (NIND) as controls were submitted to protein expression profiles of 47 inflammatory biomarkers by multiplex Luminex bead assay to investigate possible differences in the inflammatory process for MS and CIDP. Our results showed differences in CSF cytokine levels in MS and CIDP; in particular, IL12 (p40) was significantly highly expressed in MS in comparison with CIDP and NIND, while SDF-1α and SCGF-β were significantly highly expressed in CIDP cohort when compared to MS and NIND. IL-9, IL-13, and IL-17 had higher expression levels in NIND if compared with the other groups. Our study showed that, despite some common pathogenic mechanisms, central and peripheral nervous system demyelinating diseases, such as MS and CIDP, differ in some specific inflammatory soluble proteins in CSF, underlining differences in the immune response involved in those autoimmune diseases.

Transcriptome profiling in Arabidopsis inflorescence stems grown under hypergravity in terms of cell walls and plant hormones

NASA Astrophysics Data System (ADS)

Tamaoki, D.; Karahara, I.; Nishiuchi, T.; De Oliveira, S.; Schreiber, L.; Wakasugi, T.; Yamada, K.; Yamaguchi, K.; Kamisaka, S.

2009-07-01

Land plants rely on lignified secondary cell walls in supporting their body weight on the Earth. Although gravity influences the formation of the secondary cell walls, the regulatory mechanism of their formation by gravity is not yet understood. We carried out a comprehensive analysis of gene expression in inflorescence stems of Arabidopsis thaliana L. using microarray (22 K) to identify genes whose expression is modulated under hypergravity condition (300 g). Total RNA was isolated from the basal region of inflorescence stems of plants grown for 24 h at 300 g or 1 g. Microarray analysis showed that hypergravity up-regulated the expression of 403 genes to more than 2-fold. Hypergravity up-regulated the genes responsible for the biosynthesis or modification of cell wall components such as lignin, xyloglucan, pectin and structural proteins. In addition, hypergravity altered the expression of genes related to the biosynthesis of plant hormones such as auxin and ethylene and that of genes encoding hormone-responsive proteins. Our transcriptome profiling indicates that hypergravity influences the formation of secondary cell walls by modulating the pattern of gene expression, and that auxin and/or ethylene play an important role in signaling hypergravity stimulus.

Dose-response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol.

PubMed

Hook, Sharon E; Skillman, Ann D; Small, Jack A; Schultz, Irvin R

2006-07-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss,were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as "expression signatures". Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that different doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose.

A Gata2-Dependent Transcription Network Regulates Uterine Progesterone Responsiveness and Endometrial Function.

PubMed

Rubel, Cory A; Wu, San-Pin; Lin, Lin; Wang, Tianyuan; Lanz, Rainer B; Li, Xilong; Kommagani, Ramakrishna; Franco, Heather L; Camper, Sally A; Tong, Qiang; Jeong, Jae-Wook; Lydon, John P; DeMayo, Francesco J

2016-10-25

Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage. Published by Elsevier Inc.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2012-01-01

Economically viable production of solvents through acetone butanol ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of fivemore » proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.« less

The interactive effects of mercury and selenium on metabolic profiles, gene expression and antioxidant enzymes in halophyte Suaeda salsa.

PubMed

Liu, Xiaoli; Lai, Yongkai; Sun, Hushan; Wang, Yiyan; Zou, Ning

2016-04-01

Suaeda salsa is the pioneer halophyte in the Yellow River Delta and was consumed as a popular vegetable. Mercury has become a highly risky contaminant in the sediment of intertidal zones of the Yellow River Delta. In this work, we investigated the interactive effects of mercury and selenium in S. salsa on the basis of metabolic profiling, antioxidant enzyme activities and gene expression quantification. Our results showed that mercury exposure (20 μg L(-1)) inhibited plant growth of S. salsa and induced significant metabolic responses and altered expression levels of INPS, CMO, and MDH in S. salsa samples, together with the increased activities of antioxidant enzymes including SOD and POD. Overall, these results indicated osmotic and oxidative stresses, disturbed protein degradation and energy metabolism change in S. salsa after mercury exposures. Additionally, the addition of selenium could induce both antagonistic and synergistic effects including alleviating protein degradation and aggravating osmotic stress caused by mercury. © 2014 Wiley Periodicals, Inc.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Impacts of temperature and lunar day on gene expression profiles during a monthly reproductive cycle in the brooding coral Pocillopora damicornis.

PubMed

Crowder, Camerron M; Meyer, Eli; Fan, Tung-Yung; Weis, Virginia M

2017-08-01

Reproductive timing in brooding corals has been correlated to temperature and lunar irradiance, but the mechanisms by which corals transduce these environmental variables into molecular signals are unknown. To gain insight into these processes, global gene expression profiles in the coral Pocillopora damicornis were examined (via RNA-Seq) across lunar phases and between temperature treatments, during a monthly planulation cycle. The interaction of temperature and lunar day together had the largest influence on gene expression. Mean timing of planulation, which occurred at lunar days 7.4 and 12.5 for 28- and 23°C-treated corals, respectively, was associated with an upregulation of transcripts in individual temperature treatments. Expression profiles of planulation-associated genes were compared between temperature treatments, revealing that elevated temperatures disrupted expression profiles associated with planulation. Gene functions inferred from homologous matches to online databases suggest complex neuropeptide signalling, with calcium as a central mediator, acting through tyrosine kinase and G protein-coupled receptor pathways. This work contributes to our understanding of coral reproductive physiology and the impacts of environmental variables on coral reproductive pathways. © 2017 John Wiley & Sons Ltd.

Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds

PubMed Central

Lehnert, Sigrid A; Reverter, Antonio; Byrne, Keren A; Wang, Yonghong; Nattrass, Greg S; Hudson, Nicholas J; Greenwood, Paul L

2007-01-01

Background The muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60), secondary myogenesis (d 135), as well as beginning (d 195) and final stages (birth) of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24) were from Hereford dams and crossed with either Wagyu (high intramuscular fat) or Piedmontese (GDF8 mutant) sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. Results We obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5) RNA at birth. The developing longissimus muscle of fetuses carrying the Piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. We also document evidence for timing differences in differentiation events between the two breeds. Conclusion Taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. In addition, this study has highlighted a non-coding RNA, which is abundantly expressed and developmentally regulated in bovine fetal muscle. PMID:17697390

Small RNA Profiling of Influenza A Virus-Infected Cells Identifies miR-449b as a Regulator of Histone Deacetylase 1 and Interferon Beta

PubMed Central

Buggele, William A.; Krause, Katherine E.; Horvath, Curt M.

2013-01-01

The mammalian antiviral response relies on the alteration of cellular gene expression, to induce the production of antiviral effectors and regulate their activities. Recent research has indicated that virus infections can induce the accumulation of cellular microRNA (miRNA) species that influence the stability of host mRNAs and their protein products. To determine the potential for miRNA regulation of cellular responses to influenza A virus infection, small RNA profiling was carried out using next generation sequencing. Comparison of miRNA expression profiles in uninfected human A549 cells to cells infected with influenza A virus strains A/Udorn/72 and A/WSN/33, revealed virus-induced changes in miRNA abundance. Gene expression analysis identified mRNA targets for a cohort of highly inducible miRNAs linked to diverse cellular functions. Experiments demonstrate that the histone deacetylase, HDAC1, can be regulated by influenza-inducible miR-449b, resulting in altered mRNA and protein levels. Expression of miR-449b enhances virus and poly(I:C) activation of the IFNβ promoter, a process known to be negatively regulated by HDAC1. These findings demonstrate miRNA induction by influenza A virus infection and elucidate an example of miRNA control of antiviral gene expression in human cells, defining a role for miR-449b in regulation of HDAC1 and antiviral cytokine signaling. PMID:24086750

Divergent selection for residual feed intake affects the transcriptomic and proteomic profiles of pig skeletal muscle.

PubMed

Vincent, A; Louveau, I; Gondret, F; Tréfeu, C; Gilbert, H; Lefaucheur, L

2015-06-01

Improving feed efficiency is a relevant strategy to reduce feed cost and environmental waste in livestock production. Selection experiments on residual feed intake (RFI), a measure of feed efficiency, previously indicated that low RFI was associated with lower feed intake, similar growth rate, and greater lean meat content compared with high RFI. To gain insights into the molecular mechanisms underlying these differences, 24 Large White females from 2 lines divergently selected for RFI were examined. Pigs from a low-RFI ("efficient") and high-RFI ("inefficient") line were individually fed ad libitum from 67 d of age (27 kg BW) to slaughter at 115 kg BW (n = 8 per group). Additional pigs of the high-RFI line were feed restricted to the daily feed intake of the ad libitum low-RFI pigs (n = 8) to investigate the impact of selection independently of feed intake. Global gene and protein expression profiles were assessed in the LM collected at slaughter. The analyses involved a porcine commercial microarray and 2-dimensional gel electrophoresis. About 1,000 probes were differentially expressed (P < 0.01) between RFI lines. Only 10% of those probes were also affected by feed restriction. Gene functional classification indicated a greater expression of genes involved in protein synthesis and a lower expression of genes associated with mitochondrial energy metabolism in the low-RFI pigs compared with the high-RFI pigs. At the protein level, 11 unique identified proteins exhibited a differential abundance (P < 0.05) between RFI lines. Differentially expressed proteins were generally not significantly affected by feed restriction. Mitochondrial oxidative proteins such as aconitase hydratase, ATP synthase subunit α, and creatine kinase S-type had a lower abundance in the low-RFI pigs, whereas fructose-biphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase, 2 proteins involved in glycolysis, had a greater abundance in those pigs compared with high-RFI pigs. Antioxidant proteins such as superoxide dismutase and glutathione peroxidase 3 at the mRNA level and peroxiredoxin-6 at the protein level were also less expressed in LM of the most efficient pigs, likely related to lower oxidative molecule production. Collectively, both the transcriptomic and proteomic approaches revealed a lower oxidative metabolism in muscle of the low-RFI pigs and all these modifications were largely independent of differences in feed intake.

Microarray profiling of gene expression in human adipocytes in response to anthocyanins.

PubMed

Tsuda, Takanori; Ueno, Yuki; Yoshikawa, Toshikazu; Kojo, Hitoshi; Osawa, Toshihiko

2006-04-14

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and its gene expression in adipocytes. In this study, we have shown the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100 microM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the significant changes of adipocytokine expression (up-regulation of adiponectin and down-regulation of plasminogen activator inhibitor-1 and interleukin-6). Some of lipid metabolism related genes (uncoupling protein2, acylCoA oxidase1 and perilipin) also significantly induced in both common the C3G or Cy treatment groups. These studies have provided an overview of the gene expression profiles in human adipocytes treated with anthocyanins and demonstrated that anthocyanins can regulate adipocytokine gene expression to ameliorate adipocyte function related with obesity and diabetes that merit further investigation.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

PubMed

Palomares, Laura A; Srivastava, Indresh K; Ramírez, Octavio T; Cox, Manon M J

2018-06-10

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

Body Fluids as a Source of Diagnostic Biomarkers: Prostate — EDRN Public Portal

Cancer.gov

Recent advances in high-throughput protein expression profiling of bodily fluids has generated great enthusiasm and hope for this approach as a potent diagnostic tool. At the center of these efforts is the application of SELDI-TOF-MS and artificial intelligence algorithms by the EDRN BDL site at Eastern Virginia Medical School and the DMCC respectively. When the expression profiling process was applied to sera from individuals with prostate cancer (N=197), BPH (N=92) or from otherwise healthy donors (N=97) we achieved an overall misclassification rate of 90% sensitivity. Since this represents a noticeable improvement in current clinical approach we are proposing to embark upon a validation process. The described studies are designed to address validation issues and include three phases. Phase 1; Synchronization of SELDI Output within the EDRN-Prostate-SELDI Investigational Collaboration (EPSIC); addressing portability (A) Synchronize SELDI instrumentation and robotic sample processing across the EPSIC using pooled serum(QC); (B) Establish the portability and reproducibility of the SELDI protein profiling approach within the EPSIC using normal and prostate cancer patient’s serum from a single site; (C) Establish robustness of the approach toward geographic, sample collection and processing differences within EPSIC using case and control serum from five different sites. Phase 2; Population Validation Establish geographic variability and robustness in a large cross-sectional study among different sample population. Phase 3; Clinical Validation; validate the serum protein expression profiling coupled with a learning algorithm as a means for early detection of prostate cancer using longitudinal PCPT samples. We have assembled a cohesive multi-institutional team for completing these studies in a timely and efficient manner. The team consists of five EDRN laboratories, DMCC and CBI and the proposed budget reflects the total involvement.

Feasibility of investigating differential proteomic expression in depression: implications for biomarker development in mood disorders

PubMed Central

Frye, M A; Nassan, M; Jenkins, G D; Kung, S; Veldic, M; Palmer, B A; Feeder, S E; Tye, S J; Choi, D S; Biernacka, J M

2015-01-01

The objective of this study was to determine whether proteomic profiling in serum samples can be utilized in identifying and differentiating mood disorders. A consecutive sample of patients with a confirmed diagnosis of unipolar (UP n=52) or bipolar depression (BP-I n=46, BP-II n=49) and controls (n=141) were recruited. A 7.5-ml blood sample was drawn for proteomic multiplex profiling of 320 proteins utilizing the Myriad RBM Discovery Multi-Analyte Profiling platform. After correcting for multiple testing and adjusting for covariates, growth differentiation factor 15 (GDF-15), hemopexin (HPX), hepsin (HPN), matrix metalloproteinase-7 (MMP-7), retinol-binding protein 4 (RBP-4) and transthyretin (TTR) all showed statistically significant differences among groups. In a series of three post hoc analyses correcting for multiple testing, MMP-7 was significantly different in mood disorder (BP-I+BP-II+UP) vs controls, MMP-7, GDF-15, HPN were significantly different in bipolar cases (BP-I+BP-II) vs controls, and GDF-15, HPX, HPN, RBP-4 and TTR proteins were all significantly different in BP-I vs controls. Good diagnostic accuracy (ROC-AUC⩾0.8) was obtained most notably for GDF-15, RBP-4 and TTR when comparing BP-I vs controls. While based on a small sample not adjusted for medication state, this discovery sample with a conservative method of correction suggests feasibility in using proteomic panels to assist in identifying and distinguishing mood disorders, in particular bipolar I disorder. Replication studies for confirmation, consideration of state vs trait serial assays to delineate proteomic expression of bipolar depression vs previous mania, and utility studies to assess proteomic expression profiling as an advanced decision making tool or companion diagnostic are encouraged. PMID:26645624

Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry.

PubMed

Tyan, Yu-Chang; Wu, Hsin-Yi; Lai, Wu-Wei; Su, Wu-Chou; Liao, Pao-Chi

2005-01-01

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

PubMed

Ghiraldi-Lopes, Luciana D; Campanerut-Sá, Paula Az; Meneguello, Jean E; Seixas, Flávio Av; Lopes-Ortiz, Mariana A; Scodro, Regiane Bl; Pires, Claudia Ta; da Silva, Rosi Z; Siqueira, Vera Ld; Nakamura, Celso V; Cardoso, Rosilene F

2017-08-01

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus.

PubMed

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-06-09

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH.

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus

PubMed Central

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-01-01

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH. PMID:27277140

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

PubMed

Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Patterns of gene expression associated with recovery and injury in heat-stressed rats.

PubMed

Stallings, Jonathan D; Ippolito, Danielle L; Rakesh, Vineet; Baer, Christine E; Dennis, William E; Helwig, Bryan G; Jackson, David A; Leon, Lisa R; Lewis, John A; Reifman, Jaques

2014-12-03

The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model. We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response. Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

GLUT1 expression in pediatric adrenocortical tumors: a promising candidate to predict clinical behavior.

PubMed

Pinheiro, Céline; Granja, Sara; Longatto-Filho, Adhemar; Faria, André M; Fragoso, Maria C B V; Lovisolo, Silvana M; Bonatelli, Murilo; Costa, Ricardo F A; Lerário, Antonio M; Almeida, Madson Q; Baltazar, Fátima; Zerbini, Maria C N

2017-09-08

Discrimination between benign and malignant tumors is a challenging process in pediatric adrenocortical tumors. New insights in the metabolic profile of pediatric adrenocortical tumors may contribute to this distinction, predict prognosis, as well as identify new molecular targets for therapy. The aim of this work is to characterize the expression of the metabolism-related proteins MCT1, MCT2, MCT4, CD147, CD44, GLUT1 and CAIX in a series of pediatric adrenocortical tumors. A total of 50 pediatric patients presenting adrenocortical tumors, including 41 clinically benign and 9 clinically malignant tumors, were included. Protein expression was evaluated using immunohistochemistry in samples arranged in tissue microarrays. The immunohistochemical analysis showed a significant increase in plasma membrane expression of GLUT1 in malignant lesions, when compared to benign lesions ( p =0.004), being the expression of this protein associated with shorter overall and disease-free survival ( p =0.004 and p =0.001, respectively). Although significant differences were not observed for proteins other than GLUT1, MCT1, MCT4 and CD147 were highly expressed in pediatric adrenocortical neoplasias (around 90%). GLUT1 expression was differentially expressed in pediatric adrenocortical tumors, with higher expression in clinically malignant tumors, and associated with shorter survival, suggesting a metabolic remodeling towards a hyperglycolytic phenotype in this malignancy.

Comparative proteomic study on Brassica hexaploid and its parents provides new insights into the effects of polyploidization.

PubMed

Shen, Yanyue; Zhang, Yu; Zou, Jun; Meng, Jinling; Wang, Jianbo

2015-01-01

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. Although genomic and transcriptomic changes have been observed in polyploids, the effects of polyploidization on proteomic divergence are poorly understood. In this study, we reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 proteins differentially expressed between Brassica hexaploid and its parents, and 100 proteins were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins, probably providing a driving force for variation and adaptation in allopolyploids. In particular, majority of the total 452 differentially expressed proteins showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low. This study aimed to get new insights into the effects of polyploidization on proteomic divergence. Using iTRAQ LC-MS/MS technology, we identified 452 differentially expressed proteins between allopolyploid and its parents which involved in response to stimulus, multi-organism process, and immune system process, much more than previous studies using 2-DE coupled with mass spectrometry technology. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in allopolyploid and its parents, which will lead to a better understanding of novelty and plasticity of the allopolyploid genomes. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

PubMed

Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

2015-01-01

Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Proteins altered by elevated levels of palmitate or glucose implicated in impaired glucose-stimulated insulin secretion

PubMed Central

Sol, E-ri M; Hovsepyan, Meri; Bergsten, Peter

2009-01-01

Background Development of type 2 diabetes mellitus (T2DM) is characterized by aberrant insulin secretory patterns, where elevated insulin levels at non-stimulatory basal conditions and reduced hormonal levels at stimulatory conditions are major components. To delineate mechanisms responsible for these alterations we cultured INS-1E cells for 48 hours at 20 mM glucose in absence or presence of 0.5 mM palmitate, when stimulatory secretion of insulin was reduced or basal secretion was elevated, respectively. Results After culture, cells were protein profiled by SELDI-TOF-MS and 2D-PAGE. Differentially expressed proteins were discovered and identified by peptide mass fingerprinting. Complimentary protein profiles were obtained by the two approaches with SELDI-TOF-MS being more efficient in separating proteins in the low molecular range and 2D-PAGE in the high molecular range. Identified proteins included alpha glucosidase, calmodulin, gars, glucose-6-phosphate dehydrogenase, heterogenous nuclear ribonucleoprotein A3, lon peptidase, nicotineamide adenine dinucleotide hydrogen (NADH) dehydrogenase, phosphoglycerate kinase, proteasome p45, rab2, pyruvate kinase and t-complex protein. The observed glucose-induced differential protein expression pattern indicates enhanced glucose metabolism, defense against reactive oxygen species, enhanced protein translation, folding and degradation and decreased insulin granular formation and trafficking. Palmitate-induced changes could be related to altered exocytosis. Conclusion The identified altered proteins indicate mechanism important for altered β-cell function in T2DM. PMID:19607692

AFLP-based transcript profiling for cassava genome-wide expression analysis in the onset of storage root formation.

PubMed

Sojikul, Punchapat; Kongsawadworakul, Panida; Viboonjun, Unchera; Thaiprasit, Jittrawan; Intawong, Burapat; Narangajavana, Jarunya; Svasti, Mom Rajawong Jisnuson

2010-10-01

Cassava (Manihot esculenta Crantz) is a root crop that accumulates large quantities of starch, and it is an important source of carbohydrate. Study on gene expressions during storage root development provides important information on storage root formation and starch accumulation as well as unlock new traits for improving of starch yield. cDNA-Amplified Fragment Length Polymorphism (AFLP) was used to compare gene expression profiles in fibrous and storage roots of cassava cultivar Kasetsart 50. Total of 155 differentially expressed transcript-derived fragments with undetectable or low expression in leaves were characterized and classified into 11 groups regarding to their functions. The four major groups were no similarity (20%), hypothetical or unknown proteins (17%), cellular metabolism and biosynthesis (17%) and cellular communication and signaling (14%). Interestingly, sulfite reductase (MeKD82), calcium-dependent protein kinase (CDPK) (MeKD83), ent-kaurene synthase (KS) (MeKD106) and hexose transporter (HT) (MeKD154) showed root-specific expression patterns. This finding is consistent with previously reported genes involved in the initiation of potato tuber. Semi-quantitative reverse transcription polymerase chain reaction of early-developed root samples confirmed that those four genes exhibited significant expression with similar pattern in the storage root initiation and early developmental stages. We proposed that KS and HT may involve in transient induction of CDPK expression, which may play an important role in the signaling pathway of storage root initiation. Sulfite reductase, on the other hand, may involve in storage root development by facilitating sulfur-containing protein biosynthesis or detoxifying the cyanogenic glucoside content through aspartate biosynthesis. Copyright © Physiologia Plantarum 2010.

Expression profile analysis of circulating microRNAs and their effects on ion channels in Chinese atrial fibrillation patients.

PubMed

Lu, Yingmin; Hou, Shuxin; Huang, Damin; Luo, Xiaohan; Zhang, Jinchun; Chen, Jian; Xu, Weiping

2015-01-01

To investigate the changes in expression profile of circulating microRNAs (miRNAs) and the regulatory effect of atrial fibrilation (AF)-related miRNAs on ion channels. 112 patients with AF were assigned into observation group, and another 112 non-AF people were assigned into control group. Total plasma RNAs were extracted from patients' blood samples. Differentially expressed miRNA-1s were transfected into primary-cultured neonatal rat cardiac myocytes. Compared with control group, significant differences were observed in 15 kinds of miRNAs in observation group. Down-regulation of the expression of miRNAs included hsa-miR-328, hsa-miR-145, hsa-miR-222, hsa-miR-1, hsa-miR-162, hsa-miR-432, and hsa-miR-493b; Up-regulation of the expression included hsa-miR634, hsa-miR-664, hsa-miR-9, hsa-miR-152, hsa-miR-19, hsa-miR-454, hsa-miR-146, and hsa-miR-374a. The expression level of CACNB2 protein in miRNA-1 group was significantly lower than that in blank control group, negative control group, MTmiRNA-1 group, AMO-1 group and miRNA-1+AMO-1 cotransfection group (P < 0.05), while in AMO-1 group, the expression level of CACNB2 protein was significantly higher than that in other groups (P < 0.05). These results indicated that transfected miRNA-1 could significantly inhibit the expression of CACNB2 protein. Circulating miRNAs can be used in studies concerning on the regulation mechanism of the occurrence and development of AF. MiRNA-1 can decrease the intracellular Ca(2+) concentration and prevent the AF.

Expression profile analysis of circulating microRNAs and their effects on ion channels in Chinese atrial fibrillation patients

PubMed Central

Lu, Yingmin; Hou, Shuxin; Huang, Damin; Luo, Xiaohan; Zhang, Jinchun; Chen, Jian; Xu, Weiping

2015-01-01

Objective: To investigate the changes in expression profile of circulating microRNAs (miRNAs) and the regulatory effect of atrial fibrilation (AF)-related miRNAs on ion channels. Methods: 112 patients with AF were assigned into observation group, and another 112 non-AF people were assigned into control group. Total plasma RNAs were extracted from patients’ blood samples. Differentially expressed miRNA-1s were transfected into primary-cultured neonatal rat cardiac myocytes. Results: Compared with control group, significant differences were observed in 15 kinds of miRNAs in observation group. Down-regulation of the expression of miRNAs included hsa-miR-328, hsa-miR-145, hsa-miR-222, hsa-miR-1, hsa-miR-162, hsa-miR-432, and hsa-miR-493b; Up-regulation of the expression included hsa-miR634, hsa-miR-664, hsa-miR-9, hsa-miR-152, hsa-miR-19, hsa-miR-454, hsa-miR-146, and hsa-miR-374a. The expression level of CACNB2 protein in miRNA-1 group was significantly lower than that in blank control group, negative control group, MTmiRNA-1 group, AMO-1 group and miRNA-1+AMO-1 cotransfection group (P < 0.05), while in AMO-1 group, the expression level of CACNB2 protein was significantly higher than that in other groups (P < 0.05). These results indicated that transfected miRNA-1 could significantly inhibit the expression of CACNB2 protein. Conclusions: Circulating miRNAs can be used in studies concerning on the regulation mechanism of the occurrence and development of AF. MiRNA-1 can decrease the intracellular Ca2+ concentration and prevent the AF. PMID:25785065

Exploring of the molecular mechanism of rhinitis via bioinformatics methods

PubMed Central

Song, Yufen; Yan, Zhaohui

2018-01-01

The aim of this study was to analyze gene expression profiles for exploring the function and regulatory network of differentially expressed genes (DEGs) in pathogenesis of rhinitis by a bioinformatics method. The gene expression profile of GSE43523 was downloaded from the Gene Expression Omnibus database. The dataset contained 7 seasonal allergic rhinitis samples and 5 non-allergic normal samples. DEGs between rhinitis samples and normal samples were identified via the limma package of R. The webGestal database was used to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs. The differentially co-expressed pairs of the DEGs were identified via the DCGL package in R, and the differential co-expression network was constructed based on these pairs. A protein-protein interaction (PPI) network of the DEGs was constructed based on the Search Tool for the Retrieval of Interacting Genes database. A total of 263 DEGs were identified in rhinitis samples compared with normal samples, including 125 downregulated ones and 138 upregulated ones. The DEGs were enriched in 7 KEGG pathways. 308 differential co-expression gene pairs were obtained. A differential co-expression network was constructed, containing 212 nodes. In total, 148 PPI pairs of the DEGs were identified, and a PPI network was constructed based on these pairs. Bioinformatics methods could help us identify significant genes and pathways related to the pathogenesis of rhinitis. Steroid biosynthesis pathway and metabolic pathways might play important roles in the development of allergic rhinitis (AR). Genes such as CDC42 effector protein 5, solute carrier family 39 member A11 and PR/SET domain 10 might be also associated with the pathogenesis of AR, which provided references for the molecular mechanisms of AR. PMID:29257233

Impacts of ezetimibe on PCSK9 in rats: study on the expression in different organs and the potential mechanisms.

PubMed

Xu, Rui-Xia; Liu, Jun; Li, Xiao-Lin; Li, Sha; Zhang, Yan; Jia, Yan-Jun; Sun, Jing; Li, Jian-Jun

2015-03-14

Previous studies including our group have indicated the effects of ezetimibe on increased plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) concentration, while the rapid expression in different organs and the potential molecular mechanisms for this impact have not been carefully evaluated. Thirty rats were randomly divided into two groups (n = 15 for each), which were orally administrated with ezetimibe (10 mg/kg/day) or normal saline. Blood samples were obtained at day 3 after orally administration, and the PCSK9 levels were determined by ELISA. We further analyzed the mRNA expression of PCSK9, low-density lipoprotein receptor (LDLR), sterol regulator element-binding protein 2 (SREBP2), and hepatocyte nuclear factor 1 alpha (HNF-1α) by real-time PCR, as well as the protein expression by western blot, in liver, intestine and kidney respectively. Ezetimibe significantly increased plasma PCSK9 levels compared with control group, while there was no significant difference between the two groups with regard to lipid profile at day 3. Moreover, ezetimibe remarkably increased the expression of PCSK9, LDLR, SREBP2 and HNF-1α in liver. Enhanced expression of PCSK9, LDLR and SREBP2 protein were found in intestine and kidney, while no changes in the expression of HNF-1α were observed in intestine and kidney of rats with ezetimibe treatment. The data demonstrated that ezetimibe increased PCSK9 expression through the SREBP2 and HNF-1α pathways in different organs, subsequently resulting in elevated plasma PCSK9 levels prior to the alterations of lipid profile in rats.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

PubMed Central

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

2015-01-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

PubMed

Braun, Clemens; Sakamoto, Atsushi; Fuchs, Holger; Ishiguro, Naoki; Suzuki, Shinobu; Cui, Yunhai; Klinder, Klaus; Watanabe, Michitoshi; Terasaki, Tetsuya; Sauer, Achim

2017-10-02

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K p,uu,brain and K p,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, K p,uu,brain for the BCRP substrates was low. In contrast, K p,uu,CSF for both BCRP substrates was close to unity, resulting in K p,uu,CSF /K p,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

PubMed

He, Xiaocui; Zhang, Yang; Yu, Ziniu

2010-10-01

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

PubMed

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

Transcriptome profile of a bovine respiratory disease pathogen: Mannheimia haemolytica PHL213

PubMed Central

2012-01-01

Background Computational methods for structural gene annotation have propelled gene discovery but face certain drawbacks with regards to prokaryotic genome annotation. Identification of transcriptional start sites, demarcating overlapping gene boundaries, and identifying regulatory elements such as small RNA are not accurate using these approaches. In this study, we re-visit the structural annotation of Mannheimia haemolytica PHL213, a bovine respiratory disease pathogen. M. haemolytica is one of the causative agents of bovine respiratory disease that results in about $3 billion annual losses to the cattle industry. We used RNA-Seq and analyzed the data using freely-available computational methods and resources. The aim was to identify previously unannotated regions of the genome using RNA-Seq based expression profile to complement the existing annotation of this pathogen. Results Using the Illumina Genome Analyzer, we generated 9,055,826 reads (average length ~76 bp) and aligned them to the reference genome using Bowtie. The transcribed regions were analyzed using SAMTOOLS and custom Perl scripts in conjunction with BLAST searches and available gene annotation information. The single nucleotide resolution map enabled the identification of 14 novel protein coding regions as well as 44 potential novel sRNA. The basal transcription profile revealed that 2,506 of the 2,837 annotated regions were expressed in vitro, at 95.25% coverage, representing all broad functional gene categories in the genome. The expression profile also helped identify 518 potential operon structures involving 1,086 co-expressed pairs. We also identified 11 proteins with mutated/alternate start codons. Conclusions The application of RNA-Seq based transcriptome profiling to structural gene annotation helped correct existing annotation errors and identify potential novel protein coding regions and sRNA. We used computational tools to predict regulatory elements such as promoters and terminators associated with the novel expressed regions for further characterization of these novel functional elements. Our study complements the existing structural annotation of Mannheimia haemolytica PHL213 based on experimental evidence. Given the role of sRNA in virulence gene regulation and stress response, potential novel sRNA described in this study can form the framework for future studies to determine the role of sRNA, if any, in M. haemolytica pathogenesis. PMID:23046475

Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

PubMed Central

Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

An EST-based analysis identifies new genes and reveals distinctive gene expression features of Coffea arabica and Coffea canephora

PubMed Central

2011-01-01

Background Coffee is one of the world's most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency. Results Assembling the expressed sequence tags (ESTs) of C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories. Conclusion We present the first comprehensive genome-wide transcript profile study of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance. PMID:21303543

Altered gene expression changes in Arabidopsis leaf tissues and protoplasts in response to Plum pox virus infection

PubMed Central

Babu, Mohan; Griffiths, Jonathan S; Huang, Tyng-Shyan; Wang, Aiming

2008-01-01

Background Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled. Results Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q ≤ 0.05) up- (≥ 2.5 fold) and downregulated (≤ -2.5 fold), respectively. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were upregulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wall-associated components were downregulated. These gene expression changes were associated with PPV infection and symptom development. Further transcriptional profiling of protoplasts transfected with a PPV infectious clone revealed the upregulation of defence and cellular signalling genes as early as 6 hours post transfection. A cross sequence comparison analysis of genes differentially regulated by PPV-infected Arabidopsis leaves against uniEST sequences derived from PPV-infected leaves of Prunus persica, a natural host of PPV, identified orthologs related to defence, metabolism and protein synthesis. The cross comparison of genes differentially regulated by PPV infection and by the infections of other positive sense RNA viruses revealed a common set of 416 genes. These identified genes, particularly the early responsive genes, may be critical in virus infection. Conclusion Gene expression changes in PPV-infected Arabidopsis are the molecular basis of stress and defence-like responses, PPV pathogenesis and symptom development. The differentially regulated genes, particularly the early responsive genes, and a common set of genes regulated by infections of PPV and other positive sense RNA viruses identified in this study are candidates suitable for further functional characterization to shed lights on molecular virus-host interactions. PMID:18613973

Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

The expression profiles of nine genes encoding chitin deacetylase (CDA)-like proteins were studied during development and in various tissues of the red flour beetle, Tribolium castaneum, by RT-PCR. TcCDA1, TcCDA2 and TcCDA5 were expressed throughout all stages of development, while TcCDA6 – 9 were ...

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts.

PubMed

Leiss, Lina; Mutlu, Ercan; Øyan, Anne; Yan, Tao; Tsinkalovsky, Oleg; Sleire, Linda; Petersen, Kjell; Rahman, Mohummad Aminur; Johannessen, Mireille; Mitra, Sidhartha S; Jacobsen, Hege K; Talasila, Krishna M; Miletic, Hrvoje; Jonassen, Inge; Li, Xingang; Brons, Nicolaas H; Kalland, Karl-Henning; Wang, Jian; Enger, Per Øyvind

2017-02-07

Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b + immune and CD31 + endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.

Altered profile of mRNA expression in atrioventricular node of streptozotocin-induced diabetic rats

PubMed Central

Howarth, Frank Christopher; Parekh, Khatija; Jayaprakash, Petrilla; Inbaraj, Edward Samuel; Oz, Murat; Dobrzynski, Halina; Adrian, Thomas Edward

2017-01-01

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)-induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ-induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription-quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization-activated cyclic-nucleotide 2 and 3, calcium channel voltage-dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage-gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage-gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ-induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function. PMID:28731153

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells.

PubMed

Aqil, Madeeha; Naqvi, Afsar Raza; Mallik, Saurav; Bandyopadhyay, Sanghamitra; Maulik, Ujjwal; Jameel, Shahid

2014-01-01

The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes. Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways. Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome. This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

Comparative Proteomic Analysis of Tolerance and Adaptation of Ethanologenic Saccharomyces cerevisiae to Furfural, a Lignocellulosic Inhibitory Compound▿ †

PubMed Central

Lin, Feng-Ming; Qiao, Bin; Yuan, Ying-Jin

2009-01-01

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD+, and NADH/NAD+ were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology. PMID:19363068

Gene Expression Profiling of Soft and Firm Atlantic Salmon Fillet

PubMed Central

Larsson, Thomas; Mørkøre, Turid; Kolstad, Kari; Østbye, Tone-Kari; Afanasyev, Sergey; Krasnov, Aleksei

2012-01-01

Texture of salmon fillets is an important quality trait for consumer acceptance as well as for the suitability for processing. In the present work we measured fillet firmness in a population of farmed Atlantic salmon with known pedigree and investigated the relationship between this trait and gene expression. Transcriptomic analyses performed with a 21 K oligonucleotide microarray revealed strong correlations between firmness and a large number of genes. Highly similar expression profiles were observed in several functional groups. Positive regression was found between firmness and genes encoding proteasome components (41 genes) and mitochondrial proteins (129 genes), proteins involved in stress responses (12 genes), and lipid metabolism (30 genes). Coefficients of determination (R2) were in the range of 0.64–0.74. A weaker though highly significant negative regression was seen in sugar metabolism (26 genes, R2 = 0.66) and myofiber proteins (42 genes, R2 = 0.54). Among individual genes that showed a strong association with firmness, there were extracellular matrix proteins (negative correlation), immune genes, and intracellular proteases (positive correlation). Several genes can be regarded as candidate markers of flesh quality (coiled-coil transcriptional coactivator b, AMP deaminase 3, and oligopeptide transporter 15) though their functional roles are unclear. To conclude, fillet firmness of Atlantic salmon depends largely on metabolic properties of the skeletal muscle; where aerobic metabolism using lipids as fuel, and the rapid removal of damaged proteins, appear to play a major role. PMID:22745718

Gene expression profiling of soft and firm Atlantic salmon fillet.

PubMed

Larsson, Thomas; Mørkøre, Turid; Kolstad, Kari; Østbye, Tone-Kari; Afanasyev, Sergey; Krasnov, Aleksei

2012-01-01

Texture of salmon fillets is an important quality trait for consumer acceptance as well as for the suitability for processing. In the present work we measured fillet firmness in a population of farmed Atlantic salmon with known pedigree and investigated the relationship between this trait and gene expression. Transcriptomic analyses performed with a 21 K oligonucleotide microarray revealed strong correlations between firmness and a large number of genes. Highly similar expression profiles were observed in several functional groups. Positive regression was found between firmness and genes encoding proteasome components (41 genes) and mitochondrial proteins (129 genes), proteins involved in stress responses (12 genes), and lipid metabolism (30 genes). Coefficients of determination (R(2)) were in the range of 0.64-0.74. A weaker though highly significant negative regression was seen in sugar metabolism (26 genes, R(2) = 0.66) and myofiber proteins (42 genes, R(2) = 0.54). Among individual genes that showed a strong association with firmness, there were extracellular matrix proteins (negative correlation), immune genes, and intracellular proteases (positive correlation). Several genes can be regarded as candidate markers of flesh quality (coiled-coil transcriptional coactivator b, AMP deaminase 3, and oligopeptide transporter 15) though their functional roles are unclear. To conclude, fillet firmness of Atlantic salmon depends largely on metabolic properties of the skeletal muscle; where aerobic metabolism using lipids as fuel, and the rapid removal of damaged proteins, appear to play a major role.

Proteomic study of differential protein expression in mouse lung tissues after aerosolized ricin poisoning.

PubMed

Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

2014-04-28

Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.

Potential hydrophobic protein markers of breast cancer in Malaysian Chinese, Malay and Indian patients.

PubMed

Liang, Seng; Singh, Manjit; Gam, Lay-Harn

Breast cancer is a leading cause of worldwide mortality in females. In Malaysia, breast cancer is the most commonly diagnosed cancer in women. Of these, the Chinese had the most number of breast cancer cases, followed by the Indian and the Malay. The most common type of breast cancer is infiltrating ductal carcinoma (IDC). A proteomic approach was used to identify protein profile changes in cancerous tissues compared with the normal tissues, the tissues were collected from patients of three different ethnicities, i.e. Chinese, Malay and Indian. Ten differentially expressed hydrophobic proteins were identified. We had evaluated the potential of these proteins as biomarker for infiltrating ducal carcinoma (IDC) and the ethnic-specific expression of these proteins was also determined. The data showed that peroxiredoxin-2, heat shock protein 60, protein disulfide isomerase and calreticulin may serve as ethnic-related potential markers for either one or combination of Chinese, Malay and Indian cohorts as their expression levels were significantly high in the cancerous tissues compared to the normal tissues in the ethnic group tested.

Application of activity-based protein profiling to study enzyme function in adipocytes.

PubMed

Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

2014-01-01

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. © 2014 Elsevier Inc. All rights reserved.

Activation of Adiponectin Receptor Regulates Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Inhibits Lesions in ApoE-Deficient Mice.

PubMed

Sun, Lei; Yang, Xiaoxiao; Li, Qi; Zeng, Peng; Liu, Ying; Liu, Lipei; Chen, Yuanli; Yu, Miao; Ma, Chuanrui; Li, Xiaoju; Li, Yan; Zhang, Rongxin; Zhu, Yan; Miao, Qing Robert; Han, Jihong; Duan, Yajun

2017-07-01

The reduced adiponectin levels are associated with atherosclerosis. Adiponectin exerts its functions by activating adiponectin receptor (AdipoR). Proprotein convertase subtilisin kexin type 9 (PCSK9) degrades LDLR protein (low-density lipoprotein receptor) to increase serum LDL-cholesterol levels. PCSK9 expression can be regulated by PPARγ (peroxisome proliferator-activated receptor γ) or SREBP2 (sterol regulatory element-binding protein 2). The effects of AdipoR agonists on PCSK9 and LDLR expression, serum lipid profiles, and atherosclerosis remain unknown. At cellular levels, AdipoR agonists (ADP355 and AdipoRon) induced PCSK9 transcription/expression that solely depended on activation of PPAR-responsive element in the PCSK9 promoter. AdipoR agonists induced PPARγ expression; thus, the AdipoR agonist-activated PCSK9 expression/production was impaired in PPARγ deficient hepatocytes. Meanwhile, AdipoR agonists transcriptionally activated LDLR expression by activating SRE in the LDLR promoter. Moreover, AMP-activated protein kinase α (AMPKα) was involved in AdipoR agonist-activated PCSK9 expression. In wild-type mice, ADP355 increased PCSK9 and LDLR expression and serum PCSK9 levels, which was associated with activation of PPARγ, AMPKα and SREBP2 and reduction of LDL-cholesterol levels. In contrast, ADP355 reduced PCSK9 expression/secretion in apoE-deficient (apoE -/- ) mice, but it still activated hepatic LDLR, PPARγ, AMPKα, and SREBP2. More importantly, ADP355 inhibited lesions in en face aortas and sinus lesions in aortic root in apoE -/- mice with amelioration of lipid profiles. Our study demonstrates that AdipoR activation by agonists regulated PCSK9 expression differently in wild-type and apoE -/- mice. However, ADP355 activated hepatic LDLR expression and ameliorated lipid metabolism in both types of mice and inhibited atherosclerosis in apoE -/- mice. © 2017 American Heart Association, Inc.

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

Transcriptome analysis of genes involved in defense against alkaline stress in roots of wild jujube (Ziziphus acidojujuba)

PubMed Central

Tian, Shan; Wang, Bei; Zhao, Xusheng

2017-01-01

Wild jujube (Ziziphus acidojujuba Mill.) is highly tolerant to alkaline, saline and drought stress; however, no studies have performed transcriptome profiling to study the response of wild jujube to these and other abiotic stresses. In this study, we examined the tolerance of wild jujube to NaHCO3-NaOH solution and analyzed gene expression profiles in response to alkaline stress. Physiological experiments revealed that H2O2 content in leaves increased significantly and root activity decreased quickly during alkaline of pH 9.5 treatment. For transcriptome analysis, wild jujube plants grown hydroponically were treated with NaHCO3-NaOH solution for 0, 1, and 12 h and six transcriptomes from roots were built. In total, 32,758 genes were generated, and 3,604 differentially expressed genes (DEGs) were identified. After 1 h, 853 genes showed significantly different expression between control and treated plants; after 12 h, expression of 2,856 genes was significantly different. The expression pattern of nine genes was validated by quantitative real-time PCR. After gene annotation and gene ontology enrichment analysis, the genes encoding transcriptional factors, serine/threonine-protein kinases, heat shock proteins, cysteine-like kinases, calmodulin-like proteins, and reactive oxygen species (ROS) scavengers were found to be closely involved in alkaline stress response. These results will provide useful insights for elucidating the mechanisms underlying alkaline tolerance in wild jujube. PMID:28976994

Targeting a KH-domain protein with RNA decoys.

PubMed

Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

2002-09-01

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

Targeting a KH-domain protein with RNA decoys.

PubMed Central

Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

2002-01-01

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins. PMID:12358435

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B▿ †

PubMed Central

Gautam, Poonam; Shankar, Jata; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Gade, Wasudev Namdeo; Basir, Seemi Farhat; Sarma, Puranam Usha

2008-01-01

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development. PMID:18838595

Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B.

PubMed

Gautam, Poonam; Shankar, Jata; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Gade, Wasudev Namdeo; Basir, Seemi Farhat; Sarma, Puranam Usha

2008-12-01

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.

Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum)

PubMed Central

Shivaraj, S. M.; Deshmukh, Rupesh K.; Rai, Rhitu; Bélanger, Richard; Agrawal, Pawan K.; Dash, Prasanta K.

2017-01-01

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax. PMID:28447607

Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum).

PubMed

Shivaraj, S M; Deshmukh, Rupesh K; Rai, Rhitu; Bélanger, Richard; Agrawal, Pawan K; Dash, Prasanta K

2017-04-27

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax.

More than royal food - Major royal jelly protein genes in sexuals and workers of the honeybee Apis mellifera.

PubMed

Buttstedt, Anja; Moritz, Robin Fa; Erler, Silvio

2013-11-27

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee's genome. We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals. The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

iTRAQ-based quantitative protein expression profiling and MRM verification of markers in type 2 diabetes.

PubMed

Kaur, Prabhjit; Rizk, Nasser M; Ibrahim, Sereen; Younes, Noura; Uppal, Arushi; Dennis, Kevin; Karve, Tejaswita; Blakeslee, Kenneth; Kwagyan, John; Zirie, Mahmoud; Ressom, Habtom W; Cheema, Amrita K

2012-11-02

The pathogenesis of Type 2 diabetes mellitus (T2DM) is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are noninformative with respect to progression of the disease to other associated pathologies. Thus, predicting the risk and development of T2DM-related complications, such as cardiovascular disease, remains a major challenge. We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of nondiabetic control subjects (n = 76) and patients diagnosed with T2DM (n = 106). In this case-control study, the samples were randomly divided as training and validation data sets. In the first step, iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western blot and QPCR analysis. Functional pathway analysis identified perturbations in lipid and small molecule metabolism as well as pathways that lead to disruption of glucose homeostasis and blood coagulation. These putative biomarkers may be clinically useful for subset stratification of T2DM patients as well as for the development of novel therapeutics targeting the specific pathology.

Proteomics analysis of melanoma metastases: association between S100A13 expression and chemotherapy resistance

PubMed Central

Azimi, A; Pernemalm, M; Frostvik Stolt, M; Hansson, J; Lehtiö, J; Egyházi Brage, S; Hertzman Johansson, C

2014-01-01

Background: Disseminated cutaneous malignant melanoma (CMM) is commonly unresponsive to standard chemotherapies, and there are as yet no predictive markers of therapy response. Methods: In the present study we collected fresh-frozen pretreatment lymph-node metastasis samples (n=14) from melanoma patients with differential response to dacarbazine (DTIC) or temozolomide (TMZ) chemotherapy, to identify proteins with an impact on treatment response. We performed quantitative protein profiling using tandem mass spectrometry and compared the proteome differences between responders (R) and non-responders (NR), matched for age, gender and histopathological type of CMM. Results: Biological pathway analyses showed several signalling pathways differing between R vs NR, including Rho signalling. Gene expression profiling data was available for a subset of the samples, and the results were compared with the proteomics data. Four proteins with differential expression between R and NR were selected for technical validation by immunoblotting (ISYNA1, F13A1, CSTB and S100A13), and CSTB and S100A13 were further validated on a larger sample set by immunohistochemistry (n=48). The calcium binding protein S100A13 was found to be significantly overexpressed in NR compared with R in all analyses performed. Conclusions: Our results suggest that S100A13 is involved in CMM resistance to DTIC/TMZ. PMID:24722184

AURKA mRNA expression is an independent predictor of poor prognosis in patients with non-small cell lung cancer.

PubMed

Al-Khafaji, Ahmed S K; Marcus, Michael W; Davies, Michael P A; Risk, Janet M; Shaw, Richard J; Field, John K; Liloglou, Triantafillos

2017-06-01

Deregulation of mitotic spindle genes has been reported to contribute to the development and progression of malignant tumours. The aim of the present study was to explore the association between the expression profiles of Aurora kinases ( AURKA , AURKB and AURKC ), cytoskeleton-associated protein 5 ( CKAP5 ), discs large-associated protein 5 ( DLGAP5 ), kinesin-like protein 11 ( KIF11 ), microtubule nucleation factor ( TPX2 ), monopolar spindle 1 kinase ( TTK ), and β-tubulins ( TUBB ) and ( TUBB3 ) genes and clinicopathological characteristics in human non-small cell lung carcinoma (NSCLC). Reverse transcription-quantitative polymerase chain reaction-based RNA gene expression profiles of 132 NSCLC and 44 adjacent wild-type tissues were generated, and Cox's proportional hazard regression was used to examine associations. With the exception of AURKC , all genes exhibited increased expression in NSCLC tissues. Of the 10 genes examined, only AURKA was significantly associated with prognosis in NSCLC. Multivariate Cox's regression analysis demonstrated that AURKA mRNA expression [hazard ratio (HR), 1.81; 95% confidence interval (CI), 1.16-2.84; P=0.009], age (HR, 1.03; 95% CI, 1.00-1.06; P=0.020), pathological tumour stage 2 (HR, 2.43; 95% CI, 1.16-5.10; P=0.019) and involvement of distal nodes (pathological node stage 2) (HR, 3.14; 95% CI, 1.24-7.99; P=0.016) were independent predictors of poor prognosis in patients with NSCLC. Poor prognosis of patients with increased AURKA expression suggests that those patients may benefit from surrogate therapy with AURKA inhibitors.

Modification of N6-methyladenosine RNA methylation on heat shock protein expression.

PubMed

Yu, Jiayao; Li, Yi; Wang, Tian; Zhong, Xiang

2018-01-01

This study was conducted to investigate effect of N6-methyladenosine (m6A) RNA methylation on Heat shock proteins (HSPs) and dissect the profile of HSP RNA methylation. The results showed that m6A methyltransferases METTL3 mRNA was decreased in responses to heat shock stress in HepG2 cells, but m6A-specific binding protein YTHDF2 mRNA was upregulated in a manner similar to HSP70 induction. Immunofluorescence staining showed that the majority of YTHDF2 was present in the cytosol, however, nearly all YTHDF2 translocated from the cytosol into the nucleus after heat shock. METTL3 knockdown significantly changed HSP70, HSP60, and HSP27 mRNA expression in HepG2 cells using siRNA, however, mRNA lifetime was not impacted. Silence of YTHDF2 using siRNA did not change expression of HSP70, but significantly increased HSP90, HSP60, and HSPB1 mRNA expression. In addition, m6A-seq revealed that HSP m6A methylation peaks are mainly enriched on exons and around stop codons, and shows a unique distribution profile in the 5'UTR and 3'UTR. Knockdown of METTL3 changed the methylation patterns of HSPs transcript. In conclusion, m6A RNA methylation regulates HSP gene expression. Differential expression of HSPs modulated by m6A may depend on the m6A site and abundance of the target gene. This finding provides insights into new regulatory mechanisms of HSPs in normal and stress situations.

Topical Application of a Bioadhesive Black Raspberry Gel Modulates Gene Expression and Reduces Cyclooxygenase 2 Protein in Human Premalignant Oral Lesions

PubMed Central

Mallery, Susan R.; Zwick, Jared C.; Pei, Ping; Tong, Meng; Larsen, Peter E.; Shumway, Brian S.; Lu, Bo; Fields, Henry W.; Mumper, Russell J.; Stoner, Gary D.

2010-01-01

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation. PMID:18559542

Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity.

PubMed

Jensen, Jeanette H; Conley, Lene N; Hedegaard, Jakob; Nielsen, Mathilde; Young, Jette F; Oksbjerg, Niels; Hornshøj, Henrik; Bendixen, Christian; Thomsen, Bo

2012-07-01

Acute physical activity elicits changes in gene expression in skeletal muscles to promote metabolic changes and to repair exercise-induced muscle injuries. In the present time-course study, pigs were submitted to an acute bout of treadmill running until near exhaustion to determine the impact of unaccustomed exercise on global transcriptional profiles in porcine skeletal muscles. Using a combined microarray and candidate gene approach, we identified a suite of genes that are differentially expressed in muscles during postexercise recovery. Several members of the heat shock protein family and proteins associated with proteolytic events, such as the muscle-specific E3 ubiquitin ligase atrogin-1, were significantly upregulated, suggesting that protein breakdown, prevention of protein aggregation and stabilization of unfolded proteins are important processes for restoration of cellular homeostasis. We also detected an upregulation of genes that are associated with muscle cell proliferation and differentiation, including MUSTN1, ASB5 and CSRP3, possibly reflecting activation, differentiation and fusion of satellite cells to facilitate repair of muscle damage. In addition, exercise increased expression of the orphan nuclear hormone receptor NR4A3, which regulates metabolic functions associated with lipid, carbohydrate and energy homeostasis. Finally, we observed an unanticipated induction of the long non-coding RNA transcript NEAT1, which has been implicated in RNA processing and nuclear retention of adenosine-to-inosine edited mRNAs in the ribonucleoprotein bodies called paraspeckles. These findings expand the complexity of pathways affected by acute contractile activity of skeletal muscle, contributing to a better understanding of the molecular processes that occur in muscle tissue in the recovery phase.

Cross-omics comparison of stress responses in mesothelial cells exposed to heat- versus filter-sterilized peritoneal dialysis fluids.

PubMed

Kratochwill, Klaus; Bender, Thorsten O; Lichtenauer, Anton M; Herzog, Rebecca; Tarantino, Silvia; Bialas, Katarzyna; Jörres, Achim; Aufricht, Christoph

2015-01-01

Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: Insights into molecular recognition of invading pathogens in Manduca sexta.

PubMed

Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R; Jiang, Haobo

2015-07-01

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: insights into molecular recognition of invading pathogens in Manduca sexta

PubMed Central

Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T.; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R.; Jiang, Haobo

2015-01-01

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. PMID:25701384

Differences in Grain Ultrastructure, Phytochemical and Proteomic Profiles between the Two Contrasting Grain Cd-Accumulation Barley Genotypes

PubMed Central

Sun, Hongyan; Cao, Fangbin; Wang, Nanbo; Zhang, Mian; Mosaddek Ahmed, Imrul; Zhang, Guoping; Wu, Feibo

2013-01-01

To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low- grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars. PMID:24260165

Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes

PubMed Central

Fu, Jia; Wei, Chengguo; Lee, Kyung; Zhang, Weijia; He, Wu; Chuang, Peter

2016-01-01

Evaluating the mRNA profile of podocytes in the diabetic kidney may indicate genes involved in the pathogenesis of diabetic nephropathy. To determine if the podocyte-specific gene information contained in mRNA profiles of the whole glomerulus of the diabetic kidney accurately reflects gene expression in the isolated podocytes, we crossed Nos3−/− IRG mice with podocin-rtTA and TetON-Cre mice for enhanced green fluorescent protein labeling of podocytes before diabetic injury. Diabetes was induced by streptozotocin, and mRNA profiles of isolated glomeruli and sorted podocytes from diabetic and control mice were examined 10 weeks later. Expression of podocyte-specific markers in glomeruli was downregulated in diabetic mice compared with controls. However, expression of these markers was not altered in sorted podocytes from diabetic mice. When mRNA levels of glomeruli were corrected for podocyte number per glomerulus, the differences in podocyte marker expression disappeared. Analysis of the differentially expressed genes in diabetic mice also revealed distinct upregulated pathways in the glomeruli (mitochondrial function, oxidative stress) and in podocytes (actin organization). In conclusion, our data suggest reduced expression of podocyte markers in glomeruli is a secondary effect of reduced podocyte number, thus podocyte-specific gene expression detected in the whole glomerulus may not represent that in podocytes in the diabetic kidney. PMID:26264855

miR-758-3p: a blood-based biomarker that’s influence on the expression of CERP/ABCA1 may contribute to the progression of obesity to metabolic syndrome

PubMed Central

O’Neill, Sadhbh; Larsen, Mette Bohl; Gregersen, Søren; Hermansen, Kjeld; O’Driscoll, Lorraine

2018-01-01

Due to increasing prevalence of obesity, a simple method or methods for the diagnosis of metabolic syndrome are urgently required to reduce the risk of associated cardiovascular disease, diabetes and cancer. This study aimed to identify a miRNA biomarker that may distinguish metabolic syndrome from obesity and to investigate if such a miRNA may have functional relevance for metabolic syndrome. 52 adults with clinical obesity (n=26) or metabolic syndrome (n=26) were recruited. Plasma specimens were procured from all and were randomly designated to discovery and validation cohorts. miRNA discovery profiling was performed, using array technology, on plasma RNA. Validation was performed by quantitative polymerase chain reaction. The functional effect of miR-758-3p on its predicted target, cholesterol efflux regulatory protein/ATP-binding cassette transporter, was investigated using HepG2 liver cells. Custom miRNA profiling of 25 miRNAs in the discovery cohort found miR-758-3p to be detected in the obese cohort but undetected in the metabolic syndrome cohort. miR-758-3p was subsequently validated as a potential biomarker for metabolic syndrome by quantitative polymerase chain reaction. Bioinformatics analysis identified cholesterol efflux regulatory protein/ATP-binding cassette transporter as miR-758-3p’s predicted target. Specifically, mimicking miR-758-3p in HepG2 cells suppressed cholesterol efflux regulatory protein/ATP-binding cassette transporter protein expression; conversely, inhibiting miR-758-3p increased cholesterol efflux regulatory protein/ATP-binding cassette transporter protein expression. miR-758-3p holds potential as a blood-based biomarker for distinguishing progression from obesity to metabolic syndrome and as a driver in controlling cholesterol efflux regulatory protein/ATP-binding cassette transporter expression, indicating it potential role in cholesterol control in metabolic syndrome. PMID:29507696

Comparative Proteomic Analysis of Susceptible and Resistant Rice Plants during Early Infestation by Small Brown Planthopper

PubMed Central

Dong, Yan; Fang, Xianping; Yang, Yong; Xue, Gang-Ping; Chen, Xian; Zhang, Weilin; Wang, Xuming; Yu, Chulang; Zhou, Jie; Mei, Qiong; Fang, Wang; Yan, Chengqi; Chen, Jianping

2017-01-01

The small brown planthopper (Laodelphax striatellus Fallén, Homoptera, Delphacidae-SBPH) is one of the major destructive pests of rice (Oryza sativa L.). Understanding on how rice responds to SBPH infestation will contribute to developing strategies for SBPH control. However, the response of rice plant to SBPH is poorly understood. In this study, two contrasting rice genotypes, Pf9279-4 (SBPH-resistant) and 02428 (SBPH-susceptible), were used for comparative analysis of protein profiles in the leaf sheath of rice plants in responses to SBPH infestation. One hundred and thirty-two protein spots that were differentially expressed between the resistant and susceptible rice lines were identified with significant intensity differences (≥2-fold, P < 0.05) at 0, 6, and 12 h after SBPH infestation. Protein expression profile analysis in the leaf sheath of SBPH-resistant and SBPH-susceptible rice lines after SBPH infestation showed that proteins induced by SBPH feeding were involved mainly in stress response, photosynthesis, protein metabolic process, carbohydrate metabolic process, energy metabolism, cell wall-related proteins, amino acid metabolism and transcriptional regulation. Gene expression analysis of 24 differentially expressed proteins (DEPs) showed that more than 50% DEPs were positively correlated with their mRNA levels. Analysis of some physiological indexes mainly involved in the removal of oxygen reactive species showed that the levels of superoxide dismutase (SOD) and glutathione (GSH) were considerably higher in Pf9279-4 than 02428 during SBPH infestation. The catalase (CAT) activity and hydroxyl radical inhibition were lower in Pf9279-4 than 02428. Analysis of enzyme activities indicates that Pf9279-4 rice plants defend against SBPH through the activation of the pathway of the salicylic acid (SA)-dependent systemic acquired resistance. In conclusion, this study provides some insights into the molecular networks involved on cellular and physiological responses to SBPH infestation. PMID:29089949

miR-758-3p: a blood-based biomarker that's influence on the expression of CERP/ABCA1 may contribute to the progression of obesity to metabolic syndrome.

PubMed

O'Neill, Sadhbh; Larsen, Mette Bohl; Gregersen, Søren; Hermansen, Kjeld; O'Driscoll, Lorraine

2018-02-06

Due to increasing prevalence of obesity, a simple method or methods for the diagnosis of metabolic syndrome are urgently required to reduce the risk of associated cardiovascular disease, diabetes and cancer. This study aimed to identify a miRNA biomarker that may distinguish metabolic syndrome from obesity and to investigate if such a miRNA may have functional relevance for metabolic syndrome. 52 adults with clinical obesity (n=26) or metabolic syndrome (n=26) were recruited. Plasma specimens were procured from all and were randomly designated to discovery and validation cohorts. miRNA discovery profiling was performed, using array technology, on plasma RNA. Validation was performed by quantitative polymerase chain reaction. The functional effect of miR-758-3p on its predicted target, cholesterol efflux regulatory protein/ATP-binding cassette transporter, was investigated using HepG2 liver cells. Custom miRNA profiling of 25 miRNAs in the discovery cohort found miR-758-3p to be detected in the obese cohort but undetected in the metabolic syndrome cohort. miR-758-3p was subsequently validated as a potential biomarker for metabolic syndrome by quantitative polymerase chain reaction. Bioinformatics analysis identified cholesterol efflux regulatory protein/ATP-binding cassette transporter as miR-758-3p's predicted target. Specifically, mimicking miR-758-3p in HepG2 cells suppressed cholesterol efflux regulatory protein/ATP-binding cassette transporter protein expression; conversely, inhibiting miR-758-3p increased cholesterol efflux regulatory protein/ATP-binding cassette transporter protein expression. miR-758-3p holds potential as a blood-based biomarker for distinguishing progression from obesity to metabolic syndrome and as a driver in controlling cholesterol efflux regulatory protein/ATP-binding cassette transporter expression, indicating it potential role in cholesterol control in metabolic syndrome.

Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas.

PubMed

Chen, Xiaochuan; Rozance, Paul J; Hay, William W; Limesand, Sean W

2012-05-01

Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

Transcriptome Analysis of Spartina pectinata in Response to Freezing Stress

PubMed Central

Nah, Gyoungju; Lee, Moonsub; Kim, Do-Soon; Rayburn, A. Lane; Voigt, Thomas; Lee, D. K.

2016-01-01

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation. PMID:27032112

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

PubMed

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Analysis of gene expression profile microarray data in complex regional pain syndrome.

PubMed

Tan, Wulin; Song, Yiyan; Mo, Chengqiang; Jiang, Shuangjian; Wang, Zhongxing

2017-09-01

The aim of the present study was to predict key genes and proteins associated with complex regional pain syndrome (CRPS) using bioinformatics analysis. The gene expression profiling microarray data, GSE47603, which included peripheral blood samples from 4 patients with CRPS and 5 healthy controls, was obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) in CRPS patients compared with healthy controls were identified using the GEO2R online tool. Functional enrichment analysis was then performed using The Database for Annotation Visualization and Integrated Discovery online tool. Protein‑protein interaction (PPI) network analysis was subsequently performed using Search Tool for the Retrieval of Interaction Genes database and analyzed with Cytoscape software. A total of 257 DEGs were identified, including 243 upregulated genes and 14 downregulated ones. Genes in the human leukocyte antigen (HLA) family were most significantly differentially expressed. Enrichment analysis demonstrated that signaling pathways, including immune response, cell motion, adhesion and angiogenesis were associated with CRPS. PPI network analysis revealed that key genes, including early region 1A binding protein p300 (EP300), CREB‑binding protein (CREBBP), signal transducer and activator of transcription (STAT)3, STAT5A and integrin α M were associated with CRPS. The results suggest that the immune response may therefore serve an important role in CRPS development. In addition, genes in the HLA family, such as HLA‑DQB1 and HLA‑DRB1, may present potential biomarkers for the diagnosis of CRPS. Furthermore, EP300, its paralog CREBBP, and the STAT family genes, STAT3 and STAT5 may be important in the development of CRPS.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A.

PubMed

Fernández, Ignacio; Darias, Maria; Andree, Karl B; Mazurais, David; Zambonino-Infante, Jose Luís; Gisbert, Enric

2011-02-09

Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream. During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

PubMed Central

2011-01-01

Background Vitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream. Results During gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp. Conclusions Present data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages. PMID:21306609

Glutamate Neonatal Excitotoxicity Modifies VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 Protein Expression Profiles During Postnatal Development of the Cerebral Cortex and Hippocampus of Male Rats.

PubMed

Castañeda-Cabral, Jose Luis; Beas-Zarate, Carlos; Gudiño-Cabrera, Graciela; Ureña-Guerrero, Monica E

2017-09-01

Vascular endothelial growth factor (VEGF) exerts both neuroprotective and proinflammatory effects in the brain, depending on the VEGF (A-E) and VEGF receptor (VEGFR1-3) types involved. Neonatal monosodium glutamate (MSG) treatment triggers an excitotoxic degenerative process associated with several neuropathological conditions, and VEGF messenger RNA (mRNA) expression is increased at postnatal day (PD) 14 in rat hippocampus (Hp) following the treatment. The aim of this work was to establish the changes in immunoreactivity to VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 proteins induced by neonatal MSG treatment (4 g/kg, subcutaneous, at PD1, 3, 5 and 7) in the cerebral motor cortex (CMC) and Hp. Samples collected from PD2 to PD60 from control and MSG-treated male Wistar rats were assessed by western blotting for each protein. Considering that immunoreactivity measured by western blotting is related to the protein expression level, we found that each protein in each cerebral region has a specific expression profile throughout the studied ages, and all profiles were differentially modified by MSG. Specifically, neonatal MSG treatment significantly increased the immunoreactivity to the following: (1) VEGF-A at PD8-PD10 in the CMC and at PD6-PD8 in the Hp; (2) VEGF-B at PD2, PD6 and PD10 in the CMC and at PD8-PD9 in the Hp; and (3) VEGFR-2 at PD6-PD8 in the CMC and at PD21-PD60 in the Hp. Also, MSG significantly reduced the immunoreactivity to the following: (1) VEGF-B at PD8-PD9 and PD45-PD60 in the CMC; and (2) VEGFR-1 at PD4-PD6 and PD14-PD21 in the CMC and at PD4, PD9-PD10 and PD60 in the Hp. Our results indicate that VEGF-mediated signalling is involved in the excitotoxic process triggered by neonatal MSG treatment and should be further characterized.

Expansion of Genes Encoding piRNA-Associated Argonaute Proteins in the Pea Aphid: Diversification of Expression Profiles in Different Plastic Morphs

PubMed Central

Lu, Hsiao-ling; Tanguy, Sylvie; Rispe, Claude; Gauthier, Jean-Pierre; Walsh, Tom; Gordon, Karl; Edwards, Owain; Tagu, Denis; Chang, Chun-che; Jaubert-Possamai, Stéphanie

2011-01-01

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are “specialised” in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction. PMID:22162754

iTRAQ-based proteomic profile analysis of ISKNV-infected CPB cells with emphasizing on glucose metabolism, apoptosis and autophagy pathways.

PubMed

Wu, Shiwei; Yu, Lujun; Fu, Xiaozhe; Yan, Xi; Lin, Qiang; Liu, Lihui; Liang, Hongru; Li, Ningqiu

2018-05-04

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant losses in the cultured mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie interaction between ISKNV and hosts are not fully understood. In this study, the proteomic profile of CPB cells at progressive time points after ISKNV infection was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2731 proteins corresponding to 6363 novel peptides (false discovery rate <0.01) were identified. In the samples harvested 24 h (early-stage) and 72 h (late-stage) post-infection, 232 and 199 differentially expressed proteins were identified comparing with mock-infected cells, respectively. Western-blotting analysis of several proteins as G6PDH, β-tubulin and RPL11 were done to validate iTRAQ data. Among those differentially expressed proteins, several glucose metabolism-related enzymes, including glucose-6-phosphate dehydrogenase (G6PDH), pyruvate dehydrogenase phosphatase (PDP) and fumarate hydratase (FH), were up-regulated, while pyruvate dehydrogenase kinase (PDK) and enolase (ENO) were down-regulated at 24 h poi, suggesting that ISKNV enhanced glucose metabolism in CPB cells in early-stage infection. Simultaneously, expression of apoptosis-related proteins including Caspase 8, phosphoinositide 3-kinases (PI3Ks), and regulatory-associated protein of mTOR-like isoform X3 changed upon ISKNV infection, indicating that ISKNV induced apoptosis of CPB cells. Autophagy-related proteins including LC3 and PI3Ks were up-regulated at 24 h poi, indicating that ISKNV induced autophagy of CPB cells in early-stage infection. These findings may improve the understanding of ISKNV and host interaction and help clarify its pathogenesis mechanisms. Copyright © 2018. Published by Elsevier Ltd.

Proteome analysis reveals a distinct molecular profile of cellular stress following incubation of DDT1-MF2 smooth muscle cells in the presence of a high concentration of hyperforin.

PubMed

Schrattenholz, André; Schroer, Klaus; Chatterjee, Shyam S; Koch, Egon

2004-04-01

The acylphloroglucinol derivative hyperforin is a major constituent of St. John's wort extracts ( Hypericum perforatum L.), which has been demonstrated to contribute to the antidepressant action of this herbal drug. In previous investigations we observed that hyperforin causes a rapid stimulation of intracellular calcium mobilization and enhances extracellular acidification in the hamster vas deferens smooth muscle cell line DDT (1)-MF2. To obtain further insight into its mode of action, we have now examined if these effects are accompanied by changes in protein expression. Cells were incubated with hyperforin for 15 min at a concentration of 1 microg/mL. Proteome analysis in cell lysates was accomplished by two-dimensional gel electrophoresis (2D-PAGE) and proteins were visualized by silver staining. Differences in the expression pattern between hyperforin- and vehicle-treated cells were displayed by computer-assisted differential display and identification of selected protein spots was performed by peptide mass fingerprinting after digestion with trypsin. Following incubation with hyperforin marked changes in the expression of several proteins were evident. A particularly strong change was observed for 6 proteins, which were identified as tubulin-beta, enolase 3, SYNCRIP, endoplasmin, elongation factor 2 and HSP84. As these proteins are known to be involved in cellular responses to stress by regulating energy metabolism as well as synthesis, intracellular transport and folding of proteins, our results suggest that the effects observed are not components of the normal pharmacological activity profile of hyperforin but are rather indicative of cellular stress promoting activity at higher concentrations.

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

PubMed Central

Leung, Fiona Wan Lun; Lam, Hugh Simon; Tam, Yuk Him; To, Ka Fai; Cheung, Hon Ming; Leung, Kam Tong; Poon, Terence Chuen Wai; Lee, Kim Hung; Li, Karen; Fok, Tai Fai

2012-01-01

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients. PMID:22606320

Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis

PubMed Central

Grgic, Ivica; Krautzberger, A. Michaela; Hofmeister, Andreas; Lalli, Matthew; DiRocco, Derek P.; Fleig, Susanne V.; Liu, Jing; Duffield, Jeremy S.; McMahon, Andrew P.; Aronow, Bruce

2014-01-01

Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)–tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies. PMID:24652793