Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida

PubMed Central

2012-01-01

Background Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. Results We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. Conclusions We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin. PMID:22272603

Analysis of the Liver Soluble Proteome from Bull Terriers Affected with Inherited Lethal Acrodermatitis

PubMed Central

Mouat, Michael F.; Mauldin, Elizabeth A.; Casal, Margret L.

2012-01-01

Lethal acrodermatitis (LAD) is a genetic disease affecting bull terrier dogs. The phenotype is similar to that for acrodermatitis enteropathica in humans, but is currently without treatment. The purpose of the research presented here is to determine the biochemical defects associated with LAD using proteomic methodologies. Two affected (male and female) and one unaffected (male) bull terrier pups were euthanized at 14 weeks of age, their livers dissected and prepared for two-dimensional gel electrophoresis (2DE) and densitometry. Approximately 200 protein spots were observed. The density of the spots within each gel was normalized to the total spot volume of the gel; only those soluble liver protein spots that were consistently different in both of the livers of the affected pups compared to the unaffected pup were excised manually and submitted for MALDI mass spectrometry. Thirteen proteins were identified as differentially expressed in the affected, compared to the unaffected, pups. The proteins were involved in numerous cellular physiological functions, including chaperones, calcium binding, and energy metabolism, as well as being associated with the inflammatory response. Of note were haptoglobin, glutamine synthetase, prohibitin and keratin 10 which exhibited at least a 4-fold level of differential expression. These data represent the first proteomic analysis of this mutation. The differentially expressed proteins that were identified may be key in understanding the etiology of LAD, and may lead to diagnostic tools for its identification within the bull terrier population. PMID:17693109

Differential Expression of R-genes to Associate Leaf Spot Resistance in Cultivated Peanut

USDA-ARS?s Scientific Manuscript database

Breeding for acceptable levels of Early (ELS) or Late Leaf Spot (LLS) resistance in cultivated peanut has been elusive due to extreme variability of plant response in the field and the proper combinations of resistance (R)-genes in any particular peanut line. R-genes have been shown to be involved ...

The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

PubMed

Sheffield, Jeanne; Taylor, Nigel; Fauquet, Claude; Chen, Sixue

2006-03-01

Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots.

Transcriptome Profiling of Buffalograss Challenged with the Leaf Spot Pathogen Curvularia inaequalis.

PubMed

Amaradasa, Bimal S; Amundsen, Keenan

2016-01-01

Buffalograss (Bouteloua dactyloides) is a low maintenance U. S. native turfgrass species with exceptional drought, heat, and cold tolerance. Leaf spot caused by Curvularia inaequalis negatively impacts buffalograss visual quality. Two leaf spot susceptible and two resistant buffalograss lines were challenged with C. inaequalis. Samples were collected from treated and untreated leaves when susceptible lines showed symptoms. Transcriptome sequencing was done and differentially expressed genes were identified. Approximately 27 million raw sequencing reads were produced per sample. More than 86% of the sequencing reads mapped to an existing buffalograss reference transcriptome. De novo assembly of unmapped reads was merged with the existing reference to produce a more complete transcriptome. There were 461 differentially expressed transcripts between the resistant and susceptible lines when challenged with the pathogen and 1552 in its absence. Previously characterized defense-related genes were identified among the differentially expressed transcripts. Twenty one resistant line transcripts were similar to genes regulating pattern triggered immunity and 20 transcripts were similar to genes regulating effector triggered immunity. There were also nine up-regulated transcripts in resistance lines which showed potential to initiate systemic acquired resistance (SAR) and three transcripts encoding pathogenesis-related proteins which are downstream products of SAR. This is the first study characterizing changes in the buffalograss transcriptome when challenged with C. inaequalis.

Transcriptomic and Proteomic Analyses of Resistant Host Responses in Arachis diogoi Challenged with Late Leaf Spot Pathogen, Phaeoisariopsis personata

PubMed Central

Kumar, Dilip; Kirti, Pulugurtha Bharadwaja

2015-01-01

Late leaf spot is a serious disease of peanut caused by the imperfect fungus, Phaeoisariopsis personata. Wild diploid species, Arachis diogoi. is reported to be highly resistant to this disease and asymptomatic. The objective of this study is to investigate the molecular responses of the wild peanut challenged with the late leaf spot pathogen using cDNA-AFLP and 2D proteomic study. A total of 233 reliable, differentially expressed genes were identified in Arachis diogoi. About one third of the TDFs exhibit no significant similarity with the known sequences in the data bases. Expressed sequence tag data showed that the characterized genes are involved in conferring resistance in the wild peanut to the pathogen challenge. Several genes for proteins involved in cell wall strengthening, hypersensitive cell death and resistance related proteins have been identified. Genes identified for other proteins appear to function in metabolism, signal transduction and defence. Nineteen TDFs based on the homology analysis of genes associated with defence, signal transduction and metabolism were further validated by quantitative real time PCR (qRT-PCR) analyses in resistant wild species in comparison with a susceptible peanut genotype in time course experiments. The proteins corresponding to six TDFs were differentially expressed at protein level also. Differentially expressed TDFs and proteins in wild peanut indicate its defence mechanism upon pathogen challenge and provide initial breakthrough of genes possibly involved in recognition events and early signalling responses to combat the pathogen through subsequent development of resistivity. This is the first attempt to elucidate the molecular basis of the response of the resistant genotype to the late leaf spot pathogen, and its defence mechanism. PMID:25646800

Serum proteomic MRM identify peptide ions of transferrin as new fibrosis markers in chronic hepatitis B.

PubMed

Xu, Ming-Yi; Qu, Ying; Jia, Xiao-Fang; Wang, Mei-Ling; Liu, Heng; Wang, Xing-Peng; Zhang, Li-Jun; Lu, Lun-Gen

2013-09-01

Because of the limitations of liver biopsy, reliable non-invasive serum biomarkers of liver fibrosis are needed. The aim of this study was to identify such markers by the use of serum proteomics in chronic hepatitis B (CHB). Two-dimensional gel electrophoresis (2-DE) was used to identify differentially expressed protein spots in sera from 40 CHB patients [20 with mild fibrosis (S0-S1) and 20 with severe fibrosis (S3-S4)]. Mass spectrometry (MS) based multiple reaction monitoring (MRM) was used to quantify peptide ions of differential protein spots in another set of sera from 86 CHB patients with different liver fibrosis (S0-S4). Seven differentially expressed protein spots were found by 2-DE. Fourteen peptide ions of seven target protein spots were quantified by MS-based MRM. Summed peak areas ratio (SPAR) values of peptide ions from protein spot 1, 4 and 8, identified as apo serum transferrin, complement component C3c and transferrin, were significantly different from non-fibrosis (S0) to fibrosis stage 4. AUROCs of models established by peptide ions (protein spot 1, 4, 8) and model consisting of a combination of all ions were 0.848∼0.966 (S2-S4 versus S0-S1) and 0.785∼0.875 (S3-S4 versus S0-S2). Only the peptide ions model of transferrin had better sensitivity and specificity for predicting fibrosis stages than did aspartate aminotransferase-to-platelet ratio index (APRI), FIB-4 and Forn's index. Serum peptide ions of transferrin, detected by proteomic MRM, are new and promising biomarkers for staging liver fibrosis in CHB patients. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Comparative proteomic analysis in Aphis glycines Mutsumura under lambda-cyhalothrin insecticide stress.

PubMed

Bi, Rui; Pan, Yiou; Shang, Qingli; Peng, Tianfei; Yang, Shuang; Wang, Shang; Xin, Xuecheng; Liu, Yan; Xi, Jinghui

2016-09-01

Lambda-cyhalothrin is now widely used in China to control the soybean aphid Aphis glycines. To dissect the resistance mechanism, a laboratory-selected resistant soybean aphid strain (CRR) was established with a 43.42-fold resistance ratio to λ-cyhalothrin than the susceptible strain (CSS) in adult aphids. In this study, a comparative proteomic analysis between the CRR and CSS strains revealed important differences between the susceptible and resistant strains of soybean aphids for λ-cyhalothrin. Approximately 493 protein spots were detected in two-dimensional polyacrylamide gel electrophoresis (2-DE). Thirty-six protein spots displayed differential expression of >2-fold in the CRR strain compared to the CSS strain. Out of these 36 protein spots, 21 had elevated and 15 had decreased expression. Twenty-four differentially expressed proteins were identified by MALDI TOF MS/MS and categorized into the functional groups cytoskeleton-related protein, carbohydrate and energy metabolism, protein folding, antioxidant system, and nucleotide and amino acid metabolism. Function analysis showed that cytoskeleton-related proteins and energy metabolism proteins have been associated with the λ-cyhalothrin resistance of A. glycines. The differential expression of λ-cyhalothrin responsive proteins reflected the overall change in cellular structure and metabolism after insecticide treatment in aphids. In summary, our studies improve understanding of the molecular mechanism resistance of soybean aphid to lambda-cyhalothrin, which will facilitate the development of rational approaches to improve the management of this pest and to improve the yield of soybean. Copyright © 2016. Published by Elsevier Inc.

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.

Microarray analysis of differentially expressed genes engaged in fruit development between Prunus mume and Prunus armeniaca.

PubMed

Li, Xiaoying; Korir, Nicholas Kibet; Liu, Lili; Shangguan, Lingfei; Wang, Yuzhu; Han, Jian; Chen, Ming; Fang, Jinggui

2012-11-15

Microarray analysis is a technique that can be employed to provide expression profiles of single genes and new insights to elucidate the biological mechanisms responsible for fruit development. To evaluate expression of genes mostly engaged in fruit development between Prunus mume and Prunus armeniaca, we first identified differentially expressed transcripts along the entire fruit life cycle by using microarrays spotted with 10,641 ESTs collected from P. mume and other Prunus EST sequences. A total of 1418 ESTs were selected after quality control of microarray spots and analysis for differential gene expression patterns during fruit development of P. mume and P. Armeniaca. From these, 707 up-regulated and 711 down-regulated genes showing more than two-fold differences in expression level were annotated by GO based on biological processes, molecular functions and cellular components. These differentially expressed genes were found to be involved in several important pathways of carbohydrate, galactose, and starch and sucrose metabolism as well as in biosynthesis of other secondary metabolites via KEGG. This could provide detailed information on the fruit quality differences during development and ripening of these two species. With the results obtained, we provide a practical database for comprehensive understanding of molecular events during fruit development and also lay a theoretical foundation for the cloning of genes regulating in a series of important rate-limiting enzymes involved in vital metabolic pathways during fruit development. Copyright © 2012 Elsevier GmbH. All rights reserved.

Proteomic changes in female rat hippocampus following exposure to a terrified sound stress.

PubMed

Yang, Juan; Hu, Lili; Song, Tusheng; Liu, Yong; Wu, Qiuhua; Zhao, Lingyu; Liu, Liying; Zhao, Xiaoge; Zhang, Dianzeng; Huang, Chen

2014-06-01

Stress plays a profound role in the onset of affective disorders, including an elevation in risk factors for depression and anxiety. Women are twice as vulnerable to stress as men because of greater sensitivity to a substance produced during times of anxiety. To better define the abnormal proteins implicated in cognitive deficits and other stress-induced dysfunction, female rats were exposed to terrified sound stress, and two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were utilized to determine the differential protein expression in the hippocampus in sound-stressed female rats compared with controls. Quantitative differences were found in 44 protein spots which were differentially expressed between the stressed and control groups (fold change of >2; p < 0.01). Eighteen protein spots were downregulated, and 26 protein spots were upregulated in the stressed group. The seven most differentially expressed proteins were identified and validated as follows: dihydropyrimidinase-related protein 2 (DRP-2), creatine kinase B type, dynamin-1 protein, alpha-internexin, glial fibrillary acidic protein beta, gamma-enolase, and peptidyl-prolyl cis-trans isomerase A. Changes in protein levels were detected in the hippocampus of female rats subjected to terrified sound stress. The findings herein may open new opportunities for further investigations on the modulation induced in the hippocampus by stress at the molecular level, especially with respect to females stress.

Influence of white spot syndrome virus infection on hepatopancreas gene expression of `Huanghai No. 2' shrimp ( Fenneropenaeus chinensis)

NASA Astrophysics Data System (ADS)

Meng, Xianhong; Shi, Xiaoli; Kong, Jie; Luan, Sheng; Luo, Kun; Cao, Baoxiang; Liu, Ning; Lu, Xia; Li, Xupeng; Deng, Kangyu; Cao, Jiawang; Zhang, Yingxue; Zhang, Hengheng

2017-10-01

To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus (WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of `Huanghai No. 2' ( Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early (48-96 h), peak (168-192 h) and late (264-288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes (1916 annotated) expressed at all three phases, and most of the annotated were either up- or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

The heterogeneity of human mesenchymal stem cell preparations--evidence from simultaneous analysis of proteomes and transcriptomes.

PubMed

Wagner, Wolfgang; Feldmann, Robert E; Seckinger, Anja; Maurer, Martin H; Wein, Frederik; Blake, Jonathon; Krause, Ulf; Kalenka, Armin; Bürgers, Heinrich F; Saffrich, Rainer; Wuchter, Patrick; Kuschinsky, Wolfgang; Ho, Anthony D

2006-04-01

Mesenchymal stem cells (MSC) raise high hopes in clinical applications. However, the lack of common standards and a precise definition of MSC preparations remains a major obstacle in research and application of MSC. Whereas surface antigen markers have failed to precisely define this population, a combination of proteomic data and microarray data provides a new dimension for the definition of MSC preparations. In our continuing effort to characterize MSC, we have analyzed the differential transcriptome and proteome expression profiles of MSC preparations isolated from human bone marrow under two different expansion media (BM-MSC-M1 and BM-MSC-M2). In proteomics, 136 protein spots were unambiguously identified by MALDI-TOF-MS and corresponding cDNA spots were selected on our "Human Transcriptome cDNA Microarray." Combination of datasets revealed a correlation in differential gene expression and protein expression of BM-MSC-M1 vs BM-MSC-M2. Genes involved in metabolism were more highly expressed in BM-MSC-M1, whereas genes involved in development, morphogenesis, extracellular matrix, and differentiation were more highly expressed in BM-MSC-M2. Interchanging culture conditions for 8 days revealed that differential expression was retained in several genes whereas it was altered in others. Our results have provided evidence that homogeneous BM-MSC preparations can reproducibly be isolated under standardized conditions, whereas culture conditions exert a prominent impact on transcriptome, proteome, and cellular organization of BM-MSC.

Differential protein expression in Tree Shrew sclera during development of lens-induced myopia and recovery

PubMed Central

Norton, Thomas T.

2007-01-01

Purpose The tree shrew model of refractive development is particularly useful because, like humans, tree shrews have a fibrous sclera. Selective changes in some candidate extracellular matrix proteins and mRNAs have been found in the sclera during the development of, and recovery from, induced myopia. We undertook a more neutral proteomic analysis using two-dimensional gel electrophoresis and mass spectrometry to identify scleral proteins that are differentially expressed during the development of, and recovery from, lens-induced myopia. Methods Five tree shrews (Tupaia glis belangeri) wore a monocular –5 D lens for 4 days, starting 24 days after natural eye opening. At the end of this time, all treated eyes had partially compensated for the lens and were –3.5±0.7 D (mean ± SEM) myopic relative to the untreated fellow control eyes. An additional five animals wore a –5 D lens for 11–13 days, followed by 4 days of recovery without the –5 D lens. The amount of recovery was 1.6±0.4 D. Scleral proteins from both groups were then isolated and resolved by two-dimensional gel electrophoresis and spots that were differentially expressed were identified by mass spectrometry. Results The scleral protein profile typically displayed ~700 distinct protein spots within the pH 5–8 range. Comparison of the treated-eye and control-eye scleras of the lens-compensation animals revealed five spots that were significantly differentially expressed in all five pairs of eyes; all were downregulated 1.2 to 1.7 fold in the treated eye. These proteins were identified as: pigment epithelium-derived factor (PEDF), procollagen I α1, procollagen I α2, and thrombospondin I (two spots). In the recovering eyes, the two thrombospondin I spots remained lower in abundance while PEDF and the procollagens were no longer downregulated. In addition, 78 kDa glucose-regulated protein (GRP 78), a member of the heat shock protein 70 family, was slightly upregulated 1.3 fold. Conclusions We found consistent results across animals that were of a magnitude consistent with the physiologically small changes to the focal plane of these eyes. Changes in collagen confirm previous findings, but downregulation of thrombospondin I adds detail to our understanding of the chain of signals that regulates scleral creep rate. The differential changes in PEDF and GRP 78 were not expected, based on previous studies, and demonstrate the utility of the proteomic approach in tree shrew sclera. PMID:17893659

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

Proteomic analysis of plasma proteins in diabetic retinopathy patients by two dimensional electrophoresis and MALDI-Tof-MS.

PubMed

Gopalakrishnan, Vidhya; Purushothaman, Parthiban; Bhaskar, Anusha

2015-01-01

Diabetic retinopathy is a highly specific vascular complication of diabetes mellitus and progresses from mild non-proliferative abnormalities characterized by increased vascular permeability to moderate and severe proliferative diabetic retinopathy characterized by the growth of blood vessels on the retina. The aim of the study was to identify the differentially expressed proteins in diabetic retinopathy using two-dimensional electrophoresis. Blood sample was drawn from subjects with diabetes mellitus (without retinopathy) who served as controls and patients with diabetic retinopathy in tubes containing EDTA as anticoagulant. Albumin and immunoglobulin IgG collectively removed to enrich proteins of lower abundance. 2de was carried out to see if there are any differentially expressed proteins. Approximately 48 and 61 spots were identified in control and diabetic retinopathy respectively, of which three protein spots RBP1 (retinol-binding protein 1), NUD10 (Diphosphoinositol polyphosphohydrolase 3 alpha), NGB (neuroglobin) were down regulated and HBG2 (hemoglobin) and BY55 (CD 160 antigen) were upregulated in diabetic retinopathy. These five protein spots were excised and were subjected to in-gel tryptic digestion, and their identities were determined by ultraflex MALDI-TOF-MS. We report a comprehensive patient-based plasma proteomic approach to the identification of potential biomarkers for diabetic retinopathy screening and detection. We identified 5 different proteins that were differentially expressed in the plasma of control diabetic patients (without retinopathy). Among these five proteins the expression of neuroglobin (NGB) protein varied significantly and may be a potential biomarker in diabetic retinopathy. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential expression of genes and proteins associated with wool follicle cycling.

PubMed

Liu, Nan; Li, Hegang; Liu, Kaidong; Yu, Juanjuan; Cheng, Ming; De, Wei; Liu, Jifeng; Shi, Shuyan; He, Yanghua; Zhao, Jinshan

2014-08-01

Sheep are valuable resources for the wool industry. Wool growth of Aohan fine wool sheep has cycled during different seasons in 1 year. Therefore, identifying genes that control wool growth cycling might lead to ways for improving the quality and yield of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side skins at August and December time points in Aohan fine wool sheep (a Chinese indigenous breed). Microarray study revealed that 2,223 transcripts were differentially expressed, including 1,162 up-regulated and 1,061 down-regulated transcripts, comparing body side skin at the August time point to the December one (A/D) in Aohan fine wool sheep. Then seven differentially expressed genes were selected to validated the reliability of the gene chip data. The majority of the genes possibly related to follicle development and wool growth could be assigned into the categories including regulation of receptor binding, extracellular region, protein binding and extracellular space. Proteomic study revealed that 84 protein spots showed significant differences in expression levels. Of the 84, 63 protein spots were upregulated and 21 were downregulated in A/D. Finally, 55 protein points were determined through MALDI-TOF/MS analyses. Furthermore, the regulation mechanism of hair follicle might resemble that of fetation.

Alterations of proteins in MDCK cells during acute potassium deficiency.

PubMed

Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

2016-06-01

Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury. Copyright © 2016 Elsevier B.V. All rights reserved.

Mercury-induced biochemical and proteomic changes in rice roots.

PubMed

Chen, Yun-An; Chi, Wen-Chang; Huang, Tsai-Lien; Lin, Chung-Yi; Quynh Nguyeh, Thi Thuy; Hsiung, Yu-Chywan; Chia, Li-Chiao; Huang, Hao-Jen

2012-06-01

Mercury (Hg) is a serious environmental pollution threats to the planet. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. We investigated cellular, biochemical and proteomic changes in rice roots under Hg stress. Root growth rate was decreased and Hg, reactive oxygen species (ROS), and malondialdehyde (MDA) content and lipoxygenase activity were increased significantly with increasing Hg concentration in roots. We revealed a time-dependent alteration in total glutathione content and enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) during Hg stress. 2-D electrophoresis revealed differential expression of 25 spots with Hg treatment of roots: 14 spots were upregulated and 11 spots downregulated. These differentially expressed proteins were identified by ESI-MS/MS to be involved in cellular functions including redox and hormone homeostasis, chaperone activity, metabolism, and transcription regulation. These results may provide new insights into the molecular basis of the Hg stress response in plants. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

PubMed

Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

PubMed Central

Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569

Identification and proteomic analysis of a novel gossypol-degrading fungal strain.

PubMed

Yang, Xia; Sun, Jian-Yi; Guo, Jian-Lin; Weng, Xiao-Yan

2012-03-15

Cottonseed meal, an important source of feed raw materials, has limited use in the feed industry because of the presence of the highly toxic gossypol. The aim of the current work was to isolate the gossypol-degrading fungus from a soil microcosm and investigate the proteins involved in gossypol degradation. A fungal strain, AN-1, that uses gossypol as its sole carbon source was isolated and identified as Aspergillus niger. A large number of intracellular proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no significant difference was observed between the glucose-containing and gossypol-containing mycelium extracts. Two-dimensional gel electrophoresis results showed that the protein spots were concentrated in the 25.0-66.2 kDa range and distributed in different pI gradients. PDQuest software showed that 51 protein spots in the gels were differentially expressed. Of these, 20 differential protein spots, including six special spots expressed in gossypol, were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The fungus AN-1 biodegraded gossypol and the proteomic analysis results indicate that some proteins were involved in the gossypol biodegradation during fungus survival, using gossypol as its sole carbon source. Copyright © 2011 Society of Chemical Industry.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

PubMed

Cordeiro, Fabio Aparecido; Tadra-Sfeir, Michelle Zibetti; Huergo, Luciano Fernandes; de Oliveira Pedrosa, Fábio; Monteiro, Rose Adele; de Souza, Emanuel Maltempi

2013-03-01

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.

Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

PubMed Central

Aldor, Ilana S.; Krawitz, Denise C.; Forrest, William; Chen, Christina; Nishihara, Julie C.; Joly, John C.; Champion, Kathleen M.

2005-01-01

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield. PMID:15811994

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo.

PubMed

Novoa-Herran, Susana; Umaña-Perez, Adriana; Canals, Francesc; Sanchez-Gomez, Myriam

2016-01-01

How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line. The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test ( n  = 3, significance level 0.10, D > 0.642) and/or ANOVA ( n  = 3, p  < 0.05). The results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells. This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.

[Relationship of GSTP1 lower expression and multidrug resistance reversing of curcumin on human colon carcinoma cells].

PubMed

Li, He; Li, Lei

2015-08-11

To explore the proteomic differences among with and without curcumin treatment of vincristine-resistance HCT-8/VCR cells of human colon carcinoma by using mass spectrometry and two-dimensional gel electrophore-sis (2-DE). The total proteins of the both groups were extracted from serum were run in immobilized pH gradient isoelectic focusing (IPG-IEF) at the first dimension.The proteins pots in gels were visualized by silver staining protocol, scanned by using a molecular imager GS-800 calibrated densitometer. The differentially expressed proteins were identified and analyzed by PDQuest 8.0 software. The diferentially displayed protein spots were searched and identifiyed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), the interested proteins were further validated by RT-PCR and Western blot. The 2-DE HCT-8/VCR cells patterns were acquired with clear background, well-resolution and reproduction. And 1 070±96 protein spots were detected in control HCT-8/VCR cells and 1 030±69 in curcumin-treated HCT-8/VCR cells. Twenty-nine differential protein spots were found to be differentially expressed. Glutathione S-transferase pi1 gene (GSTP1), a diferentiaI expression protein was identified which one of these proteins. RT-PCR and Western blotting results showed that the expressions of GSTP1 mRNA (0.49±0.09) and protein (0.29±0.07) in curcumin-treated group were significantly lower than in control group (GSTP1 mRNA 1.19±0.21 and protein 0.70±0.13, both P<0.05), indicating that curcumin down regulated these expressions. The suppression of GSTP1 by curcumin could enhance the vincristine chemosensitivity in HCT-8/VCR. GSTP1 overexpression may be involved in the vincristine -resistance of human colon carcinoma cells.

Proteome changes in rat serum after a chronic ingestion of enriched uranium: Toward a biological signature of internal contamination and radiological effect.

PubMed

Petitot, F; Frelon, S; Chambon, C; Paquet, F; Guipaud, O

2016-08-22

The civilian and military use of uranium results in an increased risk of human exposure. The toxicity of uranium results from both its chemical and radiological properties that vary with isotopic composition. Validated biomarkers of health effects associated with exposure to uranium are neither sensitive nor specific to uranium radiotoxicity and/or radiological effect. This study aimed at investigating if serum proteins could be useful as biomarkers of both uranium exposure and radiological effect. Male Sprague-Dawley rats were chronically exposed through drinking water to low levels (40mg/L, corresponding to 1mg of uranium per animal per day) of either 4% (235)U-enriched uranium (EU) or 12% EU during 6 weeks. A proteomics approach based on two-dimensional electrophoresis (2D-DIGE) and mass spectrometry (MS) was used to establish protein expression profiles that could be relevant for discriminating between groups, and to identify some differentially expressed proteins following uranium ingestion. It demonstrated that the expressions of 174 protein spots over 1045 quantified spots were altered after uranium exposure (p<0.05). Using both inferential and non-supervised multivariate statistics, we show sets of spots features that lead to a clear discrimination between controls and EU exposed groups on the one hand (21 spots), and between 4% EU and 12% EU on the other hand (7 spots), showing that investigation of the serum proteome may possibly be of relevance to address both uranium contamination and radiological effect. Finally, using bioinformatics tools, pathway analyses of differentially expressed MS-identified proteins find that acute phase, inflammatory and immune responses as well as oxidative stress are likely involved in the response to contamination, suggesting a physiological perturbation, but that does not necessarily lead to a toxic effect. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Molecular and histological characterization of age spots

PubMed Central

Choi, Wonseon; Yin, Lanlan; Smuda, Christoph; Batzer, Jan; Hearing, Vincent J.; Kolbe, Ludger

2016-01-01

Age spots, also called solar lentigines and lentigo senilis, are light brown to black pigmented lesions of various sizes that typically develop in chronically sun-exposed skin. It is well known that age spots are strongly related to chronic sun exposure and are associated with photodamage and an increased risk for skin cancer, however, the mechanism(s) underlying their development remain poorly understood. We used immunohistochemical analysis and microarray analysis to investigate the processes involved in their formation, focusing on specific markers associated with the functions and proliferation of melanocytes and keratinocytes. A total of 193 genes were differentially expressed in age spots but melanocyte pigment genes were not among them. The increased expression of keratins 5 and 10, markers of basal and suprabasal keratinocytes, respectively, in age spots suggests that the increased proliferation of basal keratinocytes combined with the decreased turnover of suprabasal keratinocytes leads to the exaggerated formation of rete ridges in lesional epidermis which in turn disrupts the normal processing of melanin upwards from the basal layer. Based on our results, we propose a model for the development of age spots that explains the accumulation of melanin and the development of extensive rete ridges in those hyperpigmented lesions. PMID:27621222

Differential expression proteins associated with bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

PubMed

Zhang, Y X; Yu, D; Tian, X L; Liu, C Y; Gai, S P; Zheng, G S

2015-01-01

Endo-dormant flower buds of tree peony must have sufficient chilling duration to reinitiate growth, which is a major obstacle to the forcing culture of tree peony in winter. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) to identify the differentially expressed proteins of tree peony after three different chilling treatments: endo-dormancy, endo-dormancy release and eco-dormancy stages. More than 200 highly reproducible protein spots were detected, and 31 differentially expressed spots (P < 0.05) were selected for further analysis. Finally, 20 protein spots were confidently identified from databases, which were annotated and classified into seven functional categories: response to abiotic or biotic stimulus (four), metabolic processes (four), other binding (three), transcription or transcription regulation (two), biological processes (one), cell biogenesis (one) and unclassified (five). The results of qPCR of five genes were mainly consistent with that of the protein accumulation analysis as determined by 2-DE. This indicated that most of these genes were mainly regulated at transcriptional level. The activity of nitrate reductase and pyruvate dehydrogenase E1 was consistent with the 2-DE results. The proteomic profiles indicated activation of citrate cycle, amino acid metabolism, lipid metabolism, energy production, calcium signalling and cell growth processes by chilling fulfilment to facilitate dormancy release in tree peony. Analysis of functions of identified proteins will increase our knowledge of endo-dormancy release in tree peony. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Plasma immune protein analysis in the orange-spotted grouper Epinephelus coioides: Evidence for altered expressions of immune factors associated with a choline-supplemented diet.

PubMed

Shiu, Ya-Li; Chiu, Kuo-Hsun; Huynh, Truong-Giang; Liu, Ping-Chung; Liu, Chun-Hung

2017-06-01

This study aimed to unravel the regulatory roles of choline in activating immune responses and disease resistance of the orange-spotted grouper Epinephelus coioides. Fish were fed a choline-supplemented diet at 1 g kg -1 of feed for 30 days. Fish fed a fish meal basal diet without choline-supplement served as controls. At the end of the feeding trial, fish were challenged with Vibrio alginolyticus. Meanwhile, plasma proteomics of fish in each group were also evaluated by two-dimensional gel electrophoresis (2-DE), and differentially expressed proteins were identified by tandem mass spectrophotometry (MS/MS), then a Western blot analysis or real-time polymerase chain reaction was used to confirm differential expressions of immune-enhancing proteins. Results showed that choline significantly increased survival of E. coioides 48 days after being injected with V. alginolyticus. From maps of plasma proteins, a comparative analysis between the control and choline groups revealed that 111 spots matched, with 26 altered expression spots in the choline group. Of these 26 spots, 16 were upregulated and 10 downregulated. After protein identification by reverse-phase nano-high-performance liquid chromatography-electrospray ionization MS/MS analysis, eight of 26 proteins were found to be immune-related proteins, all of which were upregulated, including complement 3 (C3), alpha-2-macroglobulin-P-like isoform (A2M), fibrinogen beta chain precursor (FBG), and immunoglobulin heavy constant mu (Ighm) proteins. Expression of the A2M protein and A2M enzyme activity in plasma of fish fed choline significantly increased compared to the control group. Additionally, A2M messenger (m)RNA transcripts were also upregulated in the liver and kidneys. Significantly higher C3 expressions at both the mRNA and protein levels were detected in the liver of fish in the choline group. Moreover, FBG gene expressions in the liver and kidneys significantly increased, while Ighm increased in the kidneys and spleen of fish in the choline group. Our results suggest that dietary administration of choline can protect grouper against bacterial infections through activating the complement system, thereby inducing antiprotease activity and natural antibodies that play important roles in the innate immune system of fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

The changes of serum proteome and tissular pathology in mouse induced by botulinum toxin E injection.

PubMed

Wang, J F; Mao, X Y; Zhao, C

2014-01-01

The experiment were performed to investigate the poisoning-related proteins and main pathological changes after mouse suffered from injection of botulinum toxin serotype E. Dose of 0.75 LD50 botulinum toxin serotype E per mice were administrated by intraperitoneal injection. Survival mouse were picked as experimental group. The blood were collected from orbital blood and serum sample was separated by centrifugation. The heart, liver, spleen, lung, kidney were fixed in 10 % neutral buffered formalin and then developed paraffin sections. Serum protein components were analyzed by SDS-PAGE gel electrophoresis coupled with 2-DE SDS-PAGE gel electrophoresis. Differentially expressed proteins were analyzed by PDQUest8.0 software and subjected to ion trap mass spectrometry equipped with a high performance liquid chromatography system. The observation of pathological section showed that heart, liver, spleen, lung, kidney exhibited pathological changes in different degree, especially in heart, liver and lung tissues. Heart muscle tissue display serious inflammatory response, heart muscle fiber compulsively expanded and filled with erythrocyte and inflammatory exudates, some heart muscle fiber ruptured, even necrosis; hepatic cell in edge of liver occur apoptosis and some hepatic cell have disintegrated, and even died; pulmonary alveoli broken and partial vein filled with blood. Serum proteins component present a significant changes between control serum and botulism in 24 h by SDS-PAGE gel electrophoresis and 2-DE-SDS-PAGE gel electrophoresis. Twenty differentially expressed protein spots were observed in 2-DE profiles, in which 14 protein spots were undetectable in serum proteome under botulism, 3 protein spots exclusively expressed in state of botulism, 3 protein spots were low-expressed in serum proteome under botulism. Fourteen proteins have been identified among 20 spots elected on two-dimensional electrophoresis gels. Crystal proteins family exclusively expressed in control group serum. Haptoglobin were low-expressed under botulism in serum protein components, however, serum amyloid A only expressed in serum sample under botulism in 24 h, which were verified by Western-blot. Identified proteins involved in energy metabolism, cellular stress response, transcription, body defense and cell proliferation. These findings represent the first report of BoNT-induced changes in serum proteome and histopathology, and reinforce the utility of applying proteomic tools to the study of system-wide biological processes in normal and botulism.

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium

PubMed Central

Silvestre, Frédéric; Linares-Casenave, Javier; Doroshov, Serge I.; Kültz, Dietmar

2012-01-01

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay / Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26°C and micro-injected with Seleno-L-Methionine to reach 8 μg g-1 selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P<0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65 kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline. PMID:20435339

Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development

PubMed Central

Zhang, Ning; Chen, Feng; Huo, Wang; Cui, Dangqun

2015-01-01

Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and six cultivar-specific protein spots including serpin, small heat shock protein, β-amylase, α-amylase inhibitor, dimeric α-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of middle and late grain development. PMID:26442048

Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda)

PubMed Central

Cortés, Alba; Sotillo, Javier; Muñoz-Antoli, Carla; Fried, Bernard; Esteban, J. Guillermo; Toledo, Rafael

2015-01-01

Background Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. Methodology/Principal Findings To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. Conclusion/Significance Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection. PMID:26390031

Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies.

PubMed

Monteiro, Antónia; Chen, Bin; Ramos, Diane M; Oliver, Jeffrey C; Tong, Xiaoling; Guo, Min; Wang, Wen-Kai; Fazzino, Lisa; Kamal, Firdous

2013-07-01

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies. Copyright © 2013 Wiley Periodicals, Inc.

From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph)

PubMed Central

Liu, Hongbin; Al Nasr, Ibrahim; Liu, Xianyong; Suo, Xun; Barta, John

2015-01-01

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3–10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima. PMID:26641262

From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph).

PubMed

El-Ashram, Saeed; Yin, Qing; Liu, Hongbin; Al Nasr, Ibrahim; Liu, Xianyong; Suo, Xun; Barta, John

2015-01-01

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3-10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima.

Proteome alteration induced by hTERT transfection of human fibroblast cells.

PubMed

Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions.

Expression profile of doublesex/male abnormal-3-related transcription factor-1 during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus.

PubMed

Nozu, Ryo; Horiguchi, Ryo; Kobayashi, Yasuhisa; Nakamura, Masaru

2015-11-01

Sex change in fish involves a dramatic transformation of gonadal tissue and a switch in gametogenesis. Doublesex/male abnormal-3-related transcription factor-1 (DMRT1), encoded by the DMRT1 gene, is involved in testicular differentiation in a wide range of vertebrates as well as in sexual differentiation and gonadal sex change. In the present study, we investigated changes in the expression of dmrt1 during artificial gonadal sex change in the three-spot wrasse, Halichoeres trimaculatus, by real-time quantitative PCR and immunolocalization, using an anti-wrasse-Dmrt1 antibody that we prepared. We found that dmrt1 expression was predominantly observed in the testes, and that Dmrt1 was expressed in Sertoli cells of testes and a few granulosa cells surrounding vitellogenic oocytes of the ovary. Additionally, the upregulation of dmrt1 expression was consistent with an increase in spermatogenic cyst quantity rather than proliferation of presumptive spermatogonia, suggesting that dmrt1 is involved in the progression of spermatogenesis during sex change. Changes in the localization of Dmrt1 during gonadal sex change further implied that Sertoli cells originate from somatic cells adjacent to gonial germ cells during testicular formation in the three-spot wrasse. © 2015 Wiley Periodicals, Inc.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

PubMed

Cal, Laura; MegÍas, Manuel; Cerdá-Reverter, José Miguel; Postlethwait, John H; Braasch, Ingo; Rotllant, Josep

2017-11-01

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts. © 2017 Wiley Periodicals, Inc.

Wound outcome in combat injuries is associated with a unique set of protein biomarkers

PubMed Central

2013-01-01

Background The ability to forecast whether a wound will heal after closure without further debridement(s), would provide substantial benefits to patients with severe extremity trauma. Methods Wound effluent is a readily available material which can be collected without disturbing healthy tissue. For analysis of potential host response biomarkers, forty four serial combat wound effluent samples from 19 patients with either healing or failing traumatic- and other combat-related wounds were examined by 2-D DIGE. Spot map patterns were correlated to eventual wound outcome (healed or wound failure) and analyzed using DeCyder 7.0 and differential proteins identified via LC-MS/MS. Results This approach identified 52 protein spots that were differentially expressed and thus represent candidate biomarkers for this clinical application. Many of these proteins are intimately involved in inflammatory and immune responses. Furthermore, discriminate analysis further refined the 52 differential protein spots to a smaller subset of which successfully differentiate between wounds that will heal and those that will fail and require further surgical intervention with greater than 83% accuracy. Conclusion These results suggest candidates for a panel of protein biomarkers that may aid traumatic wound care prognosis and treatment. We recommend that this strategy be refined, and then externally validated, in future studies of traumatic wounds. PMID:24192341

[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.

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms.

PubMed

Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Almeida, Igor C; Fernandes, Patricia M B; Zingali, Russolina B

2011-07-01

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA-Seq analysis of salinity stress-responsive transcriptome in the liver of spotted sea bass (Lateolabrax maculatus).

PubMed

Zhang, Xiaoyan; Wen, Haishen; Wang, Hailiang; Ren, Yuanyuan; Zhao, Ji; Li, Yun

2017-01-01

Salinity is one of the most prominent abiotic factors, which greatly influence reproduction, development, growth, physiological and metabolic activities of fishes. Spotted sea bass (Lateolabrax maculatus), as a euryhaline marine teleost, has extraordinary ability to deal with a wide range of salinity changes. However, this species is devoid of genomic resources, and no study has been conducted at the transcriptomic level to determine genes responsible for salinity regulation, which impedes the understanding of the fundamental mechanism conferring tolerance to salinity fluctuations. Liver, as the major metabolic organ, is the key source supplying energy for iono- and osmoregulation in fish, however, little attention has been paid to its salinity-related functions but which should not be ignored. In this study, we perform RNA-Seq analysis to identify genes involved in salinity adaptation and osmoregulation in liver of spotted sea bass, generating from the fishes exposed to low and high salinity water (5 vs 30ppt). After de novo assembly, annotation and differential gene expression analysis, a total of 455 genes were differentially expressed, including 184 up-regulated and 271 down-regulated transcripts in low salinity-acclimated fish group compared with that in high salinity-acclimated group. A number of genes with a potential role in salinity adaptation for spotted sea bass were classified into five functional categories based on the gene ontology (GO) and enrichment analysis, which include genes involved in metabolites and ion transporters, energy metabolism, signal transduction, immune response and structure reorganization. The candidate genes identified in L. maculates liver provide valuable information to explore new pathways related to fish salinity and osmotic regulation. Besides, the transcriptomic sequencing data supplies significant resources for identification of novel genes and further studying biological questions in spotted sea bass.

RNA-Seq analysis of salinity stress–responsive transcriptome in the liver of spotted sea bass (Lateolabrax maculatus)

PubMed Central

Zhang, Xiaoyan; Wen, Haishen; Wang, Hailiang; Ren, Yuanyuan; Zhao, Ji; Li, Yun

2017-01-01

Salinity is one of the most prominent abiotic factors, which greatly influence reproduction, development, growth, physiological and metabolic activities of fishes. Spotted sea bass (Lateolabrax maculatus), as a euryhaline marine teleost, has extraordinary ability to deal with a wide range of salinity changes. However, this species is devoid of genomic resources, and no study has been conducted at the transcriptomic level to determine genes responsible for salinity regulation, which impedes the understanding of the fundamental mechanism conferring tolerance to salinity fluctuations. Liver, as the major metabolic organ, is the key source supplying energy for iono- and osmoregulation in fish, however, little attention has been paid to its salinity-related functions but which should not be ignored. In this study, we perform RNA-Seq analysis to identify genes involved in salinity adaptation and osmoregulation in liver of spotted sea bass, generating from the fishes exposed to low and high salinity water (5 vs 30ppt). After de novo assembly, annotation and differential gene expression analysis, a total of 455 genes were differentially expressed, including 184 up-regulated and 271 down-regulated transcripts in low salinity-acclimated fish group compared with that in high salinity-acclimated group. A number of genes with a potential role in salinity adaptation for spotted sea bass were classified into five functional categories based on the gene ontology (GO) and enrichment analysis, which include genes involved in metabolites and ion transporters, energy metabolism, signal transduction, immune response and structure reorganization. The candidate genes identified in L. maculates liver provide valuable information to explore new pathways related to fish salinity and osmotic regulation. Besides, the transcriptomic sequencing data supplies significant resources for identification of novel genes and further studying biological questions in spotted sea bass. PMID:28253338

Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain.

PubMed

Monoyios, Andreas; Patzl, Martina; Schlosser, Sarah; Hess, Michael; Bilic, Ivana

2018-02-01

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P < 0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

EPA Science Inventory

Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

Proteomic analysis of Aedes aegypti midgut during post-embryonic development and of the female mosquitoes fed different diets.

PubMed

Fernandes, Kenner Morais; de Magalhães-Júnior, Marcos Jorge; Baracat-Pereira, Maria Cristina; Martins, Gustavo Ferreira

2016-12-01

In this work we analyzed protein expression in the Aedes aegypti midgut during the larval (fourth instar, L4), pupal, and adult stages [including newly emerged (NE), sugar-fed (SF) and blood-fed (BF) females]. Two-dimensional electrophoresis showed 13 spots in the midgut of larvae, 95 in the midgut of pupae, 90 in the midgut of NE, and 76 in the midgut of SF or BF females. In the larval midguts, high serpin expression was noted, while in the pupae, protein abundance was lower than in the NE, SF, and BF females. The spots related to proteins linked to energy production, protein metabolism, signaling, and transport were highly expressed in the NE stage, while spots related proteins involved in translation were abundant in SF and BF females. The differential abundance of proteins in the midgut of A. aegypti at different developmental stages supports the necessity for midgut development during immature stage followed by the necessity of proteins related to digestion in adults. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Spotting and validation of a genome wide oligonucleotide chip with duplicate measurement of each gene

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

Thomassen, Mads; Skov, Vibe; Eiriksdottir, Freyja

2006-06-16

The quality of DNA microarray based gene expression data relies on the reproducibility of several steps in a microarray experiment. We have developed a spotted genome wide microarray chip with oligonucleotides printed in duplicate in order to minimise undesirable biases, thereby optimising detection of true differential expression. The validation study design consisted of an assessment of the microarray chip performance using the MessageAmp and FairPlay labelling kits. Intraclass correlation coefficient (ICC) was used to demonstrate that MessageAmp was significantly more reproducible than FairPlay. Further examinations with MessageAmp revealed the applicability of the system. The linear range of the chips wasmore » three orders of magnitude, the precision was high, as 95% of measurements deviated less than 1.24-fold from the expected value, and the coefficient of variation for relative expression was 13.6%. Relative quantitation was more reproducible than absolute quantitation and substantial reduction of variance was attained with duplicate spotting. An analysis of variance (ANOVA) demonstrated no significant day-to-day variation.« less

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

PubMed

Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2014-07-01

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative Proteomics of Tumor and Paired Normal Breast Tissue Highlights Potential Biomarkers in Breast Cancer.

PubMed

Da Costa, Gustavo Góes; Gomig, Talita Helen Bombardelli; Kaviski, Rodrigo; Santos Sousa, Karla; Kukolj, Caroline; De Lima, Rubens Silveira; De Andrade Urban, Cicero; Cavalli, Iglenir J; Ribeiro, Enilze M S F

2015-01-01

Breast cancer is the most common type of cancer among women worldwide, and about 57,000 new cases are expected for the Brazilian population in 2015. Elucidation of protein expression and modification is essential for the biological understanding, early diagnosis and therapeutics of breast cancer. The main objectives of the study are comparison between the proteome of tumor and paired non-tumor breast cancer tissues, describing all identified proteins, highlighting the ones most differentially expressed and comparing the data with existing literature. The five paired samples from patients with invasive ductal carcinoma were analyzed by 2-DE and MS. We collected 161 identified spots corresponding to 110 distinct proteins. Forty-three differentially-expressed spots were common to at least two samples, and the ten proteins with the highest-fold changes were CASPE, ENOG, TPM1, CAPG, VIME, TPM3, TRFE, PDIA6, WDR61 and PDIA3. Metabolic enzymes and proteins with binding functions were the most representative functional classes of proteins with increased and decreased expression in tumor tissue respectively. Taking the fold change as a parameter, we point to future targets to be studied by functional methods in a search for biomarkers for initiation and progress of breast cancer. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Effect of nano-sized, elemental selenium supplement on the proteome of chicken liver.

PubMed

Gulyas, G; Csosz, E; Prokisch, J; Javor, A; Mezes, M; Erdelyi, M; Balogh, K; Janaky, T; Szabo, Z; Simon, A; Czegledi, L

2017-06-01

The nano-sized (100-500 nm) selenium has higher bioavailability and relatively lower toxicity compared to other selenium forms. The objective of the present study was to compare liver proteome profiles of broiler chicken fed with control diet without Se supplementation and diet supplemented with nano-Se with 4.25 mg/kg DM. Differential proteome analyses were performed by two-dimensional gel electrophoresis (2D-PAGE) followed by tryptic digestion and protein identification by liquid chromatography-mass spectrometry (LC-MS). Seven hundred and eight spots were detected, and 18 protein spots showed significant difference in their intensity (p < 0.05) between the two groups. In response to nano-Se supplementation, the expression of 8 proteins was higher, and 5 proteins were lower in nano-Se supplemented group compared to control group. The functions of the differentially expressed proteins indicate that the high dose of selenium supplementation induced a dietary stress. Selenium supplementation may influence the metabolism of fatty acids and carbohydrates and antioxidant system, and increase the quantity of cytoskeletal actin and the expression of actin regulatory protein as well. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer).

PubMed

Truong, D-H; Bauwens, J; Delaplace, P; Mazzucchelli, G; Lognay, G; Francis, F

2015-11-01

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2-DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI-TOF-MS and LC-ESI-MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

Identification of highly expressed host microRNAs that respond to white spot syndrome virus infection in the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

PubMed

Zeng, D G; Chen, X L; Xie, D X; Zhao, Y Z; Yang, Q; Wang, H; Li, Y M; Chen, X H

2015-05-11

MicroRNAs (miRNAs) are known to play an important role in regulating both adaptive and innate immunity. Pacific white shrimp (Litopenaeus vannamei) is the most widely farmed crustacean species in the world. However, little is known about the role miRNAs play in shrimp immunity. To understand the impact of viral infection on miRNA expression in shrimp, we used high-throughput sequencing technology to sequence two small RNA libraries prepared from L. vannamei under normal and white spot syndrome virus (WSSV) challenged conditions. Approximately 19,312,189 and 39,763,551 raw reads corresponding to 17,414,787 and 28,633,379 high-quality mappable reads were obtained from the two libraries, respectively. Twelve conserved miRNAs and one novel miRNA that were highly expressed (>100 RPM) in L. vannamei were identified. Of the identified miRNAs, 8 were differentially expressed in response to the virus infection, of which 1 was upregulated and 7 were downregulated. The prediction of miRNA targets showed that the target genes of the differentially expressed miRNAs were related to immunity, apoptosis, and development functions. Our study provides the first characterization of L. vannamei miRNAs in response to WSSV infection, which will help to reveal the roles of miRNAs in the antiviral mechanisms of shrimp.

Proteomic analysis of muscle between hybrid abalone and parental lines Haliotis gigantea Reeve and Haliotis discus hannai Ino

PubMed Central

Di, G; Luo, X; You, W; Zhao, J; Kong, X; Ke, C

2015-01-01

To understand the potential molecular mechanism of heterosis, protein expression patterns were compared from hybrids of Haliotis gigantea (G) and Haliotis discus hannai (D) using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analyses. Expression differences were observed in muscle samples from the four groups with 673±21.0 stained spots for H. discus hannai ♀ × H. discus hannai ♂ (DD), 692±25.6 for H. gigantea ♀ × H. gigantea ♂ (GG), 679±16.2 for H. discus hannai ♀ × H. gigantea ♂ (DG) (F1 hybrid) and 700±19 for H. gigantea ♀ × H. discus hannai ♂ (GD) (F1 hybrid). Different 2-DE image muscle protein spots had a mirrored relationship between purebreds and the F1 hybrid, suggesting that all stained spots in F1 hybrid muscle were on 2-DEs from parents. DD and DG clustered together first, and then clustered with GD, whereas the distance of DD and GG was maximal according to hierarchical cluster analysis. We identified 136 differentially expressed protein spots involved in major biological processes, including energy metabolism and stress response. Most energy metabolism proteins were additive, and stress-induced proteins displayed additivity or over-dominance. In these 136 identified protein spots, hybrid offspring with additivity or over-dominance accounted for 68.38%. Data show that a proteomic approach can provide functional prediction of abalone interspecific hybridization. PMID:25669609

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress.

PubMed

Mohammadi, Payam Pour; Moieni, Ahmad; Komatsu, Setsuko

2012-11-01

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H(+) ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.

Insights from ecological niche modeling on the taxonomic distinction and niche differentiation between the black-spotted and red-spotted tokay geckoes (Gekko gecko).

PubMed

Zhang, Yueyun; Chen, Chongtao; Li, Li; Zhao, Chengjian; Chen, Weicai; Huang, Yong

2014-09-01

The black-spotted tokay and the red-spotted tokay are morphologically distinct and have largely allopatric distributions. The black-spotted tokay is characterized by a small body size and dark skin with sundry spots, while the red-spotted tokay has a relatively large body size and red spots. Based on morphological, karyotypic, genetic, and distribution differences, recent studies suggested their species status; however, their classifications remain controversial, and additional data such as ecological niches are necessary to establish firm hypotheses regarding their taxonomic status. We reconstructed their ecological niches models using climatic and geographic data. We then performed niche similarity tests (niche identity and background tests) and point-based analyses to explore whether ecological differentiation has occurred, and whether such differences are sufficient to explain the maintenance of their separate segments of environmental ranges. We found that both niche models of the black- and the red-spotted tokay had a good fit and a robust performance, as indicated by the high area under the curve (AUC) values ("black" = 0.982, SD = ± 0.002, "red" = 0.966 ± 0.02). Significant ecological differentiation across the entire geographic range was found, indicating that the involvement of ecological differentiation is important for species differentiation. Divergence along the environmental axes is highly associated with climatic conditions, with isothermality being important for the "black" form, while temperature seasonality, precipitation of warmest quarter, and annual temperature range together being important for the "red" form. These factors are likely important factors in niche differentiation between the two forms, which result in morphological replacement. Overall, beside morphological and genetic differentiation information, our results contribute to additional insights into taxonomic distinction and niche differentiation between the black- and the red-spotted tokay.

THE INFORMATION CONTENT IN ANALYTIC SPOT MODELS OF BROADBAND PRECISION LIGHT CURVES

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

Walkowicz, Lucianne M.; Basri, Gibor; Valenti, Jeff A.

2013-04-01

We present the results of numerical experiments to assess degeneracies in light curve models of starspots. Using synthetic light curves generated with the Cheetah starspot modeling code, we explore the extent to which photometric light curves constrain spot model parameters, including spot latitudes and stellar inclination. We also investigate the effects of spot parameters and differential rotation on one's ability to correctly recover rotation periods and differential rotation in the Kepler light curves. We confirm that in the absence of additional constraints on the stellar inclination, such as spectroscopic measurements of vsin i or occultations of starspots by planetary transits,more » the spot latitude and stellar inclination are difficult to determine uniquely from the photometry alone. We find that for models with no differential rotation, spots that appear on opposite hemispheres of the star may cause one to interpret the rotation period to be half of the true period. When differential rotation is included, the changing longitude separation between spots breaks the symmetry of the hemispheres and the correct rotation period is more likely to be found. The dominant period found via periodogram analysis is typically that of the largest spot. Even when multiple spots with periods representative of the star's differential rotation exist, if one spot dominates the light curve the signal of differential rotation may not be detectable from the periodogram alone. Starspot modeling is applicable to stars with a wider range of rotation rates than other surface imaging techniques (such as Doppler imaging), allows subtle signatures of differential rotation to be measured, and may provide valuable information on the distribution of stellar spots. However, given the inherent degeneracies and uncertainty present in starspot models, caution should be exercised in their interpretation.« less

The Information Content in Analytic Spot Models of Broadband Precision Light Curves

NASA Astrophysics Data System (ADS)

Walkowicz, Lucianne M.; Basri, Gibor; Valenti, Jeff A.

2013-04-01

We present the results of numerical experiments to assess degeneracies in light curve models of starspots. Using synthetic light curves generated with the Cheetah starspot modeling code, we explore the extent to which photometric light curves constrain spot model parameters, including spot latitudes and stellar inclination. We also investigate the effects of spot parameters and differential rotation on one's ability to correctly recover rotation periods and differential rotation in the Kepler light curves. We confirm that in the absence of additional constraints on the stellar inclination, such as spectroscopic measurements of vsin i or occultations of starspots by planetary transits, the spot latitude and stellar inclination are difficult to determine uniquely from the photometry alone. We find that for models with no differential rotation, spots that appear on opposite hemispheres of the star may cause one to interpret the rotation period to be half of the true period. When differential rotation is included, the changing longitude separation between spots breaks the symmetry of the hemispheres and the correct rotation period is more likely to be found. The dominant period found via periodogram analysis is typically that of the largest spot. Even when multiple spots with periods representative of the star's differential rotation exist, if one spot dominates the light curve the signal of differential rotation may not be detectable from the periodogram alone. Starspot modeling is applicable to stars with a wider range of rotation rates than other surface imaging techniques (such as Doppler imaging), allows subtle signatures of differential rotation to be measured, and may provide valuable information on the distribution of stellar spots. However, given the inherent degeneracies and uncertainty present in starspot models, caution should be exercised in their interpretation.

Proteomics Analysis of Tissue Samples Reveals Changes in Mitochondrial Protein Levels in Parathyroid Hyperplasia over Adenoma

PubMed Central

AKPINAR, GURLER; KASAP, MURAT; CANTURK, NUH ZAFER; ZULFIGAROVA, MEHIN; ISLEK, EYLÜL ECE; GULER, SERTAC ATA; SIMSEK, TURGAY; CANTURK, ZEYNEP

2017-01-01

Background/Aim: To unveil the pathophysiology of primary hyperparathyroidism, molecular details of parathyroid hyperplasia and adenoma have to be revealed. Such details will provide the tools necessary for differentiation of these two look-alike diseases. Therefore, in the present study, a comparative proteomic study using postoperative tissue samples from the parathyroid adenoma and parathyroid hyperplasia patients was performed. Materials and Methods: Protein extracts were prepared from tissue samples (n=8 per group). Protein pools were created for each group and subjected to DIGE and conventional 2DE. Following image analysis, spots representing the differentially regulated proteins were excised from the and used for identification via MALDI-TOF/TOF analysis. Results: The identities of 40 differentially-expressed proteins were revealed. Fourteen of these proteins were over-expressed in the hyperplasia while 26 of them were over-expressed in the adenoma. Conclusion: Most proteins found to be over-expressed in the hyperplasia samples were mitochondrial, underlying the importance of the mitochondrial activity as a potential biomarker for differentiation of parathyroid hyperplasia from adenoma. PMID:28446534

Time-series photometric spot modeling. 2: Fifteen years of photometry of the bright RS CVn binary HR 7275

NASA Technical Reports Server (NTRS)

Strassmeier, K. G.; Hall, D. S.; Henry, G. W.

1994-01-01

We present a time-dependent spot modeling analysis of 15 consecutive years of V-band photometry of the long-period (P(sub orb) = 28.6 days) RS CVn binary HR 7275. This baseline in time is one of the longest, uninterrupted intervals a spotted star has been observed. The spot modeling analysis yields a total of 20 different spots throughout the time span of our observations. The distribution of the observed spot migration rates is consistent with solar-type differential rotation and suggests a lower limit of the differential-rotation coefficient of 0.022 +/-0.004. The observed, maximum lifetime of a single spot (or spot group) is 4.5 years, the minimum lifetime is approximately one year, but an average spot lives for 2.2 years. If we assume that the mechanical shear by differential rotation sets the upper limit to the spot lifetime, the observed maximum lifetime in turn sets an upper limit to the differential-rotation coefficient, namely 0.04 +/- 0.01. This would be differential rotation just 5 to 8 times less than the solar value and one of the strongest among active binaries. We found no conclusive evidence for the existence of a periodic phenomenon that could be attributed to a stellar magnetic cycle.

Proteomic study of the yeast Rhodotorula mucilaginosa RCL-11 under copper stress.

PubMed

Irazusta, Verónica; Estévez, Cristina; Amoroso, María Julia; de Figueroa, Lucía I C

2012-06-01

In order to understand the mechanism involved in Rhodotorula mucilaginosa RCL-11 resistance to copper a proteomic study was conducted. Atomic absorption spectroscopy showed that the copper concentration in the medium decreased from 0.5 to 0.19 mM 48 h after inoculation of the yeast. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed expression of differential bands between cells with and without copper. In order to study this difference, two-dimensional electrophoresis of R. mucilaginosa RCL-11 exposed to Cu for 16, 24, and 48 h was carried out. Identification of differentially expressed proteins was performed by MALDI-TOF/TOF. Ten of the 16 spots identified belonged to heat shock proteins. Superoxide dismutase, methionine synthase and beta-glucosidase were also found over-expressed at high copper concentrations. The results obtained in the present work show that when R. mucilaginosa RCL-11 is exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, are expressed.

Differential proteomics analysis of Frankliniella occidentalis immune response after infection with Tomato spotted wilt virus (Tospovirus).

PubMed

Ogada, Pamella Akoth; Kiirika, Leonard Muriithi; Lorenz, Christin; Senkler, Jennifer; Braun, Hans-Peter; Poehling, Hans-Michael

2017-02-01

Tomato spotted wilt virus (TSWV) is mainly vectored by Frankliniella occidentalis Pergande, and it potentially activates the vector's immune response. However, molecular background of the altered immune response is not clearly understood. Therefore, using a proteomic approach, we investigated the immune pathways that are activated in F. occidentalis larvae after 24 h exposure to TSWV. Two-dimensional isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-IEF/SDS/PAGE) combined with mass spectrometry (MS), were used to identify proteins that were differentially expressed upon viral infection. High numbers of proteins were abundantly expressed in F. occidentalis exposed to TSWV (73%) compared to the non-exposed (27%), with the majority functionally linked to the innate immune system such as: signaling, stress response, defense response, translation, cellular lipids and nucleotide metabolism. Key proteins included: 70 kDa heat shock proteins, Ubiquitin and Dermcidin, among others, indicative of a responsive pattern of the vector's innate immune system to viral infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Situ Hot-Spot Assembly as a General Strategy for Probing Single Biomolecules.

PubMed

Liu, Huiqiao; Li, Qiang; Li, Mingmin; Ma, Sisi; Liu, Dingbin

2017-05-02

Single-molecule detection using surface-enhanced Raman spectroscopy (SERS) has attracted increasing attention in chemical and biomedical analysis. However, it remains a major challenge to probe single biomolecules by means of SERS hot spots owing to the small volume of hot spots and their random distribution on substrates. We here report an in situ hot-spot assembly method as a general strategy for probing single biomolecules. As a proof-of-concept, this proposed strategy was successfully used for the detection of single microRNA-21 (miRNA-21, a potential cancer biomarker) at the single-cell level, showing great capability in differentiating the expression of miRNA-21 in single cancer cells from normal cells. This approach was further extended to single-protein detection. The versatility of the strategy opens an exciting avenue for single-molecule detection of biomarkers of interest and thus holds great promise in a variety of biological and biomedical applications.

Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors.

PubMed

Zhao, Guozhong; Hou, Lihua; Yao, Yunping; Wang, Chunling; Cao, Xiaohong

2012-07-16

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

PubMed Central

Sioson, Allan A; Mane, Shrinivasrao P; Li, Pinghua; Sha, Wei; Heath, Lenwood S; Bohnert, Hans J; Grene, Ruth

2006-01-01

Background Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data. Results The Expresso analysis using MIV data consistently identifies more genes as differentially expressed, when compared to Expresso analysis with IIV data. The typical TM4 normalization and filtering pipeline corrects systematic intensity-specific bias on a per microarray basis. Subsequent statistical analysis with Expresso or a TM4 t-test can effectively identify differentially expressed genes. The best agreement with qRT-PCR data is obtained through the use of Expresso analysis and MIV data. Conclusion The results of this research are of practical value to biologists who analyze microarray data sets. The TM4 normalization and filtering pipeline corrects microarray-specific systematic bias and complements the normalization stage in Expresso analysis. The results of Expresso using MIV data have the best agreement with qRT-PCR results. In one experiment, MIV is a better choice than IIV as input to data normalization and statistical analysis methods, as it yields as greater number of statistically significant differentially expressed genes; TM4 does not support the choice of MIV input data. Overall, the more flexible and extensive statistical models of Expresso achieve more accurate analytical results, when judged by the yardstick of qRT-PCR data, in the context of an experimental design of modest complexity. PMID:16626497

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

PubMed Central

Hayashi, Gohei; Moro, Carlo F; Rohila, Jai Singh; Shibato, Junko; Kubo, Akihiro; Imanaka, Tetsuji; Kimura, Shinzo; Ozawa, Shoji; Fukutani, Satoshi; Endo, Satoru; Ichikawa, Katsuki; Agrawal, Ganesh Kumar; Shioda, Seiji; Hori, Motohide; Fukumoto, Manabu; Rakwal, Randeep

2015-01-01

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves. PMID:26451896

Proteomic analysis of cerebrospinal fluid in canine cervical spondylomyelopathy.

PubMed

Martin-Vaquero, Paula; da Costa, Ronaldo C; Allen, Matthew J; Moore, Sarah A; Keirsey, Jeremy K; Green, Kari B

2015-05-01

Prospective study. To identify proteins with differential expression in the cerebrospinal fluid (CSF) from 15 clinically normal (control) dogs and 15 dogs with cervical spondylomyelopathy (CSM). Canine CSM is a spontaneous, chronic, compressive cervical myelopathy similar to human cervical spondylotic myelopathy. There is a limited knowledge of the molecular mechanisms underlying these conditions. Differentially expressed CSF proteins may contribute with novel information about the disease pathogenesis in both dogs and humans. Protein separation was performed with 2-dimensional electrophoresis. A Student t test was used to detect significant differences between groups (P < 0.05). Three comparisons were made: (1) control versus CSM-affected dogs, (2) control versus non-corticosteroid-treated CSM-affected dogs, and (3) non-corticosteroid-treated CSM-affected versus corticosteroid-treated CSM-affected dogs. Protein spots exhibiting at least a statistically significant 1.25-fold change between groups were selected for subsequent identification with capillary-liquid chromatography tandem mass spectrometry. A total of 96 spots had a significant average change of at least 1.25-fold in 1 of the 3 comparisons. Compared with the CSF of control dogs, CSM-affected dogs demonstrated increased CSF expression of 8 proteins including vitamin D-binding protein, gelsolin, creatine kinase B-type, angiotensinogen, α-2-HS-glycoprotein, SPARC (secreted protein, acidic, rich in cysteine), calsyntenin-1, and complement C3, and decreased expression of pigment epithelium-derived factor, prostaglandin-H2 D-isomerase, apolipoprotein E, and clusterin. In the CSF of CSM-affected dogs, corticosteroid treatment increased the expression of haptoglobin, transthyretin isoform 2, cystatin C-like, apolipoprotein E, and clusterin, and decreased the expression of angiotensinogen, α-2-HS-glycoprotein, and gelsolin. Many of the differentially expressed proteins are associated with damaged neural tissue, bone turnover, and/or compromised blood-spinal cord barrier. The knowledge of the protein changes that occur in CSM and upon corticosteroid treatment of CSM-affected patients will aid in further understanding the pathomechanisms underlying this disease. N/A.

ADGO: analysis of differentially expressed gene sets using composite GO annotation.

PubMed

Nam, Dougu; Kim, Sang-Bae; Kim, Seon-Kyu; Yang, Sungjin; Kim, Seon-Young; Chu, In-Sun

2006-09-15

Genes are typically expressed in modular manners in biological processes. Recent studies reflect such features in analyzing gene expression patterns by directly scoring gene sets. Gene annotations have been used to define the gene sets, which have served to reveal specific biological themes from expression data. However, current annotations have limited analytical power, because they are classified by single categories providing only unary information for the gene sets. Here we propose a method for discovering composite biological themes from expression data. We intersected two annotated gene sets from different categories of Gene Ontology (GO). We then scored the expression changes of all the single and intersected sets. In this way, we were able to uncover, for example, a gene set with the molecular function F and the cellular component C that showed significant expression change, while the changes in individual gene sets were not significant. We provided an exemplary analysis for HIV-1 immune response. In addition, we tested the method on 20 public datasets where we found many 'filtered' composite terms the number of which reached approximately 34% (a strong criterion, 5% significance) of the number of significant unary terms on average. By using composite annotation, we can derive new and improved information about disease and biological processes from expression data. We provide a web application (ADGO: http://array.kobic.re.kr/ADGO) for the analysis of differentially expressed gene sets with composite GO annotations. The user can analyze Affymetrix and dual channel array (spotted cDNA and spotted oligo microarray) data for four species: human, mouse, rat and yeast. chu@kribb.re.kr http://array.kobic.re.kr/ADGO.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

PubMed

Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

2013-05-01

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

PubMed Central

2013-01-01

Background Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Results Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. Conclusions SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights. PMID:23635033

Cochliobolus lunatus down-regulates proteome at late stage of colonization and transiently alters StNPR1 expression in Solanum tuberosum L.

PubMed

Louis, Bengyella; Waikhom, Sayanika D; Jose, Robinson C; Goyari, Sailendra; Bhardwaj, Pardeep Kumar; Talukdar, Narayan C; Roy, Pranab

2017-03-01

Cochliobolus lunatus abundantly produces four-celled conidia at high temperatures (>30 °C) and under suitable conditions; the fungus colonizes potato (Solanum tuberosum L.) cultivars by adopting different invasion strategies at the microscopic level. Long-lasting defence during infection requires an upsurge in proteome changes particularly pathogenesis-related proteins chiefly under the control of nonexpresser of pathogenesis-related proteins. In order to gain molecular insights, we profiled the changes in proteome and potato nonexpresser of pathogenesis-related proteins (StNPR1) during the infection process. It is found that C. lunatus significantly (P < 0.05) suppressed the host functional proteome by 96 h after infection (hai), principally, affecting the expression of ribulose bisphosphate carboxylase enzyme, plastidic aldolase enzyme, alcohol dehydrogenase 2 and photosystem II protein prior to the formation of brown-to-black leaf spot disease. Strongest host response was observed at 24 hai hallmarked by 307 differentially expressed peptide spots concurring with the active phase of production of penetrating hyphae. Additionally, C. lunatus differentially down-regulated StNPR1 transcript by 8.19 fold by 24 hai. This study is the first to elucidate that C. lunatus transiently down-regulates the expression of StNPR1 at the onset of infection, and as a whole, infection negatively affects the expression of proteome components involved in photosynthesis, carbon fixation and light assimilation. This study contributes towards better understanding of the mechanism underlining the invasion strategies of C. lunatus.

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.

The Effect of Selenium Enrichment on Baker s Yeast Proteome

PubMed Central

El-Bayoumy, Karam; Das, Arunangshu; Russell, Stephen; Wolfe, Steven; Jordan, Rick; Renganathan, Kutralanathan; Loughran, Thomas P.; Somiari, Richard

2011-01-01

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE) – tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p≤0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially were verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY. PMID:22067702

Proteome Analysis of Date Palm (Phoenix dactylifera L.) under Severe Drought and Salt Stress.

PubMed

El Rabey, Haddad A; Al-Malki, Abdulrahman L; Abulnaja, Khalid O

2016-01-01

Date palm cultivars differently tolerate salinity and drought stress. This study was carried out to study the response of date palm to severe salinity and drought based on leaf proteome analysis. Eighteen-month-old date palm plants were subjected to severe salt (48 g/L NaCl) and drought (82.5 g/L PEG or no irrigation) conditions for one month. Using a protein 2D electrophoresis method, 55 protein spots were analyzed using mass spectrometry. ATP synthase CF1 alpha chains were significantly upregulated under all three stress conditions. Changes in the abundance of RubisCO activase and one of the RubisCO fragments were significant in the same spots only for salt stress and drought stress with no irrigation, and oxygen-evolving enhancer protein 2 was changed in different spots. Transketolase was significantly changed only in drought stress with PEG. The expression of salt and drought stress genes of the chosen protein spots was either overexpressed or downexpressed as revealed by the high or low protein abundance, respectively. In addition, all drought tolerance genes due to no irrigation were downregulated. In conclusion, the proteome analysis of date palm under salinity and drought conditions indicated that both salinity and drought tolerance genes were differentially expressed resulting in high or low protein abundance of the chosen protein spots as a result of exposure to drought and salinity stress condition.

Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation.

PubMed

Shojaei Saadi, Habib A; van Riemsdijk, Evine; Dance, Alysha L; Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

2013-04-26

The objective was to investigate expression patterns of proteins in pyriform sperm, a common morphological abnormality in bull sperm. Ejaculates were collected from sexually mature Holstein bulls (n=3) twice weekly for 10 weeks (pre-thermal insult samples). Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during week 2. Total sperm proteins were extracted from pre- and post-thermal insult sperm samples and subjected to two-dimensional gel electrophoresis. Among the protein spots detected, 131 spots were significantly expressed (False Detection Rate <0.01) with ≥ 2 fold changes between normal and pyriform sperm. Among them, 25 spots with ≥ 4 fold difference in expression patterns were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins regulating antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the molecular basis of functional deficiencies in sperm with specific morphological abnormalities, comparing normal versus morphologically abnormal sperm appeared to be a suitable experimental model for identifying important sperm functional proteins. To our knowledge, this study is the first report on differential expression of proteins in pyriform bovine sperm versus morphologically normal sperm. We report that expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins which regulate antioxidant activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the molecular basis of functional deficiencies in sperm with specific morphological abnormalities, our results suggest that comparing normal versus morphologically abnormal sperm appeared to be a suitable experimental model for identifying important sperm functional proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach.

PubMed

Hammad, Ghania; Legrain, Yona; Touat-Hamici, Zahia; Duhieu, Stéphane; Cornu, David; Bulteau, Anne-Laure; Chavatte, Laurent

2018-01-20

Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented), moderate (control), or low (depleted) concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE) to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value < 0.05) to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i) spots varying between young and presenescent cells, (ii) spots varying in response to selenium concentration in young cells, and (iii) spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between selenium, selenoproteins, and replicative senescence.

New Fks hot spot for acquired echinocandin resistance in Saccharomyces cerevisiae and its contribution to intrinsic resistance of Scedosporium species.

PubMed

Johnson, Michael E; Katiyar, Santosh K; Edlind, Thomas D

2011-08-01

Echinocandins represent a new antifungal group with potent activity against Candida species. These lipopeptides inhibit the synthesis of β-1,3-glucan, the major cell wall polysaccharide. Acquired resistance or reduced echinocandin susceptibility (RES) is rare and associated with mutations in two "hot spot" regions of Fks1 or Fks2, the probable β-1,3-glucan synthases. In contrast, many fungi demonstrate intrinsic RES for reasons that remain unclear. We are using Saccharomyces cerevisiae to understand the basis for RES by modeling echinocandin-Fks interaction. Previously characterized mutations confer cross-RES; we screened for mutations conferring differential RES, implying direct interaction of that Fks residue with a variable echinocandin side chain. One mutant (in an fks1Δ background) exhibited ≥16-fold micafungin and anidulafungin versus caspofungin RES. Sequencing identified a novel Fks2 mutation, W714L/Y715N. Equivalent W695L/Y696N and related W695L/F/C mutations in Fks1 generated by site-directed mutagenesis and the isolation of a W695L-equivalent mutation in Candida glabrata confirmed the role of the new "hot spot 3" in RES. Further mutagenesis expanded hot spot 3 to Fks1 residues 690 to 700, yielding phenotypes ranging from cross-RES to differential hypersusceptibility. Fks1 sequences from intrinsically RES Scedosporium species revealed W695F-equivalent substitutions; Fks1 hybrids expressing Scedosporium prolificans hot spot 3 confirmed that this substitution imparts RES.

Global transcriptional analysis of psoriatic skin and blood confirms known disease-associated pathways and highlights novel genomic "hot spots" for differentially expressed genes.

PubMed

Coda, Alvin B; Icen, Murat; Smith, Jason R; Sinha, Animesh A

2012-07-01

There are major gaps in our knowledge regarding the exact mechanisms and genetic basis of psoriasis. To investigate the pathogenesis of psoriasis, gene expression in 10 skin (5 lesional, 5 nonlesional) and 11 blood (6 psoriatic, 5 nonpsoriatic) samples were examined using Affymetrix HG-U95A microarrays. We detected 535 (425 upregulated, 110 downregulated) DEGs in lesional skin at 1% false discovery rate (FDR). Combining nine microarray studies comparing lesional and nonlesional psoriatic skin, 34.5% of dysregulated genes were overlapped in multiple studies. We further identified 20 skin and 2 blood associated transcriptional "hot spots" at specified genomic locations. At 5% FDR, 11.8% skin and 10.4% blood DEGs in our study mapped to one of the 12 PSORS loci. DEGs that overlap with PSORS loci may offer prioritized targets for downstream genetic fine mapping studies. Novel DEG "hot spots" may provide new targets for defining susceptibility loci in future studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cells.

PubMed

Ha, Moon Kyung; Chung, Kee Yang; Lee, Ju Hee; Bang, Dongsik; Park, Yoon Kee; Lee, Kwang Hoon

2004-09-01

Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3-10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14-15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging.

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

Proteomic analysis of mature soybean seeds from the Chernobyl area suggests plant adaptation to the contaminated environment.

PubMed

Danchenko, Maksym; Skultety, Ludovit; Rashydov, Namik M; Berezhna, Valentyna V; Mátel, L'ubomír; Salaj, Terézia; Pret'ová, Anna; Hajduch, Martin

2009-06-01

The explosion in one of the four reactors of the Chernobyl Nuclear Power Plant (CNPP, Chernobyl) caused the worst nuclear environmental disaster ever seen. Currently, 23 years after the accident, the soil in the close vicinity of CNPP is still significantly contaminated with long-living radioisotopes, such as (137)Cs. Despite this contamination, the plants growing in Chernobyl area were able to adapt to the radioactivity, and survive. The aim of this study was to investigate plant adaptation mechanisms toward permanently increased level of radiation using a quantitative high-throughput proteomics approach. Soybeans of a local variety (Soniachna) were sown in contaminated and control fields in the Chernobyl region. Mature seeds were harvested and the extracted proteins were subjected to two-dimensional gel electrophoresis (2-DE). In total, 9.2% of 698 quantified protein spots on 2-D gel were found to be differentially expressed with a p-value

Nitrogen-Deficiency Stress Induces Protein Expression Differentially in Low-N Tolerant and Low-N Sensitive Maize Genotypes

PubMed Central

Nazir, Muslima; Pandey, Renu; Siddiqi, Tariq O.; Ibrahim, Mohamed M.; Qureshi, Mohammad I.; Abraham, Gerard; Vengavasi, Krishnapriya; Ahmad, Altaf

2016-01-01

Nitrogen (N) is essential for proper plant growth and its application has proven to be critical for agricultural produce. However, for unavoidable economic and environmental problems associated with excessive use of N-fertilizers, it is an urgent demand to manage application of fertilizers. Improving the N-use efficiency (NUE) of crop plants to sustain productivity even at low N levels is the possible solution. In the present investigation, contrasting low-N sensitive (HM-4) and low-N tolerant (PEHM-2) genotypes were identified and used for comparative proteome-profiling of leaves under optimum and low N as well as restoration of low N on 3rd (NR3) and 5th (NR5) days after re-supplying N. The analysis of differential expression pattern of proteins was performed by 2-D gel electrophoresis. Significant variations in the expression of proteins were observed under low N, which were genotype specific. In the leaf proteome, 25 spots were influenced by N treatment and four spots were different between the two genotypes. Most of the proteins that were differentially accumulated in response to N level and were involved in photosynthesis and metabolism, affirming the relationship between N and carbon metabolism. In addition to this, greater intensity of some defense proteins in the low N tolerant genotype was found that may have a possible role in imparting it tolerance under N starvation conditions. The new insights generated on maize proteome in response to N-starvation and restoration would be useful toward improvement of NUE in maize. PMID:27047497

Proteome analysis of the cardiac and pericardial tissue of Biomphalaria tenagophila populations susceptible and resistant to Schistosoma mansoni infection.

PubMed

Jannotti-Passos, Liana K; Andrade, Hélida M; Caldeira, Roberta L; Romanha, Alvaro J; Murta, Silvane M F; Chapeaurouge, Donat A; Perales, Jonas; Coelho, Paulo Marcos Z; Carvalho, Omar S

2008-03-01

For a better comprehension of the parasite-host interaction, proteins expressed by the cardiac and pericardial tissues were compared between susceptible (Cabo Frio) and resistant (Taim) Biomphalaria tenagophila populations, challenged (c) and non-challenged (nc) with Schistosoma mansoni. Proteins were separated by two-dimensional gel electrophoresis (2DE) and stained with Coomassie blue. A total of 146 and 135 spots were observed in Cabo Frio (CFnc) and in Taim (Tnc) non-challenged populations, respectively, whereas 153 spots were detected in both Cabo Frio (CFc) and Taim (Tc) challenged populations. Regarding comparisons between CFnc and CFc, the numbers of exclusive spots obtained were one and nine, respectively, whereas Tnc yielded 17 and Tc eight exclusive spots. By comparing the total of spots in CF (nc+c) with T (nc+c) populations, we obtained: four exclusive spots for CFc; zero for CFnc; four for Tc and; one for Tnc. A quantitative comparison (reason>2.5) of the total spots of CF (nc+c) with T (nc+c) populations allowed us to distinguish five more intense spots for Tc, 14 for Tnc, 15 for CFnc and 11 for CFc. In the CFnc population, two proteins were identified: actin and ATP synthase alpha chain; in the CFc population, four proteins: actin, calmodulin, HSP70, and dehydrogenase; in the Tnc population, five proteins: matrilin, HSP70, actin, ATP synthase alpha chain and intermediate filament of the protein; and in the Tc population, three proteins: actin, alpha-S1 casein and ATP synthase alpha chain. Out of a total of 79 spots, only nine proteins were identified due to the low number of available nucleotide sequences in the GenBank. Nevertheless, knowing proteins regarded as differentially expressed is indispensable for hitherto unidentified genes implicated in B. tenagophila resistance and or susceptibility to S. mansoni infection.

Proteomic analysis by iTRAQ in red claw crayfish, Cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection.

PubMed

Jeswin, Joseph; Xie, Xiao-lu; Ji, Qiao-lin; Wang, Ke-jian; Liu, Hai-peng

2016-03-01

To elucidate proteomic changes of Hpt cells from red claw crayfish, Cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (iTRAQ) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (WSSV) infection. Protein database search revealed 594 protein hits by Mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. Generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, DNA replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small GTPases, G-protein-alpha stimulatory subunit, proteins bearing PDZ- or 14-3-3-domains that help holding together and organize signaling complexes, casein kinase I and proteins of the MAP-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response RNA-binding protein 1. Taken together, these protein information shed new light on the host cellular response against WSSV infection in a crustacean cell culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of calorie restriction on the zebrafish liver proteome

PubMed Central

Jury, David R.; Kaveti, Suma; Duan, Zhong-Hui; Willard, Belinda; Kinter, Michael; Londraville, Richard

2012-01-01

A proteomic approach was taken to study how fish respond to changes in calorie availability, with the longer-term goal of understanding the evolution of lipid metabolism in vertebrates. Zebrafish (Danio rerio) were fed either high (3 rations/day) or low (1 ration/7 days) calorie diets for 5 weeks and liver proteins extracted for proteomic analyses. Proteins were separated on two-dimensional electrophoresis gels and homologous spots compared between treatments to determine which proteins were up-regulated with high-calorie diet. Fifty-five spots were excised from the gel and analyzed via LC–ESI MS/MS, which resulted in the identification of 69 unique proteins (via multiple peptides). Twenty-nine of these proteins were differentially expressed between treatments. Differentially expressed proteins were mapped to Gene Ontology (GO) terms, and these terms compared to the entire zebrafish GO annotation set by Fisher's exact test. The most significant GO terms associated with high-calorie diet are related to a decrease in oxygen-binding activity in the high-calorie treatment. This response is consistent with a well-characterized response in obese humans, indicating there may be a link between lipid storage and hypoxia sensitivity in vertebrates. PMID:20494847

Biomarkers identified from serum proteomic analysis for the differential diagnosis of systemic lupus erythematosus.

PubMed

Kazemipour, N; Qazizadeh, H; Sepehrimanesh, M; Salimi, S

2015-05-01

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that involves different organs. Its most important feature is the production of specific autoantibodies against nuclear or cytoplasmic antigens. Proteomic analysis of serum, as one of the most readily available body fluids, can be used as a method for clarifying the pathogenesis of SLE. In this study the serum proteome of 13 patients with SLE was evaluated and compared with seven healthy control participants. A specific kit was used to remove high-abundance proteins. After depletion, the protein expression patterns created by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF-MS were used to identify disease-associated proteins. We found differential expression of 15 protein spots, including seven up-regulated and eight down-regulated proteins in SLE samples, in comparison with healthy participants. These spots were identified by MALDI-TOF/TOF-MS and classified into three groups include keratins, apolipoproteins and albumin, and individual proteins such as transthyretin, haptoglobin and prothrombin. These findings can help to clarify the pathophysiology and mechanism of SLE. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

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.

Biochemical traits and proteomic changes in postharvest flowers of medicinal chrysanthemum exposed to enhanced UV-B radiation.

PubMed

Yao, Xiaoqin; Chu, Jian-Zhou; Ma, Chun-Hui; Si, Chao; Li, Ji-Gang; Shi, Xiao-Fei; Liu, Chao-Nan

2015-08-01

The article studied UV-B effects on biochemical traits and proteomic changes in postharvest flowers of medicinal chrysanthemum. The experiment about UV-B effects on biochemical traits in flowers included six levels of UV-B treatments (0 (UV0), 50 (UV50), 200 (UV200), 400 (UV400), 600 (UV600) and 800 (UV800) μWcm(-2)). UV400, UV600 and UV800 treatments significantly increased the contents of hydrogen peroxide, malondialdehyde and UV-B absorbing compounds, and the activity of phenylalanine ammonia lyase enzyme over the control. The contents of chlorogenic acid and flavone in flowers were significantly increased by UV-B treatments (except for UV50 and UV800). Two-dimensional gel electrophoresis was utilized to analyze proteomic changes in flowers with or without UV-B radiation. Results indicated that 43 protein spots (>1.5-fold difference in volume) were detected, including 19 spots with a decreasing trend and 24 spots with an increasing trend, and 19 differentially expressed protein spots were successfully indentified by MALDI-TOF MS. The indentified proteins were classified based on functions, the most of which were involved in photosynthesis, respiration, protein biosynthesis and degradation and defence. An overall assessment using biochemical and differential proteomic data revealed that UV-B radiation could affect biochemical reaction and promote secondary metabolism processes in postharvest flowers. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic analysis of kidney in rats chronically exposed to monosodium glutamate.

PubMed

Sharma, Amod; Wongkham, Chaisiri; Prasongwattana, Vitoon; Boonnate, Piyanard; Thanan, Raynoo; Reungjui, Sirirat; Cha'on, Ubon

2014-01-01

Chronic monosodium glutamate (MSG) intake causes kidney dysfunction and renal oxidative stress in the animal model. To gain insight into the renal changes induced by MSG, proteomic analysis of the kidneys was performed. Six week old male Wistar rats were given drinking water with or without MSG (2 mg/g body weight, n = 10 per group) for 9 months. Kidneys were removed, frozen, and stored at -75°C. After protein extraction, 2-D gel electrophoresis was performed and renal proteome profiles were examined with Colloidal Coomassie Brilliant Blue staining. Statistically significant protein spots (ANOVA, p<0.05) with 1.2-fold difference were excised and analyzed by LC-MS. Proteomic data were confirmed by immunohistochemistry and Western blot analyses. The differential image analysis showed 157 changed spots, of which 71 spots were higher and 86 spots were lower in the MSG-treated group compared with those in the control group. Eight statistically significant and differentially expressed proteins were identified: glutathione S-transferase class-pi, heat shock cognate 71 kDa, phosphoserine phosphatase, phosphoglycerate kinase, cytosolic glycerol-3-phosphate dehydrogenase, 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, α-ketoglutarate dehydrogenase and succinyl-CoA ligase. The identified proteins are mainly related to oxidative stress and metabolism. They provide a valuable clue to explore the mechanism of renal handling and toxicity on chronic MSG intake.

Proteomic Analysis of Kidney in Rats Chronically Exposed to Monosodium Glutamate

PubMed Central

Sharma, Amod; Wongkham, Chaisiri; Prasongwattana, Vitoon; Boonnate, Piyanard; Thanan, Raynoo; Reungjui, Sirirat; Cha’on, Ubon

2014-01-01

Background Chronic monosodium glutamate (MSG) intake causes kidney dysfunction and renal oxidative stress in the animal model. To gain insight into the renal changes induced by MSG, proteomic analysis of the kidneys was performed. Methods Six week old male Wistar rats were given drinking water with or without MSG (2 mg/g body weight, n = 10 per group) for 9 months. Kidneys were removed, frozen, and stored at –75°C. After protein extraction, 2-D gel electrophoresis was performed and renal proteome profiles were examined with Colloidal Coomassie Brilliant Blue staining. Statistically significant protein spots (ANOVA, p<0.05) with 1.2-fold difference were excised and analyzed by LC-MS. Proteomic data were confirmed by immunohistochemistry and Western blot analyses. Results The differential image analysis showed 157 changed spots, of which 71 spots were higher and 86 spots were lower in the MSG-treated group compared with those in the control group. Eight statistically significant and differentially expressed proteins were identified: glutathione S-transferase class-pi, heat shock cognate 71 kDa, phosphoserine phosphatase, phosphoglycerate kinase, cytosolic glycerol-3-phosphate dehydrogenase, 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, α-ketoglutarate dehydrogenase and succinyl-CoA ligase. Conclusion The identified proteins are mainly related to oxidative stress and metabolism. They provide a valuable clue to explore the mechanism of renal handling and toxicity on chronic MSG intake. PMID:25551610

Proteomic analysis on mangrove plant Avicennia marina leaves reveals nitric oxide enhances the salt tolerance by up-regulating photosynthetic and energy metabolic protein expression.

PubMed

Shen, Zhi-Jun; Chen, Juan; Ghoto, Kabir; Hu, Wen-Jun; Gao, Gui-Feng; Luo, Mei-Rong; Li, Zan; Simon, Martin; Zhu, Xue-Yi; Zheng, Hai-Lei

2018-06-15

Avicennia marina (Forsk.) Vierh is one of the most salt-tolerant mangrove species. Our previous study revealed that nitric oxide (NO) enhanced the salt tolerance of A. marina by promoting salt secretion and Na+ sequestration under salt stress. However, little is known about the regulation of NO on proteomic profiling for this mangrove species. In this study, we used sodium nitroprusside (SNP), an NO donor, to investigate the regulatory mechanism of NO on salt tolerance of A. marina according to physiological and proteomic aspects. Photosynthesis data showed that the reduction in photosynthesis caused by high salinity treatment (400 mM NaCl) could be partially recovered by addition of SNP (100 μM). Further analysis revealed that the high salinity treatment could induce not only the stomatal limitation but also non-stomatal limitation on photosynthetic reduction, while SNP addition could restore the non-stomatal limitation, implying that the application of SNP was beneficial to the metabolic process in leaves. Proteomic analysis identified 49 differentially expressed proteins involved in various biological processes such as photosynthesis, energy metabolism, primary metabolism, RNA transcription, protein translation and stress response proteins. Under high salinity treatment, the abundances of proteins related to photosynthesis, such as ribulose-phosphate 3-epimerase (RPE, spot 3), RuBisCO large subunit (RBCL, spot 4, 5, 24), RuBisCO activase A (RCA, spot 17, 18) and quinine oxidoreductase-like protein isoform 1 (QOR1, spot 23), were significantly decreased. However, the abundance of proteins such as RBCL (spot 5, 9) and QOR1 (spot 23) were increased by SNP addition. In addition, exogenous NO supply alleviated salt tolerance by increasing the accumulation of some proteins involved in energy metabolism (spot 15), primary metabolism (spot 25, 45, 46), RNA transcription (spot 36) and stress response proteins (spot 12, 21, 26, 37, 43). The transcriptional levels of nine selected proteins were mostly consistent with their protein abundance except spot 46. Overall, the presented data demonstrated that NO has a positive effect on improving salt tolerance in A. marina by regulating the protein abundance involved in photosynthesis, energy metabolism, primary metabolism and stress response.

Identification of differentially expressed proteins during human urinary bladder cancer progression.

PubMed

Memon, Ashfaque A; Chang, Jong W; Oh, Bong R; Yoo, Yung J

2005-01-01

Comparative proteome analysis was performed between RT4 (grade-1) and T24 (grade-3) bladder cancer cell lines, in an attempt to identify differentially expressed proteins during bladder cancer progression. Among those relatively abundant proteins, seven spots changed more than two-fold reproducibly and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may involve in tumor progression and metastasis. However, additional investigations are needed on large number of human samples to further verify these findings.

Divergent Expression Patterns and Function Implications of Four nanos Genes in a Hermaphroditic Fish, Epinephelus coioides.

PubMed

Sun, Zhi-Hui; Wang, Yang; Lu, Wei-Jia; Li, Zhi; Liu, Xiao-Chun; Li, Shui-Sheng; Zhou, Li; Gui, Jian-Fang

2017-03-23

Multiple nanos genes have been characterized in several fishes, but the functional implications of their various expression patterns remain unclear. In this study, we identified and characterized four nanos genes from a hermaphroditic fish orange-spotted grouper, Epinephelus coioides . Ecnanos1a and Ecnanos1b show divergent expression patterns, and the dynamic expression change of Ecnanos1a in pituitaries during sex change is associated with testis differentiation and spermatogenesis. Ecnanos2 and Ecnanos3 might be germline stem cells (GSCs) and primordial germ cells (PGCs)-specific markers, respectively. Significantly, Ecnanos3 3'-untranslated region (UTR) is necessary for PGC specific expression, where a non-canonical "GCACGTTT" sequence is required for miR-430-mediated repression of Ecnanos3 RNA. Furthermore, grouper Dead end (Dnd) can relieve miR-430 repression in PGCs by associating with a 23 bp U-rich region (URR) in Ecnanos3 3'-UTR. The current study revealed the functional association of multiple nanos genes with PGC formation and germ cell development in orange-spotted grouper, and opened up new possibilities for developing biotechnologies through utilizing the associations between Ecnanos3 and PGCs or between Ecnanos2 and GSCs in the hermaphroditic fish.

Altered Gene Expression in Three Plant Species in Response to Treatment with Nep1, a Fungal Protein That Causes Necrosis

PubMed Central

Keates, Sarah E.; Kostman, Todd A.; Anderson, James D.; Bailey, Bryan A.

2003-01-01

Nep1 is an extracellular fungal protein that causes necrosis when applied to many dicotyledonous plants, including invasive weed species. Using transmission electron microscopy, it was determined that application of Nep1 (1.0 μg mL–1, 0.1% [v/v] Silwet-L77) to Arabidopsis and two invasive weed species, spotted knapweed (Centaurea maculosa) and dandelion (Taraxacum officinale), caused a reduction in the thickness of the cuticle and a breakdown of chloroplasts 1 to 4 h after treatment. Membrane breakdown was most severe in cells closest to the surface of application. Differential display was used to isolate cDNA clones from the three species showing differential expression in response to Nep1 treatment. Differential gene expression was observed for a putative serpin (CmSER-1) and a calmodulin-like (CmCAL-1) protein from spotted knapweed, and a putative protein phosphatase 2C (ToPP2C-1) and cytochrome P-450 (ToCYP-1) protein from dandelion. In addition, differential expression was observed for genes coding for a putative protein kinase (AtPK-1), a homolog (AtWI-12) of wound-induced WI12, a homolog (AtLEA-1) of late embryogenesis abundant LEA-5, a WRKY-18 DNA-binding protein (AtWRKY-18), and a phospholipase D (AtPLD-1) from Arabidopsis. Genes showing elevated mRNA levels in Nep1-treated (5 μg mL–1, 0.1% [v/v] Silwet-L77) leaves 15 min after Nep1 treatment included CmSER-1 and CmCAL-1 for spotted knapweed, ToCYP-1 and CmCAL-1 for dandelion, and AtPK-1, AtWRKY-18, AtWI-12, and AtLEA-1 for Arabidopsis. Levels of mRNA for AtPLD-1 (Arabidopsis) and ToPP2C-1 (dandelion) decreased rapidly in Silwet-l77-treated plants between 15 min and 4 h of treatment, but were maintained or decreased more slowly over time in Nep1-treated (5 μg mL–1, 0.1% [v/v] Silwet-L77) leaves. In general, increases in mRNA band intensities were in the range of two to five times, with only ToCYP-1 in dandelion exceeding an increase of 10 times. The identified genes have been shown to be involved or are related to gene families that are involved in plant stress responses, including wounding, drought, senescence, and disease resistance. PMID:12857840

Liver proteomics in progressive alcoholic steatosis

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

Fernando, Harshica; Wiktorowicz, John E.; Soman, Kizhake V.

2013-02-01

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved inmore » alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3) and 3 (4–6) months. C and E represent pair-fed control and ethanol-fed rats, respectively. Highlights: ► Proteins related to ethanol-induced steatosis and mild steatohepatitis are identified. ► ADH1C and ALDH2 involved in alcohol metabolism are differentially expressed at 1 and 3 months. ► Discovery proteomics identified a group of proteins to serve as potential biomarkers. ► Using nonparametric analysis DDT is identified as a possible marker for liver damage.« less

Expression patterns of sex differentiation-related genes during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus.

PubMed

Horiguchi, Ryo; Nozu, Ryo; Hirai, Toshiaki; Kobayashi, Yasuhisa; Nakamura, Masaru

2018-02-01

The three-spot wrasse, Halichoeres trimaculatus, can change sex from female to male (i.e. protogyny) due to sharp decrease in endogenous estrogen. During the sex change, ovarian tissue degenerates and testicular tissue arises newly. Finally, ovarian tissue disappears completely and replaces into mature testis. In order to predict the molecular mechanisms controlling the processes of sex change, we investigated the expression patterns of four genes (rspo1, figla, sox9b and amh), which have been thought to be associated with ovarian/testicular differentiation in vertebrates. Expression levels of rspo1 and figla, which play important roles for ovarian differentiation in vertebrates, were stable until the middle stage of the sex change, and subsequently down-regulated. Therefore, it was indicated that decrease in rspo1 and figla could result from ovarian degeneration. On the other hand, basis on the expression pattern, it was indicated that sox9b and amh, which are involved in testicular differentiation in vertebrates, were implicated in testicular formation and spermatogenesis during the sex change as well. The present results could be fundamental information for investigating the relationship between these factors and E2 depletion, which is crucial trigger for sex change. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative proteomic analysis in children with idiopathic short stature (ISS) before and after short-term recombinant human growth hormone (rhGH) therapy.

PubMed

Heo, Sun Hee; Choi, Jin-Ho; Kim, Yoo-Mi; Jung, Chang-Woo; Lee, Jin; Jin, Hye Young; Kim, Gu-Hwan; Lee, Beom Hee; Shin, Choong Ho; Yoo, Han-Wook

2013-04-01

This study was undertaken to identify growth hormone (GH) responsive proteins and protein expression patterns by short-term recombinant human growth hormone (rhGH) therapy in patients with idiopathic short stature (ISS) using proteomic analysis. Seventeen children (14 males and three females) with ISS were included. They were treated with rhGH at a dose of 0.31 ± 0.078 mg/kg/week for 3 months. Immunodepletion of six highly-abundant serum proteins followed by 2D DIGE analysis, and subsequent MALDI TOF MS, were employed to generate a panel of proteins differentially expressed after short-term rhGH therapy and verify the differences in serum levels of specific proteins by rhGH therapy. Fourteen spots were differentially expressed after rhGH treatment. Among them, apo E and apo L-1 expression were consistently enhanced, whereas serum amyloid A was reduced after rhGH therapy. The differential expressions of these proteins were subsequently verified by Western blot analysis using sera of the before and after rhGH treatment. This study suggests that rhGH therapy influences lipoprotein metabolism and enhances apo L-1 protein expression in ISS patients. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

30 CFR 206.104 - What publications are acceptable to MMS?

Code of Federal Regulations, 2010 CFR

2010-07-01

... price and ANS spot price based on certain criteria, including, but not limited to: (1) Publications... daily surveys of buyers and sellers of crude oil, and, for ANS spot prices, buyers and sellers of ANS... represent NYMEX prices or differentials or ANS spot market prices or differentials. [65 FR 14088, Mar. 15...

Proteomic changes during adult stage in pre-optic, hypothalamus, hippocampus and pituitary regions of female rat brain following neonatal exposure to estradiol-17β.

PubMed

Govindaraj, Vijayakumar; Shridharan, Radhika Nagamangalam; Rao, Addicam Jagannadha

2018-05-16

Although neonatal exposure to estrogen or estrogenic compounds results in irreversible changes in the brain function and reproductive abnormalities during adulthood but the underlying mechanisms are still largely unknown. The present study has attempted to compare the protein profiles of sexually dimorphic brain regions of adult female rats which were exposed to estradiol- 17β during neonatal period. The total proteins extracted from pre-optic area (POA), hypothalamus, hippocampus and pituitary of control and neonatally E2 treated female rats was subjected to 2D-SDS-PAGE and differentially expressed proteins were identified by MALDI TOF/TOF-MS. Our results revealed that a total of 21 protein spots which were identified as differentially expressed in all the four regions analyzed; the differential expression was further validated by RT-PCR and western blotting. The differentially expressed proteins such as 14-3-3 zeta/delta (POA), LMNA (hippocampus), Axin2 (hypothalamus), Syntaxin-7 (hippocampus), prolactin and somatotropin (pituitary) which have very important functions in the process of neuronal differentiation, migration, axon outgrowth, formation of dendritic spine density and synaptic plasticity and memory have not been previously reported in association with neonatal estrogen exposure. The affected brain functions are very important for the establishment of sex specific brain morphology and behavior. Our results suggest that the differentially expressed proteins may play an important role in irreversible changes in the brain function as well as reproductive abnormalities observed in the female rats during adulthood. Copyright © 2018 Elsevier Inc. All rights reserved.

Gene Profiling in Experimental Models of Eye Growth: Clues to Myopia Pathogenesis

PubMed Central

Stone, Richard A.; Khurana, Tejvir S.

2010-01-01

To understand the complex regulatory pathways that underlie the development of refractive errors, expression profiling has evaluated gene expression in ocular tissues of well-characterized experimental models that alter postnatal eye growth and induce refractive errors. Derived from a variety of platforms (e.g. differential display, spotted microarrays or Affymetrix GeneChips), gene expression patterns are now being identified in species that include chicken, mouse and primate. Reconciling available results is hindered by varied experimental designs and analytical/statistical features. Continued application of these methods offers promise to provide the much-needed mechanistic framework to develop therapies to normalize refractive development in children. PMID:20363242

[The usefulness of in vitro interferon-gamma assay for differential diagnosis between intestinal tuberculosis and Crohns disease].

PubMed

Lee, Jung Nam; Ryu, Dong Yup; Park, Sung Han; You, Hyun Seok; Lee, Bong Eun; Kim, Dong Uk; Kim, Tae Oh; Heo, Jeong; Kim, Gwang Ha; Song, Geun Am; Kim, Suk; Park, Do Youn

2010-06-01

It is difficult to clinically and endoscopically differentiate intestinal tuberculosis (ITB) and Crohns disease (CD). The aim of this study was to evaluate the usefulness of in vitro interferon-gamma (INF-gamma) assay for differential diagnosis between ITB and CD. Sixty patients for whom differential diagnosis between ITB and CD was difficult were enrolled between January 2007 and January 2009. The INF-gamma-producing T-cell response to early secreted antigenic target 6 and culture filtrate protein 10 were measured by T-SPOT.TB blood test in vitro. We evaluated the usefulness of T-SPOT.TB blood test by comparing its results with the final diagnosis. Twenty and forty patients were revealed to be positive and negative in T-SPOT.TB blood test, respectively. Of the 20 patients found to be positive, 12 patients (60%) were finally diagnosed as ITB, 6 patients as CD, and 2 patients as Behcets enterocolitis. Of the 40 patients with negative results, 38 patients (95%) were diagnosed as CD; one as Behcets enterocolitis; one as nonspecific colitis; none as ITB. The sensitivity and specificity of T-SPOT.TB blood test for ITB were 100% and 83.3%, respectively. Positive and negative predictive values of T-SPOT.TB blood test for ITB were 60.0% and 100%, respectively. When differential diagnosis between ITB and CD is difficult, T-SPOT.TB blood test may be a helpful and rapid diagnostic tool to exclude ITB. Prospective large-scaled studies are required for further evaluation of the usefulness of T-SPOT.TB blood test for differential diagnosis between ITB and CD.

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.

Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis.

PubMed

Degroote, Roxane L; Hauck, Stefanie M; Amann, Barbara; Hirmer, Sieglinde; Ueffing, Marius; Deeg, Cornelia A

2014-01-01

Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease.

Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

PubMed

Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

2015-08-01

Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

PubMed Central

Leal, Mariana Ferreira; Chung, Janete; Calcagno, Danielle Queiroz; Assumpção, Paulo Pimentel; Demachki, Samia; da Silva, Ismael Dale Cotrim Guerreiro; Chammas, Roger; Burbano, Rommel Rodríguez; de Arruda Cardoso Smith, Marília

2012-01-01

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets. PMID:22860099

Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio.

PubMed

Dorts, Jennifer; Kestemont, Patrick; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

2011-02-04

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.

Comparative analysis of the in vitro and in planta secretomes from Mycosphaerella fijiensis isolates.

PubMed

Escobar-Tovar, Lina; Guzmán-Quesada, Mauricio; Sandoval-Fernández, Jorge A; Gómez-Lim, Miguel A

2015-06-01

Black Sigatoka, a devastating disease of bananas and plantains worldwide, is caused by the fungus Mycosphaerella fijiensis. Several banana cultivars such as 'Yangambi Km 5' and Calcutta IV, have been known to be resistant to the fungus, but the resistance has been broken in 'Yangambi Km 5' in Costa Rica. Since the resistance of this variety still persists in Mexico, the aim of this study was to compare the in vitro and in planta secretomes from two avirulent and virulent M. fijiensis isolates using proteomics and bioinformatics approaches. We aimed to identify differentially expressed proteins in fungal isolates that differ in pathogenicity and that might be responsible for breaking the resistance in 'Yangambi Km 5'. We were able to identify 90 protein spots in the secretomes of fungal isolates encoding 42 unique proteins and 35 differential spots between them. Proteins involved in carbohydrate transport and metabolism were more prevalent. Several proteases, pathogenicity-related, ROS detoxification and unknown proteins were also highly or specifically expressed by the virulent isolate in vitro or during in planta infection. An unknown protein representing a virulence factor candidate was also identified. These results demonstrated that the secretome reflects major differences between both M. fijiensis isolates. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Leaf proteomic analysis in cassava (Manihot esculenta, Crantz) during plant development, from planting of stem cutting to storage root formation.

PubMed

Mitprasat, Mashamon; Roytrakul, Sittiruk; Jiemsup, Surasak; Boonseng, Opas; Yokthongwattana, Kittisak

2011-06-01

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student's t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P < 0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC-MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.

A probabilistic framework for microarray data analysis: fundamental probability models and statistical inference.

PubMed

Ogunnaike, Babatunde A; Gelmi, Claudio A; Edwards, Jeremy S

2010-05-21

Gene expression studies generate large quantities of data with the defining characteristic that the number of genes (whose expression profiles are to be determined) exceed the number of available replicates by several orders of magnitude. Standard spot-by-spot analysis still seeks to extract useful information for each gene on the basis of the number of available replicates, and thus plays to the weakness of microarrays. On the other hand, because of the data volume, treating the entire data set as an ensemble, and developing theoretical distributions for these ensembles provides a framework that plays instead to the strength of microarrays. We present theoretical results that under reasonable assumptions, the distribution of microarray intensities follows the Gamma model, with the biological interpretations of the model parameters emerging naturally. We subsequently establish that for each microarray data set, the fractional intensities can be represented as a mixture of Beta densities, and develop a procedure for using these results to draw statistical inference regarding differential gene expression. We illustrate the results with experimental data from gene expression studies on Deinococcus radiodurans following DNA damage using cDNA microarrays. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Comparison of the longissimus muscle proteome between obese and lean pigs at 180 days.

PubMed

Li, Anning; Mo, Delin; Zhao, Xiao; Jiang, Wei; Cong, Peiqing; He, Zuyong; Xiao, Shuqi; Liu, Xiaohong; Chen, Yaosheng

2013-02-01

Production of high-quality meat is important to satisfy the consumer and make the pig industry competitive. Obese and lean breeds of pig show clear differences in adipogenic capacity and meat quality, but the underlying molecular mechanism remains unclear. We have compared protein expression of the longissimus muscle between Lantang (LT, obese) and Landrace (LR, lean) pigs at the age of 180 days using two-dimensional fluorescence difference gel electrophoresis. Of the 1,400 protein spots detected per gel, 18 were differentially expressed between the two breeds. Using peptide mass fingerprint and tandem mass spectrometry, 17 protein spots were identified, corresponding to ten different proteins that could be divided into four groups: metabolism-related, structure-related, stress-related, and other (unclassified). Among the metabolism-related proteins, COX5A and ATP5B, which participate in oxidative phosphorylation, were highly expressed in LT, whereas ENO3, which is involved in glycolysis, was highly expressed in LR. These results may contribute valuable information to our understanding of the molecular mechanism responsible for differences between obese and lean pigs, such as growth rate and meat quality.

Evaluation of different methods of protein extraction and identification of differentially expressed proteins upon ethylene-induced early-ripening in banana peels.

PubMed

Zhang, Li-Li; Feng, Ren-Jun; Zhang, Yin-Dong

2012-08-15

Banana peels (Musa spp.) are a good example of a plant tissue where protein extraction is challenging due to the abundance of interfering metabolites. Sample preparation is a critical step in proteomic research and is critical for good results. We sought to evaluate three methods of protein extraction: trichloroacetic acid (TCA)-acetone precipitation, phenol extraction, and TCA precipitation. We found that a modified phenol extraction protocol was the most optimal method. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) demonstrated good protein separation and distinct spots of high quality protein. Approximately 300 and 550 protein spots were detected on 2-DE gels at pH values of 3-10 and 4-7, respectively. Several spots were excised from the 2-DE gels and identified by mass spectrometry. The protein spots identified were found to be involved in glycolysis, the tricarboxylic acid cycle, and the biosynthesis of ethylene. Several of the identified proteins may play important roles in banana ripening. Copyright © 2012 Society of Chemical Industry.

Expression profile of amh/Amh during bi-directional sex change in the protogynous orange-spotted grouper Epinephelus coioides.

PubMed

Wu, Guan-Chung; Li, Hau-Wen; Tey, Wei-Guan; Lin, Chien-Ju; Chang, Ching-Fong

2017-01-01

Gonadal differentiation is tightly regulated by the initial sex determining gene and the downstream sex-related genes in vertebrates. However, sex change in fish can alter the sexual fate from one sex to the other. Chemical-induced maleness in the protogynous orange-spotted grouper is transient, and a reversible sex change occurs after the chemical treatment is withdrawn. We used these characteristics to study Amh signaling during bi-directional sex change in the grouper. We successfully induced the female-to-male sex change by chemical (aromatase inhibitor, AI, or methyltestosterone, MT) treatment. A dormant gonad (a low proliferation rate of early germ cells and no characteristics of both sexes) was found during the transient phase of reversible male-to-female sex change after the withdrawal of chemical administration. Our results showed that amh (anti-mullerian hormone) and its receptor amhr2 (anti-mullerian hormone receptor type 2) were significantly increased in the gonads during the process of female-to-male sex change. Amh is expressed in the Sertoli cells surrounding the type A spermatogonia in the female-to-male grouper. Male-related gene (dmrt1 and sox9) expression was immediately decreased in MT-terminated males during the reversible male-to-female sex change. However, Amh expression was found in the surrounding cells of type A spermatogonia-like cells during the transient phase of reversible male-to-female sex change. This phenomenon is correlated with the dormancy of type A spermatogonia-like cells. Thus, Amh signaling is suggested to play roles in regulating male differentiation during the female-to-male sex change and in inhibiting type-A spermatogonia-like cell proliferation/differentiation during the reversible male-to-female sex change. We suggest that Amh signaling might play dual roles during bi-directional sex change in grouper.

Expression profile of amh/Amh during bi-directional sex change in the protogynous orange-spotted grouper Epinephelus coioides

PubMed Central

Wu, Guan-Chung; Li, Hau-Wen; Tey, Wei-Guan; Lin, Chien-Ju; Chang, Ching-Fong

2017-01-01

Gonadal differentiation is tightly regulated by the initial sex determining gene and the downstream sex-related genes in vertebrates. However, sex change in fish can alter the sexual fate from one sex to the other. Chemical-induced maleness in the protogynous orange-spotted grouper is transient, and a reversible sex change occurs after the chemical treatment is withdrawn. We used these characteristics to study Amh signaling during bi-directional sex change in the grouper. We successfully induced the female-to-male sex change by chemical (aromatase inhibitor, AI, or methyltestosterone, MT) treatment. A dormant gonad (a low proliferation rate of early germ cells and no characteristics of both sexes) was found during the transient phase of reversible male-to-female sex change after the withdrawal of chemical administration. Our results showed that amh (anti-mullerian hormone) and its receptor amhr2 (anti-mullerian hormone receptor type 2) were significantly increased in the gonads during the process of female-to-male sex change. Amh is expressed in the Sertoli cells surrounding the type A spermatogonia in the female-to-male grouper. Male-related gene (dmrt1 and sox9) expression was immediately decreased in MT-terminated males during the reversible male-to-female sex change. However, Amh expression was found in the surrounding cells of type A spermatogonia-like cells during the transient phase of reversible male-to-female sex change. This phenomenon is correlated with the dormancy of type A spermatogonia-like cells. Thus, Amh signaling is suggested to play roles in regulating male differentiation during the female-to-male sex change and in inhibiting type-A spermatogonia-like cell proliferation/differentiation during the reversible male-to-female sex change. We suggest that Amh signaling might play dual roles during bi-directional sex change in grouper. PMID:29016690

Acute high-altitude hypoxic brain injury: Identification of ten differential proteins

PubMed Central

Li, Jianyu; Qi, Yuting; Liu, Hui; Cui, Ying; Zhang, Li; Gong, Haiying; Li, Yaxiao; Li, Lingzhi; Zhang, Yongliang

2013-01-01

Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain injury. However, little is currently known about the mechanisms responsible for mitochondrial dysfunction in hypobaric hypoxic brain damage. In this study, a rat model of hypobaric hypoxic brain injury was established to investigate the molecular mechanisms associated with mitochondrial dysfunction. As revealed by two-dimensional electrophoresis analysis, 16, 21, and 36 differential protein spots in cerebral mitochondria were observed at 6, 12, and 24 hours post-hypobaric hypoxia, respectively. Furthermore, ten protein spots selected from each hypobaric hypoxia subgroup were similarly regulated and were identified by mass spectrometry. These detected proteins included dihydropyrimidinase-related protein 2, creatine kinase B-type, isovaleryl-CoA dehydrogenase, elongation factor Ts, ATP synthase beta-subunit, 3-mercaptopyruvate sulfurtransferase, electron transfer flavoprotein alpha-subunit, Chain A of 2-enoyl-CoA hydratase, NADH dehydrogenase iron-sulfur protein 8 and tropomyosin beta chain. These ten proteins are all involved in the electron transport chain and the function of ATP synthase. Our findings indicate that hypobaric hypoxia can induce the differential expression of several cerebral mitochondrial proteins, which are involved in the regulation of mitochondrial energy production. PMID:25206614

Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk.

PubMed

Chen, Y F; Zhao, W J; Wu, R N; Sun, Z H; Zhang, W Y; Wang, J C; Bilige, M; Zhang, H P

2014-12-01

Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

PubMed Central

Sui, Zhiwei; Wen, Bo; Gao, Zhimin; Chen, Quanjiao

2014-01-01

Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses. PMID:25153908

Up-regulation of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in sentinel lymph nodes of colorectal cancer

PubMed Central

He, Zhen-Yu; Wen, Hao; Shi, Chuan-Bing; Wang, Jie

2010-01-01

AIM: To investigate the early metastasis-associated proteins in sentinel lymph node micrometastasis (SLNMM) of colorectal cancer (CRC) through comparative proteome. METHODS: Hydrophobic protein samples were extracted from individual-matched normal lymph nodes (NLN) and SLNMM of CRC. Differentially expressed protein spots were detected by two-dimensional electrophoresis and image analysis, and subsequently identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry-mass spectrometry and Western blotting, respectively. RESULTS: Forty proteins were differentially expressed in NLN and SLNMM, and 4 metastasis-concerned proteins highly expressed in SLNMM were identified to be hnRNP A1, Ezrin, tubulin β-2C and Annexin A1. Further immunohistochemistry staining of these four proteins showed their clinicopathological characteristics in lymph node metastasis of CRC. CONCLUSION: Variations of hydrophobic protein expression in NLN and SLNMM of CRC and increased expression of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in SLNMM suggest a significantly elevated early CRC metastasis. PMID:20872967

Recurrent star-spot activity and differential rotation in KIC 11560447

NASA Astrophysics Data System (ADS)

Özavcı, I.; Şenavcı, H. V.; Işık, E.; Hussain, G. A. J.; O'Neal, D.; Yılmaz, M.; Selam, S. O.

2018-03-01

We present a detailed analysis of surface inhomogeneities on the K1-type subgiant component of the rapidly rotating eclipsing binary KIC 11560447, using high-precision Kepler light curves spanning nearly 4 yr, which corresponds to about 2800 orbital revolutions. We determine the system parameters precisely, using high-resolution spectra from the 2.1-m Otto Struve Telescope at the McDonald Observatory. We apply the maximum entropy method to reconstruct the relative longitudinal spot occupancy. Our numerical tests show that the procedure can recover large-scale random distributions of individually unresolved spots, and it can track the phase migration of up to three major spot clusters. By determining the drift rates of various spotted regions in orbital longitude, we suggest a way to constrain surface differential rotation and we show that the results are consistent with periodograms. The K1IV star exhibits two mildly preferred longitudes of emergence, indications of solar-like differential rotation, and a 0.5-1.3-yr recurrence period in star-spot emergence, accompanied by a secular increase in the axisymmetric component of spot occupancy.

Divergent Expression Patterns and Function Implications of Four nanos Genes in a Hermaphroditic Fish, Epinephelus coioides

PubMed Central

Sun, Zhi-Hui; Wang, Yang; Lu, Wei-Jia; Li, Zhi; Liu, Xiao-Chun; Li, Shui-Sheng; Zhou, Li; Gui, Jian-Fang

2017-01-01

Multiple nanos genes have been characterized in several fishes, but the functional implications of their various expression patterns remain unclear. In this study, we identified and characterized four nanos genes from a hermaphroditic fish orange-spotted grouper, Epinephelus coioides. Ecnanos1a and Ecnanos1b show divergent expression patterns, and the dynamic expression change of Ecnanos1a in pituitaries during sex change is associated with testis differentiation and spermatogenesis. Ecnanos2 and Ecnanos3 might be germline stem cells (GSCs) and primordial germ cells (PGCs)-specific markers, respectively. Significantly, Ecnanos3 3′-untranslated region (UTR) is necessary for PGC specific expression, where a non-canonical “GCACGTTT” sequence is required for miR-430-mediated repression of Ecnanos3 RNA. Furthermore, grouper Dead end (Dnd) can relieve miR-430 repression in PGCs by associating with a 23 bp U-rich region (URR) in Ecnanos3 3′-UTR. The current study revealed the functional association of multiple nanos genes with PGC formation and germ cell development in orange-spotted grouper, and opened up new possibilities for developing biotechnologies through utilizing the associations between Ecnanos3 and PGCs or between Ecnanos2 and GSCs in the hermaphroditic fish. PMID:28333083

Variation-preserving normalization unveils blind spots in gene expression profiling

PubMed Central

Roca, Carlos P.; Gomes, Susana I. L.; Amorim, Mónica J. B.; Scott-Fordsmand, Janeck J.

2017-01-01

RNA-Seq and gene expression microarrays provide comprehensive profiles of gene activity, but lack of reproducibility has hindered their application. A key challenge in the data analysis is the normalization of gene expression levels, which is currently performed following the implicit assumption that most genes are not differentially expressed. Here, we present a mathematical approach to normalization that makes no assumption of this sort. We have found that variation in gene expression is much larger than currently believed, and that it can be measured with available assays. Our results also explain, at least partially, the reproducibility problems encountered in transcriptomics studies. We expect that this improvement in detection will help efforts to realize the full potential of gene expression profiling, especially in analyses of cellular processes involving complex modulations of gene expression. PMID:28276435

Under the influence of the active deodorant ingredient 4-hydroxy-3-methoxybenzyl alcohol, the skin bacterium Corynebacterium jeikeium moderately responds with differential gene expression.

PubMed

Brune, Iris; Becker, Anke; Paarmann, Daniel; Albersmeier, Andreas; Kalinowski, Jörn; Pühler, Alfred; Tauch, Andreas

2006-12-15

A 70mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Corynebacterium jeikeium, a skin bacterium that is predominantly present in the human axilla and involved in axillary odor formation. Oligonucleotides representing 100% of the predicted coding regions of the C. jeikeium K411 genome were designed and spotted in quadruplicate onto epoxy-coated glass slides. The quality of the printed microarray was demonstrated by co-hybridization with fluorescently labeled cDNA probes obtained from exponentially growing C. jeikeium cultures. Accordingly, genes detected with different intensities resulting in log(2) transformed ratios greater than 0.8 or smaller than -0.8 can be regarded as differentially expressed with a confidence level greater than 99%. In an application example, we measured global changes of gene expression during growth of C. jeikeium in the presence of different concentrations of the deodorant component 4-hydroxy-3-methoxybenzyl alcohol that is active in preventing body odor formation. Global expression profiling revealed that low concentrations of 4-hydroxy-3-methoxybenzyl alcohol (0.5 and 2.5mg/ml) had almost no detectable effect on the transcriptome of C. jeikeium. A slightly higher concentration of 4-hydroxy-3-methoxybenzyl alcohol (5mg/ml) resulted in differential expression of 95 genes, 86 of which showed an enhanced expression when compared to a control culture. Besides many genes encoding proteins that apparently participate in transcription and translation, the drug resistance determinant cmx and the predicted virulence factors sapA and sapD showed significantly enhanced expression levels. Differential expression of relevant genes was validated by real-time reverse transcription PCR assays.

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.

Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals.

PubMed

Nally, Jarlath E; Grassmann, Andre A; Planchon, Sébastien; Sergeant, Kjell; Renaut, Jenny; Seshu, Janakiram; McBride, Alan J; Caimano, Melissa J

2017-01-01

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p < 0.05, fold change >1.25 or < -1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.

Sperm membrane proteins associated with the boar semen cryopreservation.

PubMed

Guimarães, Daianny B; Barros, Tatyane B; van Tilburg, Maurício F; Martins, Jorge A M; Moura, Arlindo A; Moreno, Frederico B; Monteiro-Moreira, Ana C; Moreira, Renato A; Toniolli, Ricardo

2017-08-01

This study aimed to define sperm membrane protein markers of semen freezability of boars with the aid of a proteomic approach. Semen from fourteen adult boars were subjected to slow freezing and rapid thawing. After thawing, sperm vigor and motility were analyzed, and based on these results, animals were separated into two groups: good (GFEs) and poor freezability (PFEs). Sperm membrane proteins were extracted and subjected to two-dimensional electrophoresis. Stained gels were analyzed by computerized resources to indicate differentially expressed protein spots, that were identified by mass spectrometry. Six animals showed good freezability with average sperm vigor and motility of 2.2±0.8 and 41.8±22.9, respectively, whereas eight boars showed poor freezability, with 1.9±0.6 and 26.8±17.5 of sperm vigor sperm motility, respectively. An average of 263±62.2 spots per gel and 234.2±54.6 of spots consistently present in all gels were detected. The intensities of five spots were significantly different between groups. Fc fragment of IgG binding protein and lactadherin were more intense in the PFE group, while Arylsulfatase A and F-actin capping protein subunit alpha 1 were more expressed in the GEF group. Based on their functions and interactions with other proteins, we conclude that these four sperm membrane proteins may act as potential markers of boar semen freezability. Copyright © 2017. Published by Elsevier B.V.

New Fks Hot Spot for Acquired Echinocandin Resistance in Saccharomyces cerevisiae and Its Contribution to Intrinsic Resistance of Scedosporium Species▿

PubMed Central

Johnson, Michael E.; Katiyar, Santosh K.; Edlind, Thomas D.

2011-01-01

Echinocandins represent a new antifungal group with potent activity against Candida species. These lipopeptides inhibit the synthesis of β-1,3-glucan, the major cell wall polysaccharide. Acquired resistance or reduced echinocandin susceptibility (RES) is rare and associated with mutations in two “hot spot” regions of Fks1 or Fks2, the probable β-1,3-glucan synthases. In contrast, many fungi demonstrate intrinsic RES for reasons that remain unclear. We are using Saccharomyces cerevisiae to understand the basis for RES by modeling echinocandin-Fks interaction. Previously characterized mutations confer cross-RES; we screened for mutations conferring differential RES, implying direct interaction of that Fks residue with a variable echinocandin side chain. One mutant (in an fks1Δ background) exhibited ≥16-fold micafungin and anidulafungin versus caspofungin RES. Sequencing identified a novel Fks2 mutation, W714L/Y715N. Equivalent W695L/Y696N and related W695L/F/C mutations in Fks1 generated by site-directed mutagenesis and the isolation of a W695L-equivalent mutation in Candida glabrata confirmed the role of the new “hot spot 3” in RES. Further mutagenesis expanded hot spot 3 to Fks1 residues 690 to 700, yielding phenotypes ranging from cross-RES to differential hypersusceptibility. Fks1 sequences from intrinsically RES Scedosporium species revealed W695F-equivalent substitutions; Fks1 hybrids expressing Scedosporium prolificans hot spot 3 confirmed that this substitution imparts RES. PMID:21576441

[Herbs for calming liver and suppressing yang in treatment of hyperthyroidism with hyperactive liver yang: herbal effects on lymphocyte protein expression].

PubMed

Li, Xiangping; Yin, Tao; Zhong, Guangwei; Li, Wei; Luo, Yanhong; Xiang, Lingli; Liu, Zhehao

2011-07-01

To observe the herbal effects on hyperthyroidism patients with syndrome of hyperactivity of liver-Yang by method for calming the liver and suppressing Yang and investigate its effects on the lymphocyte protein expression. This approach may lay a foundation for the further investigation of the curative mechanisms of calming the liver and suppressing Yang treatment. A total of 48 hyperthyroidism patients with syndrome of hyperactivity of liver-Yang were randomly divided into treatment group and control group. The treatment group was treated by method for calming the liver and suppressing Yang in accordance with traditional Chinese medicine (TCM) and the control group with thiamazole tablets for three periods of treatment The therapeutic effects, the score of TCM symptom, electrocardiogram (P wave), thyroid hormones and ultrasound were observed in both groups before and after the treatment. The side effects in the treatment course were observed in both groups. The level of differential protein expression was analyzed by two-dimensional electrphoresis and matrix assisted laser desorption/ionizaton time-of-flight mass spectrometry. The treatment group has the effect on stepping down the heart rate, cutting down the P wave amplitude changes, regulating the level of thyroid hormones and decreasing the volume of thyromegaly. There are not statistically significant between the treatment group and control group. However, the treatment group has obviously better effect on regulating TCM symptom and decreasing the side reaction than the control group (P<0.05). There are not statistically significant on the total effective between the treatment group and control group. The average spots in lymphocyte for normal people, before and after treating hyperthyroidism patients with syndrome of hyperactivity of liver-Yang were (429 +/- 31), (452 +/- 28) and (437 +/- 36) spots respectively. Eight down-regulated protein expressions and 11 up-regulated protein expressions were obtained in the hyperthyroidism patients with syndrome of hyperactivity of liver-Yang and normal people. Five strengthened expressions of protein were also obtained in 8 down-regulated expressions of protein and 8 lower expressions of protein in 11 up-regulated expressions of protein before and after treating the migraine patients with syndrome of hyperactivity of liver-Yang. Ten of the total 8 differential protein spots were successfully identified by MALDI-TOF-MS. The functions of these proteins were involved in metabolism associated, transportation, antioxidation, sigal transduction and immume associated protein, etc. according to information provided by NCBI and MSDB database. In this study, the TCM complex prescription with herbs for calming the liver and suppressing Yang can regulate the thyroid hormones, improves TCM symptoms, and decrease the adverse reaction. It can possibly regulate lymphocyte protein expression.

Gene expression profiling of choline-deprived neural precursor cells isolated from mouse brain.

PubMed

Niculescu, Mihai D; Craciunescu, Corneliu N; Zeisel, Steven H

2005-04-04

Choline is an essential nutrient and an important methyl donor. Choline deficiency alters fetal development of the hippocampus in rodents and these changes are associated with decreased memory function lasting throughout life. Also, choline deficiency alters global and gene-specific DNA methylation in several models. This gene expression profiling study describes changes in cortical neural precursor cells from embryonic day 14 mice, after 48 h of exposure to a choline-deficient medium. Using Significance Analysis of Microarrays, we found the expression of 1003 genes to be significantly changed (from a total of 16,000 total genes spotted on the array), with a false discovery rate below 5%. A total of 846 genes were overexpressed while 157 were underexpressed. Classification by gene ontology revealed that 331 of these genes modulate cell proliferation, apoptosis, neuronal and glial differentiation, methyl metabolism, and calcium-binding protein classes. Twenty-seven genes that had changed expression have previously been reported to be regulated by promoter or intron methylation. These findings support our previous work suggesting that choline deficiency decreases the proliferation of neural precursors and possibly increases premature neuronal differentiation and apoptosis.

Differential expression proteomics to investigate responses and resistance to Orobanche crenata in Medicago truncatula

PubMed Central

Castillejo, Ma Ángeles; Maldonado, Ana M; Dumas-Gaudot, Eliane; Fernández-Aparicio, Mónica; Susín, Rafael; Diego, Rubiales; Jorrín, Jesús V

2009-01-01

Background Parasitic angiosperm Orobanche crenata infection represents a major constraint for the cultivation of legumes worldwide. The level of protection achieved to date is either incomplete or ephemeral. Hence, an efficient control of the parasite requires a better understanding of its interaction and associated resistance mechanisms at molecular levels. Results In order to study the plant response to this parasitic plant and the molecular basis of the resistance we have used a proteomic approach. The root proteome of two accessions of the model legume Medicago truncatula displaying differences in their resistance phenotype, in control as well as in inoculated plants, over two time points (21 and 25 days post infection), has been compared. We report quantitative as well as qualitative differences in the 2-DE maps between early- (SA 27774) and late-resistant (SA 4087) genotypes after Coomassie and silver-staining: 69 differential spots were observed between non-inoculated genotypes, and 42 and 25 spots for SA 4087 and SA 27774 non-inoculated and inoculated plants, respectively. In all, 49 differential spots were identified by peptide mass fingerprinting (PMF) following MALDI-TOF/TOF mass spectrometry. Many of the proteins showing significant differences between genotypes and after parasitic infection belong to the functional category of defense and stress-related proteins. A number of spots correspond to proteins with the same function, and might represent members of a multigenic family or post-transcriptional forms of the same protein. Conclusion The results obtained suggest the existence of a generic defense mechanism operating during the early stages of infection and differing in both genotypes. The faster response to the infection observed in the SA 27774 genotype might be due to the action of proteins targeted against key elements needed for the parasite's successful infection, such as protease inhibitors. Our data are discussed and compared with those previously obtained with pea [1] and transcriptomic analysis of other plant-pathogen and plant-parasitic plant systems. PMID:19575787

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep

PubMed Central

Fowler, Paul A.; Dorà, Natalie J.; McFerran, Helen; Amezaga, Maria R.; Miller, David W.; Lea, Richard G.; Cash, Phillip; McNeilly, Alan S.; Evans, Neil P.; Cotinot, Corinne; Sharpe, Richard M.; Rhind, Stewart M.

2008-01-01

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using ‘real life’ in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25–26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome. PMID:18436539

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

PubMed Central

2014-01-01

Background Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms. Methods Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry. Results A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Conclusions Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations. PMID:24904238

Differential expression of the skeletal muscle proteome in grazed cattle.

PubMed

Shibata, M; Matsumoto, K; Oe, M; Ohnishi-Kameyama, M; Ojima, K; Nakajima, I; Muroya, S; Chikuni, K

2009-08-01

The objective of this study was to investigate the differences in the muscle proteome of grass-fed and grain-fed cattle. Eight Japanese Black Cattle 10 mo of age were separated randomly into 2 groups: 1) grazing (grass-fed) and 2) concentrate (grain-fed) groups. All cattle were first housed individually in a stall barn and fed a combination of concentrate ad libitum and Italian ryegrass hay until 21 mo of age. After this control period, the 4 grass-fed cattle were placed on outdoor pasture, whereas the other 4 grain-fed cattle continued on the concentrate diet. The cattle were slaughtered at 27 mo of age, and tissues from the semitendinosus muscle were obtained for use in proteome analysis. Differential expression of muscle proteins in the 2 groups was carried out using 2-dimensional gel electrophoresis (2DE) and Western blot analyses, with subsequent mass spectrometry. Approximately 200 individual protein spots were detected and compared in each group using 2DE, of which 20 and 9 spots, respectively, showed differences in the spot intensity for the sarcoplasmic fraction and myofibrillar fraction. In the grazing group, the relative intensity of spots was significantly greater for adenylate kinase 1 and myoglobin in the sarcoplasmic fraction, and for slow-twitch myosin light chain 2 in the myofibrillar fraction (P < 0.05), than the concentrate group. The relative spot intensity of several glycolytic enzymes was significantly greater in the grazing group, such as beta-enolase 3, fructose-1,6-bisphosphate aldolase A, triosephosphate isomerase, and heat shock 27 kDa protein (P < 0.05). Moreover, significantly greater slow twitch of troponin T, troponin I, and myosin heavy chain of semitendinosus muscle was detected in the grazing group than in the concentrate group using Western blot analysis (P < 0.05). Several previous reports have described that the slow-twitch muscle contents affect elements of nutrition, flavor, and food texture of meat. This study revealed muscle fiber type conversion to slow-twitch tissues from fast-twitch tissues occurring with change in the energy metabolic enzyme when cattle were grazed in the latter fattening period. Although analyses of the influence on elements of nutrition, flavor, and food texture were not done for this study, these results show that slow-twitch converted muscle resulting from the grazing of cattle might modify several meat characteristics.

Proteomics Analysis of Alfalfa Response to Heat Stress

PubMed Central

Li, Weimin; Wei, Zhenwu; Qiao, Zhihong; Wu, Zinian; Cheng, Lixiang; Wang, Yuyang

2013-01-01

The proteome responses to heat stress have not been well understood. In this study, alfalfa (Medicago sativa L. cv. Huaiyin) seedlings were exposed to 25°C (control) and 40°C (heat stress) in growth chambers, and leaves were collected at 24, 48 and 72 h after treatment, respectively. The morphological, physiological and proteomic processes were negatively affected under heat stress. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and differentially expressed protein spots were identified by mass spectrometry (MS). Totally, 81 differentially expressed proteins were identified successfully by MALDI-TOF/TOF. These proteins were categorized into nine classes: including metabolism, energy, protein synthesis, protein destination/storage, transporters, intracellular traffic, cell structure, signal transduction and disease/defence. Five proteins were further analyzed for mRNA levels. The results of the proteomics analyses provide a better understanding of the molecular basis of heat-stress responses in alfalfa. PMID:24324825

Response by anglers to a differential harvest regulation on three black bass species at Skiatook Lake, Oklahoma

USGS Publications Warehouse

Long, James M.; Hyler, Randy G.; Fisher, William L.

2012-01-01

Angler responses to a differential harvest regulation on black bass, Micropterus spp. at Skiatook Lake, Oklahoma was assessed from 1997 to 1999. This regulation allowed anglers to harvest 15 spotted bass, M. punctulatus (Rafinesque) of any size and six largemouth bass, M. salmoides (Lacepède) and smallmouth bass, M. dolomieu Lacepède greater than 356 mm (in aggregate) per day. Anglers’ ability to differentiate spotted bass increased after the first year of the study, but their willingness to target or harvest spotted bass declined. Mean angler catch rates (number of fish per angling hour) for all three species remained steady throughout the study. Total harvest of largemouth bass and smallmouth bass was reduced by 1999 while total harvest of spotted bass remained steady throughout the study period. Despite the more liberal regulations as incentive, the regulation failed to accomplish the primary objective of increasing angler harvest of spotted bass because of high rates of voluntary catch and release.

Proteomic Characterization of Yersinia pestis Virulence

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

Chromy, B; Murphy, G; Gonzales, A

2005-01-05

Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditionsmore » were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.« less

Macroarray expression analysis of barley susceptibility and nonhost resistance to Blumeria graminis.

PubMed

Eichmann, Ruth; Biemelt, Sophia; Schäfer, Patrick; Scholz, Uwe; Jansen, Carin; Felk, Angelika; Schäfer, Wilhelm; Langen, Gregor; Sonnewald, Uwe; Kogel, Karl-Heinz; Hückelhoven, Ralph

2006-04-01

Different formae speciales of the grass powdery mildew fungus Blumeria graminis undergo basic-compatible or basic-incompatible (nonhost) interactions with barley. Background resistance in compatible interactions and nonhost resistance require common genetic and mechanistic elements of plant defense. To build resources for differential screening for genes that potentially distinguish a compatible from an incompatible interaction on the level of differential gene expression of the plant, we constructed eight dedicated cDNA libraries, established 13.000 expressed sequence tag (EST) sequences and designed DNA macroarrays. Using macroarrays based on cDNAs derived from epidermal peels of plants pretreated with the chemical resistance activating compound acibenzolar-S-methyl, we compared the expression of barley gene transcripts in the early host interaction with B. graminis f.sp. hordei or the nonhost pathogen B. graminis f.sp. tritici, respectively. We identified 102 spots corresponding to 94 genes on the macroarray that gave significant B. graminis-responsive signals at 12 and/or 24 h after inoculation. In independent expression analyses, we confirmed the macroarray results for 11 selected genes. Although the majority of genes showed a similar expression profile in compatible versus incompatible interactions, about 30 of the 94 genes were expressed on slightly different levels in compatible versus incompatible interactions.

Comparison of camel tear proteins between summer and winter

PubMed Central

Chen, Ziyan; Shamsi, Farrukh A.; Li, Kaijun; Huang, Qiang; Al-Rajhi, Ali A.; Chaudhry, Imtiaz A.

2011-01-01

Purpose Proteins in the tear fluid have positive effects on maintaining the integrity and stabilization of the tear film, which is affected by several environmental factors. The aim of this study is to investigate seasonal variation of protein patterns in camel tears collected during the summer and winter season. Methods Tears from both eyes of 50 clinically normal camels (Camelus dromedarius) were collected in the summer (June – July) and in the winter (December – January) respectively. Pooled tear protein samples from two seasons were separated by SDS–PAGE and two-dimensional electrophoresis (2-DE). Protein spots of differential expression in two season gels were excised and subjected to in-gel digestion and identification by matrix assisted laser desorption/ionization-time of flight/time of flight-mass spectrum (MALDI-TOF/TOF-MS) analysis. Two differentially expressed proteins, lactoferrin (LF) and vitelline membrane outer layer protein 1 homolog (VMO1 homolog), were validated by western blotting. Results Thirteen well resolved bands were detected in SDS–PAGE gels of both summer and winter camel tears. By band densitometry, significantly higher intensities of band 6, 7, 11, and lower intensity of band 13 were observed in the summer group compared to the winter group. In 2-DE profiles of camel tears, four protein spots were found expressed differentially in two seasons. Further protein identification by MALDI-TOF/TOF-MS and confirmation by western blotting indicated that there was a significant decrease in LF (p=0.002) and an increase in VMO1 homolog (p=0.042) in tears in the summer compared to the winter. Conclusions The seasonal variation of camel tear fluids has been found in the composition of proteins, including LF and VMO1 homolog. This result will expand our knowledge of physiologic characteristics of tear fluids and establish a foundation for the mechanistic studies and clinical practices on ocular surface disorders. PMID:21293736

Microarray Analysis of Tomato Plants Exposed to the Nonviruliferous or Viruliferous Whitefly Vector Harboring Pepper golden mosaic virus

PubMed Central

Musser, Richard O.; Hum-Musser, Sue M.; Gallucci, Matthew; DesRochers, Brittany; Brown, Judith K.

2014-01-01

Abstract Plants are routinely exposed to biotic and abiotic stresses to which they have evolved by synthesizing constitutive and induced defense compounds. Induced defense compounds are usually made, initially, at low levels; however, following further stimulation by specific kinds of biotic and abiotic stresses, they can be synthesized in relatively large amounts to abate the particular stress. cDNA microarray hybridization was used to identify an array of genes that were differentially expressed in tomato plants 15 d after they were exposed to feeding by nonviruliferous whiteflies or by viruliferous whiteflies carrying Pepper golden mosaic virus (PepGMV) ( Begomovirus, Geminiviridae ). Tomato plants inoculated by viruliferous whiteflies developed symptoms characteristic of PepGMV, whereas plants exposed to nonviruliferous whitefly feeding or nonwounded (negative) control plants exhibited no disease symptoms. The microarray analysis yielded over 290 spotted probes, with significantly altered expression of 161 putative annotated gene targets, and 129 spotted probes of unknown identities. The majority of the differentially regulated “known” genes were associated with the plants exposed to viruliferous compared with nonviruliferous whitefly feeding. Overall, significant differences in gene expression were represented by major physiological functions including defense-, pathogen-, photosynthesis-, and signaling-related responses and were similar to genes identified for other insect–plant systems. Viruliferous whitefly-stimulated gene expression was validated by real-time quantitative polymerase chain reaction of selected, representative candidate genes (messenger RNA): arginase, dehydrin, pathogenesis-related proteins 1 and -4, polyphenol oxidase, and several protease inhibitors. This is the first comparative profiling of the expression of tomato plants portraying different responses to biotic stress induced by viruliferous whitefly feeding (with resultant virus infection) compared with whitefly feeding only and negative control nonwounded plants exposed to neither. These results may be applicable to many other plant–insect–pathogen system interactions. PMID:25525099

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer's disease

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

Chang, Seong-Hun; Jung, In-Soo; Han, Gi-Yeon

Highlights: Black-Right-Pointing-Pointer A transgenic mouse model expressing NSE-htau23 was used. Black-Right-Pointing-Pointer 2D-gel electrophoresis to analyze the cortex proteins of transgenic mice was used. Black-Right-Pointing-Pointer Differentially expressed spots in different stages of AD were identified. Black-Right-Pointing-Pointer GSTP1 and CAII were downregulated with the progression of AD. Black-Right-Pointing-Pointer SCRN1 and ATP6VE1 were up regulated and down regulated differentially. -- Abstract: Alzheimer's disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between taumore » and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal degeneration in AD.« less

Proteomic analysis of Frankliniella occidentalis and differentially expressed proteins in response to tomato spotted wilt virus infection.

PubMed

Badillo-Vargas, I E; Rotenberg, D; Schneweis, D J; Hiromasa, Y; Tomich, J M; Whitfield, A E

2012-08-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response to Tomato Spotted Wilt Virus Infection

PubMed Central

Badillo-Vargas, I. E.; Rotenberg, D.; Schneweis, D. J.; Hiromasa, Y.; Tomich, J. M.

2012-01-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction. PMID:22696645

Increased AAA-TOB3 correlates with lymph node metastasis and advanced stage of lung adenocarcinoma.

PubMed

Liu, Yanfeng; Bu, Lina; Li, Wei; Wu, Wei; Wang, Shengyu; Diao, Xin; Zhou, Jing; Chen, Guoan; Yang, Shuanying

2017-07-24

This study was to investigate the differential mitochondrial protein expressions in human lung adenocarcinoma and provide preliminary data for further exploration of the carcinogenic mechanism. Total proteins of A549 and 16HBE mitochondria were extracted through 2D polyacrylamide gel electrophoresis (2-DE). The differential mitochondria proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were further confirmed by Western blot, immunoelectron microscopy and immunohistochemistry (IHC) in A549 cells as well as lung adenocarcinoma tissues. A total of 41 differentially expressed protein spots were found in A549 mitochondria. Of them, 15 proteins were highly expressed and 26 proteins were lowly expressed in the mitochondria of A549 (by more than 1.5 times). Among the 15 more highly expressed proteins, AAA-TOB3 (by more than 3 times) was highly expressed in the mitochondria of A549 compared with the 16HBE, by LC-MS/MS identification. High electron density and clear circular colloidal gold-marked AAA-TOB3 particles were observed in the A549 cells via immunoelectron microscopy. Besides, AAA-TOB3 was confirmed to be elevated in lung adenocarcinoma by Western blot and IHC. Moreover, increased AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma (p<0.05). AAA-TOB3 was highly expressed in lung adenocarcinoma, and the up-regulation of AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma, which suggested that it could serve as a potential molecular marker for lung adenocarcinoma.

Genetic changes in muscle protein following hybridization between Haliotis diversicolor reeve Japan and Taiwan populations revealed using a proteomic approach.

PubMed

Di, Guilan; You, Weiwei; Yu, Jinjin; Wang, Dexiang; Ke, Caihuan

2013-03-01

Protein expression patterns were compared in a Japan and Taiwan population of Haliotis diversicolor and in a hybrid between them using 2DE and MALDI-TOF-TOF analyses. Using the software PDQuest, 924 ± 7 protein spots were detected in the Japan population (RR), 861 ± 11 in the Taiwan population (TT), and 882 ± 9 in the F1 hybrid (TR). RR and TR were clustered together, but the distance between RR and TT was the maximum using hierarchical cluster analysis. A total of 46 gel spots were identified and a total of 15 spots matched with abalone proteins (a 33.6% identification rate). Hybrid exhibiting additivity or overdominance accounted for 73.9% of these 46 identified proteins. The 46 differentially expressed proteins were shown to be involved in major biological processes, including muscle contraction and regulation, energy metabolism, and stress response. The proteins involved in energy metabolism included adenosine triphosphate (ATP) synthase β subunit, fructose 1, 6-bisphosphate aldolase, triosephosphate isomerase, enolase, arginine kinase, and tauropine dehydrogenase. These proteins exhibited additivity in their offspring. The proteins involved in stress responses included HSP Hsp70 (exhibiting overdominance in the offspring) and Cu/Zn-superoxide dismutase (exhibiting additivity). These results suggested that proteomic approach is suitable for analysis of heterosis and functional prediction of abalone hybridization. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative proteomic analysis of off-type and normal phenotype somatic plantlets derived from somatic embryos of Feijoa (Acca sellowiana (O. Berg) Burret).

PubMed

Fraga, Hugo Pacheco de Freitas; Agapito-Tenfen, Sarah Zanon; Caprestano, Clarissa Alves; Nodari, Rubens Onofre; Guerra, Miguel Pedro

2013-09-01

Morphological disorders in a relevant portion of emerged somatic embryos have been a limiting factor in the true-to-type plantlet formation in Acca sellowiana. In this sense, the present study undertook a comparison between normal phenotype and off-type somatic plantlets protein profiles by means of the 2-D DIGE proteomics approach. Off-type and normal phenotype somatic plantlets obtained at 10 and 20 days conversion were evaluated. Results indicated 12 exclusive spots between normal and off-type plantlets at 10 days conversion, and 17 exclusive spots at 20 days conversion. Also at 20 days conversion, 4 spots were differentially expressed, up- or down-regulated. Two proteins related to carbohydrate metabolism were only expressed in off-types at 10 days conversion, suggesting a more active respiratory pathway. A vicilin-like storage protein was only found in off-types at 20 days conversion, indicating that plantlets may present an abnormality in the mobilization of storage compounds, causing reduced vigor in the development of derived plantlets. The presence of heat shock proteins were only observed during formation of normal phenotype somatic plantlets, indicating that these proteins may be involved in normal morphogenesis of plantlets formed. These new findings shed light on possible genetic or epigenetic mechanisms governing A. sellowiana morphogenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Quantitative proteomics analysis highlights the role of redox hemostasis and energy metabolism in human embryonic stem cell differentiation to neural cells.

PubMed

Fathi, Ali; Hatami, Maryam; Vakilian, Haghighat; Han, Chia-Li; Chen, Yu-Ju; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

2014-04-14

Neural differentiation of human embryonic stem cells (hESCs) is a unique opportunity for in vitro analyses of neurogenesis in humans. Extrinsic cues through neural plate formation are well described in the hESCs although intracellular mechanisms underlying neural development are largely unknown. Proteome analysis of hESC differentiation to neural cells will help to further define molecular mechanisms involved in neurogenesis in humans. Using a two-dimensional differential gel electrophoresis (2D-DIGE) system, we analyzed the proteome of hESC differentiation to neurons at three stages, early neural differentiation, neural ectoderm and mature neurons. Out of 137 differentially accumulated protein spots, 118 spots were identified using MALDI-TOF/TOF and LC MS/MS. We observed that proteins involved in redox hemostasis, vitamin and energy metabolism and ubiquitin dependent proteolysis were more abundant in differentiated cells, whereas the abundance of proteins associated with RNA processing and protein folding was higher in hESCs. Higher abundance of proteins involved in maintaining cellular redox state suggests the importance of redox hemostasis in neural differentiation. Furthermore, our results support the concept of a coupling mechanism between neuronal activity and glucose utilization. The protein network analysis showed that the majority of the interacting proteins were associated with the cell cycle and cellular proliferation. These results enhanced our understanding of the molecular dynamics that underlie neural commitment and differentiation. In highlighting the role of redox and unique metabolic properties of neuronal cells, the present findings add insight to our understanding of hESC differentiation to neurons. The abundance of fourteen proteins involved in maintaining cellular redox state, including 10 members of peroxiredoxin (Prdx) family, mainly increased during differentiation, thus highlighting a link of neural differentiation to redox. Our results revealed markedly higher expression of genes encoding enzymes involved in the glycolysis and amino acid synthesis during differentiation. Protein network analysis predicted a number of critical mediators in hESC differentiation. These proteins included TP53, CTNNB1, SMARCA4, TNF, TERT, E2F1, MYC, RB1, and AR. Copyright © 2014 Elsevier B.V. All rights reserved.

From a 2DE-gel spot to protein function: lesson learned from HS1 in chronic lymphocytic leukemia.

PubMed

Apollonio, Benedetta; Bertilaccio, Maria Teresa Sabrina; Restuccia, Umberto; Ranghetti, Pamela; Barbaglio, Federica; Ghia, Paolo; Caligaris-Cappio, Federico; Scielzo, Cristina

2014-10-19

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease's biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as "spots" on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

A Whole-Tumor Histogram Analysis of Apparent Diffusion Coefficient Maps for Differentiating Thymic Carcinoma from Lymphoma.

PubMed

Zhang, Wei; Zhou, Yue; Xu, Xiao-Quan; Kong, Ling-Yan; Xu, Hai; Yu, Tong-Fu; Shi, Hai-Bin; Feng, Qing

2018-01-01

To assess the performance of a whole-tumor histogram analysis of apparent diffusion coefficient (ADC) maps in differentiating thymic carcinoma from lymphoma, and compare it with that of a commonly used hot-spot region-of-interest (ROI)-based ADC measurement. Diffusion weighted imaging data of 15 patients with thymic carcinoma and 13 patients with lymphoma were retrospectively collected and processed with a mono-exponential model. ADC measurements were performed by using a histogram-based and hot-spot-ROI-based approach. In the histogram-based approach, the following parameters were generated: mean ADC (ADC mean ), median ADC (ADC median ), 10th and 90th percentile of ADC (ADC 10 and ADC 90 ), kurtosis, and skewness. The difference in ADCs between thymic carcinoma and lymphoma was compared using a t test. Receiver operating characteristic analyses were conducted to determine and compare the differentiating performance of ADCs. Lymphoma demonstrated significantly lower ADC mean , ADC median , ADC 10 , ADC 90 , and hot-spot-ROI-based mean ADC than those found in thymic carcinoma (all p values < 0.05). There were no differences found in the kurtosis ( p = 0.412) and skewness ( p = 0.273). The ADC 10 demonstrated optimal differentiating performance (cut-off value, 0.403 × 10 -3 mm 2 /s; area under the receiver operating characteristic curve [AUC], 0.977; sensitivity, 92.3%; specificity, 93.3%), followed by the ADC mean , ADC median , ADC 90 , and hot-spot-ROI-based mean ADC. The AUC of ADC 10 was significantly higher than that of the hot spot ROI based ADC (0.977 vs. 0.797, p = 0.036). Compared with the commonly used hot spot ROI based ADC measurement, a histogram analysis of ADC maps can improve the differentiating performance between thymic carcinoma and lymphoma.

Molecular cloning, characterization and functional analysis of QRFP in orange-spotted grouper (Epinephelus coioides).

PubMed

Shu, Hu; Chen, Huapu; Liu, Yun; Yang, Lidong; Yang, Yuqing; Zhang, Haifa

2014-10-01

The peptide QRFP plays an important role in the regulation of vertebrate feeding behavior. In this study, we cloned the full length cDNA of a QRFP precursor in a teleost fish, the orange-spotted grouper (Epinephelus coioides). Sequence analysis has shown that the functional regions of QRFP in other vertebrates (QRFP-25 and QRFP-7) are conserved in orange-spotted grouper. RT-PCR demonstrated that the pre-processed mRNA of QRFP is widely expressed in orange-spotted grouper. Three days of food deprivation did not change the hypothalamic pre-processed QRFP expression. However, QRFP expression significantly increased when the fish were reefed after three days of fasting. Intraperitoneal injection of QRFP-25 peptide to orange-spotted grouper suppressed expression of orexin, but elevated expression of pro-opiomelanocortin (POMC) in the hypothalamus. We also investigated the effects of QRFP-25 on the expression of reproductive genes. The peptide suppressed the expression of seabream-type gonadotropin-releasing hormones (sbGnRH), luteinizing hormone beta subunit (LHβ) and follicle-stimulating hormone beta subunit (FSHβ) in vivo, as well as inhibited the expression of LHβ and FSHβ in pituitary cells in primary culture. Our results indicate that QRFP may play an inhibitory role in the regulation of feeding behavior and reproduction in orange-spotted grouper. Copyright © 2014 Elsevier Inc. All rights reserved.

Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

PubMed

Wang, Ruo-Chiau; Huang, Chien-Yu; Pan, Tai-Long; Chen, Wei-Yu; Ho, Chun-Te; Liu, Tsan-Zon; Chang, Yu-Jia

2015-01-01

To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

Proteomic analysis of blue light-induced twining response in Cuscuta australis.

PubMed

Li, Dongxiao; Wang, Liangjiang; Yang, Xiaopo; Zhang, Guoguang; Chen, Liang

2010-01-01

The parasitic plant Cuscuta australis (dodder) invades a variety of species by entwining the stem and leaves of a host and developing haustoria. The twining response prior to haustoria formation is regarded as the first sign for dodders to parasitize host plants, and thus has been the focus of studies on the host-parasite interaction. However, the molecular mechanism is still poorly understood. In the present work, we have investigated the different effects of blue and white light on the twining response, and identified a set of proteins that were differentially expressed in dodder seedlings using a proteomic approach. Approximately 1,800 protein spots were detected on each 2-D gel, and 47 spots with increased or decreased protein levels were selected and analyzed with MALDI-TOF-MS. Peptide mass fingerprints (PMFs) obtained for these spots were used for protein identification through cross-species database searches. The results suggest that the blue light-induced twining response in dodder seedlings may be mediated by proteins involved in light signal transduction, cell wall degradation, cell structure, and metabolism.

Proteomics-based approach identified differentially expressed proteins with potential roles in endometrial carcinoma.

PubMed

Li, Zhengyu; Min, Wenjiao; Huang, Canhua; Bai, Shujun; Tang, Minghai; Zhao, Xia

2010-01-01

We used proteomic approaches to identify altered expressed proteins in endometrial carcinoma, with the aim of discovering potential biomarkers or therapeutic targets for endometrial carcinoma. The global proteins extracted from endometrial carcinoma and normal endometrial tissues were separated by 2-dimensional electrophoresis and analyzed with PDQuest (Bio-Rad, Hercules, Calif) software. The differentially expressed spots were identified by mass spectrometry and searched against NCBInr protein database. Those proteins with potential roles were confirmed by Western blotting and immunohistochemical assays. Ninety-nine proteins were identified by mass spectrometry, and a cluster diagram analysis indicated that these proteins were involved in metabolism, cell transformation, protein folding, translation and modification, proliferation and apoptosis, signal transduction, cytoskeleton, and so on. In confirmatory immunoblotting and immunohistochemical analyses, overexpressions of epidermal fatty acid-binding protein, calcyphosine, and cyclophilin A were also observed in endometrial carcinoma tissues, which were consistent with the proteomic results. Our results suggested that these identified proteins, including epidermal fatty acid-binding protein, calcyphosine, and cyclophilin A, might be of potential values in the studies of endometrial carcinogenesis or investigations of diagnostic biomarkers or treatment targets for endometrial carcinoma.

Analysis of differentially regulated proteins in TM4 cells treated with bisphenol A.

PubMed

Lee, Do-Youn; Lee, Sang-Soo; Joo, Won-A; Lee, Eun-Ju; Kim, Chan-Wha

2004-06-01

BPA, bisphenol A, a monomer of epoxy resins and polycarbonate plastic, is used in many consumer products including the plastic linings of cans for food and babies' bottles. BPA has been reported to cause reproductive toxicity and affects cells in rats and mice at high doses. In this study, the effect of BPA on protein expression in TM4 cells (a mouse Sertoli cell line) known to play an essential role in Spermatogenesis was investigated by two-dimensional electrophoresis (2-DE). After 16 h exposure to 50, 100, 150, 200, and 250 microM of BPA, the viability of TM4 cells decreased to about 90, 85, 78, 55, and 30% of control respectively. Approximately 800 protein spots in TM4 cells were analyzed by 2-DE with pH 4-7 linear immobilized pH gradient (IPG) Dry Strip, and 11 proteins which showed significantly different expression levels were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Among these, HSP 27 and placental calcium binding protein may be proteins differentially expressed by BPA exposure.

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.

Specifying and Sustaining Pigmentation Patterns in Domestic and Wild Cats

PubMed Central

Kaelin, Christopher B.; Xu, Xiao; Hong, Lewis Z.; David, Victor A.; McGowan, Kelly A.; Schmidt-Küntzel, Anne; Roelke, Melody E.; Pino, Javier; Pontius, Joan; Cooper, Gregory M.; Manuel, Hermogenes; Swanson, William F.; Marker, Laurie; Harper, Cindy K.; van Dyk, Ann; Yue, Bisong; Mullikin, James C.; Warren, Wesley C.; Eizirik, Eduardo; Kos, Lidia; O’Brien, Stephen J.; Barsh, Gregory S.; Menotti-Raymond, Marilyn

2013-01-01

Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3. PMID:22997338

Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage

PubMed Central

Shi, Yun; Jiang, Li; Zhang, Li; Kang, Ruoyi; Yu, Zhifang

2014-01-01

A proteomic study, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight, was conducted in apple fruit (cv. ‘Golden Delicious’) starting at 10 days prior to harvest through 50 days in storage. Total protein was extracted using a phenol/sodium dodecyl sulfate protocol. More than 400 protein spots were detected in each gel and 55 differentially expressed proteins (p<0.05) were subjected to matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analysis. Fifty-three of these proteins were finally identified using an apple expressed sequence tag database downloaded from Genome Database for Rosaceae and placed into six categories. The categories and the percentage of proteins placed in each category were stress response and defense (49.0%), energy and metabolism (34.0%), fruit ripening and senescence (5.6%), signal transduction (3.8%), cell structure (3.8%) and protein synthesis (3.8%). Proteins involved in several multiple metabolic pathways, including glycolysis, pentose–phosphate pathway, anti-oxidative systems, photosynthesis and cell wall synthesis, were downregulated, especially during the climacteric burst in respiration and during the senescent stages of fruit development. Proteins classified as allergens or involved in cell wall degradation were upregulated during the ripening process. Some protein spots exhibited a mixed pattern (increasing to maximal abundance followed by a decrease), such as 1-aminocyclopropane-1-carboxylate oxidase, L-ascorbate peroxidase and abscisic acid response proteins. The identification of differentially expressed proteins associated with physiological processes identified in the current study provides a baseline of information for understanding the metabolic processes and regulatory mechanisms that occur in climacteric apple fruit during ripening and senescence. PMID:26504530

Proteome profiling reveals insights into cold-tolerant growth in sea buckthorn.

PubMed

He, Caiyun; Gao, Guori; Zhang, Jianguo; Duan, Aiguo; Luo, Hongmei

2016-01-01

Low temperature is one of the crucial environmental factors limiting the productivity and distribution of plants. Sea buckthorn ( Hippophae rhamnoides L.), a well recognized multipurpose plant species, live successfully in in cold desert regions. But their molecular mechanisms underlying cold tolerance are not well understood. Physiological and biochemical responses to low-temperature stress were studied in seedlings of sea buckthorn. Differentially expressed protein spots were analyzed using multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometry (MS), the concentration of phytohormone was measured using enzyme-linked immunosorbent assay, and a spectrophotometric assay was used to measure enzymatic reactions. With the increase of cold stress intensity, the photosynthesis rate, transpiration rate, stomatal conductance in leaves and contents of abscisic acid (ABA) and indole acetic acid (IAA) in roots decreased significantly; however, water-use efficiency, ABA and zeatin riboside in leaves increased significantly, while cell membrane permeability, malondialdehyde and IAA in leaves increased at 7 d and then decreased at 14 d. DIGE and MS/MS analysis identified 32 of 39 differentially expressed protein spots under low-temperature stress, and their functions were mainly involved in metabolism, photosynthesis, signal transduction, antioxidative systems and post-translational modification. The changed protein abundance and corresponding physiological-biochemical response shed light on the molecular mechanisms related to cold tolerance in cold-tolerant plants and provide key candidate proteins for genetic improvement of plants.

Proteomic identification of fat-browning markers in cultured white adipocytes treated with curcumin.

PubMed

Kim, Sang Woo; Choi, Jae Heon; Mukherjee, Rajib; Hwang, Ki-Chul; Yun, Jong Won

2016-04-01

We previously reported that curcumin induces browning of primary white adipocytes via enhanced expression of brown adipocyte-specific genes. In this study, we attempted to identify target proteins responsible for this fat-browning effect by analyzing proteomic changes in cultured white adipocytes in response to curcumin treatment. To elucidate the role of curcumin in fat-browning, we conducted comparative proteomic analysis of primary adipocytes between control and curcumin-treated cells using two-dimensional electrophoresis combined with MALDI-TOF-MS. We also investigated fatty acid metabolic targets, mitochondrial biogenesis, and fat-browning-associated proteins using combined proteomic and network analyses. Proteomic analysis revealed that 58 protein spots from a total of 325 matched spots showed differential expression between control and curcumin-treated adipocytes. Using network analysis, most of the identified proteins were proven to be involved in various metabolic and cellular processes based on the PANTHER classification system. One of the most striking findings is that hormone-sensitive lipase (HSL) was highly correlated with main browning markers based on the STRING database. HSL and two browning markers (UCP1, PGC-1α) were co-immunoprecipitated with these markers, suggesting that HSL possibly plays a role in fat-browning of white adipocytes. Our results suggest that curcumin increased HSL levels and other browning-specific markers, suggesting its possible role in augmentation of lipolysis and suppression of lipogenesis by trans-differentiation from white adipocytes into brown adipocytes (beige).

Effect of storage time on gene expression data acquired from unfrozen archived newborn blood spots.

PubMed

Ho, Nhan T; Busik, Julia V; Resau, James H; Paneth, Nigel; Khoo, Sok Kean

2016-11-01

Unfrozen archived newborn blood spots (NBS) have been shown to retain sufficient messenger RNA (mRNA) for gene expression profiling. However, the effect of storage time at ambient temperature for NBS samples in relation to the quality of gene expression data is relatively unknown. Here, we evaluated mRNA expression from quantitative real-time PCR (qRT-PCR) and microarray data obtained from NBS samples stored at ambient temperature to determine the effect of storage time on the quality of gene expression. These data were generated in a previous case-control study examining NBS in 53 children with cerebral palsy (CP) and 53 matched controls. NBS sample storage period ranged from 3 to 16years at ambient temperature. We found persistently low RNA integrity numbers (RIN=2.3±0.71) and 28S/18S rRNA ratios (~0) across NBS samples for all storage periods. In both qRT-PCR and microarray data, the expression of three common housekeeping genes-beta cytoskeletal actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA)-decreased with increased storage time. Median values of each microarray probe intensity at log 2 scale also decreased over time. After eight years of storage, probe intensity values were largely reduced to background intensity levels. Of 21,500 genes tested, 89% significantly decreased in signal intensity, with 13,551, 10,730, and 9925 genes detected within 5years, > 5 to <10years, and >10years of storage, respectively. We also examined the expression of two gender-specific genes (X inactivation-specific transcript, XIST and lysine-specific demethylase 5D, KDM5D) and seven gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways hypothesized to be related to CP risk to determine the effect of storage time on the detection of these biologically relevant genes. We found the gender-specific genes and CP-related gene sets detectable in all storage periods, but exhibited differential expression (between male vs. female or CP vs. control) only within the first six years of storage. We concluded that gene expression data quality deteriorates in unfrozen archived NBS over time and that differential gene expression profiling and analysis is recommended for those NBS samples collected and stored within six years at ambient temperature. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel seminal fluid proteins in the seed beetle Callosobruchus maculatus identified by a proteomic and transcriptomic approach.

PubMed

Bayram, H; Sayadi, A; Goenaga, J; Immonen, E; Arnqvist, G

2017-02-01

The seed beetle Callosobruchus maculatus is a significant agricultural pest and increasingly studied model of sexual conflict. Males possess genital spines that increase the transfer of seminal fluid proteins (SFPs) into the female body. As SFPs alter female behaviour and physiology, they are likely to modulate reproduction and sexual conflict in this species. Here, we identified SFPs using proteomics combined with a de novo transcriptome. A prior 2D-sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis identified male accessory gland protein spots that were probably transferred to the female at mating. Proteomic analysis of these spots identified 98 proteins, a majority of which were also present within ejaculates collected from females. Standard annotation workflows revealed common functional groups for SFPs, including proteases and metabolic proteins. Transcriptomic analysis found 84 transcripts differentially expressed between the sexes. Notably, genes encoding 15 proteins were highly expressed in male abdomens and only negligibly expressed within females. Most of these sequences corresponded to 'unknown' proteins (nine of 15) and may represent rapidly evolving SFPs novel to seed beetles. Our combined analyses highlight 44 proteins for which there is strong evidence that they are SFPs. These results can inform further investigation, to better understand the molecular mechanisms of sexual conflict in seed beetles. © 2016 The Royal Entomological Society.

Down-regulate of Djrfc2 causes tissues hypertrophy during planarian regeneration.

PubMed

Guo, Qi; Zhao, Guixia; Ni, Jiajia; Guo, Yanan; Zhang, Yizhe; Tian, Qingnan; Zhang, Shoutao

2017-11-25

Planarians are an ideal model organism for regeneration research due to their amazing ability to regenerate. DNA replication is crucial for genome stability. Replication factor C (RFC), which is a replication factor C-like complex and plays an important role during DNA replication in eukaryotes, has been reported as a wound response factor during planarian regeneration. However, how RFC controls regeneration in planarians by regulating DNA replication remains to be explained. Here, we used a two-dimensional electrophoresis (2-DE) proteomic approach to identify differentially expressed proteins in intact and regenerated planarians. Approximately 132 protein spots showed differences between intact and regenerative tissues. We selected 21 significantly expressed protein spots and processed them using TOF MS analysis. Finally, we cloned three of these candidate genes (Djhsp70, Djrfc2, Djfaim), focusing on the function of Djrfc2 during regeneration. We found that the distribution of Djrfc2 tends toward the wound site. RNA interference (RNAi) of Djrfc2 increases the number of dividing cells and the expression level of planarian neoblast marker genes, which may result in hyper-proliferation. Our studies use an available approach to directly study the regeneration dynamic at the protein level and provide further evidence to support a function of Djrfc2 in planarian regeneration. Copyright © 2017. Published by Elsevier Inc.

Identification of serum biomarkers in dogs naturally infected with Babesia canis canis using a proteomic approach

PubMed Central

2014-01-01

Background Canine babesiosis is a tick-borne disease that is caused by the haemoprotozoan parasites of the genus Babesia. There are limited data on serum proteomics in dogs, and none of the effect of babesiosis on the serum proteome. The aim of this study was to identify the potential serum biomarkers of babesiosis using proteomic techniques in order to increase our understanding about disease pathogenesis. Results Serum samples were collected from 25 dogs of various breeds and sex with naturally occurring babesiosis caused by B. canis canis. Blood was collected on the day of admission (day 0), and subsequently on the 1st and 6th day of treatment. Two-dimensional electrophoresis (2DE) of pooled serum samples of dogs with naturally occurring babesiosis (day 0, day 1 and day 6) and healthy dogs were run in triplicate. 2DE image analysis showed 64 differentially expressed spots with p ≤ 0.05 and 49 spots with fold change ≥2. Six selected spots were excised manually and subjected to trypsin digest prior to identification by electrospray ionisation mass spectrometry on an Amazon ion trap tandem mass spectrometry (MS/MS). Mass spectrometry data was processed using Data Analysis software and the automated Matrix Science Mascot Daemon server. Protein identifications were assigned using the Mascot search engine to interrogate protein sequences in the NCBI Genbank database. A number of differentially expressed serum proteins involved in inflammation mediated acute phase response, complement and coagulation cascades, apolipoproteins and vitamin D metabolism pathway were identified in dogs with babesiosis. Conclusions Our findings confirmed two dominant pathogenic mechanisms of babesiosis, haemolysis and acute phase response. These results may provide possible serum biomarker candidates for clinical monitoring of babesiosis and this study could serve as the basis for further proteomic investigations in canine babesiosis. PMID:24885808

Identification of Isolates that Cause a Leaf Spot Disease of Brassicas as Xanthomonas campestris pv. raphani and Pathogenic and Genetic Comparison with Related Pathovars.

PubMed

Vicente, J G; Everett, B; Roberts, S J

2006-07-01

ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

PubMed

Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

2009-07-01

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

Ethylene-induced differential gene expression during abscission of citrus leaves

PubMed Central

Merelo, Paz; Cercós, Manuel; Tadeo, Francisco R.; Talón, Manuel

2008-01-01

The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission. PMID:18515267

Identification and handling of artifactual gene expression profiles emerging in microarray hybridization experiments

PubMed Central

Brodsky, Leonid; Leontovich, Andrei; Shtutman, Michael; Feinstein, Elena

2004-01-01

Mathematical methods of analysis of microarray hybridizations deal with gene expression profiles as elementary units. However, some of these profiles do not reflect a biologically relevant transcriptional response, but rather stem from technical artifacts. Here, we describe two technically independent but rationally interconnected methods for identification of such artifactual profiles. Our diagnostics are based on detection of deviations from uniformity, which is assumed as the main underlying principle of microarray design. Method 1 is based on detection of non-uniformity of microarray distribution of printed genes that are clustered based on the similarity of their expression profiles. Method 2 is based on evaluation of the presence of gene-specific microarray spots within the slides’ areas characterized by an abnormal concentration of low/high differential expression values, which we define as ‘patterns of differentials’. Applying two novel algorithms, for nested clustering (method 1) and for pattern detection (method 2), we can make a dual estimation of the profile’s quality for almost every printed gene. Genes with artifactual profiles detected by method 1 may then be removed from further analysis. Suspicious differential expression values detected by method 2 may be either removed or weighted according to the probabilities of patterns that cover them, thus diminishing their input in any further data analysis. PMID:14999086

A data processing method based on tracking light spot for the laser differential confocal component parameters measurement system

NASA Astrophysics Data System (ADS)

Shao, Rongjun; Qiu, Lirong; Yang, Jiamiao; Zhao, Weiqian; Zhang, Xin

2013-12-01

We have proposed the component parameters measuring method based on the differential confocal focusing theory. In order to improve the positioning precision of the laser differential confocal component parameters measurement system (LDDCPMS), the paper provides a data processing method based on tracking light spot. To reduce the error caused by the light point moving in collecting the axial intensity signal, the image centroiding algorithm is used to find and track the center of Airy disk of the images collected by the laser differential confocal system. For weakening the influence of higher harmonic noises during the measurement, Gaussian filter is used to process the axial intensity signal. Ultimately the zero point corresponding to the focus of the objective in a differential confocal system is achieved by linear fitting for the differential confocal axial intensity data. Preliminary experiments indicate that the method based on tracking light spot can accurately collect the axial intensity response signal of the virtual pinhole, and improve the anti-interference ability of system. Thus it improves the system positioning accuracy.

Interplay of wavelength, fluence and spot-size in free-electron laser ablation of cornea.

PubMed

Hutson, M Shane; Ivanov, Borislav; Jayasinghe, Aroshan; Adunas, Gilma; Xiao, Yaowu; Guo, Mingsheng; Kozub, John

2009-06-08

Infrared free-electron lasers ablate tissue with high efficiency and low collateral damage when tuned to the 6-microm range. This wavelength-dependence has been hypothesized to arise from a multi-step process following differential absorption by tissue water and proteins. Here, we test this hypothesis at wavelengths for which cornea has matching overall absorption, but drastically different differential absorption. We measure etch depth, collateral damage and plume images and find that the hypothesis is not confirmed. We do find larger etch depths for larger spot sizes--an effect that can lead to an apparent wavelength dependence. Plume imaging at several wavelengths and spot sizes suggests that this effect is due to increased post-pulse ablation at larger spots.

Proteomics, bioinformatics and targeted gene expression analysis reveals up-regulation of cochlin and identifies other potential biomarkers in the mouse model for deafness in usher syndrome type 1F

PubMed Central

Chance, Mark R.; Chang, Jinsook; Liu, Shuqing; Gokulrangan, Giridharan; Chen, Daniel H.-C.; Lindsay, Aaron; Geng, Ruishuang; Zheng, Qing Y.; Alagramam, Kumar

2010-01-01

Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model. PMID:20097680

Roth spots in pernicious anaemia

PubMed Central

Macauley, Mavin; Nag, Satyajit

2011-01-01

Roth spots are white-centred retinal haemorrhages, previously thought to be pathognomonic for subacute bacterial endocarditis. A number of other conditions can be associated with Roth spots. In this case, the authors describe the association of Roth spots and pernicious anaemia. This association has been rarely described in the medical literature. Correct diagnosis and treatment with intramuscular vitamin B12 injections resulted in complete resolution of the anaemia and Roth spots. The authors hope to alert clinicians to think of various differentials of Roth spots, and initiate prompt investigation and management. PMID:22696623

Roth spots in pernicious anaemia.

PubMed

Macauley, Mavin; Nag, Satyajit

2011-04-19

Roth spots are white-centred retinal haemorrhages, previously thought to be pathognomonic for subacute bacterial endocarditis. A number of other conditions can be associated with Roth spots. In this case, the authors describe the association of Roth spots and pernicious anaemia. This association has been rarely described in the medical literature. Correct diagnosis and treatment with intramuscular vitamin B(12) injections resulted in complete resolution of the anaemia and Roth spots. The authors hope to alert clinicians to think of various differentials of Roth spots, and initiate prompt investigation and management.

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

PubMed Central

Marraccini, Pierre; Vinecky, Felipe; Alves, Gabriel S.C.; Ramos, Humberto J.O.; Elbelt, Sonia; Vieira, Natalia G.; Carneiro, Fernanda A.; Sujii, Patricia S.; Alekcevetch, Jean C.; Silva, Vânia A.; DaMatta, Fábio M.; Ferrão, Maria A.G.; Leroy, Thierry; Pot, David; Vieira, Luiz G.E.; da Silva, Felipe R.; Andrade, Alan C.

2012-01-01

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora. PMID:22511801

Molecular identification of an androgen receptor and its changes in mRNA levels during 17α-methyltestosterone-induced sex reversal in the orange-spotted grouper Epinephelus coioides.

PubMed

Shi, Yu; Liu, Xiaochun; Zhang, Haifa; Zhang, Yong; Lu, Danqi; Lin, Haoran

2012-09-01

Androgens play a crucial role in sex differentiation, sexual maturation, and spermatogenesis in vertebrates. The action of androgens is mediated via androgen receptors (ARs). The present study reports the cloning of the cDNA sequence of the ar in the orange-spotted grouper, with high expression in testis and relatively low in subdivision of brain areas. The cDNA sequence of ar was 2358 bp, encoding a protein of 759 amino acids (aa). Phylogenetic analysis showed that the ar cDNA sequence was closely related to that of threespot wrasse (Halichoeres trimaculatus) and medaka (Oryzias latipes) arβ. As deduced from the phylogenetic tree and the high amino acid identity with the ARβ subtype of other teleosts, grouper ar seems to be more closely related to the beta than the alpha subtype cloned to date. In the first week after 17α-methyltestosterone (MT) implantation, the transcript levels of ar in the hypothalamus declined significantly, and consistently stayed at low level expression to the second week, but increased back to the control levels in the third and fourth week. In the gonad, the mRNA expression of ar was not changed in the first week compared with the control, but increased significantly in the second week, consistently reached the highest level in the third week, dropped slightly but still higher than that of the control in the fourth week. The expression pattern of ar in hypothalamus and gonad during MT-induced sex reversal suggests the involvement of ar in regulating this process in the orange-spotted grouper. The present study provides the data of the changes in the mRNA levels of ar during MT-induced sex reversal in detail to help understand the complicated signals under sex reversal. Copyright © 2012 Elsevier Inc. All rights reserved.

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

PubMed

Kaewkascholkul, Napol; Somboonviwat, Kulwadee; Asakawa, Shuichi; Hirono, Ikuo; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

2016-07-01

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response. Copyright © 2016. Published by Elsevier Ltd.

Proteomic Analysis of Trypanosoma cruzi Response to Ionizing Radiation Stress

PubMed Central

Vieira, Helaine Graziele Santos; Grynberg, Priscila; Bitar, Mainá; Pires, Simone da Fonseca; Hilário, Heron Oliveira; Macedo, Andrea Mara; Machado, Carlos Renato; de Andrade, Hélida Monteiro; Franco, Glória Regina

2014-01-01

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress. PMID:24842666

A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy.

PubMed

Gaupels, Frank; Knauer, Torsten; van Bel, Aart J E

2008-01-01

This study investigated advantages and drawbacks of two sieve-tube sap sampling methods for comparison of phloem proteins in powdery mildew-infested vs. non-infested Hordeum vulgare plants. In one approach, sieve tube sap was collected by stylectomy. Aphid stylets were cut and immediately covered with silicon oil to prevent any contamination or modification of exudates. In this way, a maximum of 1muL pure phloem sap could be obtained per hour. Interestingly, after pathogen infection exudation from microcauterized stylets was reduced to less than 40% of control plants, suggesting that powdery mildew induced sieve tube-occlusion mechanisms. In contrast to the laborious stylectomy, facilitated exudation using EDTA to prevent calcium-mediated callose formation is quick and easy with a large volume yield. After two-dimensional (2D) electrophoresis, a digital overlay of the protein sets extracted from EDTA solutions and stylet exudates showed that some major spots were the same with both sampling techniques. However, EDTA exudates also contained large amounts of contaminative proteins of unknown origin. A combinatory approach may be most favourable for studies in which the protein composition of phloem sap is compared between control and pathogen-infected plants. Facilitated exudation may be applied for subtractive identification of differentially expressed proteins by 2D/mass spectrometry, which requires large amounts of protein. A reference gel loaded with pure phloem sap from stylectomy may be useful for confirmation of phloem origin of candidate spots by digital overlay. The method provides a novel opportunity to study differential expression of phloem proteins in monocotyledonous plant species.

Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach.

PubMed

Acosta-Muñiz, Carlos H; Escobar-Tovar, Lina; Valdes-Rodríguez, Silvia; Fernández-Pavia, Silvia; Arias-Saucedo, Luis J; de la Cruz Espindola Barquera, Maria; Gómez Lim, Miguel Á

2012-01-01

Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques. Copyright © Physiologia Plantarum 2011.

Proteomic Analysis of Mesenchymal Stem Cells from Normal and Deep Carious Dental Pulp

PubMed Central

Gao, Jie; Yan, Wenjuan; Liu, Ying; Xu, Shuaimei; Wu, Buling

2014-01-01

Dental pulp stem cells (DPSCs), precursor cells of odontoblasts, are ideal seed cells for tooth tissue engineering and regeneration. Our previous study has demonstrated that stem cells exist in dental pulp with deep caries and are called carious dental pulp stem cells (CDPSCs). The results indicated that CDPSCs had a higher proliferative and stronger osteogenic differentiation potential than DPSCs. However, the molecular mechanisms responsible for the biological differences between DPSCs and CDPSCs are poorly understood. The aim of this study was to define the molecular features of DPSCs and CDPSCs by comparing the proteomic profiles using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results revealed that there were 18 protein spots differentially expressed between DPSCs and CDPSCs in a narrow pH range of 4 to 7. These differently expressed proteins are mostly involved in the regulation of cell proliferation, differentiation, cell cytoskeleton and motility. In addition, our results suggested that CDPSCs had a higher expression of antioxidative proteins that might protect CDPSCs from oxidative stress. This study explores some potential proteins responsible for the biological differences between DPSCs and CDPSCs and expands our understanding on the molecular mechanisms of mineralization of DPSCs in the formation of the dentin-pulp complex. PMID:24809979

Comparative and quantitative proteomic analysis of normal and degenerated human annulus fibrosus cells.

PubMed

Ye, Dongping; Liang, Weiguo; Dai, Libing; Zhou, Longqiang; Yao, Yicun; Zhong, Xin; Chen, Honghui; Xu, Jiake

2015-05-01

Degeneration of the intervertebral disc (IVD) is a major chronic medical condition associated with back pain. To better understand the pathogenesis of IVD degeneration, we performed comparative and quantitative proteomic analyses of normal and degenerated human annulus fibrosus (AF) cells and identified proteins that are differentially expressed between them. Annulus fibrosus cells were isolated and cultured from patients with lumbar disc herniation (the experimental group, degenerated AF cells) and scoliosis patients who underwent orthopaedic surgery (the control group, normal AF cells). Comparative proteomic analyses of normal and degenerated cultured AF cells were carried out using 2-D electrophoresis, mass spectrometric analyses, and database searching. Quantitative analyses of silver-stained 2-D electrophoresis gels of normal and degenerated cultured AF cells identified 10 protein spots that showed the most altered differential expression levels between the two groups. Among these, three proteins were decreased, including heat shock cognate 71-kDa protein, glucose-6-phosphate 1-dehydrogenase, and protocadherin-23, whereas seven proteins were increased, including guanine nucleotide-binding protein G(i) subunit α-2, superoxide dismutase, transmembrane protein 51, adenosine receptor A3, 26S protease regulatory subunit 8, lipid phosphate phosphatase-related protein, and fatty acyl-crotonic acid reductase 1. These differentially expressed proteins might be involved in the pathophysiological process of IVD degeneration and have potential values as biomarkers of the degeneration of IVD. © 2015 Wiley Publishing Asia Pty Ltd.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

PubMed

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

PubMed Central

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds. PMID:21994173

EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer.

PubMed

Gemoll, Timo; Kollbeck, Sophie L; Karstens, Karl F; Hò, Gia G; Hartwig, Sonja; Strohkamp, Sarah; Schillo, Katharina; Thorns, Christoph; Oberländer, Martina; Kalies, Kathrin; Lehr, Stefan; Habermann, Jens K

2017-08-15

While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis ® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. Fresh frozen tissue of UCC ( n = 10) matched with SCC ( n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort.

Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

PubMed Central

Chen, Jiandong

2016-01-01

ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

Comparative proteomic analysis of Populus trichocarpa early stem from primary to secondary growth.

PubMed

Liu, Jinwen; Hai, Guanghui; Wang, Chong; Cao, Shenquan; Xu, Wenjing; Jia, Zhigang; Yang, Chuanping; Wang, Jack P; Dai, Shaojun; Cheng, Yuxiang

2015-08-03

Wood is derived from the secondary growth of tree stems. In this study, we investigated the global changes of protein abundance in Populus early stems using a proteomic approach. Morphological and histochemical analyses revealed three typical stages during Populus early stems, which were the primary growth stage, the transition stage from primary to secondary growth and the secondary growth stage. A total of 231 spots were differentially abundant during various growth stages of Populus early stems. During Populus early stem lignifications, 87 differential spots continuously increased, while 49 spots continuously decreased. These two categories encompass 58.9% of all differential spots, which suggests significant molecular changes from primary to secondary growth. Among 231 spots, 165 unique proteins were identified using LC-ESI-Q-TOF-MS, which were classified into 14 biological function groups. The proteomic characteristics indicated that carbohydrate metabolism, oxido-reduction, protein degradation and secondary cell wall metabolism were the dominantly occurring biochemical processes during Populus early stem development. This study helps in elucidating biochemical processes and identifies potential wood formation-related proteins during tree early stem development. It is a comprehensive proteomic investigation on tree early stem development that, for the first time, reveals the overall molecular networks that occur during Populus early stem lignifications. Copyright © 2015. Published by Elsevier B.V.

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

Zhang, Ping; Wang, Ningling; Lin, Xianhua

Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesiclemore » in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.« less

Differential protein accumulations in isolates of the strawberry wilt pathogen Fusarium oxysporum f. sp. fragariae differing in virulence.

PubMed

Fang, Xiangling; Barbetti, Martin J

2014-08-28

This study was conducted to define differences in Fusarium oxysporum f. sp. fragariae (Fof) isolates with different virulence efficiency to strawberry at the proteome level, in combination with their differences in mycelial growth, conidial production and germination. Comparative proteome analyses revealed substantial differences in mycelial proteomes between Fof isolates, where the 54 differentially accumulated protein spots were consistently over-accumulated or exclusively in the highly virulent isolate. These protein spots were identified through MALDI-TOF/TOF mass spectrometry analyses, and the identified proteins were mainly related to primary and protein metabolism, antioxidation, electron transport, cell cycle and transcription based on their putative functions. Proteins of great potential as Fof virulence factors were those involved in ubiquitin/proteasome-mediated protein degradation and reactive oxygen species detoxification; the hydrolysis-related protein haloacid dehalogenase superfamily hydrolase; 3,4-dihydroxy-2-butanone 4-phosphate synthase associated with riboflavin biosynthesis; and those exclusive to the highly virulent isolate. In addition, post-translational modifications may also make an important contribution to Fof virulence. F. oxysporum f. sp. fragariae (Fof), the causal agent of Fusarium wilt in strawberry, is a serious threat to commercial strawberry production worldwide. However, factors and mechanisms contributing to Fof virulence remained unknown. This study provides knowledge of the molecular basis for the differential expression of virulence in Fof, allowing new possibilities towards developing alternative and more effective strategies to manage Fusarium wilt. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of Regulatory Factors for Mesenchymal Stem Cell-Derived Salivary Epithelial Cells in a Co-Culture System

PubMed Central

Park, Yun-Jong; Koh, Jin; Gauna, Adrienne E.; Chen, Sixue; Cha, Seunghee

2014-01-01

Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia. PMID:25402494

Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions.

PubMed

Crespo-Sempere, A; Gil, J V; Martínez-Culebras, P V

2011-06-30

Aspergillus carbonarius is an important ochratoxin A producing fungus that is responsible for mycotoxin contamination of grapes and wine. In this study, the proteomes of highly (W04-40) and weakly (W04-46) OTA-producing A. carbonarius strains were compared to identify proteins that may be involved in OTA biosynthesis. Protein samples were extracted from two biological replicates and subjected to two dimensional gel electrophoresis analysis and mass spectrometry. Expression profile comparison (PDQuest software), revealed 21 differential spots that were statistically significant and showed a two-fold change in expression, or greater. Among these, nine protein spots were identified by MALDI-MS/MS and MASCOT database and twelve remain unidentified. Of the identified proteins, seven showed a higher expression in strain W04-40 (high OTA producer) and two in strain W04-46 (low OTA producer). Some of the identified amino acid sequences shared homology with proteins involved in regulation, amino acid metabolism, oxidative stress and sporulation. It is worth noting the presence of a protein with 126.5 fold higher abundance in strain W04-40 showing homology with protein CipC, a protein with unknown function related with pathogenesis and mycotoxin production by some authors. Variations in protein expression were also further investigated at the mRNA level by real-time PCR analysis. The mRNA expression levels from three identified proteins including CipC showed correlation with protein expression levels. This study represents the first proteomic analysis for a comparison of two A. carbonarius strains with different OTA production and will contribute to a better understanding of the molecular events involved in OTA biosynthesis. Copyright © 2011 Elsevier B.V. All rights reserved.

Protein expression differs between neural progenitor cells from the adult rat brain subventricular zone and olfactory bulb.

PubMed

Maurer, Martin H; Feldmann, Robert E; Bürgers, Heinrich F; Kuschinsky, Wolfgang

2008-01-16

Neural progenitor cells can be isolated from various regions of the adult mammalian brain, including the forebrain structures of the subventricular zone and the olfactory bulb. Currently it is unknown whether functional differences in these progenitor cell populations can already be found on the molecular level. Therefore, we compared protein expression profiles between progenitor cells isolated from the subventricular zone and the olfactory bulb using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry. The subventricular zone and the olfactory bulb are connected by the Rostral Migratory Stream (RMS), in which glial fibrillary acidic protein (GFAP)-positive cells guide neuroblasts. Recent literature suggested that these GFAP-positive cells possess neurogenic potential themselves. In the current study, we therefore compared the cultured neurospheres for the fraction of GFAP-positive cells and their morphology of over a prolonged period of time. We found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures. In this study, we describe differences in protein expression of neural progenitor populations isolated from two forebrain regions, the subventricular zone and the olfactory bulb. These subpopulations can be characterized by differential expression of marker proteins. We isolated fractions of progenitor cells with GFAP expression from both regions, but the GFAP-positive cells differed in number and morphology. Whereas in vitro growth characteristics of neural progenitors are preserved in both regions, our proteomic and immunohistochemical data suggest that progenitor cells from the two regions differ in morphology and functionality, but not in their proliferative capacity.

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

PubMed

El-Bebany, Ahmed F; Rampitsch, Christof; Daayf, Fouad

2010-01-01

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

Antifungal activity, main active components and mechanism of Curcuma longa extract against Fusarium graminearum

PubMed Central

Zhang, Fusheng; Chen, Qin; Chen, Cheng; Yu, Xiaorui; Liu, Qingya; Bao, Jinku

2018-01-01

Curcuma longa possesses powerful antifungal activity, as demonstrated in many studies. In this study, the antifungal spectrum of Curcuma longa alcohol extract was determined, and the resulting EC50 values (mg/mL) of its extract on eleven fungi, including Fusarium graminearum, Fusarium chlamydosporum, Alternaria alternate, Fusarium tricinctum, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium culmorum, Rhizopus oryzae, Cladosporium cladosporioides, Fusarium oxysporum and Colletotrichum higginsianum, were 0.1088, 0.1742, 0.1888, 0.2547, 0.3135, 0.3825, 0.4229, 1.2086, 4.5176, 3.8833 and 5.0183, respectively. Among them, F. graminearum was selected to determine the inhibitory effects of the compounds (including curdione, isocurcumenol, curcumenol, curzerene, β-elemene, curcumin, germacrone and curcumol) derived from Curcuma longa. In addition, the antifungal activities of curdione, curcumenol, curzerene, curcumol and isocurcumenol and the synergies of the complexes of curdione and seven other chemicals were investigated. Differential proteomics of F. graminearum was also compared, and at least 2021 reproducible protein spots were identified. Among these spots, 46 were classified as differentially expressed proteins, and these proteins are involved in energy metabolism, tRNA synthesis and glucose metabolism. Furthermore, several fungal physiological differences were also analysed. The antifungal effect included fungal cell membrane disruption and inhibition of ergosterol synthesis, respiration, succinate dehydrogenase (SDH) and NADH oxidase. PMID:29543859

Proteomic analysis of gastrocnemius muscle in rats with streptozotocin-induced diabetes and chronically exposed to fluoride.

PubMed

Lima Leite, Aline; Gualiume Vaz Madureira Lobo, Janete; Barbosa da Silva Pereira, Heloísa Aparecida; Silva Fernandes, Mileni; Martini, Tatiani; Zucki, Fernanda; Sumida, Dóris Hissako; Rigalli, Alfredo; Buzalaf, Marília Afonso Rabelo

2014-01-01

Administration of high doses of fluoride (F) can alter glucose homeostasis and lead to insulin resistance (IR). This study determined the profile of protein expression in the gastrocnemius muscle of rats with streptozotocin-induced diabetes that were chronically exposed to F. Male Wistar rats (60 days old) were randomly distributed into two groups of 18 animals. In one group, diabetes was induced through the administration of streptozotocin. Each group (D-diabetic and ND-non-diabetic) was further divided into 3 subgroups each of which was exposed to a different F concentration via drinking water (0 ppm, 10 ppm or 50 ppm F, as NaF). After 22 days of treatment, the gastrocnemius muscle was collected and submitted to proteomic analysis (2D-PAGE followed by LC-MS/MS). Protein functions were classified by the GO biological process (ClueGO v2.0.7+Clupedia v1.0.8) and protein-protein interaction networks were constructed (PSICQUIC, Cytoscape). Quantitative intensity analysis of the proteomic data revealed differential expression of 75 spots for ND0 vs. D0, 76 for ND10 vs.D10, 58 spots for ND50 vs. D50, 52 spots for D0 vs. D10 and 38 spots for D0 vs. D50. The GO annotations with the most significant terms in the comparisons of ND0 vs. D0, ND10 vs. D10, ND50 vs. D50, D0 vs. D10 and D0 vs. D50, were muscle contraction, carbohydrate catabolic processes, generation of precursor metabolites and energy, NAD metabolic processes and gluconeogenesis, respectively. Analysis of subnetworks revealed that, in all comparisons, proteins with fold changes interacted with GLUT4. GLUT4 interacting proteins, such as MDH and the stress proteins HSPB8 and GRP78, exhibited decreased expression when D animals were exposed to F. The presence of the two stress proteins indicates an increase in IR, which might worsen diabetes. Future studies should evaluate whether diabetic animals treated with F have increased IR, as well as which molecular mechanisms are involved.

Proteomic Analysis of Gastrocnemius Muscle in Rats with Streptozotocin-Induced Diabetes and Chronically Exposed to Fluoride

PubMed Central

Lima Leite, Aline; Gualiume Vaz Madureira Lobo, Janete; Barbosa da Silva Pereira, Heloísa Aparecida; Silva Fernandes, Mileni; Martini, Tatiani; Zucki, Fernanda; Sumida, Dóris Hissako; Rigalli, Alfredo; Buzalaf, Marília Afonso Rabelo

2014-01-01

Administration of high doses of fluoride (F) can alter glucose homeostasis and lead to insulin resistance (IR). This study determined the profile of protein expression in the gastrocnemius muscle of rats with streptozotocin-induced diabetes that were chronically exposed to F. Male Wistar rats (60 days old) were randomly distributed into two groups of 18 animals. In one group, diabetes was induced through the administration of streptozotocin. Each group (D-diabetic and ND-non-diabetic) was further divided into 3 subgroups each of which was exposed to a different F concentration via drinking water (0 ppm, 10 ppm or 50 ppm F, as NaF). After 22 days of treatment, the gastrocnemius muscle was collected and submitted to proteomic analysis (2D-PAGE followed by LC-MS/MS). Protein functions were classified by the GO biological process (ClueGO v2.0.7+Clupedia v1.0.8) and protein-protein interaction networks were constructed (PSICQUIC, Cytoscape). Quantitative intensity analysis of the proteomic data revealed differential expression of 75 spots for ND0 vs. D0, 76 for ND10 vs.D10, 58 spots for ND50 vs. D50, 52 spots for D0 vs. D10 and 38 spots for D0 vs. D50. The GO annotations with the most significant terms in the comparisons of ND0 vs. D0, ND10 vs. D10, ND50 vs. D50, D0 vs. D10 and D0 vs. D50, were muscle contraction, carbohydrate catabolic processes, generation of precursor metabolites and energy, NAD metabolic processes and gluconeogenesis, respectively. Analysis of subnetworks revealed that, in all comparisons, proteins with fold changes interacted with GLUT4. GLUT4 interacting proteins, such as MDH and the stress proteins HSPB8 and GRP78, exhibited decreased expression when D animals were exposed to F. The presence of the two stress proteins indicates an increase in IR, which might worsen diabetes. Future studies should evaluate whether diabetic animals treated with F have increased IR, as well as which molecular mechanisms are involved. PMID:25180703

Sexual differentiation in three unconventional mammals: spotted hyenas, elephants and tammar wallabies.

PubMed

Glickman, Stephen E; Short, Roger V; Renfree, Marilyn B

2005-11-01

The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the "specific" requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the "expanded" version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5alpha androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion.

Hotspot electron temperature from x-ray continuum measurements on the NIF

NASA Astrophysics Data System (ADS)

Jarrott, L. C.; Benedetti, L. R.; Chen, H.; Izumi, N.; Khan, S. F.; Ma, T.; Nagel, S. R.; Landen, O. L.; Pak, A.; Patel, P. K.; Schneider, M.; Scott, H. A.

2016-11-01

We report on measurements of the electron temperature in the hotspot of inertially confined, layered, spherical implosions on the National Ignition Facility using a differential filtering diagnostic. Measurements of the DT and DD ion temperatures using neutron time-of-flight detectors are complicated by the contribution of hot spot motion to the peak width, which produce an apparent temperature higher than the thermal temperature. The electron temperature is not sensitive to this non-thermal velocity and is thus a valuable input to interpreting the stagnated hot spot conditions. Here we show that the current differential filtering diagnostic provides insufficient temperature resolution for the hot spot temperatures of interest. We then propose a new differential filter configuration utilizing larger pinhole size to increase spectral fluence, as well as thicker filtration. This new configuration will improve measurement uncertainty by more than a factor of three, allowing for a more accurate hotspot temperature.

Hotspot electron temperature from x-ray continuum measurements on the NIF.

PubMed

Jarrott, L C; Benedetti, L R; Chen, H; Izumi, N; Khan, S F; Ma, T; Nagel, S R; Landen, O L; Pak, A; Patel, P K; Schneider, M; Scott, H A

2016-11-01

We report on measurements of the electron temperature in the hotspot of inertially confined, layered, spherical implosions on the National Ignition Facility using a differential filtering diagnostic. Measurements of the DT and DD ion temperatures using neutron time-of-flight detectors are complicated by the contribution of hot spot motion to the peak width, which produce an apparent temperature higher than the thermal temperature. The electron temperature is not sensitive to this non-thermal velocity and is thus a valuable input to interpreting the stagnated hot spot conditions. Here we show that the current differential filtering diagnostic provides insufficient temperature resolution for the hot spot temperatures of interest. We then propose a new differential filter configuration utilizing larger pinhole size to increase spectral fluence, as well as thicker filtration. This new configuration will improve measurement uncertainty by more than a factor of three, allowing for a more accurate hotspot temperature.

Acclimation to cold and warm temperatures is associated with differential expression of male carp blood proteins involved in acute phase and stress responses, and lipid metabolism.

PubMed

Dietrich, Mariola A; Hliwa, Piotr; Adamek, Mikołaj; Steinhagen, Dieter; Karol, Halina; Ciereszko, Andrzej

2018-05-01

The environmental temperature affects plasma biochemical indicators, antioxidant status and hematological and immunological parameters in fish. So far, only single blood proteins have been identified in response to temperature changes. The aim of this study was to compare the proteome of carp blood plasma from males acclimated to warm (30 °C) and cold (10 °C) temperatures by two-dimensional differential gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. A total of 47 spots were found to be differentially regulated by temperature (>1.2-fold change, p < 0.05): 25 protein spots were more abundant in warm-acclimated males and 22 were enriched in cold-acclimated males. The majority of differentially regulated proteins were associated with acute phase response signalling involved in: i) activation of the complement system (complement C3-H1), ii) neutralization of proteolytic enzymes (inter-alpha inhibitor H3, fetuin, serpinA1, antithrombin, alpha2-macroglobulin), iii) scavenging of free hemoglobin and radicals (haptoglobin, Wap65 kDa), iv) clot-formation (fibrinogen beta and alpha chain, T-kininogen) and v) the host's immune response modulation (ApoA1 and ApoA2). However, quite different sets of these proteins or proteoforms were involved in response to cold and warm temperatures. In addition, cold acclimation seems to be related to the proteins involved in lipid metabolism (apolipoproteins A and 14 kDa) and stress response (corticosteroid binding globulin). We discovered a strongly regulated protein Cap31 upon cold acclimation, which can serve as a potential blood biomarker of cold response in carp. These studies significantly extend our knowledge concerning mechanisms underlying thermal adaptation in poikilotherms. Copyright © 2018. Published by Elsevier Ltd.

High seroprevalence of antibodies against spotted fever and scrub typhus bacteria in patients with febrile Illness, Kenya.

PubMed

Thiga, Jacqueline W; Mutai, Beth K; Eyako, Wurapa K; Ng'ang'a, Zipporah; Jiang, Ju; Richards, Allen L; Waitumbi, John N

2015-04-01

Serum samples from patients in Kenya with febrile illnesses were screened for antibodies against bacteria that cause spotted fever, typhus, and scrub typhus. Seroprevalence was 10% for spotted fever group, <1% for typhus group, and 5% for scrub typhus group. Results should help clinicians expand their list of differential diagnoses for undifferentiated fevers.

Differentiation of Corynespora cassiicola and Cercospora sp. in leaf-spot diseases of Hydrangea macrophylla using a PCR-mediated method

Treesearch

M. T. Mmbaga; L. Mackasmiel; Ned Klopfenstein

2015-01-01

Corynespora cassiicola and Cercospora sp. have been identified as the most prevalent and destructive leaf-spot pathogens of garden hydrangea [Hydrangea macrophylla (Thunberg) Seringe] in the southeastern USA, but they are often difficult to accurately detect and distinguish because they often occur together in a disease complex with other pathogenic leaf-spot...

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

PubMed Central

Zeng, Tao; Jiang, Xueyuan; Li, Jinjun; Wang, Deqian; Li, Guoqin; Lu, Lizhi; Wang, Genlin

2013-01-01

The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals. PMID:24116183

Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium.

PubMed

Zhu, Bo; Gao, Kun-Shan; Wang, Ke-Jian; Ke, Cai-Huan; Huang, He-Qing

2012-04-01

As mercury and lead, cadmium (Cd) is one of the highly toxic metals in both the ocean and land environments, but its toxicological mechanism in organisms including human is still unclear because of the complex toxicological pathways in vivo. Here, the alga Chlorella vulgaris were cultivated at room temperature under the stress of cadmium (1 mg L(-1)) to obtain a toxic food, and then the contaminated food were directly supplied to oyster (Saccostrea cucullata) in seawater. After feeding with C. vulgaris contaminated with Cd (C. vulgaris-Cd), the differential proteins in the oyster gonad (OG) were effectively separated and identified with proteomic approaches. Eleven protein spots were observed to be significantly changed in the OG feeding with C. vulgaris-Cd, which seven spots of these differential proteins were down-regulated while four spots were up-regulated. These altered spots were further excised in gels and identified by a combined technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and database searching. A portion of these differential proteins were further proofed by real-time PCR and Western blotting. The results indicate that the major functions of these differential proteins were described as follows: binding, protein translocation, catalysis, regulation of energy metabolism, reproductive function and skeleton structure. These differential proteins in part may effectively provide a few novel biomarkers for the evaluation of Cd pollution level via a food pathway for harming halobios, mammal and human health, and for understanding the complex mechanisms of Cd toxicity in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

Hairworm anti-predator strategy: a study of causes and consequences.

PubMed

Ponton, F; Lebarbenchon, C; Lefèvre, T; Thomas, F; Duneau, D; Marché, L; Renault, L; Hughes, D P; Biron, D G

2006-11-01

One of the most fascinating anti-predator responses displayed by parasites is that of hairworms (Nematomorpha). Following the ingestion of the insect host by fish or frogs, the parasitic worm is able to actively exit both its host and the gut of the predator. Using as a model the hairworm, Paragordius tricuspidatus, (parasitizing the cricket Nemobius sylvestris) and the fish predator Micropterus salmoïdes, we explored, with proteomics tools, the physiological basis of this anti-predator response. By examining the proteome of the parasitic worm, we detected a differential expression of 27 protein spots in those worms able to escape the predator. Peptide Mass Fingerprints of candidate protein spots suggest the existence of an intense muscular activity in escaping worms, which functions in parallel with their distinctive biology. In a second step, we attempted to determine whether the energy expended by worms to escape the predator is traded off against its reproductive potential. Remarkably, the number of offspring produced by worms having escaped a predator was not reduced compared with controls.

Butterfly Diagram and Activity Cycles in HR 1099

NASA Astrophysics Data System (ADS)

Berdyugina, Svetlana V.; Henry, Gregory W.

2007-04-01

We analyze photometric data of the active RS CVn-type star HR 1099 for the years 1975-2006 with an inversion technique and reveal the nature of two activity cycles of 15-16 yr and 5.3+/-0.1 yr duration. The 16 yr cycle is related to variations of the total spot area and is coupled with the differential rotation, while the 5.3 yr cycle is caused by the symmetric redistribution of the spotted area between the opposite stellar hemispheres (flip-flop cycle). We recover long-lived active regions comprising two active longitudes that migrate in the orbital reference frame with a variable rate because of the differential rotation along with changes in the mean spot latitudes. The migration pattern is periodic with the 16 yr cycle. Combining the longitudinal migration of the active regions with a previously measured differential rotation law, we recover the first stellar butterfly diagram without an assumption about spot shapes. We find that mean latitudes of active regions at opposite longitudes change antisymmetrically in the course of the 16 yr cycle: while one active region migrates to the pole, the other approaches the equator. This suggests a precession of the global magnetic field with respect to the stellar rotational axis.

The anti-cataract molecular mechanism study in selenium cataract rats for baicalin ophthalmic nanoparticles.

PubMed

Li, Nan; Han, Zhenzhen; Li, Lin; Zhang, Bing; Liu, Zhidong; Li, Jiawei

2018-01-01

The objective of this study was to investigate the effects of the solid lipid nanoparticles of baicalin (BA-SLNs) on an experimental cataract model and explore the molecular mechanism combined with bioinformatics analysis. The transparency of lens was observed daily by slit-lamp and photography. Lenticular opacity was graded. Two-dimensional gel electrophoresis (2-DE) was employed to analyze the differential protein expression modes in each group. Proteins of interest were subjected to protein identification by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis was performed using the Ingenuity Pathway Analysis (IPA) online software to comprehend the biological implications of the proteins identified by proteomics. At the end of the sodium selenite-induced cataract progression, almost all lenses from the model group developed partial nuclear opacity; however, all lenses were clear and normal in the blank group. There was no significant difference between the BA-SLNs group and the blank group. Many protein spots were differently expressed in 2-DE patterns of total proteins of lenses from each group, and 65 highly different protein spots were selected to be identified between the BA-SLNs group and the model group. A total of 23 proteins were identified, and 12 of which were crystalline proteins. We considered crystalline proteins to play important roles in preserving the normal expression levels of proteins and the transparency of lenses. The general trend in the BA-SLN-treated lenses' data showed that BA-SLNs regulated the protein expression mode of cataract lenses to normal lenses. Our findings suggest that BA-SLNs may be a potential therapeutic agent in treating cataract by regulating protein expression and may also be a strong candidate for future clinical research.

Influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency.

PubMed

Li, Bing-Kun; Wang, Xiang; Liu, Chun-Xiao; Zheng, Shao-Bo; Li, Hu-Lin; Li, Li-Ping; Xu, A-Bai

2013-01-01

Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends: a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 (HE2), fertilization antigen-1 (FA-1), clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency (P<0.05). Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent. Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.

In silico analysis, seasonal variation and gonadotropic regulation of jag1 and its receptor notch1 in testis of spotted snakehead Channa punctatus.

PubMed

Roy, Alivia; Basak, Reetuparna; Rai, Umesh

2018-05-14

The present study in seasonally breeding spotted snakehead Channa punctatus, for the first time in nonmammalian vertebrates, demonstrated correlation between reproductive phase-dependent testicular expression of ligand Jag1/receptor Notch1 and spermatogenic events. Testicular transcriptome sequencing data from our earlier study in C. punctatus was used in the present study to select the best transcript for jag1 (cpjag1) and notch1 (cpnotch1). The transcripts cpjag1 and cpnotch1 encoded full-length putative proteins of 1215 (cpJag1) and 2475 (cpNotch1) amino acids, respectively. A marked homology in the extracellular domains of Jag1 and Notch1 was observed following their alignment with respective proteins from different vertebrates, suggesting conservation in ligand-receptor interaction in C. punctatus. Both cpJag1 and cpNotch1 showed phylogenetic closeness with their teleostean counterparts, especially with that of Perciformes. Temporal expression of cpjag1 and cpnotch1 in testis depending on reproductive phases showed an appreciably high expression during spermatogenically inactive resting and postspawning phases when seminiferous lobules consisted of spermatogonial stem cells and undifferentiated spermatogonia. Their expression sharply declined during spermatogenically active preparatory and spawning phases. It appears that involvement of cpjag1/cpnotch1 is restricted to inactive phases when spermatogonial stem cells renew themselves and replenish undifferentiated spermatogonia. This assumption is ascertained by an experimental study in which high level of testicular cpjag1/cpnotch1 expression in control fish of resting phase markedly decreased after administration of human chorionic gonadotropin that is known to induce proliferation and differentiation of spermatogonia and spawning of spermatozoa. Copyright © 2018. Published by Elsevier Inc.

Proteomic analysis of plasma membrane proteins in wheat roots exposed to phenanthrene.

PubMed

Shen, Yu; Du, Jiangxue; Yue, Le; Zhan, Xinhua

2016-06-01

Polycyclic aromatic hydrocarbons (PAHs) are potentially carcinogenic and toxic to humans through ingestion of contaminated food crops. PAHs can enter crop roots through proton/PAH symporters; however, to date, the symporter remains unclear. Here we reveal, for the first time, the plasma membrane proteome of Triticum aestivum seedling roots in response to phenanthrene (a model PAH) exposure. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF-MS and protein database search engines were employed to analyze and identify phenanthrene-responsive proteins. Over 192 protein spots are reproducibly detected in each gel, while 8 spots are differentially expressed under phenanthrene treatment. Phenanthrene induces five up-regulated proteins distinguished as 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase 2, enolase, heat shock protein 80-2, probable mediator of RNA polymerase II transcription subunit 37e (heat shock 70-kDa protein 1), and lactoylglutathione lyase. Three proteins identified as adenosine kinase 2, 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1c, and glyceraldehyde-3-phosphate dehydrogenase 3 are down-regulated under exposure to phenanthrene. The up-regulated proteins are related to plant defense response, antioxidant system, and glycolysis. The down-regulated proteins involve the metabolism of high-energy compounds and plant growth. Magnesium, which is able to bind to enolase, can enhance the transport of phenanthrene into wheat roots. Therefore, it is concluded that phenanthrene can induce differential expression of proteins in relation to carbohydrate metabolism, self-defense, and plant growth on wheat root plasma membrane. This study not only provides novel insights into PAH uptake by plant roots and PAH stress responses, but is also a good starting point for further determination and analyses of their functions using genetic and other approaches.

Genetic differentiation and inferred dynamics of a hybrid zone between Northern Spotted Owls (Strix occidentalis caurina) and California Spotted Owls (S. o. occidentalis) in northern California

USGS Publications Warehouse

Miller, Mark P.; Mullins, Tom; Forsman, Eric D.; Haig, Susan M.

2017-01-01

Genetic differentiation among Spotted Owl (Strix occidentalis) subspecies has been established in prior studies. These investigations also provided evidence for introgression and hybridization among taxa but were limited by a lack of samples from geographic regions where subspecies came into close contact. We analyzed new sets of samples from Northern Spotted Owls (NSO: S. o. caurina) and California Spotted Owls (CSO: S. o. occidentalis) in northern California using mitochondrial DNA sequences (mtDNA) and 10 nuclear microsatellite loci to obtain a clearer depiction of genetic differentiation and hybridization in the region. Our analyses revealed that a NSO population close to the northern edge of the CSO range in northern California (the NSO Contact Zone population) is highly differentiated relative to other NSO populations throughout the remainder of their range. Phylogenetic analyses identified a unique lineage of mtDNA in the NSO Contact Zone, and Bayesian clustering analyses of the microsatellite data identified the Contact Zone as a third distinct population that is differentiated from CSO and NSO found in the remainder of the subspecies' range. Hybridization between NSO and CSO was readily detected in the NSO Contact Zone, with over 50% of individuals showing evidence of hybrid ancestry. Hybridization was also identified among 14% of CSO samples, which were dispersed across the subspecies' range in the Sierra Nevada Mountains. The asymmetry of hybridization suggested that the hybrid zone may be dynamic and moving. Although evidence of hybridization existed, we identified no F1 generation hybrid individuals. We instead found evidence for F2 or backcrossed individuals among our samples. The absence of F1 hybrids may indicate that (1) our 10 microsatellites were unable to distinguish hybrid types, (2) primary interactions between subspecies are occurring elsewhere on the landscape, or (3) dispersal between the subspecies' ranges is reduced relative to historical levels, potentially as a consequence of recent regional fires.

Analysis of Phaseolus vulgaris Response to Its Association with Trichoderma harzianum (ALL-42) in the Presence or Absence of the Phytopathogenic Fungi Rhizoctonia solani and Fusarium solani

PubMed Central

Pereira, Jackeline L.; Queiroz, Rayner M. L.; Charneau, Sébastien O.; Felix, Carlos R.; Ricart, Carlos A. O.; da Silva, Francilene Lopes; Steindorff, Andrei Stecca; Ulhoa, Cirano J.; Noronha, Eliane F.

2014-01-01

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. PMID:24878929

Analysis of Phaseolus vulgaris response to its association with Trichoderma harzianum (ALL-42) in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani.

PubMed

Pereira, Jackeline L; Queiroz, Rayner M L; Charneau, Sébastien O; Felix, Carlos R; Ricart, Carlos A O; da Silva, Francilene Lopes; Steindorff, Andrei Stecca; Ulhoa, Cirano J; Noronha, Eliane F

2014-01-01

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.

Comparative Proteomic Analysis of Differentially Expressed Proteins Induced by Hydrogen Sulfide in Spinacia oleracea Leaves

PubMed Central

Chen, Juan; Liu, Ting-Wu; Hu, Wen-Jun; Simon, Martin; Wang, Wen-Hua; Chen, Juan; Liu, Xiang; Zheng, Hai-Lei

2014-01-01

Hydrogen sulfide (H2S), as a potential gaseous messenger molecule, has been suggested to play important roles in a wide range of physiological processes in plants. The aim of present study was to investigate which set of proteins is involved in H2S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H2S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold (sixty-five were up-regulated, whereas 27 were down-regulated). These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H2S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H2S. PMID:25181351

Inhibition of glycogen phosphorylation induces changes in cellular proteome and signaling pathways in MIA pancreatic cancer cells

PubMed Central

Ma, Danjun; Wang, Jiarui; Zhao, Yingchun; Lee, Wai-Nang Paul; Xiao, Jing; Go, Vay Liang W.; Wang, Qi; Recker, Robert; Xiao, Gary Guishan

2011-01-01

Objectives Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. Methods We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (25 μM, 50 μM and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC method (mSILAC). Results A total of twenty-two protein spots and four phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of MAPK/ERK and TNF-α/NF-κB. Conclusions Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through one pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown and post-translational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways. PMID:22158071

Automated detection and differentiation of drusen, exudates, and cotton-wool spots in digital color fundus photographs for diabetic retinopathy diagnosis.

PubMed

Niemeijer, Meindert; van Ginneken, Bram; Russell, Stephen R; Suttorp-Schulten, Maria S A; Abràmoff, Michael D

2007-05-01

To describe and evaluate a machine learning-based, automated system to detect exudates and cotton-wool spots in digital color fundus photographs and differentiate them from drusen, for early diagnosis of diabetic retinopathy. Three hundred retinal images from one eye of 300 patients with diabetes were selected from a diabetic retinopathy telediagnosis database (nonmydriatic camera, two-field photography): 100 with previously diagnosed bright lesions and 200 without. A machine learning computer program was developed that can identify and differentiate among drusen, (hard) exudates, and cotton-wool spots. A human expert standard for the 300 images was obtained by consensus annotation by two retinal specialists. Sensitivities and specificities of the annotations on the 300 images by the automated system and a third retinal specialist were determined. The system achieved an area under the receiver operating characteristic (ROC) curve of 0.95 and sensitivity/specificity pairs of 0.95/0.88 for the detection of bright lesions of any type, and 0.95/0.86, 0.70/0.93, and 0.77/0.88 for the detection of exudates, cotton-wool spots, and drusen, respectively. The third retinal specialist achieved pairs of 0.95/0.74 for bright lesions and 0.90/0.98, 0.87/0.98, and 0.92/0.79 per lesion type. A machine learning-based, automated system capable of detecting exudates and cotton-wool spots and differentiating them from drusen in color images obtained in community based diabetic patients has been developed and approaches the performance level of retinal experts. If the machine learning can be improved with additional training data sets, it may be useful for detecting clinically important bright lesions, enhancing early diagnosis, and reducing visual loss in patients with diabetes.

17beta-hydroxysteroid dehydrogenase type 5 is negatively correlated to apoptosis inhibitor GRP78 and tumor-secreted protein PGK1, and modulates breast cancer cell viability and proliferation.

PubMed

Xu, Dan; Aka, Juliette A; Wang, Ruixuan; Lin, Sheng-Xiang

2017-07-01

17beta-hydroxysteroid dehydrogenase type 5 (17β-HSD5) is an important enzyme associated with sex steroid metabolism in hormone-dependent cancer. However, reports on its expression and its prognostic value in breast cancer are inconsistent. Here, we demonstrate the impact of 17β-HSD5 expression modulation on the proteome of estrogen receptor-positive (ER+) breast cancer cells. RNA interference technique (siRNA) was used to knock down 17β-HSD5 gene expression in the ER+ breast cancer cell line MCF-7 and the proteome of the 17β-HSD5-knockdown cells was compared to that of MCF-7 cells using two-dimensional (2-D) gel electrophoresis followed by mass spectrometry analysis. Ingenuity pathway analysis (IPA) was additionally used to assess functional enrichment analyses of the proteomic dataset, including protein network and canonical pathways. Our proteomic analysis revealed only four differentially expressed protein spots (fold change > 2, p<0.05) between the two cell lines. The four spots were up-regulated in 17β-HSD5-knockdown MCF-7 cells, and comprised 21 proteins involved in two networks and in functions that include apoptosis inhibition, regulation of cell growth and differentiation, signal transduction and tumor metastasis. Among the proteins are nucleoside diphosphate kinase A (NME1), 78kDa glucose-regulated protein (GRP78) and phosphoglycerate kinase 1 (PGK1). We also showed that expression of 17β-HSD5 and that of the apoptosis inhibitor GRP78 are strongly but negatively correlated. Consistent with their opposite regulation, GRP78 knockdown decreased MCF-7 cell viability whereas 17β-HSD5 knockdown or inhibition increased cell viability and proliferation. Besides, IPA analysis revealed that ubiquitination pathway is significantly affected by 17β-HSD5 knockdown. Furthermore, IPA predicted the proto-oncogene c-Myc as an upstream regulator linked to the tumor-secreted protein PGK1. The latter is over-expressed in invasive ductal breast carcinoma as compared with normal breast tissue and its expression increased following 17β-HSD5 knockdown. Our present results indicate a 17β-HSD5 role in down-regulating breast cancer development. We thus propose that 17β-HSD5 may not be a potent target for breast cancer treatment but its low expression could represent a poor prognosis factor. Copyright © 2017. Published by Elsevier Ltd.

Geographic variation and genetic structure in Spotted Owls

USGS Publications Warehouse

Haig, Susan M.; Wagner, R.S.; Forsman, E.D.; Mullins, Thomas D.

2001-01-01

We examined genetic variation, population structure, and definition of conservation units in Spotted Owls (Strix occidentalis). Spotted Owls are mostly non-migratory, long-lived, socially monogamous birds that have decreased population viability due to their occupation of highly-fragmented late successional forests in western North America. To investigate potential effects of habitat fragmentation on population structure, we used random amplified polymorphic DNA (RAPD) to examine genetic variation hierarchically among local breeding areas, subregional groups, regional groups, and subspecies via sampling of 21 breeding areas (276 individuals) among the three subspecies of Spotted Owls. Data from 11 variable bands suggest a significant relationship between geographic distance among local breeding groups and genetic distance (Mantel r = 0.53, P < 0.02) although multi-dimensional scaling of three significant axes did not identify significant grouping at any hierarchical level. Similarly, neighbor-joining clustering of Manhattan distances indicated geographic structure at all levels and identified Mexican Spotted Owls as a distinct clade. RAPD analyses did not clearly differentiate Northern Spotted Owls from California Spotted Owls. Among Northern Spotted Owls, estimates of population differentiation (FST) ranged from 0.27 among breeding areas to 0.11 among regions. Concordantly, within-group agreement values estimated via multi-response permutation procedures of Jaccarda??s distances ranged from 0.22 among local sites to 0.11 among regions. Pairwise comparisons of FST and geographic distance within regions suggested only the Klamath region was in equilibrium with respect to gene flow and genetic drift. Merging nuclear data with recent mitochondrial data provides support for designation of an Evolutionary Significant Unit for Mexican Spotted Owls and two overlapping Management Units for Northern and California Spotted Owls.

Spot Sampling and Exposure Surrogate Selection as Sources of Bias in Environmental Epidemiology Studies

EPA Science Inventory

Spot measurements of chemical biomarkers are often used as quantitative exposure surrogates in environmental epidemiology studies. These measures can be expressed a number of different ways – for example, urinary biomarkers can be expressed in units of concentration (&micr...

Regulation of Leishmania (L.) amazonensis Protein Expression by Host T Cell Dependent Responses: Differential Expression of Oligopeptidase B, Tryparedoxin Peroxidase and HSP70 Isoforms in Amastigotes Isolated from BALB/c and BALB/c Nude Mice

PubMed Central

Teixeira, Priscila Camillo; Velasquez, Leonardo Garcia; Lepique, Ana Paula; de Rezende, Eloiza; Bonatto, José Matheus Camargo; Barcinski, Marcello Andre; Cunha-Neto, Edecio; Stolf, Beatriz Simonsen

2015-01-01

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania´s antigens. This work is the first to compare modifications in amastigotes’ proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression. PMID:25692783

Transcriptional Analysis of Resistance to Low Temperatures in Bermudagrass Crown Tissues

PubMed Central

Melmaiee, Kalpalatha; Anderson, Michael; Elavarthi, Sathya; Guenzi, Arron; Canaan, Patricia

2015-01-01

Bermudagrass (Cynodon dactylon L pers.) is one of the most geographically adapted and utilized of the warm-season grasses. However, bermudagrass adaptation to the Northern USA is limited by freeze damage and winterkill. Our study provides the first large-scale analyses of gene expression in bermudagrass regenerative crown tissues during cold acclimation. We compared gene expression patterns in crown tissues from highly cold tolerant “MSU” and susceptible “Zebra” genotypes exposed to near-freezing temperatures. Suppressive subtractive hybridization was used to isolate putative cold responsive genes Approximately, 3845 transcript sequences enriched for cold acclimation were deposited in the GenBank. A total of 4589 ESTs (3184 unigenes) including 744 ESTs associated with the bermudagrass disease spring dead spot were printed on microarrays and hybridized with cold acclimated complementary Deoxyribonucleic acid (cDNA). A total of 587 differentially expressed unigenes were identified in this study. Of these only 97 (17%) showed significant NCBI matches. The overall expression pattern revealed 40% more down- than up-regulated genes, which was particularly enhanced in MSU compared to Zebra. Among the up-regulated genes 68% were uniquely expressed in MSU (36%) or Zebra (32%). Among the down-regulated genes 40% were unique to MSU, while only 15% to Zebra. Overall expression intensity was significantly higher in MSU than in Zebra (p value ≤ 0.001) and the overall number of genes expressed at 28 days was 2.7 fold greater than at 2 days. These changes in expression patterns reflect the strong genotypic and temporal response to cold temperatures. Additionally, differentially expressed genes from this study can be utilized for developing molecular markers in bermudagrass and other warm season grasses for enhancing cold hardiness. PMID:26348040

Distinct functions of neuromedin u and neuromedin s in orange-spotted grouper.

PubMed

Li, Shuisheng; Xiao, Ling; Liu, Qiongyu; Zheng, Binbin; Chen, Huapu; Liu, Xiaochun; Zhang, Yong; Lin, Haoran

2015-10-01

Neuromedin U (NMU) and neuromedin S (NMS) play inhibitory roles in the regulation of food intake and energy homeostasis in mammals. However, their functions are not clearly established in teleost fish. In the present study, nmu and nms homologs were identified in several fish species. Subsequently, their cDNA sequences were cloned from the orange-spotted grouper (Epinephelus coioides). Sequence analysis showed that the orange-spotted grouper Nmu proprotein contains a 21-amino acid mature Nmu peptide (Nmu-21). The Nms proprotein lost the typical mature Nms peptide, but it retains a putative 34-amino acid peptide (Nmsrp). In situ hybridization revealed that nmu- and nms-expressing cells are mainly localized in the hypothalamic regions associated with appetite regulation. Food deprivation decreased the hypothalamic nmu mRNA levels but induced an increase of nms mRNA levels. Periprandial expression analysis showed that hypothalamic expression of nmu increased significantly at 3 h post-feeding, while nms expression was elevated at the normal feeding time. I.p. injection of synthetic Nmu-21 peptide suppressed the hypothalamic neuropeptide y (npy) expression, while Nmsrp administration significantly increased the expression of npy and orexin in orange-spotted grouper. Furthermore, the mRNA levels of LH beta subunit (lhβ) and gh in the pituitary were significantly down-regulated after Nmu-21 peptide administration, while Nmsrp was able to significantly stimulate the expression of FSH beta subunit (fshβ), prolactin (prl), and somatolaction (sl). Our results indicate that nmu and nms possess distinct neuroendocrine functions and pituitary functions in the orange spotted grouper. © 2015 Society for Endocrinology.

Spotting effect in microarray experiments

PubMed Central

Mary-Huard, Tristan; Daudin, Jean-Jacques; Robin, Stéphane; Bitton, Frédérique; Cabannes, Eric; Hilson, Pierre

2004-01-01

Background Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array. Results Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure. Conclusions The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis. PMID:15151695

Molecular characterization of mediterranean spotted fever rickettsia isolated from a European traveler in the state of São Paulo, Brazil.

PubMed

Gehrke, Flávia S; Angerami, Rodrigo N; Marrelli, Mauro T; de Souza, Eliana R; do Nascimento, Elvira M M; Colombo, Silvia; da Silva, Luiz J; Schumaker, Teresinha T S

2013-01-01

Rickettsial spotted fever is common in southeastern Brazil. Differential diagnosis of pathogens can be performed with proper laboratory methods. A traveler arriving from Portugal developed a fatal febrile hemorrhagic syndrome diagnosed as spotted fever rickettsiosis. We isolated the agent, which was identified as Rickettsia conorii conorii by sequencing rickettsial genes. © 2013 International Society of Travel Medicine.

Radioimmunoassay of ''free thyroxin'' in dried blood spots on filter paper - preliminary observations on the effective differentiation of subjects with congenital hypothyroidism from those with subnormal thyroxin-binding globulin and normal subjects

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

Mizuta, H.; Miyai, K.; Ichihara, K.

1982-03-01

In this sensitive, simple method for measuring ''free thyroxin'' (FT/sub 4/) in eluates of dried blood spots on filter paper by use of a radioimmunoassay kit (Amerlex Free T/sub 4/ RIA), the measurable range of FT/sub 4/ is 1.8 to 57 ng/L (equivalent to the concentration in serum), or 7 to 237 fg/tube. The mean coefficients of variation for within assay-within spots, within assay-between spots, and between assays were 5.3%, 5.0%, and 6.2%, respectively. FT/sub 4/ in blood spotted on filter paper is stable for at least a month when dried and kept at either -20/sup 0/C, 4/sup 0/C, roommore » temperature (about 25/sup 0/C), or 37/sup 0/C. The results for FT/sub 4/ in dried blood spots correlated closely with the free-T/sub 4/ concentration in serum (r = 0.99). The method can be used to differentiate cases of primary and secondary hypothyroidism from normal subjects and those with subnormal thyroxin-binding globulin. This method may be useful in screening for congenital hypothyroidism, because sample-retesting is not necessary.« less

Hotspot electron temperature from x-ray continuum measurements on the NIF

DOE PAGES

Jarrott, L. C.; Benedetti, L. R.; Chen, H.; ...

2016-08-24

We report on measurements of the electron temperature in the hotspot of inertially confined, layered, spherical implosions on the National Ignition Facility using a differential filtering diagnostic. Measurements of the DT and DD ion temperatures using neutron time-of-flight detectors are complicated by the contribution of hot spot motion to the peak width, which produce an apparent temperature higher than the thermal temperature. The electron temperature is not sensitive to this non-thermal velocity and is thus a valuable input to interpreting the stagnated hot spot conditions. Here we show that the current differential filtering diagnostic provides insufficient temperature resolution for themore » hot spot temperatures of interest. We then propose a new differential filter configuration utilizing larger pinhole size to increase spectral fluence, as well as thicker filtration. In conclusion, this new configuration will improve measurement uncertainty by more than a factor of three, allowing for a more accurate hotspot temperature.« less

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

PubMed

Meade, Kieran G; Gormley, Eamonn; Park, Stephen D E; Fitzsimons, Tara; Rosa, Guilherme J M; Costello, Eamon; Keane, Joseph; Coussens, Paul M; MacHugh, David E

2006-09-15

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated PBMC provides the first description of an M. bovis-specific signature of infection that may provide insights into the molecular basis of the host response to infection. Although the present study was carried out with mixed PBMC cell populations, it will guide future studies to dissect immune cell-specific gene expression patterns in response to M. bovis infection.

Analysis and design of optical systems by use of sensitivity analysis of skew ray tracing

NASA Astrophysics Data System (ADS)

Lin, Psang Dain; Lu, Chia-Hung

2004-02-01

Optical systems are conventionally evaluated by ray-tracing techniques that extract performance quantities such as aberration and spot size. Current optical analysis software does not provide satisfactory analytical evaluation functions for the sensitivity of an optical system. Furthermore, when functions oscillate strongly, the results are of low accuracy. Thus this work extends our earlier research on an advanced treatment of reflected or refracted rays, referred to as sensitivity analysis, in which differential changes of reflected or refracted rays are expressed in terms of differential changes of incident rays. The proposed sensitivity analysis methodology for skew ray tracing of reflected or refracted rays that cross spherical or flat boundaries is demonstrated and validated by the application of a cat's eye retroreflector to the design and by the image orientation of a system with noncoplanar optical axes. The proposed sensitivity analysis is projected as the nucleus of other geometrical optical computations.

Analysis and Design of Optical Systems by Use of Sensitivity Analysis of Skew Ray Tracing

NASA Astrophysics Data System (ADS)

Dain Lin, Psang; Lu, Chia-Hung

2004-02-01

Optical systems are conventionally evaluated by ray-tracing techniques that extract performance quantities such as aberration and spot size. Current optical analysis software does not provide satisfactory analytical evaluation functions for the sensitivity of an optical system. Furthermore, when functions oscillate strongly, the results are of low accuracy. Thus this work extends our earlier research on an advanced treatment of reflected or refracted rays, referred to as sensitivity analysis, in which differential changes of reflected or refracted rays are expressed in terms of differential changes of incident rays. The proposed sensitivity analysis methodology for skew ray tracing of reflected or refracted rays that cross spherical or flat boundaries is demonstrated and validated by the application of a cat ?s eye retroreflector to the design and by the image orientation of a system with noncoplanar optical axes. The proposed sensitivity analysis is projected as the nucleus of other geometrical optical computations.

Identification and characterization of pyrokinin and CAPA peptides, and corresponding GPCRs from spotted wing drosophila, Drosophila suzukii.

PubMed

Choi, Man-Yeon; Ahn, Seung-Joon; Kim, A Young; Koh, Youngho

2017-05-15

The family of FXPRLamide peptides serves as a major insect hormone. It is characterized by a core active amino acid sequence conserved at the C-terminal ends, and provides various physiological roles across the Insecta. In this study we identified and characterized pyrokinin (PK) and CAPA cDNAs encoding two FXPRLamide peptides, pyrokinin and CAPA-DH (diapause hormone), and two corresponding G protein-coupled receptors (GPCRs) from spotted wing drosophila (SWD), Drosophila suzukii. Expressions of PK and CAPA mRNAs were differentially observed during all life stages except the embryo, and the detection of CAPA transcription was relatively strong compared with the PK gene in SWD. Both D. suzukii pyrokinin receptor (DrosuPKr) and CAPA-DH receptor (DrosuCAPA-DHr) were functionally expressed and confirmed through binding to PK and DH peptides. Differential expression of two GPCRs occurred during all life stages; a strong transcription of DrosuPKr was observed in the 3rd instar. DrosuCAPA-DHr was clearly expressed from the embryo to the larva, but not detected in the adult. Gene regulation during the life stages was not synchronized between ligand and receptor. For example, SWD CAPA mRNA has been up-regulated in the adult while CAPA-DHr was down-regulated. The difference could be from the CAPA mRNA translating multiple peptides including CAPA-DH and two CAPA-PVK (periviscerokinin) peptides to act on different receptors. Comparing the genes of SWD PK, CAPA, PKr and CAPA-DHr to four corresponding genes of D. melanogaster, SWD CAPA and the receptor are more similar to D. melanogaster than PK and the receptor. These data suggest that the CAPA gene could be evolutionally more conserved to have a common biological role in insects. In addition, the effect of Kozak sequences was investigated by the expression of the GPCRs with or without Kozak sequences in Sf9 insect cells. The Kozak sequenced PK receptor was significantly less active than the original (= no Kozak sequenced) receptor. Our results provide a knowledge for potential biological function(s) of PK and CAPA-DH peptides in SWD, and possibly offer a novel control method for this pest insect in the future. Published by Elsevier Inc.

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

Effects of acute inhalation of aerosols generated during resistance spot welding with mild-steel on pulmonary, vascular and immune responses in rats

PubMed Central

Zeidler-Erdely, Patti C.; Meighan, Terence G.; Erdely, Aaron; Fedan, Jeffrey S.; Thompson, Janet A.; Bilgesu, Suzan; Waugh, Stacey; Anderson, Stacey; Marshall, Nikki B.; Afshari, Aliakbar; McKinney, Walter; Frazer, David G.; Antonini, James M.

2015-01-01

Spot welding is used in the automotive and aircraft industries, where high-speed, repetitive welding is needed to join thin sections of metal. Epoxy adhesives are applied as sealers to the metal seams. Pulmonary function abnormalities and airway irritation have been reported in spot welders, but no animal toxicology studies exist. Therefore, the goal of this study was to investigate vascular, immune and lung toxicity measures after exposure to these metal fumes in an animal model. Male Sprague-Dawley rats were exposed by inhalation to 25 mg/m3 to either mild-steel spot welding aerosols with sparking (high metal, HM) or without sparking (low metal, LM) for 4 h/d for 3, 8 and 13 d. Shams were exposed to filtered air. Bronchoalveolar lavage (BAL), lung gene expression and ex vivo BAL cell challenge were performed to assess lung toxicity. Lung resistance (RL) was evaluated before and after challenge with inhaled methacholine (MCh). Functional assessment of the vascular endothelium in isolated rat tail arteries and leukocyte differentiation in the spleen and lymph nodes via flow cytometry was also done. Immediately after exposure, baseline RL was significantly elevated in the LM spot welding aerosols, but returned to control level by 24 h postexposure. Airway reactivity to MCh was unaffected. Lung inflammation and cytotoxicity were mild and transient. Lung epithelial permeability was significantly increased after 3 and 8 d, but not after 13 d of exposure to the HM aerosol. HM aerosols also caused vascular endothelial dysfunction and increased CD4+, CD8+ and B cells in the spleen. Only LM aerosols caused increased IL-6 and MCP-1 levels compared with sham after ex vivo LPS stimulation in BAL macrophages. Acute inhalation of mild-steel spot welding fumes at occupationally relevant concentrations may act as an irritant as evidenced by the increased RL and result in endothelial dysfunction, but otherwise had minor effects on the lung. PMID:25140454

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

PubMed

Czubinski, Jaroslaw; Montowska, Magdalena; Pospiech, Edward; Lampart-Szczapa, Eleonora

2017-12-01

Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as β-conglutin. The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Day-night and reproductive cycle profiles of melatonin receptor, kiss, and gnrh expression in orange-spotted grouper (Epinephelus coioides).

PubMed

Chai, Ke; Liu, Xiaochun; Zhang, Yong; Lin, Haoran

2013-07-01

It is suggested that the MT1 melatonin receptor mediates the effects of melatonin on reproduction in rodents. Three melatonin receptor types, MT1, MT2, and Mel1c, have been identified in fish. To understand the potential roles of each type of melatonin receptor on reproduction, we explored the day-night and reproductive cycle profiles of melatonin receptor, kiss, and gnrh expression in the orange-spotted grouper (Epinephelus coioides). cDNAs encoding melatonin receptors (MT1, MT2, and Mel1c) were first isolated from the brain of the orange-spotted grouper. Quantitative real-time PCR analysis demonstrated that the expression levels of MT1 and MT2 were higher in most of the brain areas and pituitary, while mel1c mRNA was mainly distributed in some peripheral tissues and the pituitary. The expression levels of MT1 were much higher than those of MT2 and mel1c in most of the brain regions, and the day-night expression variations of MT1 were counter to those of kiss2 and gnrh1. Reproductive cycle variations in MT1 daytime expression were different from those for kiss2 and gnrh1, and contrary to ovarian fecundity. Our results suggest that MT1 may modulate gnrh1 expression through kiss2, or may directly influence it. Together, these signal cascades may regulate the seasonal breeding of the orange-spotted grouper. As the day-length variations were consistent with the ovarian fecundity variation observed during the reproductive cycle, we infer that photoperiod affects ovarian development of the orange-spotted grouper through MT1. Copyright © 2013 Wiley Periodicals, Inc.

Systems genetics reveals a transcriptional network associated with susceptibility in the maize-grey leaf spot pathosystem

USDA-ARS?s Scientific Manuscript database

We have applied a systems genetics approach to elucidate molecular mechanisms of maize responses to gray leaf spot (GLS) disease, caused by Cercospora zeina, a major threat to maize production globally. We conducted expression QTL (eQTL) analysis of gene expression variation measured in earleaf samp...

A Comparative Analysis of Industrial Escherichia coli K–12 and B Strains in High-Glucose Batch Cultivations on Process-, Transcriptome- and Proteome Level

PubMed Central

Marisch, Karoline; Bayer, Karl; Scharl, Theresa; Mairhofer, Juergen; Krempl, Peter M.; Hummel, Karin; Razzazi-Fazeli, Ebrahim; Striedner, Gerald

2013-01-01

Escherichia coli K–12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (EttanTM DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K–12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation. PMID:23950949

A comparative analysis of industrial Escherichia coli K-12 and B strains in high-glucose batch cultivations on process-, transcriptome- and proteome level.

PubMed

Marisch, Karoline; Bayer, Karl; Scharl, Theresa; Mairhofer, Juergen; Krempl, Peter M; Hummel, Karin; Razzazi-Fazeli, Ebrahim; Striedner, Gerald

2013-01-01

Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (Ettan(TM) DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K-12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.

Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

PubMed Central

Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael

2011-01-01

Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses. PMID:21949838

Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.

PubMed

Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael

2011-01-01

Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses.

Proteomic analysis of Pinus radiata needles: 2-DE map and protein identification by LC/MS/MS and substitution-tolerant database searching.

PubMed

Valledor, Luis; Castillejo, Maria A; Lenz, Christof; Rodríguez, Roberto; Cañal, Maria J; Jorrín, Jesús

2008-07-01

Pinus radiata is one of the most economically important forest tree species, with a worldwide production of around 370 million m (3) of wood per year. Current selection of elite trees to be used in conservation and breeding programes requires the physiological and molecular characterization of available populations. To identify key proteins related to tree growth, productivity and responses to environmental factors, a proteomic approach is being utilized. In this paper, we present the first report of the 2-DE protein reference map of physiologically mature P. radiata needles, as a basis for subsequent differential expression proteomic studies related to growth, development, biomass production and responses to stresses. After TCA/acetone protein extraction of needle tissue, 549 +/- 21 well-resolved spots were detected in Coommassie-stained gels within the 5-8 pH and 10-100 kDa M(r) ranges. The analytical and biological variance determined for 450 spots were of 31 and 42%, respectively. After LC/MS/MS analysis of in-gel tryptic digested spots, proteins were identified by using the novel Paragon algorithm that tolerates amino acid substitution in the first-pass search. It allowed the confident identification of 115 out of the 150 protein spots subjected to MS, quite unusual high percentage for a poor sequence database, as is the case of P. radiata. Proteins were classified into 12 or 18 groups based on their corresponding cell component or biological process/pathway categories, respectively. Carbohydrate metabolism and photosynthetic enzymes predominate in the 2-DE protein profile of P. radiata needles.

The maintenance of pluripotency following laser direct-write of mouse embryonic stem cells.

PubMed

Raof, Nurazhani Abdul; Schiele, Nathan R; Xie, Yubing; Chrisey, Douglas B; Corr, David T

2011-03-01

The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/geometries may allow for the recreation of a stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment. Copyright © 2010 Elsevier Ltd. All rights reserved.

Hot spot detection, segmentation, and identification in PET images

NASA Astrophysics Data System (ADS)

Blaffert, Thomas; Meetz, Kirsten

2006-03-01

Positron Emission Tomography (PET) images provide functional or metabolic information from areas of high concentration of [18F]fluorodeoxyglucose (FDG) tracer, the "hot spots". These hot spots can be easily detected by the eye, but delineation and size determination required e.g. for diagnosis and staging of cancer is a tedious task that demands for automation. The approach for such an automated hot spot segmentation described in this paper comprises three steps: A region of interest detection by the watershed transform, a heart identification by an evaluation of scan lines, and the final segmentation of hot spot areas by a local threshold. The region of interest detection is the essential step, since it localizes the hot spot identification and the final segmentation. The heart identification is an example of how to differentiate between hot spots. Finally, we demonstrate the combination of PET and CT data. Our method is applicable to other techniques like SPECT.

Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2

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

Mussotter, Franz, E-mail: franz.mussotter@bfr.bund

Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2{sup +/+}) and Nrf2 knockout (nrf2{sup −/−}) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gelmore » electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100 μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100 μM CA, respectively. For 5 and 10 μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2{sup −/−} BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. - Highlights: • Contact allergens induce proteins involved in DC maturation Nrf2-dependently. • Induction of these proteins points to a functional role of Nrf2 in DC maturation. • Evidence that metabolic reprogramming enables DC activation by contact allergens. • Identification of biomarker candidates for development of in vitro testing methods.« less

Spatial Distribution of the Threshold Beam Spots of Laser Weapons Simulators

DTIC Science & Technology

1993-09-08

This paper was based on the transmission theory of elliptical Gaussian beam fluxes in deriving some transmission equations for the threshold beam...spots of laser weapon simulators, in order to revise and expand the expressions for the threshold beam spots, their maximum range, the extinction

Characterization of expressed resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs)

USDA-ARS?s Scientific Manuscript database

Cultivated peanut (Arachis hypogaea L.) is one of the most important food legume crops grown worldwide, and is a major source for edible oil and protein. However, due to low genetic variation, peanut is very vulnerable to a variety of pathogens, such as early leaf spot, late leaf spot, rust and Toma...

Pathovars of Pseudomonas syringae Causing Bacterial Brown Spot and Halo Blight in Phaseolus vulgaris L. Are Distinguishable by Ribotyping

PubMed Central

González, Ana J.; Landeras, Elena; Mendoza, M. Carmen

2000-01-01

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported. PMID:10653764

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.

Proteomic analysis of Trichoderma atroviride mycelia stressed by organophosphate pesticide dichlorvos.

PubMed

Tang, Jun; Liu, Lixing; Huang, Xiuli; Li, Yingying; Chen, Yunpeng; Chen, Jie

2010-02-01

The proteomic approach is a powerful tool to study microbial response to environmental stress. To evaluate the responses of Trichoderma spp. to the organophosphate pesticide dichlorvos, mycelia of Trichoderma atroviride T23 were exposed to dichlorvos at concentrations of 0, 100, 300, 500, and 1000 microg/mL, respectively. Changes in protein expression were investigated using two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Sixteen protein spots were differentially expressed. They were identified by MALDI-TOF/TOF MS and were found to be linked to energy metabolism, transport, signal transduction, and stress tolerance. Among stress-related proteins, glutathione peroxidase-like protein (GPX), 1,4-benzoquinone reductase, and HEX1 were upregulated by and cyclophilin A induced by 1000 microg/mL dichlorvos when compared with the control. These proteins were considered to be associated with fungal adaptation to adverse conditions. The results will help us to understand molecular mechanisms through which Trichoderma responds to organophosphate pesticides.

Meta-transcriptomics indicates biotic cross-tolerance in willow trees cultivated on petroleum hydrocarbon contaminated soil.

PubMed

Gonzalez, Emmanuel; Brereton, Nicholas J B; Marleau, Julie; Guidi Nissim, Werther; Labrecque, Michel; Pitre, Frederic E; Joly, Simon

2015-10-12

High concentrations of petroleum hydrocarbon (PHC) pollution can be hazardous to human health and leave soils incapable of supporting agricultural crops. A cheap solution, which can help restore biodiversity and bring land back to productivity, is cultivation of high biomass yielding willow trees. However, the genetic mechanisms which allow these fast-growing trees to tolerate PHCs are as yet unclear. Salix purpurea 'Fish Creek' trees were pot-grown in soil from a former petroleum refinery, either lacking or enriched with C10-C50 PHCs. De novo assembled transcriptomes were compared between tree organs and impartially annotated without a priori constraint to any organism. Over 45% of differentially expressed genes originated from foreign organisms, the majority from the two-spotted spidermite, Tetranychus urticae. Over 99% of T. urticae transcripts were differentially expressed with greater abundance in non-contaminated trees. Plant transcripts involved in the polypropanoid pathway, including phenylalanine ammonia-lyase (PAL), had greater expression in contaminated trees whereas most resistance genes showed higher expression in non-contaminated trees. The impartial approach to annotation of the de novo transcriptomes, allowing for the possibility for multiple species identification, was essential for interpretation of the crop's response treatment. The meta-transcriptomic pattern of expression suggests a cross-tolerance mechanism whereby abiotic stress resistance systems provide improved biotic resistance. These findings highlight a valuable but complex biotic and abiotic stress response to real-world, multidimensional contamination which could, in part, help explain why crops such as willow can produce uniquely high biomass yields on challenging marginal land.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain.

PubMed

Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami; Ohnishi, Jun; Ohki, Takehiro; Tsuda, Shinya

2009-08-01

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

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

Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami

2009-08-01

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the secondmore » putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.« less

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

PubMed

Choi, Hoseong; Jo, Yeonhwa; Lian, Sen; Jo, Kyoung-Min; Chu, Hyosub; Yoon, Ju-Yeon; Choi, Seung-Kook; Kim, Kook-Hyung; Cho, Won Kyong

2015-06-01

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

PubMed

Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard

2009-08-01

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Asymptomatic petechial eruption on the lower legs.

PubMed

Mendese, Gary; Grande, Donald

2013-09-01

The authors report an unusual case of Rocky Mountain spotted fever that presented as an asymptomatic petechial eruption on the lower legs. Rocky Mountain spotted fever is rare in New England and, as such, is typically not on the differential diagnosis when presented with such patients. What began as an asymptomatic eruption progressed to more classic signs of the disease, including a positive Rocky Mountain spotted fever titer. The patient was successfully treated with doxycydine and within a short period of time, was completely back at baseline.

White-centred retinal haemorrhages (Roth spots).

PubMed

Ling, R; James, B

1998-10-01

Roth spots (white-centred retinal haemorrhages) were classically described as septic emboli lodged in the retina of patients with subacute bacterial endocarditis. Indeed many have considered Roth spots pathognomonic for this condition. More recent histological evidence suggests, however, that they are not foci of bacterial abscess. Instead, they are nonspecific and may be found in many other diseases. A review of the histology and the pathogenesis of these white-centred haemorrhages will be provided, along with the work-up of the differential diagnosis.

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp.

PubMed

Thagun, Chonprakun; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Narangajavana, Jarunya; Sojikul, Punchapat

2012-09-15

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparative proteomic analysis of Cronobacter sakazakii isolates with different virulences.

PubMed

Du, Xin-jun; Han, Ran; Li, Ping; Wang, Shuo

2015-10-14

Cronobacter is a genus of widespread, opportunistic, foodborne pathogens that can result in serious illnesses in at-risk infants because of their immature immunity and high dependence on powdered formula, which is one of the foods most often contaminated by this pathogen. However, limited information is available regarding the pathogenesis and the specific virulence factors of this species. In this study, the virulences of 42 Cronobacter sakazakii isolates were analyzed by infecting neonatal SD rats. A comparison of the typing patterns of the isolates enabled groups with close relationships but that exhibited distinct pathogenesis to be identified. Among these groups, 2 strains belonging to the same group but showing distinct virulences were selected, and 2-DE was applied to identify differentially expressed proteins, focusing on virulence-related proteins. A total of 111 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), and 89 were successfully identified. Further analysis suggested that at least 11 of these proteins may be involved in the pathogenesis of this pathogen. Real-time PCR was carried out to further confirm the differential expression pattern of the genes, and the results indicated that the mRNA expression levels were consistent with the protein expression levels. The virulence factors and pathogenesis of Cronobacter are largely unknown. In combination with animal toxicological experiments and subtyping results of C. sakazakii, comparative proteomics analysis was performed to comprehensively evaluate the differentially expressed proteins of two isolates that exhibited distinct virulence but were closely related. These procedures made it possible to identify the virulence-related of factors of Cronobacter. Among the 89 total identified proteins, at least 11 show virulence-related potential. This work provides comprehensive candidates for the further investigation of the pathogenesis of Cronobacter. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic analysis of the response of α-ketoglutarate-producer Yarrowia lipolytica WSH-Z06 to environmental pH stimuli.

PubMed

Guo, Hongwei; Wan, Hui; Chen, Hongwen; Fang, Fang; Liu, Song; Zhou, Jingwen

2016-10-01

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

Adult murine CNS stem cells express aquaporin channels.

PubMed

La Porta, Caterina A M; Gena, Patrizia; Gritti, Angela; Fascio, Umberto; Svelto, Maria; Calamita, Giuseppe

2006-02-01

Fluid homoeostasis is of critical importance in many functions of the CNS (central nervous system) as indicated by the fact that dysregulation of cell volume underlies clinical conditions such as brain oedema and hypoxia. Water balance is also important during neurogenesis as neural stem cells move considerable amounts of water into or out of the cell to rapidly change their volume during differentiation. Consistent with the relevance of water transport in CNS, multiple AQP (aquaporin) water channels have been recognized and partially characterized in brain cell function. However, the presence and distribution of AQPs in CNS stem cells has not yet been assessed. In the present study, we investigate the expression and subcellular localization of AQPs in murine ANSCs (adult neural stem cells). Considerable AQP8 mRNAs were found in ANSCs where, as expected, the transcript of two additional AQPs, AQP4 and AQP9, was also detected. Immunoblotting with subcellular membrane fractions of ANSCs showed predominant expression of AQP8 in the mitochondria-enriched fraction. This result was consistent with the spotted immunoreactivity profile encountered within the ANSCs by confocal immunofluorescence. AQP8 may have a role in mitochondrial volume regulation during ANSC differentiation. Recognition of AQPs in ANSCs is a step forward in our knowledge of water homoeostasis in the CNS and provides useful information for the purposes of stem cell technology.

Image analysis tools and emerging algorithms for expression proteomics

PubMed Central

English, Jane A.; Lisacek, Frederique; Morris, Jeffrey S.; Yang, Guang-Zhong; Dunn, Michael J.

2012-01-01

Since their origins in academic endeavours in the 1970s, computational analysis tools have matured into a number of established commercial packages that underpin research in expression proteomics. In this paper we describe the image analysis pipeline for the established 2-D Gel Electrophoresis (2-DE) technique of protein separation, and by first covering signal analysis for Mass Spectrometry (MS), we also explain the current image analysis workflow for the emerging high-throughput ‘shotgun’ proteomics platform of Liquid Chromatography coupled to MS (LC/MS). The bioinformatics challenges for both methods are illustrated and compared, whilst existing commercial and academic packages and their workflows are described from both a user’s and a technical perspective. Attention is given to the importance of sound statistical treatment of the resultant quantifications in the search for differential expression. Despite wide availability of proteomics software, a number of challenges have yet to be overcome regarding algorithm accuracy, objectivity and automation, generally due to deterministic spot-centric approaches that discard information early in the pipeline, propagating errors. We review recent advances in signal and image analysis algorithms in 2-DE, MS, LC/MS and Imaging MS. Particular attention is given to wavelet techniques, automated image-based alignment and differential analysis in 2-DE, Bayesian peak mixture models and functional mixed modelling in MS, and group-wise consensus alignment methods for LC/MS. PMID:21046614

Cloning and pattern of expression of the shiro-uo vasa gene during embryogenesis and its roles in PGC development.

PubMed

Miyake, Akimitsu; Saito, Taiju; Kashiwagi, Noboru; Ando, Daisuke; Yamamoto, Akitsugu; Suzuki, Tohru; Nakatsuji, Norio; Nakatsuji, Takako

2006-01-01

The vasa genes are expressed in the germ cell lineage in many organisms, but their expression patterns show large variations. Recent studies suggest that vasa transcripts are involved in germ cell lineage development. In this paper, we isolated the vasa cDNA clone from a teleost, shiro-uo, Leucopsarion petersii and examined its expression pattern during embryogenesis. Then, we examined the functional significance of vasa mRNA during the formation of primordial germ cells (PGCs). The amino acid sequence of shiro-uo VASA is 61.1% identical to that of zebrafish. In whole-mount in situ hybridization, vasa transcripts appeared at the 4- and 8-cell stages as four spots at both ends of two cleavage planes between the lower tier of blastomeres and the yolk cell mass. At the 16-cell stage, eight spots were observed. After the blastula stage, shiro-uo vasa transcripts showed similar localization as in the zebrafish. Ultrastructural analysis of 4-cell stage embryos revealed the presence of a subcellular organelle that resembled 'nuage' in the germ cell lineage observed in the embryos of various organisms. We carried out micromanipulation of 4- or 8-cell stage embryos to remove the vasa mRNA-containing spots and then measured the number of the vasa-expressing PGCs in the genital ridge of the manipulated embryos. The numbers decreased when all of the four spots were removed, indicating that the vasa-containing spots at early cleavage stages have important functions in the development of PGCs.

MACULA: Fast Modeling of Rotational Modulations of Spotty Stars

NASA Astrophysics Data System (ADS)

Kipping, David

2015-08-01

Rotational modulations are frequently observed on stars observed by photometry surveys such as Kepler, with periodicities ranging from days to months and amplitudes of sub-parts-per-million to several percent. These variations may be studied to reveal important stellar properties such as rotational periods, inclinations and gradients of differential rotation. However, inverting the disk-integrated flux into a solution for spot number, sizes, contrasts, etc is highly degenerate and thereby necessitating an exhaustive search of the parameter space. In recognition of this, the software MACULA is designed to be a fast forward model of circular, grey spots on rotating stars, including effects such as differential rotation, spot evolution and even spot penumbra/umbra. MACULA seeks to achieve computational efficiency by using a wholly analytic description of the disk-integrated flux, which is described in Kipping (2012), leading to a computational improvement of three orders-of-magnitude over its numerical counterparts. As part of the hack day, I'll show how to simulate light curves with MACULA and provide examples with visualizations. I will also discuss the on-going development of the code, which will head towards modeling spot crossing events and radial velocity jitter and I encourage discussions amongst the participants on analytic methods to this end.

Induction of Primary Male in Juvenile Red Spotted Grouper Epinephelus akaara by Immersion of 17α-Methyltestosterone

PubMed Central

Lee, Chi-Hoon; Hur, Sang-Woo; Na, Oh-Soo; Baek, Hae-Ja; Noh, Choong-Hwan; Han, Sang-Hyun; Lee, Young-Don

2014-01-01

We investigated the androgenic effects of 17α- methyltestosterone (MT) on gonadal sex reversal in juvenile red spotted grouper Epinephelus akaara. The fish were immersed in 17α-MT at 1 and 5 mg/L. Treatment method of 17α-MT was once weekly for 4 and 8 weeks. Fish were sampled at 12 months after end of the treatment period in order to histological analysis. At the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. Formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 DAH in red spotted grouper. At 12 months after end of the treatment period, control group, 17α-MT 1 mg/L treatment group for 4 and 8 weeks, and 17α-MT 5 mg/L treatment group for 4 weeks were all female. However, sex-changed males without ovarian cavity were observed in the 17α-MT 5 mg/L treatment group for 8 weeks. In grouper, we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation. PMID:25949180

Sequence and Expression Characteristics of Long Noncoding RNAs in Honey Bee Caste Development – Potential Novel Regulators for Transgressive Ovary Size

PubMed Central

Humann, Fernanda C.; Tiberio, Gustavo J.; Hartfelder, Klaus

2013-01-01

Division of labor in social insect colonies relies on a strong reproductive bias that favors queens. Although the ecological and evolutionary success attained through caste systems is well sketched out in terms of ultimate causes, the molecular and cellular underpinnings driving the development of caste phenotypes are still far from understood. Recent genomics approaches on honey bee developmental biology revealed a set of genes that are differentially expressed genes in larval ovaries and associated with transgressive ovary size in queens and massive cell death in workers. Amongst these, two contigs called special attention, both being over 200 bp in size and lacking apparent coding potential. Herein, we obtained their full cDNA sequences. These and their secondary structure characteristics placed in evidence that they are bona fide long noncoding RNAs (lncRNA) differentially expressed in larval ovaries, thus named lncov1 and lncov2. Genomically, both map within a previously identified QTL on chromosome 11, associated with transgressive ovary size in honey bee workers. As lncov1 was over-expressed in worker ovaries we focused on this gene. Real-time qPCR analysis on larval worker ovaries evidenced an expression peak coinciding with the onset of autophagic cell death. Cellular localization analysis through fluorescence in situ hybridization revealed perinuclear spots resembling omega speckles known to regulate trafficking of RNA-binding proteins. With only four lncRNAs known so far in honey bees, two expressed in the ovaries, these findings open a novel perspective on regulatory factors acting in the fine tuning of developmental processes underlying phenotypic plasticity related to social life histories. PMID:24205350

Effects of genotype and isolate on expression of dollar spot in seashore paspalum

USDA-ARS?s Scientific Manuscript database

Seashore paspalum (Paspalum vaginatum Swartz) is a warm-season turfgrass species primarily utilized on golf courses and athletic fields and is often impacted by dollar spot disease. Dollar spot, caused by Sclerotinia homoeocarpa F.T. Bennett, is a major fungal disease and the most common turfgrass p...

Analytical modeling of the temporal evolution of hot spot temperatures in silicon solar cells

NASA Astrophysics Data System (ADS)

Wasmer, Sven; Rajsrima, Narong; Geisemeyer, Ino; Fertig, Fabian; Greulich, Johannes Michael; Rein, Stefan

2018-03-01

We present an approach to predict the equilibrium temperature of hot spots in crystalline silicon solar cells based on the analysis of their temporal evolution right after turning on a reverse bias. For this end, we derive an analytical expression for the time-dependent heat diffusion of a breakdown channel that is assumed to be cylindrical. We validate this by means of thermography imaging of hot spots right after turning on a reverse bias. The expression allows to be used to extract hot spot powers and radii from short-term measurements, targeting application in inline solar cell characterization. The extracted hot spot powers are validated at the hands of long-term dark lock-in thermography imaging. Using a look-up table of expected equilibrium temperatures determined by numerical and analytical simulations, we utilize the determined hot spot properties to predict the equilibrium temperatures of about 100 industrial aluminum back-surface field solar cells and achieve a high correlation coefficient of 0.86 and a mean absolute error of only 3.3 K.

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.

Birth-jump processes and application to forest fire spotting.

PubMed

Hillen, T; Greese, B; Martin, J; de Vries, G

2015-01-01

Birth-jump models are designed to describe population models for which growth and spatial spread cannot be decoupled. A birth-jump model is a nonlinear integro-differential equation. We present two different derivations of this equation, one based on a random walk approach and the other based on a two-compartmental reaction-diffusion model. In the case that the redistribution kernels are highly concentrated, we show that the integro-differential equation can be approximated by a reaction-diffusion equation, in which the proliferation rate contributes to both the diffusion term and the reaction term. We completely solve the corresponding critical domain size problem and the minimal wave speed problem. Birth-jump models can be applied in many areas in mathematical biology. We highlight an application of our results in the context of forest fire spread through spotting. We show that spotting increases the invasion speed of a forest fire front.

Spatial variation in colour morph, spotting and allozyme frequencies in the candy-stripe spider, Enoplognatha ovata (Theridiidae) on two Swedish archipelagos.

PubMed

Oxford, G S; Gunnarsson, B

2006-01-01

The selective significance, if any, of many invertebrate visible polymorphisms is still not fully understood. Here we examine colour- and black spotting-morph frequencies in the spider Enoplognatha ovata in populations on two Swedish archipelagos with respect to different spatial scales and, in one archipelago, against the background of variation at four putative neutral allozyme marker loci. Every population studied was polymorphic for colour and 28 out of 30 contained all three colour morphs--lineata, redimita and ovata. We found no evidence for a breakdown in the traditional colour morph designation previously suggested for other northern European populations of this species. For colour there is no significant heterogeneity at spatial scales greater than between local populations within islands. Black spotting frequencies show a similar lack of pattern over larger spatial scales except that there are significant differences between the Stockholm and Göteborg archipelagos. Measures of population differentiation (theta) within the Stockholm islands for the two visible systems show them to be significantly more differentiated than the neutral markers, suggesting local selection acting on them in a population-specific manner. On the basis of previous observations and the distribution of spotting phenotypes on a European scale, it is argued that thermal selection might operate on black spotting during the juvenile stages favouring more spots in continental climates. It is not clear what selective forces act on colour.

Magnetic activity and differential rotation in the young Sun-like stars KIC 7985370 and KIC 7765135

NASA Astrophysics Data System (ADS)

Fröhlich, H.-E.; Frasca, A.; Catanzaro, G.; Bonanno, A.; Corsaro, E.; Molenda-Żakowicz, J.; Klutsch, A.; Montes, D.

2012-07-01

Aims: We present a detailed study of the two Sun-like stars KIC 7985370 and KIC 7765135, to determine their activity level, spot distribution, and differential rotation. Both stars were previously discovered by us to be young stars and were observed by the NASA Kepler mission. Methods: The fundamental stellar parameters (vsini, spectral type, Teff, log g, and [Fe/H]) were derived from optical spectroscopy by comparison with both standard-star and synthetic spectra. The spectra of the targets allowed us to study the chromospheric activity based on the emission in the core of hydrogen Hα and Ca ii infrared triplet (IRT) lines, which was revealed by the subtraction of inactive templates. The high-precision Kepler photometric data spanning over 229 days were then fitted with a robust spot model. Model selection and parameter estimation were performed in a Bayesian manner, using a Markov chain Monte Carlo method. Results: We find that both stars are Sun-like (of G1.5 V spectral type) and have an age of about 100-200 Myr, based on their lithium content and kinematics. Their youth is confirmed by their high level of chromospheric activity, which is comparable to that displayed by the early G-type stars in the Pleiades cluster. The Balmer decrement and flux ratio of their Ca ii-IRT lines suggest that the formation of the core of these lines occurs mainly in optically thick regions that are analogous to solar plages. The spot model applied to the Kepler photometry requires at least seven persistent spots in the case of KIC 7985370 and nine spots in the case of KIC 7765135 to provide a satisfactory fit to the data. The assumption of the longevity of the star spots, whose area is allowed to evolve with time, is at the heart of our spot-modelling approach. On both stars, the surface differential rotation is Sun-like, with the high-latitude spots rotating slower than the low-latitude ones. We found, for both stars, a rather high value of the equator-to-pole differential rotation (dΩ ≈ 0.18 rad d-1), which disagrees with the predictions of some mean-field models of differential rotation for rapidly rotating stars. Our results agree instead with previous works on solar-type stars and other models that predict a higher latitudinal shear, increasing with equatorial angular velocity, that can vary during the magnetic cycle. Based on public Kepler data, on observations made with the Italian Telescopio Nazionale Galileo (TNG) operated by the Fundación Galileo Galilei - INAF at the Observatorio del Roque del los Muchachos, La Palma (Canary Islands), on observations collected at the 2.2-m telescope of the Centro Astronómico Hispano Alemán (CAHA) at Calar Alto (Almería, Spain), operated jointly by the Max-Planck-Institut für Astronomie and the Instituto de Astrofísica de Andalucía (CSIC), and on observations collected at the Catania Astrophysical Observatory (Italy).

A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency

PubMed Central

Khandakar, Jebunnahar; Haraguchi, Izumi; Yamaguchi, Kenichi; Kitamura, Yoshie

2013-01-01

Hyoscyamus albus is a well-known source of the tropane alkaloids, hyoscyamine and scopolamine, which are biosynthesized in the roots. To assess the major biochemical adaptations that occur in the roots of this plant in response to iron deficiency, we used a small-scale proteomic approach in which 100 mg of root tips were treated with and without Fe, respectively, for 5 days. Two-dimensional mini gels showed that 48 spots were differentially accumulated between the two conditions of Fe availability and a further 36 proteins were identified from these spots using MALDI-QIT-TOF mass spectrometry. The proteins that showed elevated levels in the roots lacking Fe were found to be associated variously with carbohydrate metabolism, cell differentiation, secondary metabolism, and oxidative defense. Most of the proteins involved in carbohydrate metabolism were increased in abundance, but mitochondrial NAD-dependent malate dehydrogenase was decreased, possibly resulting in malate secretion. Otherwise, all the proteins showing diminished levels in the roots were identified as either Fe-containing or ATP-requiring. For example, a significant decrease was observed in the levels of hyoscyamine 6β-hydroxylase (H6H), which requires Fe and is involved in the conversion of hyoscyamine to scopolamine. To investigate the effects of Fe deficiency on alkaloid biosynthesis, gene expression studies were undertaken both for H6H and for another Fe-dependent protein, Cyp80F1, which is involved in the final stage of hyoscyamine biosynthesis. In addition, tropane alkaloid contents were determined. Reduced gene expression was observed in the case of both of these proteins and was accompanied by a decrease in the content of both hyoscyamine and scopolamine. Finally, we have discussed energetic and Fe-conservation strategies that might be adopted by the roots of H. albus to maintain iron homeostasis under Fe-limiting conditions. PMID:24009619

The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism.

PubMed

Bækkedal, Cecilie; Haugen, Peik

2015-01-01

The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding.

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

PubMed Central

Wang, Xun; Ji, Sang Chun; Jeon, Heung Jin; Lee, Yonho; Lim, Heon M.

2015-01-01

The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT. PMID:26045496

Characterization of gonadal soma-derived factor expression during sex change in the protogynous wrasse, Halichoeres trimaculatus.

PubMed

Horiguchi, Ryo; Nozu, Ryo; Hirai, Toshiaki; Kobayashi, Yasuhisa; Nagahama, Yoshitaka; Nakamura, Masaru

2013-04-01

Sex change in fishes provides a good experimental model for understanding the mechanisms and plasticity of sex determination and differentiation. The three-spot wrasse, Halichoeres trimaculatus is a protogynous hermaphrodite. During sex change from female to male, the ovary is replaced by the testis through the degeneration of oocytes and subsequent spermatogenesis. In the present study, we cloned a cDNA-encoding gonadal soma-derived factor (GSDF) from protogynous wrasse and examined its expression pattern in the sexually mature gonads and the sex-changing gonad induced experimentally by aromatase inhibition. Expression of gsdf was predominantly observed in the testis, and it was mainly localized to the supporting cells surrounding the spermatogonia. In the ovary, only slight expression of gsdf was observed in morphologically undifferentiated supporting cells in contact with oogonia. During sex change, strong expression of gsdf appeared first in the supporting cells surrounding the gonial germ cells before the onset of spermatogenesis. Thereafter, the expression of gsdf continually increased in the supporting cells surrounding the proliferating spermatogonia throughout the sex change. These results suggest that gsdf is involved in the proliferation of spermatogonia and subsequent spermatogenesis in both the testis and the gonad in the early stages of sex change. Copyright © 2013 Wiley Periodicals, Inc.

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

PubMed

Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass ( Lateolabrax maculatus)

NASA Astrophysics Data System (ADS)

Shin, Moon-Kyeong; Park, Ho-Ra; Yeo, Won-Jun; Han, Kyung-Nam

2018-03-01

The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass ( Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

Changes in the leaf proteome profile of Withania somnifera (L.) Dunal in response to Alternaria alternata infection

PubMed Central

Singh, Varinder; Singh, Baldev; Joshi, Robin; Jaju, Puneet

2017-01-01

Withania somnifera is a high value medicinal plant which is used against large number of ailments. The medicinal properties of the plant attributes to a wide array of important secondary metabolites. The plant is predominantly infected with leaf spot pathogen Alternaria alternata, which leads to substantial biodeterioration of pharmaceutically important metabolites. To develop an effective strategy to combat this disease, proteomics based approach could be useful. Hence, in the present study, three different protein extraction methods tris-buffer based, phenol based and trichloroacetic acid-acetone (TCA-acetone) based method were comparatively evaluated for two-dimensional electrophoresis (2-DE) analysis of W. somnifera. TCA-acetone method was found to be most effective and was further used to identify differentially expressed proteins in response to fungal infection. Thirty-eight differentially expressed proteins were identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry (MALDI TOF/TOF MS/MS). The known proteins were categorized into eight different groups based on their function and maximum proteins belonged to energy and metabolism, cell structure, stress and defense and RNA/DNA categories. Differential expression of some key proteins were also crosschecked at transcriptomic level by using qRT-PCR and were found to be consistent with the 2-DE data. These outcomes enable us to evaluate modifications that take place at the proteomic level during a compatible host pathogen interaction. The comparative proteome analysis conducted in this paper revealed the involvement of many key proteins in the process of pathogenesis and further investigation of these identified proteins could assist in the discovery of new strategies for the development of pathogen resistance in the plant. PMID:28575108

Architecture of thermal adaptation in an Exiguobacterium sibiricum strain isolated from 3 million year old permafrost: A genome and transcriptome approach

PubMed Central

Rodrigues, Debora F; Ivanova, Natalia; He, Zhili; Huebner, Marianne; Zhou, Jizhong; Tiedje, James M

2008-01-01

Background Many microorganisms have a wide temperature growth range and versatility to tolerate large thermal fluctuations in diverse environments, however not many have been fully explored over their entire growth temperature range through a holistic view of its physiology, genome, and transcriptome. We used Exiguobacterium sibiricum strain 255-15, a psychrotrophic bacterium from 3 million year old Siberian permafrost that grows from -5°C to 39°C to study its thermal adaptation. Results The E. sibiricum genome has one chromosome and two small plasmids with a total of 3,015 protein-encoding genes (CDS), and a GC content of 47.7%. The genome and transcriptome analysis along with the organism's known physiology was used to better understand its thermal adaptation. A total of 27%, 3.2%, and 5.2% of E. sibiricum CDS spotted on the DNA microarray detected differentially expressed genes in cells grown at -2.5°C, 10°C, and 39°C, respectively, when compared to cells grown at 28°C. The hypothetical and unknown genes represented 10.6%, 0.89%, and 2.3% of the CDS differentially expressed when grown at -2.5°C, 10°C, and 39°C versus 28°C, respectively. Conclusion The results show that E. sibiricum is constitutively adapted to cold temperatures stressful to mesophiles since little differential gene expression was observed between 4°C and 28°C, but at the extremities of its Arrhenius growth profile, namely -2.5°C and 39°C, several physiological and metabolic adaptations associated with stress responses were observed. PMID:19019206

Asymptomatic Petechial Eruption on the Lower Legs

PubMed Central

Grande, Donald

2013-01-01

The authors report an unusual case of Rocky Mountain spotted fever that presented as an asymptomatic petechial eruption on the lower legs. Rocky Mountain spotted fever is rare in New England and, as such, is typically not on the differential diagnosis when presented with such patients. What began as an asymptomatic eruption progressed to more classic signs of the disease, including a positive Rocky Mountain spotted fever titer. The patient was successfully treated with doxycydine and within a short period of time, was completely back at baseline. PMID:24062875

White-centred retinal haemorrhages (Roth spots).

PubMed Central

Ling, R.; James, B.

1998-01-01

Roth spots (white-centred retinal haemorrhages) were classically described as septic emboli lodged in the retina of patients with subacute bacterial endocarditis. Indeed many have considered Roth spots pathognomonic for this condition. More recent histological evidence suggests, however, that they are not foci of bacterial abscess. Instead, they are nonspecific and may be found in many other diseases. A review of the histology and the pathogenesis of these white-centred haemorrhages will be provided, along with the work-up of the differential diagnosis. Images Figure 1 Figure 2 PMID:10211348

RING domain is essential for the antiviral activity of TRIM25 from orange spotted grouper.

PubMed

Yang, Ying; Huang, Youhua; Yu, Yepin; Yang, Min; Zhou, Sheng; Qin, Qiwei; Huang, Xiaohong

2016-08-01

Tripartite motif-containing 25 (TRIM25) has been demonstrated to exert crucial roles in the regulation of innate immune signaling. However, the roles of fish TRIM25 in antiviral immune response still remained uncertain. Here, a novel fish TRIM25 gene from orange spotted grouper (EcTRIM25) was cloned and its roles in grouper virus infection were elucidated. EcTRIM25 encoded a 734-aa protein which shared 68% identity to large yellow croaker (Larimichthys crocea). Amino acid alignment showed that EcTRIM25 contained three conserved domains, including a RING-finger domain, a B box/coiled-coil domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM25 was predominantly detected in skin, spleen and intestine. After stimulation with Singapore grouper iridovirus (SGIV) or poly I:C, the relative expression of EcTRIM25 in grouper spleen was significantly increased at the early stage of injection. Subcellular localization analysis showed that EcTRIM25 distributed throughout the cytoplasm in grouper cells. Notably, the deletion RING domain affected its accurate localization and displayed microtubule like structures or bright aggregates in GS cells. After incubation with SGIV or red spotted grouper nervous necrosis virus (RGNNV), overexpression of full length of EcTRIM25 in vitro significantly decreased the viral gene transcription of SGIV and RGNNV. Consistently, the deletion of RING domain obviously affected the inhibitory effect of EcTRIM25. Furthermore, overexpression of EcTRIM25 significantly increased the expression level of interferon related signaling molecules, including interferon regulatory factor (IRF) 3, interferon-induced 35-kDa protein (IFP35), MXI, IRF7 and myeloid differentiation factor 88 (MyD88), suggesting that the positive regulation of interferon immune response by EcTRIM25 might affected RGNNV replication directly. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcTRIM25. We proposed that the regulation of IRF7, MyD88 and pro-inflammation cytokines might contribute more important roles in SGIV infection. In addition, the RING domain of EcTRIM25 also played critical roles in the regulation of interferon immune and inflammation response. Together, our results will provide new evidences that the RING domain was essential for the antiviral action of fish TRIM25 against iridovirus and nodavirus infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Progressive outer retinal necrosis presenting as cherry red spot.

PubMed

Yiu, Glenn; Young, Lucy H

2012-10-01

To report a case of progressive outer retinal necrosis (PORN) presenting as a cherry red spot. Case report. A 53-year-old woman with recently diagnosed HIV and varicella-zoster virus (VZV) aseptic meningitis developed rapid sequential vision loss in both eyes over 2 months. Her exam showed a "cherry red spot" in both maculae with peripheral atrophy and pigmentary changes, consistent with PORN. Due to her late presentation and the rapid progression of her condition, she quickly developed end-stage vision loss in both eyes. PORN should be considered within the differential diagnosis of a "cherry red spot." Immune-deficient patients with a history of herpetic infection who present with visual loss warrant prompt ophthalmological evaluation.

Dynamic imaging model and parameter optimization for a star tracker.

PubMed

Yan, Jinyun; Jiang, Jie; Zhang, Guangjun

2016-03-21

Under dynamic conditions, star spots move across the image plane of a star tracker and form a smeared star image. This smearing effect increases errors in star position estimation and degrades attitude accuracy. First, an analytical energy distribution model of a smeared star spot is established based on a line segment spread function because the dynamic imaging process of a star tracker is equivalent to the static imaging process of linear light sources. The proposed model, which has a clear physical meaning, explicitly reflects the key parameters of the imaging process, including incident flux, exposure time, velocity of a star spot in an image plane, and Gaussian radius. Furthermore, an analytical expression of the centroiding error of the smeared star spot is derived using the proposed model. An accurate and comprehensive evaluation of centroiding accuracy is obtained based on the expression. Moreover, analytical solutions of the optimal parameters are derived to achieve the best performance in centroid estimation. Finally, we perform numerical simulations and a night sky experiment to validate the correctness of the dynamic imaging model, the centroiding error expression, and the optimal parameters.

Starspot evolution, differential rotation, and magnetic cycles in the chromospherically active binaries lambda andromedae, sigma Geminorum, II Pegasi, and V711 Tauri

NASA Technical Reports Server (NTRS)

Henry, Gregory W.; Eaton, Joel A.; Hamer, Jamesia; Hall, Douglas S.

1995-01-01

We have analyzed 15-19 yr of photoelectric photometry, obtained manually and with automated telescopes, of the chromospherically active binaries lambda And, sigma Gem, II Peg, and V711 Tau. These observations let us identify individual dark starspots on the stellar surfaces from periodic dimming of the starlight, follow the evolution of these spots, and search for long-term cyclic changes in the properties of these starspots that might reveal magnetic cycles analogous to the Sun's 11 yr sunspot cycle. We developed a computer code to fit a simple two-spot model to our observed light curves that allows us to extract the most easily determinable and most reliable spot parameters from the light curves, i.e., spot longitudes and radii. We then used these measured properties to identify individual spots and to chart their life histories by constructing migration and amplitude curves. We identified and followed 11 spots in lambda And, 16 in sigma Gem, 12 in II Peg, and 15 in V711 Tau. Lifetimes of individual spots ranged from a few months to longer than 6 yr. Differential rotation coefficients, estimated from the observed range of spot rotation periods for each star and defined by equation (2), were 0.04 for lambda And, 0.038 for sigma Gem, 0.005 for II Peg, and 0.006 for V711 Tau, versus 0.19 for the Sun. We searched for cyclic changes in mean brightness, B-V color index, and spot rotation period as evidence for long-term cycles. Of these, long-term variability in mean brightness appears to offer the best evidence for such cycles in these four stars. Cycles of 11.1 yr for lambda And, 8.5 yr for sigma Gem, 11 yr for II Peg, and 16 yr V711 Tau are implied by these mean brightness changes. Cyclic changes in spot rotation period were found in lambda And and possibly II Peg. Errors in B-V were too large for any long-term changes to be detectable.

Effects of acute inhalation of aerosols generated during resistance spot welding with mild-steel on pulmonary, vascular and immune responses in rats.

PubMed

Zeidler-Erdely, Patti C; Meighan, Terence G; Erdely, Aaron; Fedan, Jeffrey S; Thompson, Janet A; Bilgesu, Suzan; Waugh, Stacey; Anderson, Stacey; Marshall, Nikki B; Afshari, Aliakbar; McKinney, Walter; Frazer, David G; Antonini, James M

2014-10-01

Spot welding is used in the automotive and aircraft industries, where high-speed, repetitive welding is needed to join thin sections of metal. Epoxy adhesives are applied as sealers to the metal seams. Pulmonary function abnormalities and airway irritation have been reported in spot welders, but no animal toxicology studies exist. Therefore, the goal of this study was to investigate vascular, immune and lung toxicity measures after exposure to these metal fumes in an animal model. Male Sprague-Dawley rats were exposed by inhalation to 25 mg/m³ to either mild-steel spot welding aerosols with sparking (high metal, HM) or without sparking (low metal, LM) for 4 h/d for 3, 8 and 13 d. Shams were exposed to filtered air. Bronchoalveolar lavage (BAL), lung gene expression and ex vivo BAL cell challenge were performed to assess lung toxicity. Lung resistance (R(L)) was evaluated before and after challenge with inhaled methacholine (MCh). Functional assessment of the vascular endothelium in isolated rat tail arteries and leukocyte differentiation in the spleen and lymph nodes via flow cytometry was also done. Immediately after exposure, baseline R(L) was significantly elevated in the LM spot welding aerosols, but returned to control level by 24 h postexposure. Airway reactivity to MCh was unaffected. Lung inflammation and cytotoxicity were mild and transient. Lung epithelial permeability was significantly increased after 3 and 8 d, but not after 13 d of exposure to the HM aerosol. HM aerosols also caused vascular endothelial dysfunction and increased CD4+, CD8+ and B cells in the spleen. Only LM aerosols caused increased IL-6 and MCP-1 levels compared with sham after ex vivo LPS stimulation in BAL macrophages. Acute inhalation of mild-steel spot welding fumes at occupationally relevant concentrations may act as an irritant as evidenced by the increased R(L) and result in endothelial dysfunction, but otherwise had minor effects on the lung.

Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

PubMed Central

Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

2015-01-01

Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965

Salinity-induced inhibition of growth in the aquatic pteridophyte Azolla microphylla primarily involves inhibition of photosynthetic components and signaling molecules as revealed by proteome analysis.

PubMed

Thagela, Preeti; Yadav, Ravindra Kumar; Mishra, Vagish; Dahuja, Anil; Ahmad, Altaf; Singh, Pawan Kumar; Tiwari, Budhi Sagar; Abraham, Gerard

2017-01-01

Salinity stress causes adverse physiological and biochemical changes in the growth and productivity of a plant. Azolla, a symbiotic pteridophyte and potent candidate for biofertilizer due to its nitrogen fixation ability, shows reduced growth and nitrogen fixation during saline stress. To better understand regulatory components involved in salinity-induced physiological changes, in the present study, Azolla microphylla plants were exposed to NaCl (6.74 and 8.61 ds/m) and growth, photochemical reactions of photosynthesis, ion accumulation, and changes in cellular proteome were studied. Maximum dry weight was accumulated in control and untreated plant while a substantial decrease in dry weight was observed in the plants exposed to salinity. Exposure of the organism to different concentrations of salt in hydroponic conditions resulted in differential level of Na + and K + ion accumulation. Comparative analysis of salinity-induced proteome changes in A. microphylla revealed 58 salt responsive proteins which were differentially expressed during the salt exposure. Moreover, 42 % spots among differentially expressed proteins were involved in different signaling events. The identified proteins are involved in photosynthesis, energy metabolism, amino acid biosynthesis, protein synthesis, and defense. Downregulation of these key metabolic proteins appears to inhibit the growth of A. microphylla in response to salinity. Altogether, the study revealed that in Azolla, increased salinity primarily affected signaling and photosynthesis that in turn leads to reduced biomass.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

PubMed

Graser, Stephanie; Mentrup, Birgit; Schneider, Doris; Klein-Hitpass, Ludger; Jakob, Franz; Hofmann, Christine

2015-10-01

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteomic analysis of pollination-induced corolla senescence in petunia.

PubMed

Bai, Shuangyi; Willard, Belinda; Chapin, Laura J; Kinter, Michael T; Francis, David M; Stead, Anthony D; Jones, Michelle L

2010-02-01

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petuniaxhybrida 'Mitchell Diploid' corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

Preliminary evidence of mitochondrial dysfunction associated with post-infective fatigue after acute infection with Epstein Barr virus.

PubMed

Vernon, Suzanne D; Whistler, Toni; Cameron, Barbara; Hickie, Ian B; Reeves, William C; Lloyd, Andrew

2006-01-31

Acute infectious diseases are typically accompanied by non-specific symptoms including fever, malaise, irritability and somnolence that usually resolve on recovery. However, in some individuals these symptoms persist in what is commonly termed post-infective fatigue. The objective of this pilot study was to determine the gene expression correlates of post-infective fatigue following acute Epstein Barr virus (EBV) infection. We followed 5 people with acute mononucleosis who developed post-infective fatigue of more than 6 months duration and 5 HLA-matched control subjects who recovered within 3 months. Subjects had peripheral blood mononuclear cell (PBMC) samples collected at varying time points including at diagnosis, then every 2 weeks for 3 months, then every 3 months for a year. Total RNA was extracted from the PBMC samples and hybridized to microarrays spotted with 3,800 oligonucleotides. Those who developed post-infective fatigue had gene expression profiles indicative of an altered host response during acute mononucleosis compared to those who recovered uneventfully. Several genes including ISG20 (interferon stimulated gene), DNAJB2 (DnaJ [Hsp40] homolog and CD99), CDK8 (cyclin-dependent kinase 8), E2F2 (E2F transcription factor 2), CDK8 (cyclin-dependent kinase 8), and ACTN2 (actinin, alpha 2), known to be regulated during EBV infection, were differentially expressed in post-infective fatigue cases. Several of the differentially expressed genes affect mitochondrial functions including fatty acid metabolism and the cell cycle. These preliminary data provide insights into alterations in gene transcripts associated with the varied clinical outcomes from acute infectious mononucleosis.

Treatment of Idiopathic Toe-Walking in Children with Autism Using GaitSpot Auditory Speakers and Simplified Habit Reversal

ERIC Educational Resources Information Center

Marcus, Ann; Sinnott, Brigit; Bradley, Stephen; Grey, Ian

2010-01-01

This study aimed to examine the effectiveness of a simplified habit reversal procedure (SHR) using differential reinforcement of incompatible behaviour (DRI) and a stimulus prompt (GaitSpot Auditory Squeakers) to reduce the frequency of idiopathic toe-walking (ITW) and increase the frequency of correct heel-to-toe-walking in three children with…

New Insights into the Roles of Host Gene-Necrotrophic Effector Interactions in Governing Susceptibility of Durum Wheat to Tan Spot and Septoria nodorum Blotch.

PubMed

Virdi, Simerjot K; Liu, Zhaohui; Overlander, Megan E; Zhang, Zengcui; Xu, Steven S; Friesen, Timothy L; Faris, Justin D

2016-12-07

Tan spot and Septoria nodorum blotch (SNB) are important diseases of wheat caused by the necrotrophic fungi Pyrenophora tritici-repentis and Parastagonospora nodorum, respectively. The P. tritici-repentis necrotrophic effector (NE) Ptr ToxB causes tan spot when recognized by the Tsc2 gene. The NE ToxA is produced by both pathogens and has been associated with the development of both tan spot and SNB when recognized by the wheat Tsn1 gene. Most work to study these interactions has been conducted in common wheat, but little has been done in durum wheat. Here, quantitative trait loci (QTL) analysis of a segregating biparental population indicated that the Tsc2-Ptr ToxB interaction plays a prominent role in the development of tan spot in durum. However, analysis of two biparental populations indicated that the Tsn1-ToxA interaction was not associated with the development of tan spot, but was strongly associated with the development of SNB. Pa. nodorum expressed ToxA at high levels in infected Tsn1 plants, whereas ToxA expression in P. tritici-repentis was barely detectable, suggesting that the differences in disease levels associated with the Tsn1-ToxA interaction were due to differences in pathogen expression of ToxA These and previous results together indicate that: (1) the effects of Tsn1-ToxA on tan spot in common wheat can range from nonsignificant to highly significant depending on the host genetic background; (2) Tsn1-ToxA is not a significant factor for tan spot development in durum wheat; and (3) Tsn1-ToxA plays a major role in SNB development in both common and durum wheat. Durum and common wheat breeders alike should strive to remove both Tsc2 and Tsn1 from their materials to achieve disease resistance. Copyright © 2016 Virdi et al.

Preliminary study on plasma proteins in pregnant and non-pregnant female dogs.

PubMed

Szczubiał, Marek; Wawrzykowski, Jacek; Dąbrowski, Roman; Krawczyk, Magdalena; Kankofer, Marta

2017-07-15

In this study, we used a combined approach based on 2-dimensional electrophoresis (2-DE), difference in gel electrophoresis (DIGE), and mass spectrometry (MS) to identify the plasma protein composition in pregnant female dogs and compared it with non-pregnant female dogs. We used the plasma samples obtained from four female dogs during I, II, and III thirds of pregnancy, three days after parturition, as well as from four non-pregnant female dogs in diestrus phase. Analysis of 2-DE gel image exhibited of 249 protein spots. The intensity of staining of 35 spots differed significantly (P < 0.05) between the non-pregnant and pregnant female dogs. We used matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI TOF MS) to identify 47 spots corresponding to 52 different proteins. Five identified protein spots, including zinc finger BED domain-containing protein 5, hemoglobin subunit beta-2, integrator complex subunit 7, apolipoprotein A-I, and glutamyl aminopeptidase were differentially presented in the plasma of pregnant and non-pregnant female dogs. To the best of our knowledge, this is the first report on the plasma protein profile of pregnant and non-pregnant female dogs. In this study, we identified proteins that have not been previously identified in dogs. Our findings showed that numerous protein spots were differentially presented in the plasma of female dogs during normal pregnancy. Although we identified only a limited number of differentially presented proteins, our study demonstrated that the plasma protein profile changed during pregnancy in female dogs, which suggests its importance in maintaining pregnancy. Further studies are necessary to define complete plasma protein profile of pregnant female dogs and to identify all proteins that are differentially presented in the pregnant animals compared with the non-pregnant ones. In addition, studies are warranted to explain the role of those proteins in maintaining the pregnancy and their usefulness in detection of early pregnancy. Furthermore, our results indicated that DIGE technique is useful in the comparison of samples originated from different states and time points in dogs. Copyright © 2017 Elsevier Inc. All rights reserved.

TvMP50 is an Immunogenic Metalloproteinase during Male Trichomoniasis*

PubMed Central

Quintas-Granados, Laura Itzel; Villalpando, José Luis; Vázquez-Carrillo, Laura Isabel; Arroyo, Rossana; Mendoza-Hernández, Guillermo; Álvarez-Sánchez, María Elizbeth

2013-01-01

Trichomonas vaginalis, a human urogenital tract parasite, is capable of surviving in the male microenvironment, despite of the presence of Zn2+. Concentrations > 1.6 mm of Zn2+ have a trichomonacidal effect; however, in the presence of ≤1.6 mm Zn2+, several trichomonad proteins are up- or down-regulated. Herein, we analyzed the proteome of a T. vaginalis male isolate (HGMN01) grown in the presence of Zn2+ and found 32 protein spots that were immunorecognized by male trichomoniasis patient serum. Using mass spectrometry (MS), the proteins were identified and compared with 23 spots that were immunorecognized in the proteome of a female isolate using the same serum. Interestingly, we found a 50-kDa metallopeptidase (TvMP50). Unexpectedly, this proteinase was immunodetected by the serum of male trichomoniasis patients but not by the female patient serum or sera from healthy men and women. We analyzed the T. vaginalis genome and localized the mp50 gene in locus TVAG_403460. Using an RT-PCR assay, we amplified a 1320-bp mp50 mRNA transcript that was expressed in the presence of Zn2+ in the HGMN01 and CNCD147 T. vaginalis isolates. According to a Western blot assay, native TvMP50 was differentially expressed in the presence of Zn2+. The TvMP50 proteolytic activity increased in the presence of Zn2+ in both isolates and was inhibited by EDTA but not by ptosyl-L-lysine chloromethyl ketone (TLCK), E64, leupeptin, or phenylmethane sulfonyl fluoride. Furthermore, the recombinant TvMP50 had proteolytic activity that was inhibited by EDTA. These data suggested that TvMP50 is immunogenic during male trichomoniasis, and Zn2+ induces its expression. PMID:23579185

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.

Stathmin decreases cholangiocarcinoma cell line sensitivity to staurosporine-triggered apoptosis via the induction of ERK and Akt signaling.

PubMed

Wang, Yueqi; Gao, Zhihui; Zhang, Dexiang; Bo, Xiaobo; Wang, Yaojie; Wang, Jiwen; Shen, Sheng; Liu, Han; Suo, Tao; Pan, Hongtao; Ai, Zhilong; Liu, Houbao

2017-02-28

Cholangiocarcinoma is a rare, but highly fatal malignancy. However, the intrinsic mechanism involved in its tumorigenesis remains obscure. An urgent need remains for a promising target for cholangiocarcinoma biological therapies. Based on comparative proteomical technologies, we found 253 and 231 different spots in gallbladder tumor cell lines and cholangiocarcinoma cell lines, respectively, relative to non-malignant cells. Using Mass Spectrometry (MS) and database searching, we chose seven differentially expressed proteins. High Stathmin expression was found in both cholangiocarcinoma and gallbladder carcinoma cells. Stathmin expression was validated using immunohistochemistry and western blot in cholangiocarcinoma tissue samples and peritumoral tissue. It was further revealed that high Stathmin expression was associated with the repression of staurosporine-induced apoptosis in the cholangiocarcinoma cell. Moreover, we found that Stathmin promoted cancer cell proliferation and inhibited its apoptosis through protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) signaling. Integrin, β1 appears to serve as a partner of Stathmin induction of ERK and Akt signaling by inhibiting apoptosis in the cholangiocarcinoma cell. Understanding the regulation of anti-apoptosis effect by Stathmin might provide new insight into how to overcome therapeutic resistance in cholangiocarcinoma.

Comparative proteomics of leaves found at different stem positions of maize seedlings.

PubMed

Chen, Yi-Bo; Wang, Dan; Ge, Xuan-Liang; Zhao, Biligen-Gaowa; Wang, Xu-Chu; Wang, Bai-Chen

2016-07-01

To better understand the roles of leaves at different stem positions during plant development, we measured the physiological properties of leaves 1-4 on maize seedling stems, and performed a proteomics study to investigate the differences in protein expression in the four leaves using two-dimensional difference gel electrophoresis and tandem mass spectrometry in conjunction with database searching. A total of 167 significantly differentially expressed protein spots were found and identified. Of these, 35% are involved in photosynthesis. By further analysis of the data, we speculated that in leaf 1 the seedling has started to transition from a heterotroph to an autotroph, development of leaf 2 is the time at which the seedling fully transitions from a heterotroph to an autotroph, and leaf maturity was reached only with fully expanded leaves 3 and 4, although there were still some protein expression differences in the two leaves. These results suggest that the different leaves make different contributions to maize seedling growth via modulation of the expression of the photosynthetic proteins. Together, these results provide insight into the roles of the different maize leaves as the plant develops from a heterotroph to an autotroph. Copyright © 2016 Elsevier GmbH. All rights reserved.

Differential expression proteomics of human colorectal cancer based on a syngeneic cellular model for the progression of adenoma to carcinoma.

PubMed

Roth, Udo; Razawi, Hanieh; Hommer, Julia; Engelmann, Katja; Schwientek, Tilo; Müller, Stefan; Baldus, Stephan E; Patsos, Georgios; Corfield, Anthony P; Paraskeva, Christos; Hanisch, Franz-Georg

2010-01-01

This is the first differential expression proteomics study on a human syngeneic cellular in vitro progression model of the colorectal adenoma-to-carcinoma sequence, the anchorage-dependent non-tumorigenic adenoma derived cell line AA/C1 and the derived anchorage-independent and tumorigenic carcinoma cell line AA/C1/SB10C. The study is based on quantitative 2-DE and is complemented by Western blot validation. Excluding redundancies due to proteolysis and post-translational modified isoforms of over 2000 protein spots, 13 proteins were revealed as regulated with statistical variance being within the 95th confidence level and were identified by peptide mass fingerprinting in MALDI MS. Progression-associated proteins belong to the functional complexes of anaerobic glycolysis/gluconeogenesis, steroid biosynthesis, prostaglandin biosynthesis, the regulation and maintenance of the cytoskeleton, protein biosynthesis and degradation, the regulation of apoptosis or other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in colorectal carcinoma. Among upregulated proteins we identified 3-HMG-CoA synthase, protein phosphatase 1, prostaglandin E synthase 2, villin 1, annexin A1, triosephosphate isomerase, phosphoserine aminotransferase 1, fumarylacetoacetate hydrolase and pyrroline-5-carboxylate reductase 1 (PYCR1), while glucose-regulated protein 78, cathepsin D, lamin A/C and quinolate phosphoribosyltransferase were downregulated.

Analysis of differential protein expression by cisplatin treatment in cervical carcinoma cells.

PubMed

Yim, E-K; Lee, K-H; Kim, C-J; Park, J-S

2006-01-01

Cisplatin (cis-diaminedichloroplatinum), a DNA-damaging agent, which readily induces apoptosis in vitro, is one of the widely used anticancer drug in the treatment of human malignancies. Cisplatin has played an important role in cervical cancer management for effective chemotherapeutic regimen, but the underlying mechanisms inducing cell death at protein level are unknown. Using proteome analysis, an investigation aimed at a better understanding of the antiproliferative mechanisms by cisplatin was carried out in HeLa cervical carcinoma cells. In total, 21 protein spots were found to be differentially expressed following cisplatin treatment, of which 12 were upregulated (eg, regulator of G-protein signaling, TRAF:TNF (tumor necrosis factor) receptor-associated factor-interacting protein [I-TRAF], and cyclin-dependent kinase inhibitor p27 [p27(kip1)]) and 9 were downregulated (eg, myc proto-oncoprotein [c-myc] and proliferating cell nuclear antigen). Interestingly, we found the upregulation of proliferating cell nuclear antigen, which used molecular marker in cervical cancer screening. On the basis of proteomic data, we showed that cisplatin induced TRAF2-mediated NF-kappaB downregulation. In addition, our study demonstrated that cisplatin induced membrane death receptor-mediated and mitochondria-mediated apoptosis pathway. Our findings may offer new insights into the antiproliferative mechanism by cisplatin and its mode of action in cervical carcinoma cells.

Genetic variation and biogeography of the spotted gar Lepisosteus oculatus from core and peripheral populations.

PubMed

David, Solomon R; Wright, Jeremy J

2017-11-01

The spotted gar (Lepisosteus oculatus) shows a disjunct natural distribution, with a core population extending from the central Mississippi River Basin to the U.S. gulf coast and a peripheral population in the southern Great Lakes Basin. Despite significant conservation concerns for this species in the Great Lakes watersheds where it occurs, few genetic examinations and comparisons of these populations have been performed. We investigated inter- and intrapopulational variation in several mitochondrial genetic markers (cytochrome oxidase subunit I, COI; cytochrome oxidase subunit II, COII; and 16S rRNA, 16S) from spotted gars taken from core and peripheral populations. Genetic diversity was highest in the Mississippi River Basin and lowest in the Great Lakes Basin, while the Nueces River Basin (Texas) population showed the greatest level of divergence from other populations. Average genetic distance among core and peripheral populations was over an order of magnitude less than that seen between L. oculatus and its sister species, the Florida gar (L. platyrhincus), although a significant correlation was found between genetic and geographical distance in L. oculatus. Genetic divergence in spotted gars is likely to be related to a combination of geographic isolation and founder effects associated with recent colonization following glacial retreat. Despite its apparent lack of significant genetic differentiation or haplotype diversity, the Great Lakes population of spotted gars has previously been shown to be a unique component of the species, and additional studies are needed to determine the genetic mechanisms underlying regional adaptations as well as potential morphological differentiation among spotted gar populations. © 2017 Wiley Periodicals, Inc.

The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

PubMed Central

Bækkedal, Cecilie; Haugen, Peik

2015-01-01

The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding. PMID:26327359

Elucidation of defense-related signaling responses to spot blotch infection in bread wheat (Triticum aestivum L.).

PubMed

Sahu, Ranabir; Sharaff, Murali; Pradhan, Maitree; Sethi, Avinash; Bandyopadhyay, Tirthankar; Mishra, Vinod K; Chand, Ramesh; Chowdhury, Apurba K; Joshi, Arun K; Pandey, Shree P

2016-04-01

Spot blotch disease, caused by Bipolaris sorokiniana, is an important threat to wheat, causing an annual loss of ~17%. Under epidemic conditions, these losses may be 100%, yet the molecular responses of wheat to spot blotch remain almost uncharacterized. Moreover, defense-related phytohormone signaling genes have been poorly characterized in wheat. Here, we have identified 18 central components of salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and enhanced disease susceptibility 1 (EDS1) signaling pathways as well as the genes of the phenylpropanoid pathway in wheat. In time-course experiments, we characterized the reprogramming of expression of these pathways in two contrasting genotypes: Yangmai #6 (resistant to spot blotch) and Sonalika (susceptible to spot blotch). We further evaluated the performance of a population of recombinant inbred lines (RILs) by crossing Yangmai#6 and Sonalika (parents) and subsequent selfing to F10 under field conditions in trials at multiple locations. We characterized the reprogramming of defense-related signaling in these RILs as a consequence of spot blotch attack. During resistance to spot blotch attack, wheat strongly elicits SA signaling (SA biogenesis as well as the NPR1-dependent signaling pathway), along with WRKY33 transcription factor, followed by an enhanced expression of phenylpropanoid pathway genes. These may lead to accumulation of phenolics-based defense metabolites that may render resistance against spot blotch. JA signaling may synergistically contribute to the resistance. Failure to elicit SA (and possibly JA) signaling may lead to susceptibility against spot blotch infection in wheat. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The base pairing RNA Spot 42 participates in a multi-output feedforward loop to help enact catabolite repression in Escherichia coli

PubMed Central

Beisel, Chase L.; Storz, Gisela

2011-01-01

SUMMARY Bacteria selectively consume some carbon sources over others through a regulatory mechanism termed catabolite repression. Here, we show that the base pairing RNA Spot 42 plays a broad role in catabolite repression in Escherichia coli by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse non-preferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multi-output feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression. PMID:21292161

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits

PubMed Central

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-01-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain-dead donors. In the present study, two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis were conducted to profile the differentially-expressed proteins between brain death and the control group renal tissues. A total of 40 age- and sex-matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two-dimensional gel electrophoresis, >2-fold alterations were identified by MALDI-TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3-N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b-c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre-mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V-type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin-3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time-dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain-dead donors. PMID:28534953

Differentially expressed proteins in ER+ MCF7 and ER- MDA- MB-231 human breast cancer cells by RhoGDI-α silencing and overexpression.

PubMed

Hooshmand, Somayeh; Ghaderi, Abbas; Yusoff, Khatijah; Thilakavathy, Karuppiah; Rosli, Rozita; Mojtahedi, Zahra

2014-01-01

The consequence of Rho GDP dissociation inhibitor alpha (RhoGDIα) activity on migration and invasion of estrogen receptor positive (ER+) and negative (ER-) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDIα and other proteins interacting directly or indirectly with RhoGDIα in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα. The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

Testing the recovery of stellar rotation signals from Kepler light curves using a blind hare-and-hounds exercise

NASA Astrophysics Data System (ADS)

Aigrain, S.; Llama, J.; Ceillier, T.; Chagas, M. L. das; Davenport, J. R. A.; García, R. A.; Hay, K. L.; Lanza, A. F.; McQuillan, A.; Mazeh, T.; de Medeiros, J. R.; Nielsen, M. B.; Reinhold, T.

2015-07-01

We present the results of a blind exercise to test the recoverability of stellar rotation and differential rotation in Kepler light curves. The simulated light curves lasted 1000 d and included activity cycles, Sun-like butterfly patterns, differential rotation and spot evolution. The range of rotation periods, activity levels and spot lifetime were chosen to be representative of the Kepler data of solar-like stars. Of the 1000 simulated light curves, 770 were injected into actual quiescent Kepler light curves to simulate Kepler noise. The test also included five 1000-d segments of the Sun's total irradiance variations at different points in the Sun's activity cycle. Five teams took part in the blind exercise, plus two teams who participated after the content of the light curves had been released. The methods used included Lomb-Scargle periodograms and variants thereof, autocorrelation function and wavelet-based analyses, plus spot modelling to search for differential rotation. The results show that the `overall' period is well recovered for stars exhibiting low and moderate activity levels. Most teams reported values within 10 per cent of the true value in 70 per cent of the cases. There was, however, little correlation between the reported and simulated values of the differential rotation shear, suggesting that differential rotation studies based on full-disc light curves alone need to be treated with caution, at least for solar-type stars. The simulated light curves and associated parameters are available online for the community to test their own methods.

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.

Biomarker Discovery for Early Detection of Hepatocellular Carcinoma in Hepatitis C–infected Patients*

PubMed Central

Mustafa, Mehnaz G.; Petersen, John R.; Ju, Hyunsu; Cicalese, Luca; Snyder, Ned; Haidacher, Sigmund J.; Denner, Larry; Elferink, Cornelis

2013-01-01

Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of 18O/16O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using 18O/16O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies. PMID:24008390

Gene expression and growth as indicators of effects of the BP Deepwater Horizon oil spill on spotted seatrout (Cynoscion nebulosus).

PubMed

Brewton, Rachel Aileen; Fulford, Richard; Griffitt, Robert J

2013-01-01

The BP Deepwater Horizon oil spill has great potential to negatively affect estuarine fish populations. In order to assess possible impacts of this event, a series of sublethal lab experiments were performed, using the economically and ecologically important species spotted seatrout (Cynoscion nebulosus). Larval and juvenile spotted seatrout were exposed to sublethal concentrations of high energy water accommodated fraction (HEWAF), chemically enhanced water accommodated fraction (CEWAF), or dispersant alone in an acute exposure. Response to exposure was evaluated with quantative polymerase chain reaction (qPCR) to examine expression of cytochrome P-4501A (CYP1A). Growth of larvae and juveniles over the duration of the experiment was measured as an index of physiological response. Our data showed that the different life stages respond differently to crude and dispersed oil, with larval spotted seatrout affected most by CEWAF, while juvenile spotted seatrout were affected to a greater extent by HEWAF. In both cases, the treatment with the highest CYP1A levels resulted in the greatest reductions in growth.

Growth shapes and turbulent spots in unstable systems

NASA Astrophysics Data System (ADS)

Conrado, Claudine V.; Bohr, Tomas

1995-05-01

We study the growth shapes of localized turbulent patches (turbulent spots) in a class of partial differential equations (PDEs) in two spatial dimensions, of first order in time and in one scalar field u. The PDE's are chosen such that the ``laminar'' state u=0 is unstable, at least convectively (i.e., in a moving frame). We show which symmetry breaking terms are required to generate the characteristic nonconvex growth shapes found in turbulent spots in boundary layers, and how the shapes transform when we vary the coefficients of these terms. Finally, we show how it is possible, in certain cases, to find exact solutions for the nonliear growth shapes and how to measure Lyapunov exponents of growing turbulent spots.

Expression of Skin Patterns in Metamorphosing Tadpoles.

ERIC Educational Resources Information Center

Bagnara, Joseph T.; And Others

1985-01-01

Explains an experiment in which the rotation of a patch of skin on a tadpole results in a frog with rotated spots, proving that the spot pattern is determined long before it becomes visible. Procedures used are included. (DH)

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea.

PubMed

Azumi, Kaoru; Usami, Takeshi; Kamimura, Akiko; Sabau, Sorin V; Miki, Yasufumi; Fujie, Manabu; Jung, Sung-Ju; Kitamura, Shin-Ichi; Suzuki, Satoru; Yokosawa, Hideyoshi

2007-12-01

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.

Comparison of spot esculin hydrolysis with the PathoTec strip test for rapid differentiation of anaerobic bacteria.

PubMed Central

Qadri, S M; Johnson, S; Smith, J C; Zubairi, S; Gillum, R L

1981-01-01

The ability of several anaerobic bacteria to hydrolyze esculin to esculetin is used by clinical microbiologists and taxonomists in the differentiation and identification of both gram-positive and gram-negative microorganisms. Conventional methods used for determining esculin hydrolysis by anaerobic bacteria require 24 to 48 h for completion. In this paper we evaluate two procedures which yield rapid results. A total of 738 anaerobic bacteria were used in this study. A total of 99% of the esculin-hydrolyzing anaerobic bacteria gave positive results with the spot test in 1 h, whereas the other test method, the PathoTec strip test (General Diagnostics, Morris Plains, N.J.), required 4 h for 96% of the strains tested to yield positive reactions. Both tests showed a 100% specificity when compared with the standard broth test and are easy to perform, accurate, and economical. The spot test is superior to the PathoTec strip test in yielding results more rapidly. PMID:7016896

Large and Small-Scale Cropland Classification on the Foothills of Mount Kenya Based on SPOT-5 Take-5 Data Time Series

NASA Astrophysics Data System (ADS)

Eckert, Sandra

2016-08-01

The SPOT-5 Take 5 campaign provided SPOT time series data of an unprecedented spatial and temporal resolution. We analysed 29 scenes acquired between May and September 2015 of a semi-arid region in the foothills of Mount Kenya, with two aims: first, to distinguish rainfed from irrigated cropland and cropland from natural vegetation covers, which show similar reflectance patterns; and second, to identify individual crop types. We tested several input data sets in different combinations: the spectral bands and the normalized difference vegetation index (NDVI) time series, principal components of NDVI time series, and selected NDVI time series statistics. For the classification we used random forests (RF). In the test differentiating rainfed cropland, irrigated cropland, and natural vegetation covers, the best classification accuracies were achieved using spectral bands. For the differentiation of crop types, we analysed the phenology of selected crop types based on NDVI time series. First results are promising.

Acute Heat Stress Changes Protein Expression in the Testes of a Broiler-Type Strain of Taiwan Country Chickens.

PubMed

Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chan, Hong-Lin; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2018-03-19

Heat stress leads to decreased fertility in roosters. This study investigated the global protein expression in response to acute heat stress in the testes of a broiler-type strain of Taiwan country chickens (TCCs). Twelve 45-week-old roosters were randomly allocated to the control group maintained at 25°C, and three groups subjected to acute heat stress at 38°C for 4 h, with 0, 2, and 6 h of recovery, respectively. Testis samples were collected for hematoxylin and eosin staining, apoptosis assay, and protein analysis. The results revealed 101 protein spots that differed significantly from the control following exposure to acute heat stress. The proteins that were differentially expressed participated mainly in protein metabolism and other metabolic processes, responses to stimuli, apoptosis, cellular organization, and spermatogenesis. Proteins that negatively regulate apoptosis were downregulated and proteins involved in autophagy and major heat shock proteins (HSP90α, HSPA5, and HSPA8) were upregulated in the testes of heat-stressed chickens. In conclusion, acute heat stress causes a change in protein expression in the testes of broiler-type B strain TCCs and may thus impair cell morphology, spermatogenesis, and apoptosis. The expression of heat shock proteins increased to attenuate the testicular injury induced by acute heat stress.

p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity.

PubMed

Chaudhari, Aditi; Krumlinde, Daniel; Lundqvist, Annika; Akyürek, Levent M; Bandaru, Sashidhar; Skålén, Kristina; Ståhlman, Marcus; Borén, Jan; Wettergren, Yvonne; Ejeskär, Katarina; Rotter Sopasakis, Victoria

2015-10-01

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Spotting handwritten words and REGEX using a two stage BLSTM-HMM architecture

NASA Astrophysics Data System (ADS)

Bideault, Gautier; Mioulet, Luc; Chatelain, Clément; Paquet, Thierry

2015-01-01

In this article, we propose a hybrid model for spotting words and regular expressions (REGEX) in handwritten documents. The model is made of the state-of-the-art BLSTM (Bidirectional Long Short Time Memory) neural network for recognizing and segmenting characters, coupled with a HMM to build line models able to spot the desired sequences. Experiments on the Rimes database show very promising results.

Clustering-based spot segmentation of cDNA microarray images.

PubMed

Uslan, Volkan; Bucak, Ihsan Ömür

2010-01-01

Microarrays are utilized as that they provide useful information about thousands of gene expressions simultaneously. In this study segmentation step of microarray image processing has been implemented. Clustering-based methods, fuzzy c-means and k-means, have been applied for the segmentation step that separates the spots from the background. The experiments show that fuzzy c-means have segmented spots of the microarray image more accurately than the k-means.

2D-Difference Gel Electrophoretic Proteomic Analysis of a Cell Culture Model of Alveolar Rhabdomyosarcoma

PubMed Central

Pressey, Joseph G.; Pressey, Christine S.; Robinson, Gloria; Herring, Richie; Wilson, Landon; Kelly, David R.; Kim, Helen

2011-01-01

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2 dimensional difference gel electrophoresis (2D-DIGE) we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder™ image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 non-redundant proteins. 2D DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 non-redundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by western blot analysis and immunohistochemistry (IHC). Thus, the 2D DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies. PMID:21110518

2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma.

PubMed

Pressey, Joseph G; Pressey, Christine S; Robinson, Gloria; Herring, Richie; Wilson, Landon; Kelly, David R; Kim, Helen

2011-02-04

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.

Damage threshold from large retinal spot size repetitive-pulse laser exposures.

PubMed

Lund, Brian J; Lund, David J; Edsall, Peter R

2014-10-01

The retinal damage thresholds for large spot size, multiple-pulse exposures to a Q-switched, frequency doubled Nd:YAG laser (532 nm wavelength, 7 ns pulses) have been measured for 100 μm and 500 μm retinal irradiance diameters. The ED50, expressed as energy per pulse, varies only weakly with the number of pulses, n, for these extended spot sizes. The previously reported threshold for a multiple-pulse exposure for a 900 μm retinal spot size also shows the same weak dependence on the number of pulses. The multiple-pulse ED50 for an extended spot-size exposure does not follow the n dependence exhibited by small spot size exposures produced by a collimated beam. Curves derived by using probability-summation models provide a better fit to the data.

Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males.

PubMed

Silva, M H; Nascimento, M D S B; Leonardo, F S; Rebêlo, J M M; Pereira, S R F

2011-01-01

Entomological surveys in the state of Maranhão have recorded morphologically distinct populations of Lutzomyia longipalpis (Lutz & Neiva). Some populations have one pair of spots (1S) on the fourth tergite, while others have two pairs (2S) on the third and fourth tergites of males. In the present study we investigated the degree of genetic polymorphism among four populations in the municipalities of Caxias, Codó and Raposa, in the state of Maranhão, Brazil, by using RAPD (Random Amplified Polymorphic DNA) markers. A total of 35 loci were identified, of which 30 were polymorphic. The highest polymorphism was observed with primer OPA 4, which produced 11 different profiles. Genetic diversity was assessed using grouping methods that produced a dendrogram in which the genotypes could be clearly separated into two main clades according to the number of spots on the male abdominal tergites. One cluster contained the populations from Caxias and Codó, and the other was formed by the populations from Raposa and Codó. The results of our RAPD analysis showed a clear separation between the populations with one and two pairs of spots. The epidemiologic significance of this genetic differentiation should be investigated in future studies.

Differentially delayed root proteome responses to salt stress in sugar cane varieties.

PubMed

Pacheco, Cinthya Mirella; Pestana-Calsa, Maria Clara; Gozzo, Fabio Cesar; Mansur Custodio Nogueira, Rejane Jurema; Menossi, Marcelo; Calsa, Tercilio

2013-12-06

Soil salinity is a limiting factor to sugar cane crop development, although in general plants present variable mechanisms of tolerance to salinity stress. The molecular basis underlying these mechanisms can be inferred by using proteomic analysis. Thus, the objective of this work was to identify differentially expressed proteins in sugar cane plants submitted to salinity stress. For that, a greenhouse experiment was established with four sugar cane varieties and two salt conditions, 0 mM (control) and 200 mM NaCl. Physiological and proteomics analyses were performed after 2 and 72 h of stress induction by salt. Distinct physiological responses to salinity stress were observed in the varieties and linked to tolerance mechanisms. In proteomic analysis, the roots soluble protein fraction was extracted, quantified, and analyzed through bidimensional electrophoresis. Gel images analyses were done computationally, where in each contrast only one variable was considered (salinity condition or variety). Differential spots were excised, digested by trypsin, and identified via mass spectrometry. The tolerant variety RB867515 showed the highest accumulation of proteins involved in growth, development, carbohydrate and energy metabolism, reactive oxygen species metabolization, protein protection, and membrane stabilization after 2 h of stress. On the other hand, the presence of these proteins in the sensitive variety was verified only in stress treatment after 72 h. These data indicate that these stress responses pathways play a role in the tolerance to salinity in sugar cane, and their effectiveness for phenotypical tolerance depends on early stress detection and activation of the coding genes expression.

Preliminary evidence of mitochondrial dysfunction associated with post-infective fatigue after acute infection with Epstein Barr Virus

PubMed Central

Vernon, Suzanne D; Whistler, Toni; Cameron, Barbara; Hickie, Ian B; Reeves, William C; Lloyd, Andrew

2006-01-01

Background Acute infectious diseases are typically accompanied by non-specific symptoms including fever, malaise, irritability and somnolence that usually resolve on recovery. However, in some individuals these symptoms persist in what is commonly termed post-infective fatigue. The objective of this pilot study was to determine the gene expression correlates of post-infective fatigue following acute Epstein Barr virus (EBV) infection. Methods We followed 5 people with acute mononucleosis who developed post-infective fatigue of more than 6 months duration and 5 HLA-matched control subjects who recovered within 3 months. Subjects had peripheral blood mononuclear cell (PBMC) samples collected at varying time points including at diagnosis, then every 2 weeks for 3 months, then every 3 months for a year. Total RNA was extracted from the PBMC samples and hybridized to microarrays spotted with 3,800 oligonucleotides. Results Those who developed post-infective fatigue had gene expression profiles indicative of an altered host response during acute mononucleosis compared to those who recovered uneventfully. Several genes including ISG20 (interferon stimulated gene), DNAJB2 (DnaJ [Hsp40] homolog and CD99), CDK8 (cyclin-dependent kinase 8), E2F2 (E2F transcription factor 2), CDK8 (cyclin-dependent kinase 8), and ACTN2 (actinin, alpha 2), known to be regulated during EBV infection, were differentially expressed in post-infective fatigue cases. Several of the differentially expressed genes affect mitochondrial functions including fatty acid metabolism and the cell cycle. Conclusion These preliminary data provide insights into alterations in gene transcripts associated with the varied clinical outcomes from acute infectious mononucleosis. PMID:16448567

Identifying the mechanism of Escherichia coli O157:H7 survival by the addition of salt in the treatment with organic acids.

PubMed

Bae, Y-M; Yoon, J-H; Kim, J-Y; Lee, S-Y

2018-01-01

In this study, the effects of the addition of salt to treatment with acids (one of several organic acids and salt in various solutions including rich or minimal broth, buffer, or distilled water) on the reduction of Escherichia coli O157:H7 were investigated. The protein expression profiles corresponding to acid stress (acetic acid) with or without salt addition were studied using a comparative proteomic analysis of E. coli O157:H7. When acetic, lactic, or propionic acid was combined with 3% NaCl, mutually antagonistic effects of acid and salt on viability of E. coli O157:H7 were observed only in tryptone and yeast extract broth. After exposure to acetic acid alone or in combination with salt, approximately 851 and 916 protein spots were detected, respectively. Analysis of 10 statistically significant differentially expressed proteins revealed that these proteins are mainly related to energy metabolism. When we compared protein expression of E. coli O157:H7 treated with acetic acid and the combination of the acid and salt, the differentially expressed proteins were not related to acid stress- and salt stress-inducible proteins such as stress shock proteins. According to these results, the increased resistance of E. coli O157:H7 to acetic acid after the addition of salt may not be the result of synthesis of proteins related to these phenomena; therefore, further research needs to be conducted to identify the mechanism of the mutually antagonistic effect of some organic acids and salt. © 2017 The Society for Applied Microbiology.

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

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

PubMed

Müller, Gabrielle do Amaral E Silva; Lüchmann, Karim Hahn; Razzera, Guilherme; Toledo-Silva, Guilherme; Bebianno, Maria João; Marques, Maria Risoleta Freire; Bainy, Afonso Celso Dias

2018-06-06

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, β-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Classification of fecal contamination on leafy greens by hyperspectral imaging

NASA Astrophysics Data System (ADS)

Yang, Chun-Chieh; Jun, Won; Kim, Moon S.; Chao, Kaunglin; Kang, Sukwon; Chan, Diane E.; Lefcourt, Alan

2010-04-01

This paper reported the development of hyperspectral fluorescence imaging system using ultraviolet-A excitation (320-400 nm) for detection of bovine fecal contaminants on the abaxial and adaxial surfaces of romaine lettuce and baby spinach leaves. Six spots of fecal contamination were applied to each of 40 lettuce and 40 spinach leaves. In this study, the wavebands at 666 nm and 680 nm were selected by the correlation analysis. The two-band ratio, 666 nm / 680 nm, of fluorescence intensity was used to differentiate the contaminated spots from uncontaminated leaf area. The proposed method could accurately detect all of the contaminated spots.

Impact of range shifter material on proton pencil beam spot characteristics.

PubMed

Shen, Jiajian; Liu, Wei; Anand, Aman; Stoker, Joshua B; Ding, Xiaoning; Fatyga, Mirek; Herman, Michael G; Bues, Martin

2015-03-01

To quantitatively investigate the effect of range shifter materials on single-spot characteristics of a proton pencil beam. An analytic approximation for multiple Coulomb scattering ("differential Moliere" formula) was adopted to calculate spot sizes of proton spot scanning beams impinging on a range shifter. The calculations cover a range of delivery parameters: six range shifter materials (acrylonitrile butadiene styrene, Lexan, Lucite, polyethylene, polystyrene, and wax) and water as reference material, proton beam energies ranging from 75 to 200 MeV, range shifter thicknesses of 4.5 and 7.0 g/cm(2), and range shifter positions from 5 to 50 cm. The analytic method was validated by comparing calculation results with the measurements reported in the literature. Relative to a water-equivalent reference, the spot size distal to a wax or polyethylene range shifter is 15% smaller, while the spot size distal to a range shifter made of Lexan or Lucite is about 6% smaller. The relative spot size variations are nearly independent of beam energy and range shifter thickness and decrease with smaller air gaps. Among the six material investigated, wax and polyethylene are desirable range shifter materials when the spot size is kept small. Lexan and Lucite are the desirable range shifter materials when the scattering power is kept similar to water.

QTL for white spot syndrome virus resistance and the sex-determining locus in the Indian black tiger shrimp (Penaeus monodon).

PubMed

Robinson, Nicholas A; Gopikrishna, Gopalapillay; Baranski, Matthew; Katneni, Vinaya Kumar; Shekhar, Mudagandur S; Shanmugakarthik, Jayakani; Jothivel, Sarangapani; Gopal, Chavali; Ravichandran, Pitchaiyappan; Gitterle, Thomas; Ponniah, Alphis G

2014-08-28

Shrimp culture is a fast growing aquaculture sector, but in recent years there has been a shift away from tiger shrimp Penaeus monodon to other species. This is largely due to the susceptibility of P. monodon to white spot syndrome virus disease (Whispovirus sp.) which has impacted production around the world. As female penaeid shrimp grow more rapidly than males, mono-sex production would be advantageous, however little is known about genes controlling or markers associated with sex determination in shrimp. In this study, a mapped set of 3959 transcribed single nucleotide polymorphisms were used to scan the P. monodon genome for loci associated with resistance to white-spot syndrome virus and sex in seven full-sibling tiger shrimp families challenged with white spot syndrome virus. Linkage groups 2, 3, 5, 6, 17, 18, 19, 22, 27 and 43 were found to contain quantitative trait loci significantly associated with hours of survival after white spot syndrome virus infection (P < 0.05 after Bonferroni correction). Nine QTL were significantly associated with hours of survival. Of the SNPs mapping to these and other regions with suggestive associations, many were found to occur in transcripts showing homology to genes with putative immune functions of interest, including genes affecting the action of the ubiquitin-proteasome pathway, lymphocyte-cell function, heat shock proteins, the TOLL pathway, protein kinase signal transduction pathways, mRNA binding proteins, lectins and genes affecting the development and differentiation of the immune system (eg. RUNT protein 1A). Several SNPs significantly associated with sex were mapped to linkage group 30, the strongest associations (P < 0.001 after Bonferroni correction) for 3 SNPs located in a 0.8 cM stretch between positions 43.5 and 44.3 cM where the feminisation gene (FEM-1, affecting sexual differentiation in Caenorhabditis elegans) mapped. The markers for disease resistance and sexual differentiation identified by this study could be useful for marker assisted selection to improve resistance to WSSV and for identifying homogametic female individuals for mono-sex (all female) production. The genes with putative functions affecting immunity and sexual differentiation that were found to closely map to these loci provide leads about the mechanisms affecting these important economic traits in shrimp.

Proteomic analysis of two divergently responding potato genotypes (Solanum tuberosum L.) following osmotic stress treatment in vitro.

PubMed

Bündig, Christin; Jozefowicz, Anna Maria; Mock, Hans-Peter; Winkelmann, Traud

2016-06-30

Starch potatoes (Solanum tuberosum L.) are of interest for production of starch, ethanol, and biopolymers. Due to the predicted increase in drought periods, the breeding of starch potatoes for drought tolerance is essential. This study aims to elucidate the physiological mechanisms that give rise to drought tolerance. Two genotypes contrasting in drought tolerance were compared. We applied osmotic stress which is a known component of drought stress under in vitro conditions. Shoot tips were harvested after 11days of culture on control medium and medium supplied with 0.2M sorbitol. Their proteomes were analyzed using two-dimensional isoelectric focussing sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D-IEF/SDS-PAGE). Of a total of 679 distinct protein spots, 118 and 20 spots with differential abundance were found in the sensitive and the tolerant genotype, respectively, after the application of stress. Using mass spectrometry, the proteins in 100 differentially abundant spots were identified; a majority of these proteins were from the chloroplast. For the sensitive genotype, an increase in the abundance of proteinase inhibitors and their precursors, changes in stress responsive proteins and an altered RNA/DNA-binding response were observed. The differentially abundant spots of the tolerant genotype comprised one chaperone and one hydrogen peroxide detoxifying protein. Our findings reveal that the two genotypes have different responses to osmotic stress in terms of protein degradation and reactive oxygen species (ROS) scavenging and production. Our data suggest that the tolerant genotype might adjust to the applied stress more quickly. A comparative temporal analysis might provide further insights into these rapid changes and assist in the development of biomarkers. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative analysis of proteome extracted from barley crowns grown under different drought conditions

PubMed Central

Vítámvás, Pavel; Urban, Milan O.; Škodáček, Zbynek; Kosová, Klára; Pitelková, Iva; Vítámvás, Jan; Renaut, Jenny; Prášil, Ilja T.

2015-01-01

Barley cultivar Amulet was used to study the quantitative proteome changes through different drought conditions utilizing two-dimensional difference gel electrophoresis (2D-DIGE). Plants were cultivated for 10 days under different drought conditions. To obtain control and differentially drought-treated plants, the soil water content was kept at 65, 35, and 30% of soil water capacity (SWC), respectively. Osmotic potential, water saturation deficit, 13C discrimination, and dehydrin accumulation were monitored during sampling of the crowns for proteome analysis. Analysis of the 2D-DIGE gels revealed 105 differentially abundant spots; most were differentially abundant between the controls and drought-treated plants, and 25 spots displayed changes between both drought conditions. Seventy-six protein spots were successfully identified by tandem mass spectrometry. The most frequent functional categories of the identified proteins can be put into the groups of: stress-associated proteins, amino acid metabolism, carbohydrate metabolism, as well as DNA and RNA regulation and processing. Their possible role in the response of barley to drought stress is discussed. Our study has shown that under drought conditions barley cv. Amulet decreased its growth and developmental rates, displayed a shift from aerobic to anaerobic metabolism, and exhibited increased levels of several protective proteins. Comparison of the two drought treatments revealed plant acclimation to milder drought (35% SWC); but plant damage under more severe drought treatment (30% SWC). The results obtained revealed that cv. Amulet is sensitive to drought stress. Additionally, four spots revealing a continuous and significant increase with decreasing SWC (UDP-glucose 6-dehydrogenase, glutathione peroxidase, and two non-identified) could be good candidates for testing of their protein phenotyping capacity together with proteins that were significantly distinguished in both drought treatments. PMID:26175745

Evaluation of Fuel Character Effects on the F101 Engine Combustion System

DTIC Science & Technology

1979-06-01

inner liner. The dome, outer liner, and outer cowl are a single welded assembly. Provisions for differential expansion between the fuel injectors and...Electric smoke measurement console shown in Figure 20 contains standard test equipment which fully conforms to SAE ARP 1179 (Reference 5). Smoke spot...spot samples are later delivered to the data processing area, where the reflectances are measured and the SAE Smoke Number is calculated. The gaseous

Eclipse cooling of selected lunar features

NASA Technical Reports Server (NTRS)

Shorthill, R. W.; Saari, J. M.; Baird, F. E.; Lecompte, J. R.

1970-01-01

Thermal measurements were made in the 10 to 12 micron band of the lunar surface during the total eclipse of December19, 1964. A normalized differential thermal contour map is included, showing the location of the thermal anomalies or hot spots on the disk and the eclipse cooling curves of 400 sites, of which more than 300 were hot spots. The eclipse cooling data is compared to a particulate thermophysical model of the soil.

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.

Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

PubMed

Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

2016-02-02

The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes. Copyright © 2015 Elsevier B.V. All rights reserved.

The mRNA expression of P450 aromatase, gonadotropin beta-subunits and FTZ-F1 in the orange-spotted grouper (Epinephelus Coioides) during 17alpha-methyltestosterone-induced precocious sex change.

PubMed

Zhang, Weimin; Zhang, Yong; Zhang, Lihong; Zhao, Huihong; Li, Xin; Huang, He; Lin, Haoran

2007-06-01

The orange-spotted grouper Epinephelus coioides is a protogynous hermaphroditic fish, but the physiological basis of its sex change remains largely unknown. In the present study, the 2-year-old orange-spotted grouper was induced to change sex precociously by oral administration of 17alpha-methyltestosterone (MT, 50 mg/Kg diet, twice a day at daily ration of 5% bodyweight) for 60 days. The serum testosterone levels were significantly elevated after MT treatment for 20 and 40 days as compared to control, but the levels of serum estradiol (E(2)) remained unchanged. The expression of P450aromA in the gonad significantly decreased after MT treatment for 20, 40, and 60 days. Accordingly, the enzyme activity of gonadal aromatase was also lower. The expression of FSHbeta subunit in the pituitary was significantly decreased after MT treatment for 20 days, but returned to the control levels after 40 and 60 days; however, the expression of LHbeta subunit was not altered significantly by MT treatment. The expression of FTZ-F1 in the gonad also decreased significantly in response to MT treatment for 40 and 60 days, but its expression in the pituitary was not altered significantly. Interestingly, when tested in vitro on ovarian fragments, MT had no direct effect on the expression of P450aromA and FTZ-F1 as well as the activity of gonadal aromatase, suggesting that the inhibition of gonadal P450aromatase and FTZ-F1 by MT may be mediated at upper levels of the brain-pituitary-gonadal axis. Taken together, these results indicated that FSH, P450aromA, FTZ-F1, and serum testosterone are associated with the MT-induced sex change of the orange-spotted grouper, but the cause-effect relationship between these factors and sex change in this species remains to be characterized. (c) 2006 Wiley-Liss, Inc.

Localization and expression of MreB in Vibrio parahaemolyticus under different stresses.

PubMed

Chiu, Shen-Wen; Chen, Shau-Yan; Wong, Hin-chung

2008-11-01

MreB, the homolog of eukaryotic actin, may play a vital role when prokaryotes cope with stress by altering their spatial organization, including their morphology, subcellular architecture, and localization of macromolecules. This study investigates the behavior of MreB in Vibrio parahaemolyticus under various stresses. The behavior of MreB was probed using a yellow fluorescent protein-MreB conjugate in merodiploid strain SC9. Under normal growth conditions, MreB formed helical filaments in exponential-phase cells. The shape of starved or stationary-phase cells changed from rods to small spheroids. The cells differentiated into the viable but nonculturable (VBNC) state with small spherical cells via a "swelling-waning" process. In all cases, drastic remodeling of the MreB cytoskeleton was observed. MreB helices typically were loosened and fragmented into short filaments, arcs, and spots in bacteria under these stresses. The disintegrated MreB exhibited a strong tendency to attach to the cytoplasmic membrane. The expression of mreB generally declined in bacteria in the stationary phase and under starvation but was upregulated during the initial periods of cold shock and VBNC state differentiation and decreased afterwards. Our findings demonstrated the behavior of MreB in the morphological changes of V. parahaemolyticus under intrinsic or extrinsic stresses and may have important implications for studying the cellular stress response and aging.

Analysis of A549 cell proteome alteration in response to recombinant influenza A virus nucleoprotein and its interaction with cellular proteins, a preliminary study.

PubMed

Kumar, D; Tiwari, K; Rajala, M S

Influenza A virus undergoes frequent changes of antigenicity and contributes to seasonal epidemics or unpredictable pandemics. Nucleoprotein, encoded by gene segment 5, is an internal protein of the virus and is conserved among strains of different host origins. In the current study, we analyzed the differentially expressed proteins in A549 cells transiently transfected with the recombinant nucleoprotein of influenza A virus by 2D gel electrophoresis. The resolved protein spots on gel were identified by MALDI-TOF/Mass spectrometry analysis. The majority of the host proteins detected to be differentially abundant in recombinant nucleoprotein-expressing cells as compared to vector-transfected cells are the proteins of metabolic pathways, glycolytic enzymes, molecular chaperones and cytoskeletal proteins. We further demonstrated the interaction of virus nucleoprotein with some of the identified host cellular proteins. In vitro binding assay carried out using the purified recombinant nucleoprotein (pET29a+NP-His) and A549 cell lysate confirmed the interaction between nucleoprotein and host proteins, such as alpha enolase 1, pyruvate kinase and β-actin. The preliminary data of our study provides the information on virus nucleoprotein interaction with proteins involved in glycolysis. However, studies are ongoing to understand the significance of these interactions in modulating the host factors during virus replication.

Unraveling the Protein Network of Tomato Fruit in Response to Necrotrophic Phytopathogenic Rhizopus nigricans

PubMed Central

Pan, Xiaoqi; Zhu, Benzhong; Luo, Yunbo; Fu, Daqi

2013-01-01

Plants are endowed with a sophisticated defense mechanism that gives signals to plant cells about the immediate danger from surroundings and protects them from pathogen invasion. In the search for the particular proteins involved in fruit defense responses, we report here a comparative analysis of tomato fruit (Solanum lycopersicum cv. Ailsa Craig) infected by Rhizopus nigricans Ehrenb, which is a significant contributor to postharvest rot disease in fresh tomato fruits. In total, four hundred forty-five tomato proteins were detected in common between the non-infected group and infected tomato fruit of mature green. Forty-nine differentially expressed spots in 2-D gels were identified, and were sorted into fifteen functional groups. Most of these proteins participate directly in the stress response process, while others were found to be involved in several equally important biological processes: protein metabolic process, carbohydrate metabolic process, ethylene biosynthesis, and cell death and so on. These responses occur in different cellular components, both intra- and extracellular spaces. The differentially expressed proteins were integrated into several pathways to show the regulation style existing in tomato fruit host. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their roles in pathogen-plant interactions. Collectively results provide evidence that several regulatory pathways contribute to the resistance of tomato fruit to pathogen. PMID:24023804

Functional proteomics of nonalcoholic steatohepatitis: Mitochondrial proteins as targets of S-adenosylmethionine

PubMed Central

Santamaría, Enrique; Avila, Matías A.; Latasa, M. Ujue; Rubio, Angel; Martín-Duce, Antonio; Lu, Shelly C.; Mato, José M.; Corrales, Fernando J.

2003-01-01

Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis (MAT1A−/−) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when MAT1A−/− expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at ≈8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase β-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in ob/ob mice and obese patients who are at risk for nonalcoholic steatohepatitis. PMID:12631701

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

Hemolymph proteome changes during worker brood development match the biological divergences between western honey bees (Apis mellifera) and eastern honey bees (Apis cerana).

PubMed

Feng, Mao; Ramadan, Haitham; Han, Bin; Fang, Yu; Li, Jianke

2014-07-05

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages. The brood body weight of Apis mellifera was significantly heavier than that of Apis cerana at each developmental stage. Significantly, different protein expression patterns and metabolic pathways were observed in 74 proteins (166 spots) that were differentially abundant between the two bee species. The function of hemolymph in energy storage, odor communication, and antioxidation is of equal importance for the western and eastern bees, indicated by the enhanced expression of different protein species. However, stronger expression of protein folding, cytoskeletal and developmental proteins, and more highly activated energy producing pathways in western bees suggests that the different bee species have developed unique strategies to match their specific physiology using hemolymph to deliver nutrients and in immune defense. Our disparate findings constitute a proof-of-concept of molecular details that the ecologically shaped different physiological conditions of different bee species match with the hemolymph proteome during the brood stage. This also provides a starting point for future research on the specific hemolymph proteins or pathways related to the differential phenotypes or physiology.

Differential expression of hepatic genes with embryonic exposure to an environmentally relevant PCB mixture in Japanese quail (Coturnix japonica).

PubMed

Bohannon, Meredith E; Porter, Tom E; Lavoie, Emma T; Ottinger, Mary Ann

2018-06-22

The upper Hudson River was contaminated with polychlorinated biphenyls (PCB) Aroclor mixtures from the 1940s until the late 1970s. Several well-established biomarkers, such as induction of hepatic cytochrome P450 monooxygenases, were used to measure exposure to PCBs and similar contaminants in birds. In the present study, Japanese quail eggs were injected with a PCB mixture based upon a congener profile found in spotted sandpiper eggs at the upper Hudson River and subsequently, RNA was extracted from hatchling liver tissue for hybridization to a customized chicken cDNA microarray. Nominal concentrations of the mixture used for microarray hybridization were 0, 6, 12, or 49 μg/g egg. Hepatic gene expression profiles were analyzed using cluster and pathway analyses. Results showed potentially useful biomarkers of both exposure and effect attributed to PCB mixture. Biorag and Ingenuity Pathway Analysis® analyses revealed differentially expressed genes including those involved in glycolysis, xenobiotic metabolism, replication, protein degradation, and tumor regulation. These genes included cytochrome P450 1A5 (CYP1A5), cytochrome b5 (CYB5), NADH-cytochrome b5 reductase, glutathione S-transferase (GSTA), fructose bisphosphate aldolase (ALDOB), glycogen phosphorylase, carbonic anhydrase, and DNA topoisomerase II. CYP1A5, CYB5, GSTA, and ALDOB were chosen for quantitative real-time polymerase chain reaction confirmation, as these genes exhibited a clear dose response on the array. Data demonstrated that an initial transcriptional profile associated with an environmentally relevant PCB mixture in Japanese quail occurred.

Regulation of Mammary Tumor Formation and Lipid Biosynthesis by Spot 14

DTIC Science & Technology

2013-10-01

1     Introduction: THRSP/Spot14/S14 is a small cytoplasmic protein that is highly expressed in tissues that synthesize fatty acids de novo...cycle regulation and also with various metabolic pathways, including glycolysis /gluconeogenesis and the pentose phosphate pathway (Table 1). The

Black-spot poison ivy: a rare phenomenon.

PubMed

Paniagua, Carmen T; Bean, Ashley S

2011-06-01

To provide an overview of the clinical presentation, diagnosis, management, and treatment with advanced practice nursing implications of black-spot poison ivy phenomenon. Case presentation and comprehensive literature review on black-spot poison ivy. Black-spot poison ivy is a rare phenomenon and usually poses a diagnostic challenge. It usually presents after exposure to a higher concentration of uroshiol on Toxicodendron plants. Patients present with black-spot deposits on the epidermis with underlying poison ivy dermatitis. The black deposits cannot be washed off the skin and are followed by itchy blisters. They eventually peel off and the skin heals without scarring. An understanding of the pathophysiology, clinical presentation, treatment, and management of this rare phenomenon is important for the nurse practitioner (NP) to be able to make an accurate diagnosis and initiate appropriate treatment without delay. The NP's recognition and differentiation of it from other skin disorders including melanoma is paramount. ©2011 The Author(s) Journal compilation ©2011 American Academy of Nurse Practitioners.

Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

PubMed

Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

2016-04-07

Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

ToF-SIMS study of differentiation of human bone-derived stromal cells: new insights into osteoporosis.

PubMed

Schaepe, Kaija; Werner, Janina; Glenske, Kristina; Bartges, Tessa; Henss, Anja; Rohnke, Marcus; Wenisch, Sabine; Janek, Jürgen

2017-07-01

Lipids have numerous important functions in the human body, as they form the cells' plasma membranes and play a key role in many disease states, presumably also in osteoporosis. Here, the fatty acid composition of the outer plasma membranes of cells differentiated into the osteogenic and adipogenic direction is studied with surface-sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). For data evaluation, principal component analysis (PCA) is applied. Human (bone-derived) mesenchymal stromal cells (hMSCs) from an osteoporotic donor and a control donor are compared to reveal differences in the fatty acid composition of the membranes. The chemical information is correlated to staining and real-time quantitative polymerase chain reaction (rt-qPCR) results to provide insight into the gene expression of several differentiation markers on the RNA level. Adipogenic differentiation of hMSCs from a non-osteoporotic donor correlates with increased relative intensities of all fatty acids under investigation. After osteogenic differentiation of non-osteoporotic cells, the relative mass signal intensities of unsaturated fatty acids such as oleic and linoleic acids are increased. However, the osteoporotic cells show increased levels of palmitic acid in the plasma membrane after exposure to osteogenic differentiation conditions, which correlates to an immature differentiation state relative to non-osteoporotic osteogenic cells. This immature differentiation state is confirmed by increased early osteogenic differentiation factor Runx2 on RNA level and by less calcium mineralization spots seen in von Kossa staining and ToF-SIMS images. Graphical abstract Time-of-flight secondary ion mass spectrometry is applied to analyze the fatty acid composition of the outer plasma membranes of cells differentiated into the adipogenic and osteogenic direction. Cells from an osteoporotic and a control donor are compared to reveal differences due to differentiation and disease stage of the cells.

The GLAS Algorithm Theoretical Basis Document for Laser Footprint Location (Geolocation) and Surface Profiles

NASA Technical Reports Server (NTRS)

Shutz, Bob E.; Urban, Timothy J.

2014-01-01

This ATBD summarizes (and links with other ATBDs) the elements used to obtain the geolocated GLAS laser spot location, with respect to the Earth Center of Mass. Because of the approach used, the reference frame used to express the geolocation is linked to the reference frame used for POD and PAD, which are related to the ITRF. The geolocated spot coordinates (which includes the elevation or height, with respect to an adopted reference ellipsoid) is the inferred position of the laser spot, since the spot location is not directly measured. This document also summarizes the GLAS operation time periods.

Laser spot dynamics.

PubMed

Postan, A

1987-03-01

The dynamics of a pulsed laser spot covering an optical aperture of a receiver is analyzed. This analysis includes the influence of diffraction, jitter, atmospheric absorption and scattering, and atmospheric turbulence. A simple expression for the probability of response of the receiver illuminated by the laser spot is derived. It is found that this probability would not always increase as the laser beam divergence decreases. Moreover, this probability has an optimum (maximum) with respect to the laser beam divergence or rather with respect to the diameter of the transmitting optics.

Characterization of Coconut cadang-cadang viroid variants from oil palm affected by orange spotting disease in Malaysia.

PubMed

Wu, Y H; Cheong, L C; Meon, S; Lau, W H; Kong, L L; Joseph, H; Vadamalai, G

2013-06-01

A 246-nt variant of Coconut cadang-cadang viroid (CCCVd) has been identified and described from oil palms with orange spotting symptoms in Malaysia. Compared with the 246-nt form of CCCVd from coconut, the oil palm variant substituted C(31)→U in the pathogenicity domain and G(70)→C in the central conserved domain. This is the first sequence reported for a 246-nt variant of CCCVd in oil palms expressing orange spotting symptoms.

Simulation and Design Methods for Free-electron Laser Systems

DTIC Science & Technology

2009-12-01

the transverse Laplacian symbol ( 2 ) denotes differentiation only in the x and y coordinates. In this new parabolic form of the wave equation... compounded over a longer time and has a greater effect. In an oscillator, each pass an optical pulse makes through the resonator is a new interaction with...phosphor screen in the beam’s path and observing the spot(s) illuminated upon it. The phosphor screen can also serve as a charge collector , with

Proteomic identification of differentially expressed proteins between male and female plants in Pistacia chinensis.

PubMed

Xiong, Erhui; Wu, Xiaolin; Shi, Jiang; Wang, Xiaoyan; Wang, Wei

2013-01-01

Pistacia chinensis is a strict dioecious plant with male and female flowers in individuals. In China, P. chinensis is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in P. chinensis, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in P. chinensis. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in P. chinensis.

Proteomic Identification of Differentially Expressed Proteins between Male and Female Plants in Pistacia chinensis

PubMed Central

Shi, Jiang; Wang, Xiaoyan; Wang, Wei

2013-01-01

Pistacia chinensis is a strict dioecious plant with male and female flowers in individuals. In China, P. chinensis is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in P. chinensis, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in P. chinensis. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in P. chinensis. PMID:23691188

Transcriptome profiling of the Macrobrachium rosenbergii lymphoid organ under the white spot syndrome virus challenge.

PubMed

Cao, Jun; Wu, Lei; Jin, Min; Li, Tingting; Hui, Kaimin; Ren, Qian

2017-08-01

Macrobrachium rosenbergii is a crustacean with economic importance, and adult prawns are generally thought to be tolerant to white spot syndrome virus (WSSV) infection. Although certain genes are known to respond to WSSV infection and lymphoid tissue is an important immune organ, the response of lymphoid organ to WSSV infection is unclear. Next-generation sequencing was employed in this study to determine the transcriptome differences between WSSV infection and mock lymphoid organs. A total of 44,606,694 and 40,384,856 clean reads were generated and assembled into 73,658 and 72,374 unigenes from the control sample and the WSSV infection sample, respectively. Based on homology searches, KEGG, GO, and COG analysis, 21,323 unigenes were annotated. Among them, 4951 differential expression genes were identified and categorized into 244 metabolic pathways. Coagulation cascades, and pattern recognition receptor signaling pathways were used as examples to discuss the response of host to WSSV infection. We also identified 12,308 simple sequence repeats, which can be further used as functional markers. Results contribute to a better understanding of the immune response of prawn lymphoid organ to WSSV and provide information for identifying novel genes in the absence of the prawn genome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic analysis of carbon concentrating chemolithotrophic bacteria Serratia sp. for sequestration of carbon dioxide.

PubMed

Bharti, Randhir K; Srivastava, Shaili; Thakur, Indu Shekhar

2014-01-01

A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO). The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC), however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials.

Proteomic Analysis of Carbon Concentrating Chemolithotrophic Bacteria Serratia sp. for Sequestration of Carbon Dioxide

PubMed Central

Bharti, Randhir K.; Srivastava, Shaili; Thakur, Indu Shekhar

2014-01-01

A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO). The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC), however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials. PMID:24619032

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.

PubMed

Della Corte, Anna; Maugeri, Norma; Pampuch, Agnieszka; Cerletti, Chiara; de Gaetano, Giovanni; Rotilio, Domenico

2008-02-01

Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.

White spotting phenotype induced by targeted REST disruption during neural crest specification to a melanocyte cell lineage.

PubMed

Aoki, Hitomi; Hara, Akira; Kunisada, Takahiro

2015-05-01

Neural crest cells (NCCs) emerge from the dorsal region of the neural tube of vertebrate embryos and have the pluripotency to differentiate into both neuronal and non-neuronal lineages including melanocytes. Rest, also known as NRSF (neuro-restrictive silencer factor), is a regulator of neuronal development and function and suggested to be involved in the lineage specification of NCCs. However, further investigations of Rest gene functions in vivo have been hampered by the fact that Rest null mice show early embryonic lethality. To investigate the function of Rest in NCC development, we recently established NCC-specific Rest conditional knockout (CKO) mice and observed their neonatal death. Here, we have established viable heterozygous NCC-specific Rest CKO mice to analyze the function of Rest in an NCC-derived melanocyte cell lineage and found that the white spotting phenotype was associated with the reduction in the number of melanoblasts in the embryonic skin. The Rest deletion induced after the specification to melanocytes did not reduce the number of melanoblasts; therefore, the expression of REST during the early neural crest specification stage was necessary for the normal development of melanoblasts to cover all of the skin. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

A Search for Planetary Transits of the Star HD 187123 by Spot Filter CCD Differential Photometry

NASA Technical Reports Server (NTRS)

Castellano, T.; DeVincenzi, D. (Technical Monitor)

2000-01-01

A novel method for performing high precision, time series CCD differential photometry of bright stars using a spot filter, is demonstrated. Results for several nights of observing of the 51 Pegasi b-type planet bearing star HD 187123 are presented. Photometric precision of 0.0015 - 0.0023 magnitudes is achieved. No transits are observed at the epochs predicted from the radial velocity observation. If the planet orbiting HD 187123 at 0.0415 AU is an inflated Jupiter similar in radius to HD 209458b it would have been detected at the greater than 6(sigma), level if the orbital inclination is near 90 degrees and at the greater than 3(sigma), level if the orbital inclination is as small as 82.7 degrees.

Differential gene expression profile from haematopoietic tissue stem cells of red claw crayfish, Cherax quadricarinatus, in response to WSSV infection.

PubMed

Liu, Hai-peng; Chen, Rong-yuan; Zhang, Qiu-xia; Peng, Hui; Wang, Ke-jian

2011-07-01

White spot syndrome virus (WSSV) is one of the most important viral pathogens in crustaceans. During WSSV infection, multiple cell signaling cascades are activated, leading to the generation of antiviral molecules and initiation of programmed cell death of the virus infected cells. To gain novel insight into cell signaling mechanisms employed in WSSV infection, we have used suppression subtractive hybridization (SSH) to elucidate the cellular response to WSSV challenge at the gene level in red claw crayfish haematopoietic tissue (Hpt) stem cell cultures. Red claw crayfish Hpt cells were infected with WSSV for 1h (L1 library) and 12h (L12 library), respectively, after which the cell RNA was prepared for SSH using uninfected cells as drivers. By screening the L1 and L12 forward libraries, we have isolated the differentially expressed genes of crayfish Hpt cells upon WSSV infection. Among these genes, the level of many key molecules showed clearly up-regulated expression, including the genes involved in immune responses, cytoskeletal system, signal transduction molecules, stress, metabolism and homestasis related genes, and unknown genes in both L1 and L12 libraries. Importantly, of the 2123 clones screened, 176 novel genes were found the first time to be up-regulated in WSSV infection in crustaceans. To further confirm the up-regulation of differentially expressed genes, the semi-quantitative RT-PCR were performed to test twenty randomly selected genes, in which eight of the selected genes exhibited clear up-regulation upon WSSV infection in red claw crayfish Hpt cells, including DNA helicase B-like, multiprotein bridging factor 1, apoptosis-linked gene 2 and an unknown gene-L1635 from L1 library; coatomer gamma subunit, gabarap protein gene, tripartite motif-containing 32 and an unknown gene-L12-254 from L2 library, respectively. Taken together, as well as in immune and stress responses are regulated during WSSV infection of crayfish Hpt cells, our results also light the significance of cytoskeletal system, signal transduction and other unknown genes in the regulation of antiviral signals during WSSV infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Rapid differentiation of rocky mountain spotted fever from chickenpox, measles, and enterovirus infections and bacterial meningitis by frequency-pulsed electron capture gas-liquid chromatographic analysis of sera.

PubMed Central

Brooks, J B; McDade, J E; Alley, C C

1981-01-01

Normal sera and sera from patients with Rocky Mountain spotted fever, chickenpox, enterovirus infections, measles, and Neisseria meningitidis infections were extracted with organic solvents under acidic and basic conditions and then derivatized with trichloroethanol or heptafluorobutyric anhydride-ethanol to form electron-capturing derivatives of organic acids, alcohols, and amines. The derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). There were unique differences in the FPEC-GLC profiles of sera obtained from patients with these respective diseases. With Rocky Mountain spotted fever patients, typical profiles were detected as early as 1 day after onset of disease and before antibody could be detected in the serum. Rapid diagnosis of Rocky Mountain spotted fever by FPEC-GLC could permit early and effective therapy, thus preventing many deaths from this disease. PMID:7276147

Roth Spots in Ocular Toxoplasmosis.

PubMed

Furtado, João M; Toscano, Marcelo; Castro, Vinicius; Rodrigues, Murilo W

2016-10-01

To report a case with unilateral preexisting ocular toxoplasmosis (OT) and newly occurred active retinochoroidal inflammation associated with white-centered retinal hemorrhages (Roth spots) in the healthy eye. Case report. A 20 year-old man presented with a decrease of visual acuity in his right eye associated with 2+ cells in the anterior chamber. Ophthalmoscopy revealed an active retinochoroidal lesion on the upper nasal border of the optic disc associated with local hemorrhage and vitritis. The posterior pole presented white-centered flame-shaped retinal hemorrhages. Toxoplasmosis serology showed an IgG titer >300IU/ml and a negative IgM titer. A complete blood count revealed no abnormalities; other serologies were negative. After 2 weeks of treatment with sulfadiazine, pyrimethamine, folinic acid and prednisone, although the peripapillary lesion was still active, the Roth spots disappeared. OT may be one differential diagnosis of patients suffering Roth spots in association with retinochoroidal inflammation.

Identification of gibberellin acid-responsive proteins in rice leaf sheath using proteomics.

PubMed

Gu, Jia-Yu; Wang, Ye; Zhang, Xu; Zhang, Shi-Hua; Gao, Yin; An, Cheng-Cai

2010-06-01

The phytohormone gibberellin acid (GA) controls many aspects of plant development. In this study, we identified proteins that are differentially expressed between the rice (Oryza sativa L.) GA-deficient cultivar, Aijiaonante, and its parental line, Nante. Proteins were extracted from rice leaf sheath and examined by 2DGE. Among more than 1200 protein spots reproducibly detected on each gel, 29 were found to be highly up-regulated by GAs in Nante, and 6 were down-regulated by GAs in Aijiaonante. These 35 proteins were identified by MALDI-TOF MS and were classified into three groups based on their putative function in metabolism, stress/defense processes and signal transduction. These data suggest that metabolic pathways are the main target of regulation by GAs during rice development. Our results provide new information about the involvement of GAs in rice development.

Transition Heat Transfer Modeling Based on the Characteristics of Turbulent Spots

NASA Technical Reports Server (NTRS)

Simon, Fred; Boyle, Robert

1998-01-01

While turbulence models are being developed which show promise for simulating the transition region on a turbine blade or vane, it is believed that the best approach with the greatest potential for practical use is the use of models which incorporate the physics of turbulent spots present in the transition region. This type of modeling results in the prediction of transition region intermittency which when incorporated in turbulence models give a good to excellent prediction of the transition region heat transfer. Some models are presented which show how turbulent spot characteristics and behavior can be employed to predict the effect of pressure gradient and Mach number on the transition region. The models predict the spot formation rate which is needed, in addition to the transition onset location, in the Narasimha concentrated breakdown intermittency equation. A simplified approach is taken for modeling turbulent spot growth and interaction in the transition region which utilizes the turbulent spot variables governing transition length and spot generation rate. The models are expressed in terms of spot spreading angle, dimensionless spot velocity, dimensionless spot area, disturbance frequency and Mach number. The models are used in conjunction with a computer code to predict the effects of pressure gradient and Mach number on the transition region and compared with VKI experimental turbine data.

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression.

PubMed

Yan, Aifen; Zhang, Lingjuang; Tang, Zhiguo; Zhang, Yanhong; Qin, Chaobin; Li, Bo; Li, Wensheng; Lin, Haoran

2011-07-01

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro. Copyright © 2011 Elsevier Inc. All rights reserved.

Proteomic analysis of S-nitrosylated and S-glutathionylated proteins in wheat seedlings with different dehydration tolerances.

PubMed

Gietler, Marta; Nykiel, Małgorzata; Orzechowski, Sławomir; Fettke, Joerg; Zagdańska, Barbara

2016-11-01

A loss of dehydration tolerance in wheat seedlings on the fifth day following imbibition is associated with a disturbance in cellular redox homeostasis, as documented by a shift of the reduced/oxidized glutathione ratio to a more oxidized state and a significant increase in the ratio of protein thiols to the total thiol group content. Therefore, the identification and characterization of redox-sensitive proteins are important steps toward understanding the molecular mechanisms of the loss of dehydration tolerance. In the present study, proteins that were differentially expressed between fully turgid (control), dehydrated tolerant (four-day-old) and dehydrated sensitive (six-day-old) wheat seedlings were analysed. Protein spots having at least a significant (p < 0.05) two-fold change in protein abundance were selected by Delta2D as differentially expressed, identified by MALDI-TOF and LC-MS/MS, and classified according to their function. The observed changes in the proteomic patterns of the differentially S-nitrosylated and S-glutathionylated proteins were highly specific in dehydration-tolerant and -sensitive wheat seedlings. The metabolic function of these proteins indicates that dehydration tolerance is mainly related to nucleic acids, protein metabolism, and energy metabolism. It has been proven that leaf-specific thionins BTH6 and DB4, chloroplastic 50S ribosomal protein L16, phospholipase A1-II delta, and chloroplastic thioredoxin M2 are both S-nitrosylated and S-glutathionylated upon water deficiency. Our results revealed the existence of interplay between S-nitrosylation and S-glutathionylation, two redox-regulated protein posttranslational modifications that could enhance plant defence mechanisms and/or facilitate the acclimation of plants to unfavourable environmental conditions. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Proteomic analysis of liver in rats chronically exposed to fluoride.

PubMed

Pereira, Heloísa Aparecida Barbosa da Silva; Leite, Aline de Lima; Charone, Senda; Lobo, Janete Gualiume Vaz Madureira; Cestari, Tania Mary; Peres-Buzalaf, Camila; Buzalaf, Marília Afonso Rabelo

2013-01-01

Fluoride (F) is a potent anti-cariogenic element, but when ingestion is excessive, systemic toxicity may be observed. This can occur as acute or chronic responses, depending on both the amount of F and the time of exposure. The present study identified the profile of protein expression possibly associated with F-induced chronic hepatotoxicity. Weanling male Wistar rats (three-weeks old) were divided into three groups and treated with drinking water containing 0, 5 or 50 mg/L F for 60 days (n=6/group). At this time point, serum and livers were collected for F analysis, which was done using the ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion. Livers were also submitted to histological and proteomic analyses (2D-PAGE followed by LC-MS/MS). Western blotting was done for confirmation of the proteomic data A dose-response was observed in serum F levels. In the livers, F levels were significantly increased in the 50 mg/L F group compared to groups treated with 0 and 5 mg/L F. Liver morphometric analysis did not reveal alterations in the cellular structures and lipid droplets were present in all groups. Proteomic quantitative intensity analysis detected 33, 44, and 29 spots differentially expressed in the comparisons between control vs. 5 mg/L F, control vs. 50 mg/L F, and 5 mg/L vs. 50 mg/L F, respectively. From these, 92 proteins were successfully identified. In addition, 18, 1, and 5 protein spots were shown to be exclusive in control, 5, and 50 mg/L F, respectively. Most of proteins were related to metabolic process and pronounced alterations were seen for the high-F level group. In F-treated rats, changes in the apolipoprotein E (ApoE) and GRP-78 expression may account for the F-induced toxicity in the liver. This can contribute to understanding the molecular mechanisms underlying hepatoxicity induced by F, by indicating key-proteins that should be better addressed in future studies.

Proteomics Analysis Identifies Molecular Targets Related to Diabetes Mellitus-associated Bladder Dysfunction *S⃞

PubMed Central

Yohannes, Elizabeth; Chang, Jinsook; Christ, George J.; Davies, Kelvin P.; Chance, Mark R.

2008-01-01

Protein expression profiles in rat bladder smooth muscle were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four age-matched control rat bladder tissues were prepared independently and analyzed together across multiple DIGE gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 100 spots were determined to be significantly changing among the four experimental groups. A subsequent mass spectrometry analysis of the 100 spots identified a total of 56 unique proteins. Of the proteins identified by two-dimensional DIGE/MS, 10 exhibited significant changes 1 week after STZ-induced hyperglycemia, whereas the rest showed differential expression after 2 months. A network analysis of these proteins using MetaCore™ suggested induction of transcriptional factors that are too low to be detected by two-dimensional DIGE and identified an enriched cluster of down-regulated proteins that are involved in cell adhesion, cell shape control, and motility, including vinculin, intermediate filaments, Ppp2r1a, and extracellular matrix proteins. The proteins that were up-regulated include proteins involved in muscle contraction (e.g. Mrlcb and Ly-GDI), in glycolysis (e.g. α-enolase and Taldo1), in mRNA processing (e.g. heterogeneous nuclear ribonucleoprotein A2/B1), in inflammatory response (e.g. S100A9, Annexin 1, and apoA-I), and in chromosome segregation and migration (e.g. Tuba1 and Vil2). Our results suggest that the development of diabetes-related complications in this model involves the down-regulation of structural and extracellular matrix proteins in smooth muscle that are essential for normal muscle contraction and relaxation but also induces proteins that are associated with cell proliferation and inflammation that may account for some of the functional deficits known to occur in diabetic complications of bladder. PMID:18337374

Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish

NASA Technical Reports Server (NTRS)

Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.

The Expression of Pre- and Postcopulatory Sexually Selected Traits Reflects Levels of Dietary Stress in Guppies

PubMed Central

Rahman, Md. Moshiur; Turchini, Giovanni M.; Gasparini, Clelia; Norambuena, Fernando; Evans, Jonathan P.

2014-01-01

Environmental and ecological conditions can shape the evolution of life history traits in many animals. Among such factors, food or nutrition availability can play an important evolutionary role in moderating an animal's life history traits, particularly sexually selected traits. Here, we test whether diet quantity and/or composition in the form of omega-3 long chain polyunsaturated fatty acids (here termed ‘n3LC’) influence the expression of pre- and postcopulatory traits in the guppy (Poecilia reticulata), a livebearing poeciliid fish. We assigned males haphazardly to one of two experimental diets supplemented with n3LC, and each of these diet treatments was further divided into two diet ‘quantity’ treatments. Our experimental design therefore explored the main and interacting effects of two factors (n3LC content and diet quantity) on the expression of precopulatory (sexual behaviour and sexual ornamentation, including the size, number and spectral properties of colour spots) and postcopulatory (the velocity, viability, number and length of sperm) sexually selected traits. Our study revealed that diet quantity had significant effects on most of the pre- and postcopulatory traits, while n3LC manipulation had a significant effect on sperm traits and in particular on sperm viability. Our analyses also revealed interacting effects of diet quantity and n3LC levels on courtship displays, and the area of orange and iridescent colour spots in the males’ colour patterns. We also confirmed that our dietary manipulations of n3LC resulted in the differential uptake of n3LC in body and testes tissues in the different n3LC groups. This study reveals the effects of diet quantity and n3LC on behavioural, ornamental and ejaculate traits in P. reticulata and underscores the likely role that diet plays in maintaining the high variability in these condition-dependent sexual traits. PMID:25170940

Differentiating gastrointestinal stromal tumors from gastric adenocarcinomas and normal mucosae using confocal Raman microspectroscopy

NASA Astrophysics Data System (ADS)

Hsu, Chih-Wei; Huang, Chia-Chi; Sheu, Jeng-Horng; Lin, Chia-Wen; Lin, Lien-Fu; Jin, Jong-Shiaw; Chen, Wenlung

2016-07-01

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and gastric adenocarcinomas are a common cancer worldwide. To differentiate GISTs from adenocarcinomas is important because the surgical processes for both are different; the former excises the tumor with negative margins, while the latter requires radical gastrectomy with lymph node dissection. Endoscopy with biopsy is used to distinguish GISTs from adenocarcinomas; however, it may cause tumor bleeding in GISTs. We reported here the confocal Raman microspectroscopy as an effective tool to differentiate GISTs, adenocarcinomas, and normal mucosae. Of 119 patients enrolled in this study, 102 patients underwent gastrectomy (40 GISTs and 62 adenocarcinomas), and 17 patients with benign lesions were obtained as normal mucosae. Raman signals were integrated for 100 s for each spot on the specimen, and 5 to 10 spots, depending on the sample size, were chosen for each specimen. There were significant differences among those tissues as evidenced by different Raman signal responding to phospholipids and protein structures. The spectral data were further processed and analyzed by using principal component analysis. A two-dimensional plot demonstrated that GISTs, adenocarcinomas, and normal gastric mucosae could be effectively differentiated from each other.

Proteomic analysis of etiolated juvenile tetraploid Robinia pseudoacacia branches during different cutting periods.

PubMed

Lu, Nan; Xu, Zhaohe; Meng, Bingnan; Sun, Yuhan; Zhang, Jiangtao; Wang, Shaoming; Li, Yun

2014-04-21

The propagation of hard-branch cuttings of tetraploid Robinia pseudoacacia (black locust) is restricted by the low rooting rate; however, etiolated juvenile tetraploid black locust branches result in a significantly higher rooting rate of cuttings compared with non-etiolated juvenile tetraploid branches. To identify proteins that influence the juvenile tetraploid branch rooting process, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectra (MALDI-TOF/TOF-MS) were used to analyze proteomic differences in the phloem of tetraploid R. pseudoacacia etiolated and non-etiolated juvenile branches during different cutting periods. A total of 58 protein spots differed in expression level, and 16 protein spots were only expressed in etiolated branches or non-etiolated ones. A total of 40 highly expressed protein spots were identified by mass spectrometry, 14 of which were accurately retrieved. They include nucleoglucoprotein metabolic proteins, signaling proteins, lignin synthesis proteins and phyllochlorin. These results help to reveal the mechanism of juvenile tetraploid R. pseudoacacia etiolated branch rooting and provide a valuable reference for the improvement of tetraploid R. pseudoacacia cutting techniques.

Identification of immunity-related genes in the larvae of Protaetia brevitarsis seulensis (Coleoptera: Cetoniidae) by a next-generation sequencing-based transcriptome analysis.

PubMed

Bang, Kyeongrin; Hwang, Sejung; Lee, Jiae; Cho, Saeyoull

2015-01-01

To identify immune-related genes in the larvae of white-spotted flower chafers, next-generation sequencing was conducted with an Illumina HiSeq2000, resulting in 100 million cDNA reads with sequence information from over 10 billion base pairs (bp) and >50× transcriptome coverage. A subset of 77,336 contigs was created, and ∼35,532 sequences matched entries against the NCBI nonredundant database (cutoff, e < 10(-5)). Statistical analysis was performed on the 35,532 contigs. For profiling of the immune response, samples were analyzed by aligning 42 base sequence tags to the de novo reference assembly, comparing levels in immunized larvae to control levels of expression. Of the differentially expressed genes, 3,440 transcripts were upregulated and 3,590 transcripts were downregulated. Many of these genes were confirmed as immune-related genes such as pattern recognition proteins, immune-related signal transduction proteins, antimicrobial peptides, and cellular response proteins, by comparison to published data. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Proteomic analysis in the Dufour's gland of Africanized Apis mellifera workers (Hymenoptera: Apidae).

PubMed

Teixeira, Aparecida das Dores; Games, Patricia D; Katz, Benjamin B; Tomich, John M; Zanuncio, José C; Serrão, José Eduardo

2017-01-01

The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour's gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour's glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour's gland contributing to a more complete understanding of the biological functions of this gland in honeybees.

Spatial cognitive deficits in an animal model of Wernicke-Korsakoff syndrome are related to changes in thalamic VDAC protein concentrations.

PubMed

Bueno, K O; de Souza Resende, L; Ribeiro, A F; Dos Santos, D M; Gonçalves, E C; Vigil, F A B; de Oliveira Silva, I F; Ferreira, L F; de Castro Pimenta, A M; Ribeiro, A M

2015-05-21

Proteomic profiles of the thalamus and the correlation between the rats' performance on a spatial learning task and differential protein expression were assessed in the thiamine deficiency (TD) rat model of Wernicke-Korsakoff syndrome. Two-dimensional gel-electrophoresis detected 320 spots and a significant increase or decrease in seven proteins. Four proteins were correlated to rat behavioral performance in the Morris Water Maze. One of the four proteins was identified by mass spectrometry as Voltage-Dependent Anion Channels (VDACs). The association of VDAC is evident in trials in which the rats' performance was worst, in which the VDAC protein was reduced, as confirmed by Western blot. No difference was observed on the mRNA of Vdac genes, indicating that the decreased VDAC expression may be related to a post-transcriptional process. The results show that TD neurodegeneration involves changes in thalamic proteins and suggest that VDAC protein activity might play an important role in an initial stage of the spatial learning process. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Genetic identification of spotted owls, barred owls, and their hybrids: Legal implications of hybrid identity

USGS Publications Warehouse

Haig, Susan M.; Wennerberg, Liv; Mullins, Thomas D.; Forsman, E.D.; Trail, P.

2004-01-01

Recent population expansion of Barred Owls ( Strix varia) into western North America has led to concern that they may compete with and further harm the Northern Spotted Owl ( S. occidentalis caurina), which is already listed as threatened under the U.S. Endangered Species Act (ESA). Because they hybridize, there is a legal need under the ESA for forensic identification of both species and their hybrids. We used mitochondrial control-region DNA and amplified fragment-length polymorphism (AFLP) analyses to assess maternal and biparental gene flow in this hybridization process. Mitochondrial DNA sequences (524 base pairs) indicated large divergence between Barred and Spotted Owls (13.9%). Further, the species formed two distinct clades with no signs of previous introgression. Fourteen diagnostic AFLP bands also indicated extensive divergence between the species, including markers differentiating them. Principal coordinate analyses and assignment tests clearly supported this differentiation. We found that hybrids had unique genetic combinations, including AFLP markers from both parental species, and identified known hybrids as well as potential hybrids with unclear taxonomic status. Our analyses corroborated the findings of extensive field studies that most hybrids genetically sampled resulted from crosses between female Barred Owls and male Spotted Owls. These genetic markers make it possible to clearly identify these species as well as hybrids and can now be used for research, conservation, and law enforcement. Several legal avenues may facilitate future conservation of Spotted Owls and other ESA-listed species that hybridize, including the ESA similarity-of-appearance clause (section 4[e]) and the Migratory Bird Treaty Act. The Migratory Bird Treaty Act appears to be the most useful route at this time.

Hierarchical Parallelization of Gene Differential Association Analysis

PubMed Central

2011-01-01

Background Microarray gene differential expression analysis is a widely used technique that deals with high dimensional data and is computationally intensive for permutation-based procedures. Microarray gene differential association analysis is even more computationally demanding and must take advantage of multicore computing technology, which is the driving force behind increasing compute power in recent years. In this paper, we present a two-layer hierarchical parallel implementation of gene differential association analysis. It takes advantage of both fine- and coarse-grain (with granularity defined by the frequency of communication) parallelism in order to effectively leverage the non-uniform nature of parallel processing available in the cutting-edge systems of today. Results Our results show that this hierarchical strategy matches data sharing behavior to the properties of the underlying hardware, thereby reducing the memory and bandwidth needs of the application. The resulting improved efficiency reduces computation time and allows the gene differential association analysis code to scale its execution with the number of processors. The code and biological data used in this study are downloadable from http://www.urmc.rochester.edu/biostat/people/faculty/hu.cfm. Conclusions The performance sweet spot occurs when using a number of threads per MPI process that allows the working sets of the corresponding MPI processes running on the multicore to fit within the machine cache. Hence, we suggest that practitioners follow this principle in selecting the appropriate number of MPI processes and threads within each MPI process for their cluster configurations. We believe that the principles of this hierarchical approach to parallelization can be utilized in the parallelization of other computationally demanding kernels. PMID:21936916

Hierarchical parallelization of gene differential association analysis.

PubMed

Needham, Mark; Hu, Rui; Dwarkadas, Sandhya; Qiu, Xing

2011-09-21

Microarray gene differential expression analysis is a widely used technique that deals with high dimensional data and is computationally intensive for permutation-based procedures. Microarray gene differential association analysis is even more computationally demanding and must take advantage of multicore computing technology, which is the driving force behind increasing compute power in recent years. In this paper, we present a two-layer hierarchical parallel implementation of gene differential association analysis. It takes advantage of both fine- and coarse-grain (with granularity defined by the frequency of communication) parallelism in order to effectively leverage the non-uniform nature of parallel processing available in the cutting-edge systems of today. Our results show that this hierarchical strategy matches data sharing behavior to the properties of the underlying hardware, thereby reducing the memory and bandwidth needs of the application. The resulting improved efficiency reduces computation time and allows the gene differential association analysis code to scale its execution with the number of processors. The code and biological data used in this study are downloadable from http://www.urmc.rochester.edu/biostat/people/faculty/hu.cfm. The performance sweet spot occurs when using a number of threads per MPI process that allows the working sets of the corresponding MPI processes running on the multicore to fit within the machine cache. Hence, we suggest that practitioners follow this principle in selecting the appropriate number of MPI processes and threads within each MPI process for their cluster configurations. We believe that the principles of this hierarchical approach to parallelization can be utilized in the parallelization of other computationally demanding kernels.

CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

PubMed Central

El Mouali, Youssef; Gaviria-Cantin, Tania; Gibert, Marta; Westermann, Alexander J.; Vogel, Jörg

2018-01-01

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression. PMID:29879120

Antisolar differential rotation with surface lithium enrichment on the single K-giant V1192 Orionis

NASA Astrophysics Data System (ADS)

Kővári, Zs.; Strassmeier, K. G.; Carroll, T. A.; Oláh, K.; Kriskovics, L.; Kővári, E.; Kovács, O.; Vida, K.; Granzer, T.; Weber, M.

2017-10-01

Context. Stars with about 1-2 solar masses at the red giant branch (RGB) represent an intriguing period of stellar evolution, I.e. when the convective envelope interacts with the fast-rotating core. During these mixing episodes freshly synthesized lithium can come up to the stellar surface along with high angular momentum material. This high angular momentum may alter the surface rotation pattern. Aims: The single rapidly rotating K-giant V1192 Ori is revisited to determine its surface differential rotation, lithium abundance, and basic stellar properties such as a precise rotation period. The aim is to independently verify the antisolar differential rotation of the star and possibly find a connection to the surface lithium abundance. Methods: We applied time-series Doppler imaging to a new multi-epoch data set. Altogether we reconstructed 11 Doppler images from spectroscopic data collected with the STELLA robotic telescope between 2007-2016. We used our inversion code iMap to reconstruct all stellar surface maps. We extracted the differential rotation from these images by tracing systematic spot migration as a function of stellar latitude from consecutive image cross-correlations. Results: The position of V1192 Ori in the Hertzsprung-Russell diagram suggests that the star is in the helium core-burning phase just leaving the RGB bump. We measure A(Li)NLTE = 1.27, I.e. a value close to the anticipated transition value of 1.5 from Li-normal to Li-rich giants. Doppler images reveal extended dark areas arranged quasi-evenly along an equatorial belt. No cool polar spot is found during the investigated epoch. Spot displacements clearly suggest antisolar surface differential rotation with α = - 0.11 ± 0.02 shear coefficient. Conclusions: The surface Li enrichment and the peculiar surface rotation pattern may indicate a common origin. Based on data obtained with the STELLA robotic observatory in Tenerife, an AIP facility jointly operated by AIP and IAC.

Focusing on butterfly eyespot focus: uncoupling of white spots from eyespot bodies in nymphalid butterflies.

PubMed

Iwata, Masaki; Otaki, Joji M

2016-01-01

Developmental studies on butterfly wing color patterns often focus on eyespots. A typical eyespot (such as that of Bicyclus anynana) has a few concentric rings of dark and light colors and a white spot (called a focus) at the center. The prospective eyespot center during the early pupal stage is known to act as an organizing center. It has often been assumed, according to gradient models for positional information, that a white spot in adult wings corresponds to an organizing center and that the size of the white spot indicates how active that organizing center was. However, there is no supporting evidence for these assumptions. To evaluate the feasibility of these assumptions in nymphalid butterflies, we studied the unique color patterns of Calisto tasajera (Nymphalidae, Satyrinae), which have not been analyzed before in the literature. In the anterior forewing, one white spot was located at the center of an eyespot, but another white spot associated with either no or only a small eyespot was present in the adjacent compartment. The anterior hindwing contained two adjacent white spots not associated with eyespots, one of which showed a sparse pattern. The posterior hindwing contained two adjacent pear-shaped eyespots, and the white spots were located at the proximal side or even outside the eyespot bodies. The successive white spots within a single compartment along the midline in the posterior hindwing showed a possible trajectory of a positional determination process for the white spots. Several cases of focus-less eyespots in other nymphalid butterflies were also presented. These results argue for the uncoupling of white spots from eyespot bodies, suggesting that an eyespot organizing center does not necessarily differentiate into a white spot and that a prospective white spot does not necessarily signify organizing activity for an eyespot. Incorporation of these results in future models for butterfly wing color pattern formation is encouraged.

Normal dimensions of the posterior pituitary bright spot on magnetic resonance imaging.

PubMed

Côté, Martin; Salzman, Karen L; Sorour, Mohammad; Couldwell, William T

2014-02-01

The normal pituitary bright spot seen on unenhanced T1-weighted MRI is thought to result from the T1-shortening effect of the vasopressin stored in the posterior pituitary. Individual variations in its size may be difficult to differentiate from pathological conditions resulting in either absence of the pituitary bright spot or in T1-hyperintense lesions of the sella. The objective of this paper was to define a range of normal dimensions of the pituitary bright spot and to illustrate some of the most commonly encountered pathologies that result in absence or enlargement of the pituitary bright spot. The authors selected normal pituitary MRI studies from 106 patients with no pituitary abnormality. The size of each pituitary bright spot was measured in the longest axis and in the dimension perpendicular to this axis to describe the typical dimensions. The authors also present cases of patients with pituitary abnormalities to highlight the differences and potential overlap between normal and pathological pituitary imaging. All of the studies evaluated were found to have pituitary bright spots, and the mean dimensions were 4.8 mm in the long axis and 2.4 mm in the short axis. The dimension of the pituitary bright spot in the long axis decreased with patient age. The distribution of dimensions of the pituitary bright spot was normal, indicating that 99.7% of patients should have a pituitary bright spot measuring between 1.2 and 8.5 mm in its long axis and between 0.4 and 4.4 mm in its short axis, an interval corresponding to 3 standard deviations below and above the mean. In cases where the dimension of the pituitary bright spot is outside this range, pathological conditions should be considered. The pituitary bright spot should always be demonstrated on T1-weighted MRI, and its dimensions should be within the identified normal range in most patients. Outside of this range, pathological conditions affecting the pituitary bright spot should be considered.

Analytical study on the self-healing property of Bessel beam

NASA Astrophysics Data System (ADS)

Chu, X.

2012-10-01

With the help of Babinet principle, an analytical expression for the self-healing of Bessel beam is derived by using the Gaussian absorption function to describe the obstacle. Based on the analytical expression, the self-healing properties of Bessel beam are studied. It shows that Bessel beam has the ability to reconstruct its beam shape disturbed by an obstacle. However, during the self-healing process, not only the intensity of the beam behind the obstacle but also the other part will be affected by the obstruction. Meanwhile, the highlight spot, which intensity is larger than that without the obstacle will appear, and the size and strength of the highlight spot is determined by the size of the obstacle. From the change of Poynting vector and Babinet principle, the physical interpretations for the self-healing ability, the effects of the obstruction on the other part and the appearance of highlight spot are given.

The inheritance of resistance to bacterial leaf spot of lettuce caused by Xanthomonas campestris pv. vitians in three lettuce cultivars

USDA-ARS?s Scientific Manuscript database

Lettuce yields can be reduced by the disease bacterial leaf spot (BLS) caused by the pathogen Xanthomonas campestris pv. vitians (Xcv) and host resistance is the most feasible method to reduce disease losses. The cultivars La Brillante, Pavane, and Little Gem express an incompatible host-pathogen in...

Association analysis of bacterial leaf spot resistance and SNP markers derived from expressed sequence tags (ESTs) in lettuce (Lactuca sativa L.)

USDA-ARS?s Scientific Manuscript database

Bacterial leaf spot of lettuce, caused by Xanthomonas campestris pv. vitians, is a devastating disease of lettuce worldwide. Since there are no chemicals available for effective control of the disease, host-plant resistance is highly desirable to protect lettuce production. A total of 179 lettuce ge...

Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat (Triticum monococcum) in response to attack by the grain aphid (Sitobion avenae).

PubMed

Guan, Wenzhu; Ferry, Natalie; Edwards, Martin G; Bell, Howard A; Othman, Hamizah; Gatehouse, John A; Gatehouse, Angharad M R

2015-01-01

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS-LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.

Non-invasive detection of candidate pregnancy protein biomarkers in the feces of captive polar bears (Ursus maritimus).

PubMed

Curry, E; Stoops, M A; Roth, T L

2012-07-15

Currently, there is no method of accurately and non-invasively diagnosing pregnancy in polar bears. Specific proteins may exhibit altered profiles in the feces of pregnant bears, but predicting appropriate candidate proteins to investigate is speculative at best. The objective of this study was to identify potential pregnancy biomarker proteins based on their increased abundance in the feces of pregnant polar bears compared to pseudopregnant females (controls) using two-dimensional in-gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Three 2D-DIGE gels were performed to evaluate fecal protein profiles from controls (n=3) and pregnant polar bears (n=3). There were 2224.67±52.39 (mean±SEM) spots resolved per gel. Of these, only five proteins were elevated in the pregnant group (P<0.05), and seven additional spots tended to be higher (0.0599.9% confidence interval. The 11 spots represented seven distinct proteins, five of which were significantly more abundant in the pregnant group: IgGFc-binding protein, filamin-C, carboxypeptidase B, transthyretin, and immunoglobulin heavy chain variable region. To our knowledge, this was the first study that employed 2D-DIGE to identify differentially expressed proteins in fecal samples to characterize a physiological condition other than those related to gastrointestinal disorders. These promising results provided a strong foundation for ensuing efforts to develop a non-invasive pregnancy assay for use in both captive and wild polar bears. Copyright © 2012 Elsevier Inc. All rights reserved.

Analysis of proteomic differences between liquefied after-cataracts and normal lenses using two-dimensional gel electrophoresis and mass spectrometry

PubMed Central

Ge, Jia-Jia; Huang, Yu-Sen

2017-01-01

AIM To analyze and identify the proteomic differences between liquefied after-cataracts and normal lenses by means of liquefied chromatography-tandem mass spectrometry (LC-MS/MS). METHODS Three normal lenses and three liquefied after-cataracts were exposed to depolymerizing reagents to extract the total proteins. Protein concentrations were separated using two-dimensional gel electrophoresis (2-DE). The digitized images obtained with a GS-800 scanner were then analyzed with PDQuest7.0 software to detect the differentially-expressed protein spots. These protein spots were cut from the gel using a proteome work spot cutter and subjected to in-gel digestion with trypsin. The digested peptide separation was conducted by LC-MS/MS. RESULTS The 2-DE maps showed that lens proteins were in a pH range of 3-10 with a relative molecular weight of 21-70 kD. The relative molecular weight of the more abundant proteins was localized at 25-50 kD, and the isoelectric points were found to lie between PI 4-9. The maps also showed that the protein level within the liquefied after-cataracts was at 29 points and significantly lower than in normal lenses. The 29 points were identified by LC-MS/MS, and ten of these proteins were identified by mass spectrometry and database queries: beta-crystallin B1, glyceraldehyde-3-phosphate dehydrogenase, carbonyl reductase (NADPH) 1, cDNA FLJ55253, gamma-crystallin D, GAS2-like protein 3, sorbitol dehydrogenase, DNA FLJ60282, phosphoglycerate kinase, and filensin. CONCLUSION The level of the ten proteins may play an important role in the development of liquefied after-cataracts. PMID:28944190

Neurotoxicity following acute inhalation of aerosols generated during resistance spot weld-bonding of carbon steel.

PubMed

Sriram, Krishnan; Jefferson, Amy M; Lin, Gary X; Afshari, Aliakbar; Zeidler-Erdely, Patti C; Meighan, Terence G; McKinney, Walter; Jackson, Mark; Cumpston, Amy; Cumpston, Jared L; Leonard, Howard D; Frazer, David G; Antonini, James M

2014-10-01

Welding generates complex metal aerosols, inhalation of which is linked to adverse health effects among welders. An important health concern of welding fume (WF) exposure is neurological dysfunction akin to Parkinson's disease (PD). Some applications in manufacturing industry employ a variant welding technology known as "weld-bonding" that utilizes resistance spot welding, in combination with adhesives, for metal-to-metal welding. The presence of adhesives raises additional concerns about worker exposure to potentially toxic components like Methyl Methacrylate, Bisphenol A and volatile organic compounds (VOCs). Here, we investigated the potential neurotoxicological effects of exposure to welding aerosols generated during weld-bonding. Male Sprague-Dawley rats were exposed (25 mg/m³ targeted concentration; 4 h/day × 13 days) by whole-body inhalation to filtered air or aerosols generated by either weld-bonding with sparking (high metal, low VOCs; HM) or without sparking (low metal; high VOCs; LM). Fumes generated under these conditions exhibited complex aerosols that contained both metal oxide particulates and VOCs. LM aerosols contained a greater fraction of VOCs than HM, which comprised largely metal particulates of ultrafine morphology. Short-term exposure to LM aerosols caused distinct changes in the levels of the neurotransmitters, dopamine (DA) and serotonin (5-HT), in various brain areas examined. LM aerosols also specifically decreased the mRNA expression of the olfactory marker protein (Omp) and tyrosine hydroxylase (Th) in the olfactory bulb. Consistent with the decrease in Th, LM also reduced the expression of dopamine transporter (Slc6a3; Dat), as well as, dopamine D2 receptor (Drd2) in the olfactory bulb. In contrast, HM aerosols induced the expression of Th and dopamine D5 receptor (Drd5) mRNAs, elicited neuroinflammation and blood-brain barrier-related changes in the olfactory bulb, but did not alter the expression of Omp. Our findings divulge the differential effects of LM and HM aerosols in the brain and suggest that exposure to weld-bonding aerosols can potentially elicit neurotoxicity following a short-term exposure. However, further investigations are warranted to determine if the aerosols generated by weld-bonding can contribute to persistent long-term neurological deficits and/or neurodegeneration.

Comparative proteomic analysis of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low temperature.

PubMed

Gu, Xianbin; Gao, Zhihong; Zhuang, Weibing; Qiao, Yushan; Wang, Xiuyun; Mi, Lin; Zhang, Zhen; Lin, Zhilin

2013-05-01

Low-temperature stress is one of the major abiotic stresses in plants worldwide, and the dehydration responsive element binding protein (DREB) transcription factor induces expression of genes involved in environmental stress tolerance in plants. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) and subsequent mass spectrometric identification was used to study the changes in the leaf proteome profiles of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low-temperature conditions. By comparing the proteomic profiles, we located 21 protein spots that were reproducibly up- or down-regulated by more than twofold between transgenic and non-transgenic strawberries. Eight identified proteins function in energy and metabolism, four in biosynthetic processes, four were stress and defense related, three spots were identified as cold-stress related expressed sequence tags (ESTs), and two were unknown proteins. The change patterns of low-temperature tolerance proteins, including photosynthetic proteins (RuBisCO large subunit and RuBisCO activase), cytoplasmic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), late embryogenesis abundant protein 14-A (Lea14-A), eukaryotic translation initiation factor 5A (eIF5A), and cold-stress related ESTs, were differentially regulated between non-transgenic and rd29A:RdreB1BI transgenic strawberries. They are likely important gene products in the regulatory network of the RdreB1BI gene. Consequently, this study provides the first characterization of the transgenic strawberry proteome and the predicted target proteins of the RdreB1BI gene by using proteomic approaches. Copyright © 2013 Elsevier GmbH. All rights reserved.

Proteomic differential display analysis for TS-1-resistant and -sensitive pancreatic cancer cells using two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Yoshida, Kanako; Kuramitsu, Yasuhiro; Murakami, Kohei; Ryozawa, Shomei; Taba, Kumiko; Kaino, Seiji; Zhang, Xiulian; Sakaida, Isao; Nakamura, Kazuyuki

2011-06-01

TS-1 is an oral anticancer agent containing two biochemical modulators for 5-fluorouracil (5-FU) and tegafur (FT), a metabolically activated prodrug of 5-FU. TS-1 has been recognized as an effective anticancer drug using standard therapies for patients with advanced pancreatic cancer along with gemcitabine. However, a high level of inherent and acquired tumor resistance to TS-1 induces difficulty in the treatment. To identify proteins linked to the TS-1-resistance of pancreatic cancer, we profiled protein expression levels in samples of TS-1-resistant and -sensitive pancreatic cancer cell lines by using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytotoxicity of a 5-FU/5-chloro-2,4-dihydroxypyridine (CDHP) combination towards pancreatic cancer cell lines was evaluated by MTS assay. Panc-1, BxPC-3, MiaPaCa-2 and PK59 showed high sensitivity to the 5-FU/CDHP combination (TS-1-sensitive), whereas PK45p and KLM-1 were much less sensitive (TS-1-resistant). Proteomic analysis showed that eleven spots, including T-complex protein 1 subunit beta, ribonuclease inhibitor, elongation factor 1-delta, peroxiredoxin-2 and superoxide dismutase (Cu-Zn), appeared to be down-regulated, and 29 spots, including hypoxia up-regulated protein 1, lamin-A/C, endoplasmin, fascin and annexin A1, appeared to be up-regulated in TS-1-resistant cells compared with -sensitive cells. These results suggest that the identified proteins showing different expression between TS-1-sensitive and -resistant pancreatic cancer cells possibly relate to TS-1-sensitivity. These findings could be useful to overcome the TS-1-resistance of pancreatic cancer cells.

Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection.

PubMed

Xue, Shuxia; Liu, Yichen; Zhang, Yichen; Sun, Yan; Geng, Xuyun; Sun, Jinsheng

2013-01-01

White spot syndrome virus (WSSV) is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei) were compared using a short-read deep sequencing method (Illumina). RNA-seq analysis generated more than 25.81 million clean pair end (PE) reads, which were assembled into 52,073 unigenes (mean size = 520 bp). Based on sequence similarity searches, 23,568 (45.3%) genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 14,941 (63.4%) unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE) analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection) of the virus, while large transcriptional differences were identified in the late infection (48 hpi) of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

Fabrication of high quality cDNA microarray using a small amount of cDNA.

PubMed

Park, Chan Hee; Jeong, Ha Jin; Jung, Jae Jun; Lee, Gui Yeon; Kim, Sang-Chul; Kim, Tae Soo; Yang, Sang Hwa; Chung, Hyun Cheol; Rha, Sun Young

2004-05-01

DNA microarray technology has become an essential part of biological research. It enables the genome-scale analysis of gene expression in various types of model systems. Manufacturing high quality cDNA microarrays of microdeposition type depends on some key factors including a printing device, spotting pins, glass slides, spotting solution, and humidity during spotting. UsingEthe Microgrid II TAS model printing device, this study defined the optimal conditions for producing high density, high quality cDNA microarrays with the least amount of cDNA product. It was observed that aminosilane-modified slides were superior to other types of surface modified-slides. A humidity of 30+/-3% in a closed environment and the overnight drying of the spotted slides gave the best conditions for arraying. In addition, the cDNA dissolved in 30% DMSO gave the optimal conditions for spotting compared to the 1X ArrayIt, 3X SSC and 50% DMSO. Lastly, cDNA in the concentration range of 100-300 ng/ micro l was determined to be best for arraying and post-processing. Currently, the printing system in this study yields reproducible 9000 spots with a spot size 150 mm diameter, and a 200 nm spot spacing.

PcToll3 was involved in anti-Vibrio response by regulating the expression of antimicrobial peptides in red swamp crayfish, Procambarus clarkii.

PubMed

Lan, Jiang-Feng; Wei, Shun; Wang, Yu-Qing; Dai, Yun-Jia; Tu, Jia-Gang; Zhao, Li-Juan; Li, Xin-Cang; Qin, Qi-Wei; Chen, Nan; Lin, Li

2016-10-01

Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus. Copyright © 2016 Elsevier Ltd. All rights reserved.

A high density of human communication-associated genes in chromosome 7q31-q36: differential expression in human and non-human primate cortices.

PubMed

Schneider, E; Jensen, L R; Farcas, R; Kondova, I; Bontrop, R E; Navarro, B; Fuchs, E; Kuss, A W; Haaf, T

2012-01-01

The human brain is distinguished by its remarkable size, high energy consumption, and cognitive abilities compared to all other mammals and non-human primates. However, little is known about what has accelerated brain evolution in the human lineage. One possible explanation is that the appearance of advanced communication skills and language has been a driving force of human brain development. The phenotypic adaptations in brain structure and function which occurred on the way to modern humans may be associated with specific molecular signatures in today's human genome and/or transcriptome. Genes that have been linked to language, reading, and/or autism spectrum disorders are prime candidates when searching for genes for human-specific communication abilities. The database and genome-wide expression analyses we present here revealed a clustering of such communication-associated genes (COAG) on human chromosomes X and 7, in particular chromosome 7q31-q36. Compared to the rest of the genome, we found a high number of COAG to be differentially expressed in the cortices of humans and non-human primates (chimpanzee, baboon, and/or marmoset). The role of X-linked genes for the development of human-specific cognitive abilities is well known. We now propose that chromosome 7q31-q36 also represents a hot spot for the evolution of human-specific communication abilities. Selective pressure on the T cell receptor beta locus on chromosome 7q34, which plays a pivotal role in the immune system, could have led to rapid dissemination of positive gene variants in hitchhiking COAG. Copyright © 2012 S. Karger AG, Basel.

Comparative Proteomic Analysis of Different Isolates of Fusarium oxysporum f.sp. lycopersici to Exploit the Differentially Expressed Proteins Responsible for Virulence on Tomato Plants

PubMed Central

Manikandan, Rajendran; Harish, Sankarasubramanian; Karthikeyan, Gandhi; Raguchander, Thiruvengadam

2018-01-01

The vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici is an important soil borne pathogen causes severe yield loss. The molecular characterization and their interaction with its host is necessary to develop a protection strategy. 20 isolates of F. oxysporum f.sp. lycopersici (FOL) were isolated from wilt infected tomato plants across Tamil Nadu. They were subjected to cultural, morphological, molecular and virulence studies. The results revealed that all the isolates produced both micro and macro conidia with different size, number of cells. The colors of the culture and growth pattern were also varied. In addition, chlamydospores were observed terminally and intercalary. The PCR analysis with F. oxysporum species-specific primer significantly amplified an amplicon of 600 bp fragment in all the isolates. Based on the above characters and pathogenicity, isolate FOL-8 was considered as virulent and FOL-20 was considered as least virulent. Proteomics strategy was adopted to determine the virulence factors between the isolates of FOL-8 and FOL-20. The 2D analyses have showed the differential expression of 17 different proteins. Among them, three proteins were down regulated and 14 proteins were significantly up regulated in FOL-8 than FOL-20 isolate. Among the 17 proteins, 10 distinct spots were analyzed by MALDI-TOF. The functions of the analyzed proteins, suggested that they were involved in pathogenicity, symptom expression and disease development, sporulation, growth, and higher penetration rate on tomato root tissue. Overall, these experiments proves the role of proteome in pathogenicity of F. oxysporum f.sp. lycopersici in tomato and unravels the mechanism behinds the virulence of the pathogen in causing wilt disease. PMID:29559969

The evolution of cichlid fish egg-spots is linked with a cis-regulatory change

PubMed Central

Santos, M. Emília; Braasch, Ingo; Boileau, Nicolas; Meyer, Britta S.; Sauteur, Loïc; Böhne, Astrid; Belting, Heinz-Georg; Affolter, Markus; Salzburger, Walter

2014-01-01

The origin of novel phenotypic characters is a key component in organismal diversification; yet, the mechanisms underlying the emergence of such evolutionary novelties are largely unknown. Here we examine the origin of egg-spots, an evolutionary innovation of the most species-rich group of cichlids, the haplochromines, where these conspicuous male fin colour markings are involved in mating. Applying a combination of RNAseq, comparative genomics and functional experiments, we identify two novel pigmentation genes, fhl2a and fhl2b, and show that especially the more rapidly evolving b-paralog is associated with egg-spot formation. We further find that egg-spot bearing haplochromines, but not other cichlids, feature a transposable element in the cis-regulatory region of fhl2b. Using transgenic zebrafish, we finally demonstrate that this region shows specific enhancer activities in iridophores, a type of pigment cells found in egg-spots, suggesting that a cis-regulatory change is causally linked to the gain of expression in egg-spot bearing haplochromines. PMID:25296686

Thermally-Driven Mantle Plumes Reconcile Hot-spot Observations

NASA Astrophysics Data System (ADS)

Davies, D.; Davies, J.

2008-12-01

Hot-spots are anomalous regions of magmatism that cannot be directly associated with plate tectonic processes (e.g. Morgan, 1972). They are widely regarded as the surface expression of upwelling mantle plumes. Hot-spots exhibit variable life-spans, magmatic productivity and fixity (e.g. Ito and van Keken, 2007). This suggests that a wide-range of upwelling structures coexist within Earth's mantle, a view supported by geochemical and seismic evidence, but, thus far, not reproduced by numerical models. Here, results from a new, global, 3-D spherical, mantle convection model are presented, which better reconcile hot-spot observations, the key modification from previous models being increased convective vigor. Model upwellings show broad-ranging dynamics; some drift slowly, while others are more mobile, displaying variable life-spans, intensities and migration velocities. Such behavior is consistent with hot-spot observations, indicating that the mantle must be simulated at the correct vigor and in the appropriate geometry to reproduce Earth-like dynamics. Thermally-driven mantle plumes can explain the principal features of hot-spot volcanism on Earth.

Exclusion of EDNRB and KIT as the basis for white spotting in Border Collies.

PubMed

Metallinos, D; Rine, J

2000-01-01

White spotting patterns in mammals can be caused by mutations in the genes for the endothelin B receptor and c-Kit, whose protein products are necessary for proper migration, differentiation or survival of the melanoblast population of cells. Although there are many different dog breeds that segregate white spotting patterns, no genes have been identified that are linked to these phenotypes. An intercross was generated from a female Newfoundland and a male Border Collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies. The Border Collie markings segregated as a simple autosomal recessive (7/25 intercross progeny had the phenotype). Two candidate genes, for the endothelin B receptor (EDNRB) and c-Kit (KIT), were evaluated for segregation with the white spotting pattern. Polymorphisms between the Border Collie and Newfoundland were identified for EDNRB using Southern analysis after a portion of the canine gene had been cloned. Polymorphisms for KIT were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene. Both EDNRB and KIT were excluded as a cause of the white spotting pattern in at least two of the intercross progeny. Although these genes have been implicated in white spotting in other mammals, including horses, pigs, cows, mice and rats, they do not appear to be responsible for the white spotting pattern found in the Border Collie breed of dog.

Exclusion of EDNRB and KIT as the basis for white spotting in Border Collies

PubMed Central

Metallinos, Danika; Rine, Jasper

2000-01-01

Background: White spotting patterns in mammals can be caused by mutations in the genes for the endothelin B receptor and c-Kit, whose protein products are necessary for proper migration, differentiation or survival of the melanoblast population of cells. Although there are many different dog breeds that segregate white spotting patterns, no genes have been identified that are linked to these phenotypes. Results: An intercross was generated from a female Newfoundland and a male Border Collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies. The Border Collie markings segregated as a simple autosomal recessive (7/25 intercross progeny had the phenotype). Two candidate genes, for the endothelin B receptor (EDNRB) and c-Kit (KIT), were evaluated for segregation with the white spotting pattern. Polymorphisms between the Border Collie and Newfoundland were identified for EDNRB using Southern analysis after a portion of the canine gene had been cloned. Polymorphisms for KIT were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene. Conclusions: Both EDNRB and KIT were excluded as a cause of the white spotting pattern in at least two of the intercross progeny. Although these genes have been implicated in white spotting in other mammals, including horses, pigs, cows, mice and rats, they do not appear to be responsible for the white spotting pattern found in the Border Collie breed of dog. PMID:11178229

Development of PCR-based assays for detecting and differentiating three species of botrytis infecting broad bean

USDA-ARS?s Scientific Manuscript database

Botrytis cinerea, B. fabae and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop PCR-based assays to detect and differentiate this three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on t...

Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

PubMed

Angeloni, Cristina; Turroni, Silvia; Bianchi, Laura; Fabbri, Daniele; Motori, Elisa; Malaguti, Marco; Leoncini, Emanuela; Maraldi, Tullia; Bini, Luca; Brigidi, Patrizia; Hrelia, Silvana

2013-01-01

Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

PubMed

Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

2015-01-01

Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

PubMed

Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

2017-05-01

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

ZmGns, a maize class I β-1,3-glucanase, is induced by biotic stresses and possesses strong antimicrobial activity.

PubMed

Xie, Yu-Rong; Raruang, Yenjit; Chen, Zhi-Yuan; Brown, Robert L; Cleveland, Thomas E

2015-03-01

Plant β-1,3-glucanases are members of the pathogenesis-related protein 2 (PR-2) family, which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses. One of the differentially expressed proteins (spot 842) identified in a recent proteomic comparison between five pairs of closely related maize (Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study. Here, the corresponding cDNA was cloned from maize and designated as ZmGns. ZmGns encodes a protein of 338 amino acids containing a potential signal peptide. The expression of ZmGns was detectible in all tissues studied with the highest level in silks. ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus. ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid. In vivo, ZmGns showed a significant inhibitory activity against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis. Its high level of expression in the silk tissue and its induced expression by phytohormone treatment, as well as by bacterial and fungal infections, suggest it plays a complex role in maize growth, development, and defense. © 2014 Institute of Botany, Chinese Academy of Sciences.

Human duodenal proteome modulations by glutamine and antioxidants.

PubMed

Thébault, Sandrine; Deniel, Nicolas; Galland, Alexandra; Lecleire, Stéphane; Charlionet, Roland; Coëffier, Moïse; Tron, François; Vaudry, David; Déchelotte, Pierre

2010-03-01

Glutamine (Gln) has protective, anti-inflammatory effects in animal models and humans. Antioxidant nutrients may exert synergistic effects on intestinal functions. Therefore, these combined nutrients may have a therapeutic potential during intestinal inflammation. This study was designed to investigate in humans the effects of a supplement composed of Gln and high-dosed antioxidant micronutrients compared to isomolar Gln only, on duodenal proteome. Enteral perfusion of Gln (0.8  mmol  x  kg(-1) x  h(-1)) or supplement was performed in two groups of six healthy volunteers during 5  h before taking endoscopic duodenal biopsies. Protein expression was analyzed by 2-DE and the relevant proteins identified by MS/MS. About 1500 protein spots were revealed in both supplement and Gln conditions. Comparative proteomics analysis indicated that 11 proteins were differentially and significantly (p≤0.05) expressed in response to the supplement. These proteins were essentially implicated in metabolism pathways, e.g. fatty acid binding protein-1 and 40S ribosomal protein SA expressions were downregulated while manganese superoxide dismutase and retinal dehydrogenase-1 expressions were upregulated. This study provides new information on human duodenal proteome and its nutritional modulation, and supports further clinical investigations designed to evaluate the effects of Gln plus antioxidants during intestinal inflammation and cancer. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium[S

PubMed Central

Rudolph, Michael C.; Wellberg, Elizabeth A.; Lewis, Andrew S.; Terrell, Kristina L.; Merz, Andrea L.; Maluf, N. Karl; Serkova, Natalie J.; Anderson, Steven M.

2014-01-01

Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. PMID:24771867

Sequencing, de novo assembly and characterization of the spotted scat Scatophagus argus (Linnaeus 1766) transcriptome for discovery of reproduction related genes and SSRs

NASA Astrophysics Data System (ADS)

Yang, Wei; Chen, Huapu; Cui, Xuefan; Zhang, Kewei; Jiang, Dongneng; Deng, Siping; Zhu, Chunhua; Li, Guangli

2017-09-01

Spotted scat (Scatophagus argus) is an economically important farmed fish, particularly in East and Southeast Asia. Because there has been little research on reproductive development and regulation in this species, the lack of a mature artificial reproduction technology remains a barrier for the sustainable development of the aquaculture industry. More genetic and genomic background knowledge is urgently needed for an in-depth understanding of the molecular mechanism of reproductive process and identification of functional genes related to sexual differentiation, gonad maturation and gametogenesis. For these reasons, we performed transcriptomic analysis on spotted scat using a multiple tissue sample mixing strategy. The Illumina RNA sequencing generated 118 510 486 raw reads. After trimming, de novo assembly was performed and yielded 99 888 unigenes with an average length of 905.75 bp. A total of 45 015 unigenes were successfully annotated to the Nr, Swiss-Prot, KOG and KEGG databases. Additionally, 23 783 and 27 183 annotated unigenes were assigned to 56 Gene Ontology (GO) functional groups and 228 KEGG pathways, respectively. Subsequently, 2 474 transcripts associated with reproduction were selected using GO term and KEGG pathway assignments, and a number of reproduction-related genes involved in sex differentiation, gonad development and gametogenesis were identified. Furthermore, 22 279 simple sequence repeat (SSR) loci were discovered and characterized. The comprehensive transcript dataset described here greatly increases the genetic information available for spotted scat and contributes valuable sequence resources for functional gene mining and analysis. Candidate transcripts involved in reproduction would make good starting points for future studies on reproductive mechanisms, and the putative sex differentiation-related genes will be helpful for sex-determining gene identification and sex-specific marker isolation. Lastly, the SSRs can serve as marker resources for future research into genetics, marker-assisted selection (MAS) and conservation biology.

The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

PubMed

de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

2015-11-01

In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

Power Subsystem In-Flight Behaviour

NASA Astrophysics Data System (ADS)

Loche, Didier; Cosculluela, Valerie

2005-05-01

A synthesis of the In-flight monitoring of the Electrical Power Subsystem (EPS) behaviour of SPOT family and Mars Express is presented.It covers the solar array and battery performance, their degradation with life compared to the expected one in order to have lessons learned for future designs but also for in-orbit satellites software improvement.The SPOT family (from SPOT1 launched in 1986 up to ENVISAT/SPOT5 launched in 2002) EPS is based on an unregulated bus hard connected to the batteries. The solar array is split in sections, some digital and others PWM controlled in order to provide an accurate battery voltage and current regulation whatever is satellite power need. This regulation is performed by hardware. Mars Express EPS provides a regulated 28V bus. The battery power is managed by Battery Charge & Discharge Regulator (BCDR). The SA power is controlled by a Maximum Power Point Tracker (MPPT) logic. A bad connection between the SA and the Power Conditioning Unit (PCU) has led to a reduction of the power by about 30% and requested a large amount of test and simulations to estimate which power could be made available to the spacecraft and to monitor the actual EPS performance.

Matrilin-1 expression is increased in the vertebral column of Atlantic salmon (Salmo salar L.) individuals displaying spinal fusions.

PubMed

Pedersen, Mona E; Takle, Harald; Ytteborg, Elisabeth; Veiseth-Kent, Eva; Enersen, Grethe; Færgestad, Ellen; Baeverfjord, Grete; Hannesson, Kirsten O

2011-12-01

We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.

Garlic (Allium sativum L.) fertility: transcriptome and proteome analyses provide insight into flower and pollen development

PubMed Central

Shemesh-Mayer, Einat; Ben-Michael, Tomer; Rotem, Neta; Rabinowitch, Haim D.; Doron-Faigenboim, Adi; Kosmala, Arkadiusz; Perlikowski, Dawid; Sherman, Amir; Kamenetsky, Rina

2015-01-01

Commercial cultivars of garlic, a popular condiment, are sterile, making genetic studies and breeding of this plant challenging. However, recent fertility restoration has enabled advanced physiological and genetic research and hybridization in this important crop. Morphophysiological studies, combined with transcriptome and proteome analyses and quantitative PCR validation, enabled the identification of genes and specific processes involved in gametogenesis in fertile and male-sterile garlic genotypes. Both genotypes exhibit normal meiosis at early stages of anther development, but in the male-sterile plants, tapetal hypertrophy after microspore release leads to pollen degeneration. Transcriptome analysis and global gene-expression profiling showed that >16,000 genes are differentially expressed in the fertile vs. male-sterile developing flowers. Proteome analysis and quantitative comparison of 2D-gel protein maps revealed 36 significantly different protein spots, 9 of which were present only in the male-sterile genotype. Bioinformatic and quantitative PCR validation of 10 candidate genes exhibited significant expression differences between male-sterile and fertile flowers. A comparison of morphophysiological and molecular traits of fertile and male-sterile garlic flowers suggests that respiratory restrictions and/or non-regulated programmed cell death of the tapetum can lead to energy deficiency and consequent pollen abortion. Potential molecular markers for male fertility and sterility in garlic are proposed. PMID:25972879

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.

Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein expression to circulative polerovirus transmission.

PubMed

Cilia, M; Tamborindeguy, C; Fish, T; Howe, K; Thannhauser, T W; Gray, S

2011-03-01

Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission.

Genetics Coupled to Quantitative Intact Proteomics Links Heritable Aphid and Endosymbiont Protein Expression to Circulative Polerovirus Transmission▿ †

PubMed Central

Cilia, M.; Tamborindeguy, C.; Fish, T.; Howe, K.; Thannhauser, T. W.; Gray, S.

2011-01-01

Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission. PMID:21159868

Differential action and differential expression of DNA polymerase I during Escherichia coli colony development.

PubMed Central

Shapiro, J A

1992-01-01

A mini-Tn10 insertion in the polA cistron (polA2099) was isolated in a search for mutations that affect patterned Mudlac replication in colonies. The polA2099 mutation had a dramatic effect on cell morphogenesis during the first few hours of microcolony development. Abnormal microcolonies containing filamentous cells were produced as a result of SOS induction. Despite gross abnormalities in early microcolonies, mature polA2099 colonies after 2 to 4 days were morphologically indistinguishable from Pol+ colonies, and 44-h polA2099 colonies displayed a cell size distribution very similar to that of Pol+ colonies. These results suggested the involvement of a protective factor produced during colony growth that compensated for the polA deficiency. The action of a diffusible substance that stimulates growth of polA2099 microcolonies was shown by spotting dilute polA2099 cultures next to established colonies. Differential transcription of polA during colony development was visualized by growing colonies containing polA-lacZ fusions on beta-galactosidase indicator agar. When polA-lacZ colonies were inoculated next to established colonies, a diffusible factor was seen to inhibit polA transcription during the earliest stages of colony development. These results show that a basic housekeeping function, DNA polymerase I, is subject to multicellular control by the changing conditions which the bacteria create as they proliferate on agar. Images PMID:1331025

Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus.

PubMed

Medina, Martha L; Kiernan, Urban A; Francisco, Wilson A

2004-03-01

Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.

Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.

PubMed

Gutiérrez-Sánchez, Gerardo; Atwood, James; Kolli, V S Kumar; Roussos, Sévastianos; Augur, Christopher

2012-04-01

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.

Hot-spot evolution and the global tectonics of Venus

NASA Astrophysics Data System (ADS)

Phillips, R. J.; Grimm, R. E.; Malin, M. C.

1991-05-01

The global tectonics of Venus may be dominated by plumes rising from the mantle and impinging on the lithosphere, giving rise to hot spots. Global sea-floor spreading does not take place, but direct convective coupling of mantle flow fields to the lithosphere leads to regional-scale deformation and may allow lithospheric transport on a limited scale. A hot-spot evolutionary sequence comprises (1) a broad domal uplift resulting from a rising mantle plume, (2) massive partial melting in the plume head and generation of a thickened crust or crustal plateau, (3) collapse of dynamic topography, and (4) creep spreading of the crustal plateau. Crust on Venus is produced by gradual vertical differentiation with little recycling rather than by the rapid horizontal creation and consumption characteristic of terrestrial sea-floor spreading.

Genèse d'un horizon tacheté par déferruginisation dans une couverture à latérite du Bassin amazonien

NASA Astrophysics Data System (ADS)

Rosolen, Vania; Lamotte, Mathieu; Boulet, René; Trichet, Jean; Rouer, Olivier; José Melfi, Adolpho

A mottled horizon in a laterite cover (without any duricrust) was studied by microscopy and quantitative chemical microanalysis. Apart from the voids, light red spots consisting of Fe-rich particles (≈2 μm) are set in clayey plasma. Dark red spots consisted of concentrations of Fe-rich particles. These patterns are inherited. On the border of structural or biological voids, where Fe-depletion features are systematic, gray or yellow spots result from dissolution of the Fe-rich particles and impregnation of the plasma by iron, respectively. The present Fe-depletion is the dominant process that explains the mottled differentiation and the absence of lateritic duricrust. To cite this article: V. Rosolen et al., C. R. Geoscience 334 (2002) 187-195.

Hot-spot evolution and the global tectonics of Venus

NASA Technical Reports Server (NTRS)

Phillips, Roger J.; Grimm, Robert E.; Malin, Michael C.

1991-01-01

The global tectonics of Venus may be dominated by plumes rising from the mantle and impinging on the lithosphere, giving rise to hot spots. Global sea-floor spreading does not take place, but direct convective coupling of mantle flow fields to the lithosphere leads to regional-scale deformation and may allow lithospheric transport on a limited scale. A hot-spot evolutionary sequence comprises (1) a broad domal uplift resulting from a rising mantle plume, (2) massive partial melting in the plume head and generation of a thickened crust or crustal plateau, (3) collapse of dynamic topography, and (4) creep spreading of the crustal plateau. Crust on Venus is produced by gradual vertical differentiation with little recycling rather than by the rapid horizontal creation and consumption characteristic of terrestrial sea-floor spreading.

Hot-spot evolution and the global tectonics of venus.

PubMed

Phillips, R J; Grimm, R E; Malin, M C

1991-05-03

The global tectonics of Venus may be dominated by plumes rising from the mantle and impinging on the lithosphere, giving rise to hot spots. Global sea-floor spreading does not take place, but direct convective coupling of mantle flow fields to the lithosphere leads to regional-scale deformation and may allow lithospheric transport on a limited scale. A hot-spot evolutionary sequence comprises (i) a broad domal uplift resulting from a rising mantle plume, (ii) massive partial melting in the plume head and generation of a thickened crust or crustal plateau, (iii) collapse of dynamic topography, and (iv) creep spreading of the crustal plateau. Crust on Venus is produced by gradual vertical differentiation with little recycling rather than by the rapid horizontal creation and consumption characteristic of terrestrial sea-floor spreading.

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits.

PubMed

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-07-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain‑dead donors. In the present study, two‑dimensional gel electrophoresis and MALDI‑TOF MS‑based comparative proteomic analysis were conducted to profile the differentially‑expressed proteins between brain death and the control group renal tissues. A total of 40 age‑ and sex‑matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two‑dimensional gel electrophoresis, >2‑fold alterations were identified by MALDI‑TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3‑N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b‑c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre‑mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V‑type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin‑3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time‑dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain‑dead donors.

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

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

Methodological study of affine transformations of gene expression data with proposed robust non-parametric multi-dimensional normalization method.

PubMed

Bengtsson, Henrik; Hössjer, Ola

2006-03-01

Low-level processing and normalization of microarray data are most important steps in microarray analysis, which have profound impact on downstream analysis. Multiple methods have been suggested to date, but it is not clear which is the best. It is therefore important to further study the different normalization methods in detail and the nature of microarray data in general. A methodological study of affine models for gene expression data is carried out. Focus is on two-channel comparative studies, but the findings generalize also to single- and multi-channel data. The discussion applies to spotted as well as in-situ synthesized microarray data. Existing normalization methods such as curve-fit ("lowess") normalization, parallel and perpendicular translation normalization, and quantile normalization, but also dye-swap normalization are revisited in the light of the affine model and their strengths and weaknesses are investigated in this context. As a direct result from this study, we propose a robust non-parametric multi-dimensional affine normalization method, which can be applied to any number of microarrays with any number of channels either individually or all at once. A high-quality cDNA microarray data set with spike-in controls is used to demonstrate the power of the affine model and the proposed normalization method. We find that an affine model can explain non-linear intensity-dependent systematic effects in observed log-ratios. Affine normalization removes such artifacts for non-differentially expressed genes and assures that symmetry between negative and positive log-ratios is obtained, which is fundamental when identifying differentially expressed genes. In addition, affine normalization makes the empirical distributions in different channels more equal, which is the purpose of quantile normalization, and may also explain why dye-swap normalization works or fails. All methods are made available in the aroma package, which is a platform-independent package for R.

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

High-Density Spot Seeding for Tissue Model Formation

NASA Technical Reports Server (NTRS)

Marquette, Michele L. (Inventor); Sognier, Marguerite A. (Inventor)

2016-01-01

A model of tissue is produced by steps comprising seeding cells at a selected concentration on a support to form a cell spot, incubating the cells to allow the cells to partially attach, rinsing the cells to remove any cells that have not partially attached, adding culture medium to enable the cells to proliferate at a periphery of the cell spot and to differentiate toward a center of the cell spot, and further incubating the cells to form the tissue. The cells may be C2C12 cells or other subclones of the C2 cell line, H9c2(2-1) cells, L6 cells, L8 cells, QM7 cells, Sol8 cells, G-7 cells, G-8 cells, other myoblast cells, cells from other tissues, or stem cells. The selected concentration is in a range from about 1 x 10(exp 5) cells/ml to about 1 x 10(exp 6) cells/ml. The tissue formed may be a muscle tissue or other tissue depending on the cells seeded.

High-Density Spot Seeding for Tissue Model Formation

NASA Technical Reports Server (NTRS)

Marquette, Michele L. (Inventor); Sognier, Marguerite A. (Inventor)

2014-01-01

A method for making a tissue includes seeding cells at a selected concentration on a support to form a cell spot, incubating the cells to allow the cells to partially attach, rinsing the cells to remove any unattached cells, adding culture medium to enable the cells to proliferate at a periphery of the cell spot and to differentiate toward a center of the cell spot, and further incubating the cells to form the tissue. The cells may be C2C12 cells or other subclones of the C2 cell line, H9c2(2-1) cells, L6 cells, L8 cells, QM7 cells, Sol8 cells, G-7 cells, G-8 cells, other myoblast cells, cells from other tissues, or stem cells. The selected concentration is in a range from about 1 x 10(exp 5) cells/ml to about 1 x 10(exp 6) cells/ml. The tissue formed may be a skeletal muscle tissue, a cardiac muscle tissue, nerve tissue, or a bone tissue.

Effect of postharvest ultraviolet-C treatment on the proteome changes in fresh cut mango (Mangifera indica L. cv. Chokanan).

PubMed

George, Dominic Soloman; Razali, Zuliana; Santhirasegaram, Vicknesha; Somasundram, Chandran

2016-06-01

Postharvest treatments of fruits using techniques such as ultraviolet-C have been linked with maintenance of the fruit quality as well as shelf-life extension. However, the effects of this treatment on the quality of fruits on a proteomic level remain unclear. This study was conducted in order to understand the response of mango fruit to postharvest UV-C irradiation. Approximately 380 reproducible spots were detected following two-dimensional gel electrophoresis. Through gel analysis, 24 spots were observed to be differentially expressed in UV-C treated fruits and 20 were successfully identified via LCMS/MS. Postharvest UV-C treatment resulted in degradative effects on these identified proteins of which 40% were related to stress response, 45% to energy and metabolism and 15% to ripening and senescence. In addition, quality and shelf-life analysis of control and irradiated mangoes was evaluated. UV-C was found to be successful in retention of quality and extension of shelf-life up to 15 days. Furthermore, UV-C was also successful in increasing antioxidants (total flavonoid, reducing power and ABTS scavenging activity) in mangoes. This study provides an overview of the effects of UV-C treatment on the quality of mango on a proteomic level as well as the potential of this treatment in shelf-life extension of fresh-cut fruits. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Transcriptome changes associated with Tomato spotted wilt virus infection in various life stages of its thrips vector, Frankliniella fusca (Hinds).

PubMed

Shrestha, Anita; Champagne, Donald E; Culbreath, Albert K; Rotenberg, Dorith; Whitfield, Anna E; Srinivasan, Rajagopalbabu

2017-08-01

Persistent propagative viruses maintain intricate interactions with their arthropod vectors. In this study, we investigated the transcriptome-level responses associated with a persistent propagative phytovirus infection in various life stages of its vector using an Illumina HiSeq sequencing platform. The pathosystem components included a Tospovirus, Tomato spotted wilt virus (TSWV), its insect vector, Frankliniella fusca (Hinds), and a plant host, Arachis hypogaea (L.). We assembled (de novo) reads from three developmental stage groups of virus-exposed and non-virus-exposed F. fusca into one transcriptome consisting of 72 366 contigs and identified 1161 differentially expressed (DE) contigs. The number of DE contigs was greatest in adults (female) (562) when compared with larvae (first and second instars) (395) and pupae (pre- and pupae) (204). Upregulated contigs in virus-exposed thrips had blastx annotations associated with intracellular transport and virus replication. Upregulated contigs were also assigned blastx annotations associated with immune responses, including apoptosis and phagocytosis. In virus-exposed larvae, Blast2GO analysis identified functional groups, such as multicellular development with downregulated contigs, while reproduction, embryo development and growth were identified with upregulated contigs in virus-exposed adults. This study provides insights into differences in transcriptome-level responses modulated by TSWV in various life stages of an important vector, F. fusca.

Gravitational stress-induced changes in the phosphoproteom of Arabidopsis thaliana cell cultures

NASA Astrophysics Data System (ADS)

Hampp, Ruediger; Hausmann, Niklas; Neef, Maren; Schuetz, Wolfgang; Madlung, Johannes; Fladerer, Claudia; Nordheim, Alfred; Costa, Alex; Barjaktarovic, Zarko

Callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly (P¡0.05) in amount after 2h of hypergravity (18 up-, 10 down-regulated) could be identified. The corre-sponding proteins were largely involved in stress responses, including detoxification of reactive oxygen species (ROS; Barjaktaroviá et al., J. Exptl. Bot. 58:4357 (2007)). In the present study, c we extended these investigations to phosphorylated proteins. For this purpose, callus cell cul-tures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded most reliable data. First changes, however, were visible as early as 10 min after start of treatment. Out of the protein spots altered in phosphorylation, we were able to identify 24 from those responding to random positioning and 12 which responded to 8 g. The respective proteins are involved in scavenging and detoxification of ROS (32Most recent data obtained from parabolic flights indicate that exposure times to g of as little as 20 s are sufficient to alter the phosphorylation of proteins pattern. This is accompanied by changes in the cellular Ca2+ and H2O2 contents.

The Transcription Factors Ets1 and Sox10 Interact During Murine Melanocyte Development

PubMed Central

Saldana-Caboverde, Amy; Perera, Erasmo M.; Watkins-Chow, Dawn; Hansen, Nancy F.; Vemulapalli, Meghana; Mullikin, James C; Pavan, William J.; Kos, Lidia

2015-01-01

Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC. PMID:25912689

THE IMPACT OF SURFACE TEMPERATURE INHOMOGENEITIES ON QUIESCENT NEUTRON STAR RADIUS MEASUREMENTS

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

Elshamouty, K. G.; Heinke, C. O.; Morsink, S. M.

Fitting the thermal X-ray spectra of neutron stars (NSs) in quiescent X-ray binaries can constrain the masses and radii of NSs. The effect of undetected hot spots on the spectrum, and thus on the inferred NS mass and radius, has not yet been explored for appropriate atmospheres and spectra. A hot spot would harden the observed spectrum, so that spectral modeling tends to infer radii that are too small. However, a hot spot may also produce detectable pulsations. We simulated the effects of a hot spot on the pulsed fraction and spectrum of the quiescent NSs X5 and X7 inmore » the globular cluster 47 Tucanae, using appropriate spectra and beaming for hydrogen atmosphere models, incorporating special and general relativistic effects, and sampling a range of system angles. We searched for pulsations in archival Chandra HRC-S observations of X5 and X7, placing 90% confidence upper limits on their pulsed fractions below 16%. We use these pulsation limits to constrain the temperature differential of any hot spots, and to then constrain the effects of possible hot spots on the X-ray spectrum and the inferred radius from spectral fitting. We find that hot spots below our pulsation limit could bias the spectroscopically inferred radius downward by up to 28%. For Cen X-4 (which has deeper published pulsation searches), an undetected hot spot could bias its inferred radius downward by up to 10%. Improving constraints on pulsations from quiescent LMXBs may be essential for progress in constraining their radii.« less

Comparative point-spread function calculations for the MOMS-1, Thematic Mapper and SPOT-HRV instruments

NASA Technical Reports Server (NTRS)

Salomonson, V. V.; Nickeson, J. E.; Bodechtel, J.; Zilger, J.

1988-01-01

Point-spread functions (PSF) comparisons were made between the Modular Optoelectronic Multispectral Scanner (MOMS-01), the LANDSAT Thematic Mapper (TM) and the SPOT-HRV instruments, principally near Lake Nakuru, Kenya. The results, expressed in terms of the width of the point spread functions at the 50 percent power points as determined from the in-scene analysis show that the TM has a PSF equal to or narrower than the MOMS-01 instrument (50 to 55 for the TM versus 50 to 68 for the MOMS). The SPOT estimates of the PSF range from 36 to 40. When the MOMS results are adjusted for differences in edge scanning as compared to the TM and SPOT, they are nearer 40 in the 575 to 625 nm band.

Loci on chromosomes 1A and 2A affect resistance to tan (yellow) spot in wheat populations not segregating for tsn1.

PubMed

Shankar, Manisha; Jorgensen, Dorthe; Taylor, Julian; Chalmers, Ken J; Fox, Rebecca; Hollaway, Grant J; Neate, Stephen M; McLean, Mark S; Vassos, Elysia; Golzar, Hossein; Loughman, Robert; Mather, Diane E

2017-12-01

QTL for tan spot resistance were mapped on wheat chromosomes 1A and 2A. Lines were developed with resistance alleles at these loci and at the tsn1 locus on chromosome 5B. These lines expressed significantly higher resistance than the parent with tsn1 only. Tan spot (syn. yellow spot and yellow leaf spot) caused by Pyrenophora tritici-repentis is an important foliar disease of wheat in Australia. Few resistance genes have been mapped in Australian germplasm and only one, known as tsn1 located on chromosome 5B, is known in Australian breeding programs. This gene confers insensitivity to the fungal effector ToxA. The main aim of this study was to map novel resistance loci in two populations: Calingiri/Wyalkatchem, which is fixed for the ToxA-insensitivity allele tsn1, and IGW2574/Annuello, which is fixed for the ToxA-sensitivity allele Tsn1. A second aim was to combine new loci with tsn1 to develop lines with improved resistance. Tan spot severity was evaluated at various growth stages and in multiple environments. Symptom severity traits exhibited quantitative variation. The most significant quantitative trait loci (QTL) were detected on chromosomes 2A and 1A. The QTL on 2A explained up to 29.2% of the genotypic variation in the Calingiri/Wyalkatchem population with the resistance allele contributed by Wyalkatchem. The QTL on 1A explained up to 28.1% of the genotypic variation in the IGW2574/Annuello population with the resistance allele contributed by Annuello. The resistance alleles at both QTL were successfully combined with tsn1 to develop lines that express significantly better resistance at both seedling and adult plant stages than Calingiri which has tsn1 only.

Mantle compositions below petit-spot volcanoes of the NW Pacific Plate

NASA Astrophysics Data System (ADS)

Hirano, N.

2017-12-01

Monogenetic petit-spot volcanoes of a few kilometers in diameter and <300 m in height form volcanic clusters on the subducting NW Pacific plate offshore from NE Japan. Three of these petit-spot provinces form clusters with extents of 1,000-10,000 km2, containing between 15 to 90 monogenetic volcanoes, respectively (Hirano et al., 2008). The magmas that form these volcanoes originate below the lithosphere and ascend along the concavely flexed zone of the outer-rise prior to plate subduction at the trench (Hirano et al., 2006). This forms a unique opportunity to geochemically examine the mantle beneath the oceanic crust in a region outside of the well-examined but spatially restricted areas of mid-oceanic ridges and hotspots, indicating that these petit-spot lavas and associated xenoliths can directly provide the information on the asthenospheric and lithospheric material within and beneath old and subducting plates. Recent research into the geochemistry of petit-spot lavas and the petrography of xenoliths within these lavas indicates that the conventional subducting lithospheric theories require some revision in terms of the nature of subducting lithospheric and asthenospheric materials (e.g., heterogeneous asthenosphere and the presence of a higher geothermal gradient than the conventional GDH1 model; Machida et al., 2015; Yamamoto et al., 2014). The fact that the majority of the petit-spot lava samples do not contain olivine phenocrysts and have differentiated compositions (45-52 wt% SiO2, Mg# values of 50-65) indicates that these magmas have undergone differentiation in a magma chamber. However, geobarometry indicates that the deepest-sourced associated peridotitic xenoliths were derived from a depth of 42 km (Yamamoto et al., 2014). This indicates that melt fractionation must have occurred at depths greater than the middle lithosphere, a situation where the depth of fractionation could correlate with the rotation of the σ3 stress axis from the extensionally lower to the compressional upper part of the lithosphere. This rotation is the result of concave flexure prior to the outer rise of the subduction zone (Valentine & Hirano, 2010). Pilet et al. (2016) and Yamamoto et al. (2009) reported that these xenoliths were derived from a metasomatized region of the mantle, with this region metasomatized by prior melts of petit-spot magmas in the province. The strategic analysis of xenocrystic olivines from several petit-spot volcanoes also indicates that more depleted compositions are located in areas more proximal to the trench. This indicates that the lithospheric mantle in this region must have been significantly metasomatized prior to the onset of trench subduction.

Photospheric Acne at The Bottom of the Main-Sequence: Doppler Images of M4.5 - M9V Stars

NASA Astrophysics Data System (ADS)

Barnes, John R.; Haswell, Carole A.; Jeffers, Sandra V.; Jones, Hugh R. A.; Pavlenko, Yakiv V.; Lohr, Marcus E.; Jenkins, James S.

2016-07-01

Starspots are an important manifestation of stellar activity and yet their distribution patterns on the lowest mass stars is notwell known. Time series spectra of fullyconvective M dwarfs taken in the red-optical with UVES reveal numerous line profiledistortions which are interpreted as starspots. New Doppler images of HU Del (GJ 791.2A; M4.5V), BL Ceti (GJ 65A; M5.5V)and UV Ceti (GJ 65B; M6V) attwoepochs separated by three nights are presented. We find that contrastratioscorrespondingto photosphere-spot temperature differences of only 100-400 Kare sufficient to model the time series spectra of M4.5V - M9Vstars. Starspotsare reconstructed at a range of phases and latitudes with mean spot filling factors of only a few per cent.The distribution and low-contrast of the spots/spot-groups that we recover are likely to be responsible for the low amplitudephotometric variability seen in late-M dwarfs. The stability of the spot patterns in the two sets of timeseries observationsenables us to measure the latitude dependent differential rotation, which we find to be consistent with zero.

DNA Double-Strand Breaks Coupled with PARP1 and HNRNPA2B1 Binding Sites Flank Coordinately Expressed Domains in Human Chromosomes

PubMed Central

Fedoseeva, Daria M.; Sosin, Dmitri V.; Grachev, Sergei A.; Serebraykova, Marina V.; Romanenko, Svetlana A.; Vorobieva, Nadezhda V.; Kravatsky, Yuri V.

2013-01-01

Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs) is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50–250 kb DNA domains. We found that about 30% of the domains (denoted forum domains) possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites. PMID:23593027

Distribution and abundance of black bass in Skiatook Lake, Oklahoma, after introduction of smallmouth bass and a liberalized harvest regulation on spotted bass

USGS Publications Warehouse

Long, James M.; Fisher, William L.

2005-01-01

We conducted a 3-year study to examine the trends in abundance and distribution of three sympatric black bass species (Micropterus) in an Oklahoma reservoir after implementation of a differential harvest regulation to reduce the abundance of spotted bass M. punctulatus and after stocking nonnative smallmouth bass M. dolomieu. Largemouth bass M. salmoides were stocked in Skiatook Lake, Oklahoma, immediately after its creation in 1984 to supplement the existing population in the watershed. Nonnative smallmouth bass were stocked in 1990, and their abundance and distribution have increased ever since. Native spotted bass, which have less fishery value than the other two black bass species, increased fivefold in abundance in 1994, became the predominant black bass species by at least 1996, and appeared to displace largemouth bass from many habitats. From boat-mounted electrofishing sampling conducted in April and May 1997–1999, we found that spotted bass abundance (proportion and catch per hour) had decreased while smallmouth bass abundance and distribution within the reservoir steadily increased. Largemouth bass abundance did not change among years. Throughout our study period, spotted bass was always the most abundant black bass species where differences in abundance were found. Our results suggest that the continually expanding smallmouth bass population is displacing spotted bass from many of their formerly used habitats, much like spotted bass had displaced largemouth bass by 1996.

Profundulus kreiseri, a new species of Profundulidae (Teleostei, Cyprinodontiformes) from northwestern Honduras

PubMed Central

Matamoros, Wilfredo A.; Schaefer, Jacob F.; Hernández, Carmen L.; Prosanta Chakrabarty

2012-01-01

Abstract A new species of Profundulus, Profundulus kreiseri (Cyprinodontiformes: Profundulidae), is described from the Chamelecón and Ulúa Rivers in the northwestern Honduran highlands. Based on a phylogenetic analysis using cytochrome b and the presence of synapomorphic characters (dark humeral spot, a scaled preorbital region and between 32-34 vertebrae), this new species is placed in the subgenus Profundulus, which also includes Profundulus (Profundulus) oaxacae, Profundulus (Profundulus) punctatus and Profundulus (Profundulus) guatemalensis. Profundulus kreiseri can be distinguished from other members of the subgenus Profundulus by having less than half of its caudal fin densely scaled. Profundulus kreiseri can further be differentiated from Profundulus (Profundulus) oaxacae and Profundulus (Profundulus) punctatus by the absence of rows of dark spots on its flanks. The new species can further be differentiated from Profundulus (Profundulus) guatemalensis by the presence of fewer caudal- and pectoral-fin rays. The new species is distinguished from congeners of the profundulid subgenus Tlaloc (viz., Profundulus (Tlaloc) hildebrandi, Profundulus (Tlaloc) labialis, Profundulus (Tlaloc) candalarius and Profundulus (Tlaloc) portillorum) by having a scaled preorbital region and a dark humeral spot. Profundulus kreiseri and Profundulus portillorum are the only two species of Profundulus that are endemic to the region south of the Motagua River drainage in southern Guatemala and northwestern Honduras. PMID:23166464

Microarray image analysis: background estimation using quantile and morphological filters.

PubMed

Bengtsson, Anders; Bengtsson, Henrik

2006-02-28

In a microarray experiment the difference in expression between genes on the same slide is up to 103 fold or more. At low expression, even a small error in the estimate will have great influence on the final test and reference ratios. In addition to the true spot intensity the scanned signal consists of different kinds of noise referred to as background. In order to assess the true spot intensity background must be subtracted. The standard approach to estimate background intensities is to assume they are equal to the intensity levels between spots. In the literature, morphological opening is suggested to be one of the best methods for estimating background this way. This paper examines fundamental properties of rank and quantile filters, which include morphological filters at the extremes, with focus on their ability to estimate between-spot intensity levels. The bias and variance of these filter estimates are driven by the number of background pixels used and their distributions. A new rank-filter algorithm is implemented and compared to methods available in Spot by CSIRO and GenePix Pro by Axon Instruments. Spot's morphological opening has a mean bias between -47 and -248 compared to a bias between 2 and -2 for the rank filter and the variability of the morphological opening estimate is 3 times higher than for the rank filter. The mean bias of Spot's second method, morph.close.open, is between -5 and -16 and the variability is approximately the same as for morphological opening. The variability of GenePix Pro's region-based estimate is more than ten times higher than the variability of the rank-filter estimate and with slightly more bias. The large variability is because the size of the background window changes with spot size. To overcome this, a non-adaptive region-based method is implemented. Its bias and variability are comparable to that of the rank filter. The performance of more advanced rank filters is equal to the best region-based methods. However, in order to get unbiased estimates these filters have to be implemented with great care. The performance of morphological opening is in general poor with a substantial spatial-dependent bias.

Global gene expression analysis in a mouse model for Norrie disease: late involvement of photoreceptor cells.

PubMed

Lenzner, Steffen; Prietz, Sandra; Feil, Silke; Nuber, Ulrike A; Ropers, H-Hilger; Berger, Wolfgang

2002-09-01

Mutations in the NDP gene give rise to a variety of eye diseases, including classic Norrie disease (ND), X-linked exudative vitreoretinopathy (EVRX), retinal telangiectasis (Coats disease), and advanced retinopathy of prematurity (ROP). The gene product is a cystine-knot-containing extracellular signaling molecule of unknown function. In the current study, gene expression was determined in a mouse model of ND, to unravel disease-associated mechanisms at the molecular level. Gene transcription in the eyes of 2-year-old Ndp knockout mice was compared with that in the eyes of age-matched wild-type control animals, by means of cDNA subtraction and microarrays. Clones (n = 3072) from the cDNA subtraction libraries were spotted onto glass slides and hybridized with fluorescently labeled RNA-derived targets. More than 230 differentially expressed clones were sequenced, and their expression patterns were verified by virtual Northern blot analysis. Numerous gene transcripts that are absent or downregulated in the eye of Ndp knockout mice are photoreceptor cell specific. In younger Ndp knockout mice (up to 1 year old), however, all these transcripts were found to be expressed at normal levels. The identification of numerous photoreceptor cell-specific transcripts with a reduced expression in 2-year-old, but not in young, Ndp knockout mice indicates that normal gene expression in these light-sensitive cells of mutant mice is established and maintained over a long period and that rods and cones are affected relatively late in the mouse model of ND. Obviously, the absence of the Ndp gene product is not compatible with long-term survival of photoreceptor cells in the mouse.

Gene Expression of Tissue-Specific Molecules in Ex vivo Dermacentor variabilis (Acari: Ixodidae) During Rickettsial Exposure

PubMed Central

SUNYAKUMTHORN, PIYANATE; PETCHAMPAI, NATTHIDA; GRASPERGE, BRITTON J.; KEARNEY, MICHAEL T.; SONENSHINE, DANIEL E.; MACALUSO, KEVIN R.

2014-01-01

Ticks serve as both vectors and the reservoir hosts capable of transmitting spotted fever group Rickettsia by horizontal and vertical transmission. Persistent maintenance of Rickettsia species in tick populations is dependent on the specificity of the tick and Rickettsia relationship that limits vertical transmission of particular Rickettsia species, suggesting host-derived mechanisms of control. Tick-derived molecules are differentially expressed in a tissue-specific manner in response to rickettsial infection; however, little is known about tick response to specific rickettsial species. To test the hypothesis that tissue-specific tick-derived molecules are uniquely responsive to rickettsial infection, a bioassay to characterize the tick tissue-specific response to different rickettsial species was used. Whole organs of Dermacentor variabilis (Say) were exposed to either Rickettsia montanensis or Rickettsia amblyommii, two Rickettsia species common, or absent, in field-collected D. variabilis, respectively, for 1 and 12 h and harvested for quantitative real time-polymerase chain reaction assays of putative immune-like tick-derived factors. The results indicated that tick genes are differently expressed in a temporal and tissue-specific manner. Genes encoding glutathione S-transferase 1 (dvgst1) and Kunitz protease inhibitor (dvkpi) were highly expressed in midgut, and rickettsial exposure downregulated the expression of both genes. Two other genes encoding glutathione S-transferase 2 (dvgst2) and β-thymosin (dvβ-thy) were highly expressed in ovary, with dvβ-thy expression significantly downregulated in ovaries exposed to R. montanensis, but not R. amblyommii, at 12-h postexposure, suggesting a selective response. Deciphering the tissue-specific molecular interactions between tick and Rickettsia will enhance our understanding of the key mechanisms that mediate rickettsial infection in ticks. PMID:24180114

Prediction of Petermann I and II Spot Sizes for Single-mode Dispersion-shifted and Dispersion-flattened Fibers by a Simple Technique

NASA Astrophysics Data System (ADS)

Kamila, Kiranmay; Panda, Anup Kumar; Gangopadhyay, Sankar

2013-09-01

Employing the series expression for the fundamental modal field of dispersion-shifted trapezoidal and dispersion-flattened graded and step W fibers, we present simple but accurate analytical expressions for Petermann I and II spot sizes of such kind of fibers. Choosing some typical dispersion-shifted trapezoidal and dispersion-flattened graded and step W fibers as examples, we show that our estimations match excellently with the exact numerical results. The evaluation of the concerned propagation parameters by our formalism needs very little computations. This accurate but simple formalism will benefit the system engineers working in the field of all optical technology.

Characterization of proteins in the muscle of limanda yokohamae from the masan bay, Korea

NASA Astrophysics Data System (ADS)

Kim, Soo Woon; Kim, Sam Moon; Lee, Dong Kun; Moon, Hyo Bang; Choi, Hee Gu; Kang, Chang Keun; Choe, Eun Sang

2007-06-01

Increasing industrial development in the Masan Bay area of Korea over the past decades increased the risk for the survival of marine organisms in the bay area by the deterioration of the water quality. Since living organisms have the ability to adapt contamination-associated stimuli by the alteration of gene expression, changes in proteins can be used as an important criterion for assessing the levels of environmental conditions. In this study, therefore, alterations of the expression of proteins in the muscle of Limanda yokohamae from Dukdong and Dotsum in the bay area were surveyed and characterized as compared with Haegumgang, which served as a control site. The results demonstrated that the twenty spots detected from Dukdong and Dotsum were similar to each other. Fifteen proteins were found to be predicted or undefined proteins, while five proteins were identified as heavy polypeptide 11 of myosin, apolipoprotein A-I, fibroblast growth factor 17b precursor, G protein-coupled receptor kinase 1 b and bonnie and clyde. These data suggest that local fish in the bay area have dysfunction in muscle physiology including contraction, lipid metabolism, proliferation and differentiation and nervous system.

Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis.

PubMed

Ye, Yingwang; Li, Hui; Ling, Na; Han, Yongjia; Wu, Qingping; Xu, Xiaoke; Jiao, Rui; Gao, Jina

2016-01-18

Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter sakazakii is the most common species in terms of isolation frequency. However, the molecular basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential virulence factors were determined using two-dimensional electrophoresis (2-DE) coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential expression (fold change>1.2), we identified 31 different proteins, of which the expression abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic analysis in the Dufour’s gland of Africanized Apis mellifera workers (Hymenoptera: Apidae)

PubMed Central

2017-01-01

The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour’s gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour’s glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour’s gland contributing to a more complete understanding of the biological functions of this gland in honeybees. PMID:28542566

Insights into salicylic acid responses in cucumber (Cucumis sativus L.) cotyledons based on a comparative proteomic analysis.

PubMed

Hao, J H; Dong, C J; Zhang, Z G; Wang, X L; Shang, Q M

2012-05-01

To investigate the response of cucumber seedlings to exogenous salicylic acid (SA) and gain a better understanding of SA action mechanism, we generated a proteomic profile of cucumber (Cucumis sativus L.) cotyledons treated with exogenous SA. Analysis of 1500 protein spots from each gel revealed 63 differentially expressed proteins, 59 of which were identified successfully. Of the identified proteins, 97% matched cucumber proteins using a whole cucumber protein database based on the newly completed genome established by our laboratory. The identified proteins were involved in various cellular responses and metabolic processes, including antioxidative reactions, cell defense, photosynthesis, carbohydrate metabolism, respiration and energy homeostasis, protein folding and biosynthesis. The two largest functional categories included proteins involved in antioxidative reactions (23.7%) and photosynthesis (18.6%). Furthermore, the SA-responsive protein interaction network revealed 13 key proteins, suggesting that the expression changes of these proteins could be critical for SA-induced resistance. An analysis of these changes suggested that SA-induced resistance and seedling growth might be regulated in part through pathways involving antioxidative reactions and photosynthesis. © 2012 Elsevier Ireland Ltd. All rights reserved.

[Rocky mountain spotted fever: report of two cases].

PubMed

Martínez-Medina, Miguel Angel; Padilla-Zamudio, Guillermo; Solís-Gallardo, Lilia Patricia; Guevara-Tovar, Marcela

2005-01-01

Rocky Mountain spotted fever (RMSF) is an acute febrile illness caused by infection with Ricketsia Rickettsii, characterized by the presence of petechial rash. Even though the etiology, clinical characteristics and availability of effective antibiotics are known, RMSF related deaths have a prevalence of 4%. In its early stages RMFS can resemble many others infectious conditions and the diagnosis can be difficult. The present paper reports two patients with RMSF; these cases underscore the importance of prompt diagnosis and appropriate antimicrobial therapy, and consider RMSF as a differential diagnosis in any patient who develops fever and rash in an endemic area.

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

PubMed

Jiang, Dong-Neng; Li, Jian-Tao; Tao, Ya-Xiong; Chen, Hua-Pu; Deng, Si-Ping; Zhu, Chun-Hua; Li, Guang-Li

2017-05-01

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle 4 , D-Phe 7 ]-α-melanocyte stimulating hormone; 10 -6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10 -7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10 -6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10 -6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.

Segmentation and detection of fluorescent 3D spots.

PubMed

Ram, Sundaresh; Rodríguez, Jeffrey J; Bosco, Giovanni

2012-03-01

The 3D spatial organization of genes and other genetic elements within the nucleus is important for regulating gene expression. Understanding how this spatial organization is established and maintained throughout the life of a cell is key to elucidating the many layers of gene regulation. Quantitative methods for studying nuclear organization will lead to insights into the molecular mechanisms that maintain gene organization as well as serve as diagnostic tools for pathologies caused by loss of nuclear structure. However, biologists currently lack automated and high throughput methods for quantitative and qualitative global analysis of 3D gene organization. In this study, we use confocal microscopy and fluorescence in-situ hybridization (FISH) as a cytogenetic technique to detect and localize the presence of specific DNA sequences in 3D. FISH uses probes that bind to specific targeted locations on the chromosomes, appearing as fluorescent spots in 3D images obtained using fluorescence microscopy. In this article, we propose an automated algorithm for segmentation and detection of 3D FISH spots. The algorithm is divided into two stages: spot segmentation and spot detection. Spot segmentation consists of 3D anisotropic smoothing to reduce the effect of noise, top-hat filtering, and intensity thresholding, followed by 3D region-growing. Spot detection uses a Bayesian classifier with spot features such as volume, average intensity, texture, and contrast to detect and classify the segmented spots as either true or false spots. Quantitative assessment of the proposed algorithm demonstrates improved segmentation and detection accuracy compared to other techniques. Copyright © 2012 International Society for Advancement of Cytometry.

Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification.

PubMed

Ou, Keli; Kesuma, Djohan; Ganesan, Kumaresan; Yu, Kun; Soon, Sou Yen; Lee, Suet Ying; Goh, Xin Pei; Hooi, Michelle; Chen, Wei; Jikuya, Hiroyuki; Ichikawa, Tetsuo; Kuyama, Hiroki; Matsuo, Ei-ichi; Nishimura, Osamu; Tan, Patrick

2006-09-01

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.

Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

2012-11-15

Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

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.

The synergistic effects of CO2 laser treatment with calcium silicate cement of antibacterial, osteogenesis and cementogenesis efficacy

NASA Astrophysics Data System (ADS)

Hsu, T.-T.; Kao, C.-T.; Chen, Y.-W.; Huang, T.-H.; Yang, J.-J.; Shie, M.-Y.

2015-05-01

Calcium silicate-based material (CS) has been successfully used in dental clinical applications. Some researches show that the antibacterial effects of CO2 laser irradiation are highly efficient when bacteria are embedded in biofilm, due to a photo-thermal mechanism. The purpose of this study was to confirm the effects of CO2 laser irradiation on CS, with regard to both material characterization and human periodontal ligament cell (hPDLs) viability. CS was irradiated with a dental CO2 laser using directly mounted fiber optics in wound healing mode with a spot area of 0.25 cm2, and then stored in an incubator at 100% relative humidity and 37 °C for 1 d to set. The hPDLs cultured on CS were analyzed, along with their proliferation and odontogenic differentiation behaviors. The results indicate that the CO2 laser irradiation increased the amount of Ca and Si ions released from the CS, and regulated cell behavior. CO2 laser-irradiated CS promoted cementogenic differentiation of hPDLs, with the increased formation of mineralized nodules on the substrate’s surface. It also up-regulated the protein expression of multiple markers of cementogenic and the expression of cementum attachment protein. The current study provides new and important data about the effects of CO2 laser irradiation on CS. Taking cell functions into account, the Si concentration released from CS with laser irradiated may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue.

Fly artifact documentation of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) - a forensically important blowfly species in Malaysia.

PubMed

Zuha, R M; Supriyani, M; Omar, B

2008-04-01

Analysis on fly artifacts produced by forensically important blowfly, Chrysomya megacephala (Fabricius) (Diptera:Calliphoridae), revealed several unique patterns. They can be divided into fecal spots, regurgitation spots and swiping stains. The characteristics of fecal spots are round with three distinct levels of pigmentation; creamy, brownish and darkly pigmented. Matrix of the spots appears cloudy. The round spots are symmetrical and non-symmetrical, delineated by irregular and darker perimeter which only visible in fairly colored fecal spots. Diameter of these artifacts ranged from 0.5 mm to 4 mm. Vomit or regurgitation spots are determined by the presence of craters due to sucking activity of blowflies and surrounded by thickly raised and darker colored perimeter. The size of these specks ranged from 1 mm to 2 mm. Matrix of the spots displays irregular surface and reflective under auxiliary microscope light. Swiping stains due to defecation by flies consists of two distinguishable segments, the body and tail. It can be seen as a tear drop-like, sperm-like, snake-like and irregular tadpole-like stain. The direction of body and tail is inconsistent and length ranged between 4.8 mm to 9.2 mm. A finding that should be highlighted in this observation is the presence of crater on tadpole-like swiping stain which is apparent by its raised border characteristic and reflective under auxiliary microscope light. The directionality of this darkly brown stain is random. This unique mix of regurgitation and swiping stain has never been reported before. Highlighting the features of artifacts produced by flies would hopefully add our understanding in differentiating them from blood spatters produced from victims at crime scenes.

Stellar Differential Rotation of F-Stars Using DI and ZDI: The Case of HR1817

NASA Astrophysics Data System (ADS)

Marsden, Stephen

2018-04-01

The measure of surface differential rotation via the motion of spots and/or magnetic features on the stellar surface is a critical part of understanding the stellar dynamo. Here we present several epochs of (Zeeman) Doppler imaging of the young late-F star HR1817 from 2001 until 2011. These results show that HR1817 exhibits a high shear of its surface features, significantly above the solar value. It would appear that F stars, with thin convective zones, have surface differential rotation rates much higher than that of low mass stars.

Acclimation of brackish water pearl spot (Etroplus suratensis) to various salinities: relative changes in abundance of branchial Na(+)/K (+)-ATPase and Na (+)/K (+)/2Cl (-) co-transporter in relation to osmoregulatory parameters.

PubMed

Chandrasekar, S; Nich, T; Tripathi, G; Sahu, N P; Pal, A K; Dasgupta, S

2014-06-01

The present study was conducted to elucidate the osmoregulatory ability of the fish pearl spot (Etroplus suratensis) to know the scope of this species for aquaculture under various salinities. Juvenile pearl spot were divided into three groups and acclimated to freshwater (FW), brackish water (BW) or seawater (SW) for 15 days. The fish exhibited effective salinity tolerance under osmotic challenges. Although the plasma osmolality and Na(+), K(+) and Cl(-) levels increased with the increasing salinities, the parameters remained within the physiological range. The muscle water contents were constant among FW-, BW- and SW-acclimated fish. Two Na+/K+-ATPase α-isoforms (NKA α) were expressed in gills during acclimation in FW, BW and SW. Abundance of one isoform was up-regulated in response to seawater acclimation, suggesting its role in ion secretion similar to NKA α1b, while expression of another isoform was simultaneously up-regulated in response to both FW and SW acclimation, suggesting the presence of isoforms switching phenomenon during acclimation to different salinities. Nevertheless, NKA enzyme activities in the gills of the SW and FW individuals were higher (p < 0.05) than in BW counterparts. Immunohistochemistry revealed that Na(+)/K(+)-ATPase immunoreactive (NKA-IR) cells were mainly distributed in the interlamellar region of the gill filaments in FW groups and in the apical portion of the filaments in BW and SW groups. The number of NKA-IR cells in the gills of the FW-acclimated fish was almost similar to that of SW individuals, which exceeded that of the BW individuals. The NKA-IR cells of BW and SW were bigger in size than their FW counterparts. Besides, the relative abundance of branchial Na(+)/K(+)/2Cl(-) co-transporter showed stronger evidence in favor of involvement of this protein in hypo-osmoregulation, requiring ion secretion by the chloride cells. To the best of our knowledge, this is the first study reporting the wide salinity tolerance of E. suratensis involving differential activation of ion transporters and thereby suggesting its potential as candidate for fish farming under different external salinities.

MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii

PubMed Central

Sahni, Abha; Narra, Hema P.; Patel, Jignesh; Sahni, Sanjeev K.

2017-01-01

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R. rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections. PMID:28698491

Transcriptome response to elevated CO2, water deficit, and thermal stress in peanut

USDA-ARS?s Scientific Manuscript database

Previously, our laboratories have performed gene expression studies using EST sequencing and spotted microarrays to investigate tissue-specific gene expression and response to abiotic stress. While these studies have provided valuable insight into these processes, they are constrained by sequencer t...

Digital image analysis of Ki67 in hot spots is superior to both manual Ki67 and mitotic counts in breast cancer.

PubMed

Stålhammar, Gustav; Robertson, Stephanie; Wedlund, Lena; Lippert, Michael; Rantalainen, Mattias; Bergh, Jonas; Hartman, Johan

2018-05-01

During pathological examination of breast tumours, proliferative activity is routinely evaluated by a count of mitoses. Adding immunohistochemical stains of Ki67 provides extra prognostic and predictive information. However, the currently used methods for these evaluations suffer from imperfect reproducibility. It is still unclear whether analysis of Ki67 should be performed in hot spots, in the tumour periphery, or as an average of the whole tumour section. The aim of this study was to compare the clinical relevance of mitoses, Ki67 and phosphohistone H3 in two cohorts of primary breast cancer specimens (total n = 294). Both manual and digital image analysis scores were evaluated for sensitivity and specificity for luminal B versus A subtype as defined by PAM50 gene expression assays, for high versus low transcriptomic grade, for axillary lymph node status, and for prognostic value in terms of prediction of overall and relapse-free survival. Digital image analysis of Ki67 outperformed the other markers, especially in hot spots. Tumours with high Ki67 expression and high numbers of phosphohistone H3-positive cells had significantly increased hazard ratios for all-cause mortality within 10 years from diagnosis. Replacing manual mitotic counts with digital image analysis of Ki67 in hot spots increased the differences in overall survival between the highest and lowest histological grades, and added significant prognostic information. Digital image analysis of Ki67 in hot spots is the marker of choice for routine analysis of proliferation in breast cancer. © 2017 John Wiley & Sons Ltd.

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community.

PubMed

Yokoi, Takahide; Kaku, Yoshiko; Suzuki, Hiroyuki; Ohta, Masayuki; Ikuta, Hajime; Isaka, Kazuichi; Sumino, Tatsuo; Wagatsuma, Masako

2007-08-01

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.

Solvent-accessible surface area: How well can be applied to hot-spot detection?

PubMed

Martins, João M; Ramos, Rui M; Pimenta, António C; Moreira, Irina S

2014-03-01

A detailed comprehension of protein-based interfaces is essential for the rational drug development. One of the key features of these interfaces is their solvent accessible surface area profile. With that in mind, we tested a group of 12 SASA-based features for their ability to correlate and differentiate hot- and null-spots. These were tested in three different data sets, explicit water MD, implicit water MD, and static PDB structure. We found no discernible improvement with the use of more comprehensive data sets obtained from molecular dynamics. The features tested were shown to be capable of discerning between hot- and null-spots, while presenting low correlations. Residue standardization such as rel SASAi or rel/res SASAi , improved the features as a tool to predict ΔΔGbinding values. A new method using support machine learning algorithms was developed: SBHD (Sasa-Based Hot-spot Detection). This method presents a precision, recall, and F1 score of 0.72, 0.81, and 0.76 for the training set and 0.91, 0.73, and 0.81 for an independent test set. Copyright © 2013 Wiley Periodicals, Inc.

Molecular targets for diabetes mellitus-associated erectile dysfunction.

PubMed

Yohannes, Elizabeth; Chang, Jinsook; Tar, Moses T; Davies, Kelvin P; Chance, Mark R

2010-03-01

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCore suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-gamma, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-gamma, and Hsp47 confirmed increased p53 and 14-3-3-gamma and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a approximately 3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment.

Molecular Targets for Diabetes Mellitus-associated Erectile Dysfunction*

PubMed Central

Yohannes, Elizabeth; Chang, Jinsook; Tar, Moses T.; Davies, Kelvin P.; Chance, Mark R.

2010-01-01

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCoreTM suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-γ, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-γ, and Hsp47 confirmed increased p53 and 14-3-3-γ and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a ∼3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment. PMID:20007950

PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells.

PubMed

Fossum, Caroline; Hjertner, Bernt; Lövgren, Tanja; Fuxler, Lisbeth; Charerntantanakul, Wasin; Wallgren, Per

2014-02-01

A porcine circovirus type 2 SPOT (PCV2-SPOT) assay was established to enumerate virus-secreting lymphocytes obtained from naturally infected pigs. The assay is based on the same principle as general ELISPOT assays but instead of detecting cytokine or immunoglobulin secretion, PCV2 particles are immobilized and detected as filter spots. The method was used to evaluate the influence of various cell activators on the PCV2 secretion in vitro and was also applied to study the PCV2 secretion by lymphocytes obtained from pigs in healthy herds and in a herd afflicted by postweaning multisystemic wasting disease (PMWS). Peripheral blood mononuclear cells (PBMCs) obtained from a pig with severe PMWS produced PCV2-SPOTs spontaneously whereas PBMCs obtained from pigs infected subclinically only generated PCV2-SPOTs upon in vitro stimulation. The PCV2 secretion potential was related to the PCV2 DNA content in the PBMCs as determined by two PCV2 real-time PCR assays, developed to differentiate between Swedish PCV2 genogroups 1 (PCV2a) and 3 (PCV2b). Besides the current application these qPCRs could simplify future epidemiological studies and allow genogroup detection/quantitation in dual infection experiments and similar studies. The developed PCV2-SPOT assay offers a semi-quantitative approach to evaluate the potential of PCV2-infected porcine cells to release PCV2 viral particles as well as a system to evaluate the ability of different cell types or compounds to affect PCV2 replication and secretion. Copyright © 2013 Elsevier B.V. All rights reserved.

Novel Gardos channel mutations linked to dehydrated hereditary stomatocytosis (xerocytosis).

PubMed

Andolfo, Immacolata; Russo, Roberta; Manna, Francesco; Shmukler, Boris E; Gambale, Antonella; Vitiello, Giuseppina; De Rosa, Gianluca; Brugnara, Carlo; Alper, Seth L; Snyder, L Michael; Iolascon, Achille

2015-10-01

Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Affected red cells are characterized by a nonspecific cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHSt cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. A recent study has identified two families with DHSt associated with a single mutation in the KCNN4 gene encoding the Gardos channel (KCa3.1), the erythroid Ca(2+) -sensitive K(+) channel of intermediate conductance, also expressed in many other cell types. We present here, in the second report of DHSt associated with KCNN4 mutations, two previously undiagnosed DHSt families. Family NA exhibited the same de novo missense mutation as that recently described, suggesting a hot spot codon for DHSt mutations. Family WO carried a novel, inherited missense mutation in the ion transport domain of the channel. The patients' mild hemolytic anemia did not improve post-splenectomy, but splenectomy led to no serious thromboembolic events. We further characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of CD34+ cells and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development. © 2015 Wiley Periodicals, Inc.

Upregulation of eIF-5A1 in the paralyzed muscle after spinal cord transection associates with spontaneous hindlimb locomotor recovery in rats by upregulation of the ErbB, MAPK and neurotrophin signal pathways.

PubMed

Shang, Fei-Fei; Zhao, Wei; Zhao, Qi; Liu, Jia; Li, Da-Wei; Zhang, Hua; Zhou, Xin-Fu; Li, Cheng-Yun; Wang, Ting-Hua

2013-10-08

It is well known that trauma is frequently accompanied by spontaneous functional recovery after spinal cord injury (SCI), but the underlying mechanisms remain elusive. In this study, BBB scores showed a gradual return of locomotor functions after SCT. Proteomics analysis revealed 16 differential protein spots in the gastrocnemius muscle between SCT and normal rats. Of these differential proteins, eukaryotic translation initiation factor 5A1 (elf-5A1), a highly conserved molecule throughout eukaryotes, exhibited marked upregulation in the gastrocnemius muscle after SCT. To study the role of eIF-5A1 in the restoration of hindlimb locomotor functions following SCT, we used siRNA to downregulate the mRNA level of eIF-5A1. Compared with untreated SCT control rats, those subjected to eIF-5A1 knockdown exhibited impaired functional recovery. Moreover, gene expression microarrays and bioinformatic analysis showed high correlation between three main signal pathways (ErbB, MAPK and neurotrophin signal pathways) and eIF-5A1. These signal pathways regulate cell proliferation, differentiation and neurocyte growth. Consequently, eIF-5A1 played a pivotal role via these signal pathways in hindlimb locomotor functional recovery after SCT, which could pave the way for the development of a new strategy for the treatment of spinal cord injury in clinical trials. Copyright © 2012. Published by Elsevier B.V.

Functional differentiation of the human lumbar perivertebral musculature revisited by means of muscle fibre type composition.

PubMed

Hesse, Bettina; Fröber, Rosemarie; Fischer, Martin S; Schilling, Nadja

2013-12-01

Human back muscles have been classified as local stabilizers, global stabilizers and global mobilizers. This concept is supported by the distribution of slow and fast muscle fibres in quadrupedal mammals, but has not been evaluated for humans because detailed information on the fibre type composition of their perivertebral musculature is rare. Moreover, such information is derived from spot samples, which are assumed to be representative for the respective muscle. In accordance with the proposed classification, numerous studies in animals indicate great differences in the fibre distribution within and among the muscles due to fibre type regionalization. The aims of this study were to (1) qualitatively explore the applicability of the proposed functional classification for human back muscles by studying their fibre type composition and (2) evaluate the representativeness of spot sampling techniques. For this, the fibre type distribution of the whole lumbar perivertebral musculature of two male cadavers was investigated three-dimensionally using immunohistochemistry. Despite great local variations (e.g., among fascicles), all muscles were composed of about 50% slow and 50% fast fibres. Thus, contradicting the concepts of lumbar muscle function, no functional differentiation of the muscles was observed in our study of the muscle contractile properties. The great similarity in fibre composition among the muscles equips each muscle equally well for a broad range of tasks and therefore has the potential to allow for great functional versatility of the human back musculature. Spot samples do not prove to be representative for the whole muscle. The great intraspecific variability observed previously in single-spot samples is potentially misleading. Copyright © 2013 Elsevier GmbH. All rights reserved.

Correlation Techniques as Applied to Pose Estimation in Space Station Docking

NASA Technical Reports Server (NTRS)

Rollins, J. Michael; Juday, Richard D.; Monroe, Stanley E., Jr.

2002-01-01

The telerobotic assembly of space-station components has become the method of choice for the International Space Station (ISS) because it offers a safe alternative to the more hazardous option of space walks. The disadvantage of telerobotic assembly is that it does not provide for direct arbitrary views of mating interfaces for the teleoperator. Unless cameras are present very close to the interface positions, such views must be generated graphically, based on calculated pose relationships derived from images. To assist in this photogrammetric pose estimation, circular targets, or spots, of high contrast have been affixed on each connecting module at carefully surveyed positions. The appearance of a subset of spots essentially must form a constellation of specific relative positions in the incoming digital image stream in order for the docking to proceed. Spot positions are expressed in terms of their apparent centroids in an image. The precision of centroid estimation is required to be as fine as 1I20th pixel, in some cases. This paper presents an approach to spot centroid estimation using cross correlation between spot images and synthetic spot models of precise centration. Techniques for obtaining sub-pixel accuracy and for shadow, obscuration and lighting irregularity compensation are discussed.

Proteomic identification of rhythmic proteins in rice seedlings.

PubMed

Hwang, Heeyoun; Cho, Man-Ho; Hahn, Bum-Soo; Lim, Hyemin; Kwon, Yong-Kook; Hahn, Tae-Ryong; Bhoo, Seong Hee

2011-04-01

Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes. 2011 Elsevier B.V. All rights reserved.

Bone Morphogenetic Protein 3 Controls Insulin Gene Expression and Is Down-regulated in INS-1 Cells Inducibly Expressing a Hepatocyte Nuclear Factor 1A–Maturity-onset Diabetes of the Young Mutation*

PubMed Central

Bonner, Caroline; Farrelly, Angela M.; Concannon, Caoimhín G.; Dussmann, Heiko; Baquié, Mathurin; Virard, Isabelle; Wobser, Hella; Kögel, Donat; Wollheim, Claes B.; Rupnik, Marjan; Byrne, Maria M.; König, Hans-Georg; Prehn, Jochen H. M.

2011-01-01

Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A–maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY. PMID:21628466

Adaptive Weibull Multiplicative Model and Multilayer Perceptron Neural Networks for Dark-Spot Detection from SAR Imagery

PubMed Central

Taravat, Alireza; Oppelt, Natascha

2014-01-01

Oil spills represent a major threat to ocean ecosystems and their environmental status. Previous studies have shown that Synthetic Aperture Radar (SAR), as its recording is independent of clouds and weather, can be effectively used for the detection and classification of oil spills. Dark formation detection is the first and critical stage in oil-spill detection procedures. In this paper, a novel approach for automated dark-spot detection in SAR imagery is presented. A new approach from the combination of adaptive Weibull Multiplicative Model (WMM) and MultiLayer Perceptron (MLP) neural networks is proposed to differentiate between dark spots and the background. The results have been compared with the results of a model combining non-adaptive WMM and pulse coupled neural networks. The presented approach overcomes the non-adaptive WMM filter setting parameters by developing an adaptive WMM model which is a step ahead towards a full automatic dark spot detection. The proposed approach was tested on 60 ENVISAT and ERS2 images which contained dark spots. For the overall dataset, an average accuracy of 94.65% was obtained. Our experimental results demonstrate that the proposed approach is very robust and effective where the non-adaptive WMM & pulse coupled neural network (PCNN) model generates poor accuracies. PMID:25474376

Proteomic analysis of testis biopsies in men treated with injectable testosterone undecanoate alone or in combination with oral levonorgestrel as potential male contraceptive.

PubMed

Cui, Yugui; Zhu, Hui; Zhu, Yefei; Guo, Xuejiang; Huo, Ran; Wang, Xinghai; Tong, Jiansun; Qian, Lixin; Zhou, Zuomin; Jia, Yue; Lue, Yan-He; Hikim, Amiya Sinha; Wang, Christina; Swerdloff, Ronald S; Sha, Jiahao

2008-09-01

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.

The Proteome of Shigella flexneri 2a 2457T Grown at 30 and 37 °C*

PubMed Central

Zhu, Li; Zhao, Ge; Stein, Robert; Zheng, Xuexue; Hu, Wei; Shang, Na; Bu, Xin; Liu, Xiankai; Wang, Jie; Feng, Erling; Wang, Bin; Zhang, Xuemin; Ye, Qinong; Huang, Peitang; Zeng, Ming; Wang, Hengliang

2010-01-01

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 °C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 °C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 °C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri. PMID:20164057

The proteome of Shigella flexneri 2a 2457T grown at 30 and 37 degrees C.

PubMed

Zhu, Li; Zhao, Ge; Stein, Robert; Zheng, Xuexue; Hu, Wei; Shang, Na; Bu, Xin; Liu, Xiankai; Wang, Jie; Feng, Erling; Wang, Bin; Zhang, Xuemin; Ye, Qinong; Huang, Peitang; Zeng, Ming; Wang, Hengliang

2010-06-01

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 degrees C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 degrees C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 degrees C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri.

Neurotoxicity following acute inhalation of aerosols generated during resistance spot weld-bonding of carbon steel

PubMed Central

Sriram, Krishnan; Jefferson, Amy M.; Lin, Gary X.; Afshari, Aliakbar; Zeidler-Erdely, Patti C.; Meighan, Terence G.; McKinney, Walter; Jackson, Mark; Cumpston, Amy; Cumpston, Jared L.; Leonard, Howard D.; Frazer, David G.; Antonini, James M.

2015-01-01

Welding generates complex metal aerosols, inhalation of which is linked to adverse health effects among welders. An important health concern of welding fume (WF) exposure is neurological dysfunction akin to Parkinson’s disease (PD). Some applications in manufacturing industry employ a variant welding technology known as “weld-bonding” that utilizes resistance spot welding, in combination with adhesives, for metal-to-metal welding. The presence of adhesives raises additional concerns about worker exposure to potentially toxic components like Methyl Methacrylate, Bisphenol A and volatile organic compounds (VOCs). Here, we investigated the potential neurotoxicological effects of exposure to welding aerosols generated during weld-bonding. Male Sprague–Dawley rats were exposed (25 mg/m3 targeted concentration; 4 h/day × 13 days) by whole-body inhalation to filtered air or aerosols generated by either weld-bonding with sparking (high metal, low VOCs; HM) or without sparking (low metal; high VOCs; LM). Fumes generated under these conditions exhibited complex aerosols that contained both metal oxide particulates and VOCs. LM aerosols contained a greater fraction of VOCs than HM, which comprised largely metal particulates of ultrafine morphology. Short-term exposure to LM aerosols caused distinct changes in the levels of the neurotransmitters, dopamine (DA) and serotonin (5-HT), in various brain areas examined. LM aerosols also specifically decreased the mRNA expression of the olfactory marker protein (Omp) and tyrosine hydroxylase (Th) in the olfactory bulb. Consistent with the decrease in Th, LM also reduced the expression of dopamine transporter (Slc6a3; Dat), as well as, dopamine D2 receptor (Drd2) in the olfactory bulb. In contrast, HM aerosols induced the expression of Th and dopamine D5 receptor (Drd5) mRNAs, elicited neuroinflammation and blood–brain barrier-related changes in the olfactory bulb, but did not alter the expression of Omp. Our findings divulge the differential effects of LM and HM aerosols in the brain and suggest that exposure to weld-bonding aerosols can potentially elicit neurotoxicity following a short-term exposure. However, further investigations are warranted to determine if the aerosols generated by weld-bonding can contribute to persistent long-term neurological deficits and/or neurodegeneration. PMID:25265048

Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets.

PubMed

Dey-Rao, Rama; Sinha, Animesh A

2017-01-28

Significant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state. We undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy. Unsupervised clustering methods of the VL-blood dataset demonstrate a "disease-state"-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as "hidden" (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional "hot spot" that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets. We examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address a major gap in knowledge regarding the systemic changes underlying skin-specific manifestation of vitiligo. Several transcriptional "hot spots" observed in both environments offer prioritized targets for identifying disease risk genes. Finally, within the transcriptional framework of VL, we identify five novel molecules (STAT1, PRKCD, PTPN6, MYC and FGFR2) that lend themselves to being targeted by drugs for future potential VL-therapy.