Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin

The TSC2 gene, mutated in patients with tuberous sclerosis complex (TSC), encodes a 200 kDa protein TSC2 (tuberin). The importance of TSC2 in the regulation of cell growth and proliferation is irrefutable. TSC2 in complex with TSC1 negatively regulates the mTOR complex 1 (mTORC1) via RHEB in the PI3K-AKT-mTOR pathway and in turn regulates cell proliferation. It shows nuclear as well as cytoplasmic localization. However, its nuclear function remains elusive. In order to identify the nuclear function of TSC2, a whole-genome expression profiling of TSC2 overexpressing cells was performed, and the results showed differential regulation of 266 genes. Interestingly, transcription was found to be the most populated functional category. EREG (Epiregulin), a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in TSC lesions, making its regulatory aspects intriguing. Using the luciferase reporter, ChIP and EMSA techniques, we show that TSC2 binds to the EREG promoter between -352 bp and -303 bp and negatively regulates its expression. This is the first evidence for the role of TSC2 as a transcription factor and of TSC2 binding to the promoter of any gene.

Profiling of Luteal Transcriptome during Prostaglandin F2-Alpha Treatment in Buffalo Cows: Analysis of Signaling Pathways Associated with Luteolysis

In several species including the buffalo cow, prostaglandin (PG) F-2 alpha is the key molecule responsible for regression of corpus luteum (CL). Experiments were carried out to characterize gene expression changes in the CL tissue at various time points after administration of luteolytic dose of PGF(2 alpha) in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF(2 alpha) treatment and evidence of apoptosis was demonstrable at 18 h post treatment. Microarray analysis indicated expression changes in several of immediate early genes and transcription factors within 3 h of treatment. Also, changes in expression of genes associated with cell to cell signaling, cytokine signaling, steroidogenesis, PG synthesis and apoptosis were observed. Analysis of various components of LH/CGR signaling in CL tissues indicated decreased LH/CGR protein expression, pCREB levels and PKA activity post PGF(2 alpha) treatment. The novel finding of this study is the down regulation of CYP19A1 gene expression accompanied by decrease in expression of E-2 receptors and circulating and intra luteal E-2 post PGF(2 alpha) treatment. Mining of microarray data revealed several differentially expressed E-2 responsive genes. Since CYP19A1 gene expression is low in the bovine CL, mining of microarray data of PGF(2 alpha)-treated macaques, the species with high luteal CYP19A1 expression, showed good correlation between differentially expressed E-2 responsive genes between both the species. Taken together, the results of this study suggest that PGF(2 alpha) interferes with luteotrophic signaling, impairs intraluteal E-2 levels and regulates various signaling pathways before the effects on structural luteolysis are manifest.

Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni2+-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 x 10(8) min(-1) M-1, 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors.

Interactome analyses of Salmonella pathogenicity islands reveal SicA indispensable for virulence

Background: Serovars of Salmonella enterica, namely Typhi and Typhimurium, reportedly, are the bacterial pathogens causing systemic infections like gastroenteritis and typhoid fever. To elucidate the role and importance in such infection, the proteins of the Type III secretion system of Salmonella pathogenicity islands and two component signal transduction systems, have been mainly focused. However, the most indispensable of these virulent ones and their hierarchical role has not yet been studied extensively. Results: We have adopted a theoretical approach to build an interactome comprising the proteins from the Salmonella pathogeneicity islands (SPI) and two component signal transduction systems. This interactome was then analyzed by using network parameters like centrality and k-core measures. An initial step to capture the fingerprint of the core network resulted in a set of proteins which are involved in the process of invasion and colonization, thereby becoming more important in the process of infection. These proteins pertained to the Inv, Org, Prg, Sip, Spa, Ssa and Sse operons along with chaperone protein SicA. Amongst them, SicA was figured out to be the most indispensable protein from different network parametric analyses. Subsequently, the gene expression levels of all these theoretically identified important proteins were confirmed by microarray data analysis. Finally, we have proposed a hierarchy of the proteins involved in the total infection process. This theoretical approach is the first of its kind to figure out potential virulence determinants encoded by SPI for therapeutic targets for enteric infection. Conclusions: A set of responsible virulent proteins was identified and the expression level of their genes was validated by using independent, published microarray data. The result was a targeted set of proteins that could serve as sensitive predictors and form the foundation for a series of trials in the wet-lab setting. Understanding these regulatory and virulent proteins would provide insight into conditions which are encountered by this intracellular enteric pathogen during the course of infection. This would further contribute in identifying novel targets for antimicrobial agents. (C) 2014 Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection. (C) 2015 The Authors. Published by Elsevier Inc.

The influence of reduced oxygen availability on gene expression in laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis

Mycobacterium tuberculosis has the ability to persist within the host in a dormant stage. One important condition believed to contribute to dormancy is reduced access to oxygen known as hypoxia. However, the response of M. tuberculosis to such hypoxia condition is not fully characterized. Virtually all dormant models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of M. tuberculosis from South India to hypoxia. Analysis of microarray results revealed that a total of 1161 genes were differentially regulated (>= 1.5 fold change) in H37Rv, among them 659 genes upregulated and 502 genes down regulated. Microarray data of clinical isolates showed that a total of 790 genes were differentially regulated in S7 among which 453 genes were upregulated and 337 down regulated. Interestingly, numerous genes were also differentially regulated in S10 (total 2805 genes) of which 1463 genes upregulated and 1342 genes down regulated during reduced oxygen condition (Wayne's model). One hundred and thirty-four genes were found common and upregulated among all three strains (H37Rv, S7, and S10) and can be targeted for drug/vaccine development against TB. (C) 2015 Published by Elsevier B.V.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

DNA methylation analysis of phenotype specific stratified Indian population

Background: DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. Methods: Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. Results: Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). Conclusion: Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

椭偏光学生物传感器及生物医学应用

椭偏光学生物传感器是识别和检测蛋白质的一种新型的高通量、快速生物分子分析技术.它可以实现无标记多元生物分子自动化检测、静态或动态测量及定性和定量测量等,已应用于生物医学与临床检测等方面,如肿瘤标志蛋白检测、乙肝五项同时检测、蛋白质竞争吸附以及多元蛋白质相互作用动态测量等,显示出了广阔的应用前景.

Geometric optimization methods for the analysis of gene expression data

DNA microarrays provide such a huge amount of data that unsupervised methods are required to reduce the dimension of the data set and to extract meaningful biological information. This work shows that Independent Component Analysis (ICA) is a promising approach for the analysis of genome-wide transcriptomic data. The paper first presents an overview of the most popular algorithms to perform ICA. These algorithms are then applied on a microarray breast-cancer data set. Some issues about the application of ICA and the evaluation of biological relevance of the results are discussed. This study indicates that ICA significantly outperforms Principal Component Analysis (PCA).

Quantitative RNA-seq analysis of the campylobacter jejuni transcriptome

Campylobacter jejuni is the most common bacterial cause of foodborne disease in the developed world. Its general physiology and biochemistry, as well as the mechanisms enabling it to colonize and cause disease in various hosts, are not well understood, and new approaches are required to understand its basic biology. High-throughput sequencing technologies provide unprecedented opportunities for functional genomic research. Recent studies have shown that direct Illumina sequencing of cDNA (RNA-seq) is a useful technique for the quantitative and qualitative examination of transcriptomes. In this study we report RNA-seq analyses of the transcriptomes of C. jejuni (NCTC11168) and its rpoN mutant. This has allowed the identification of hitherto unknown transcriptional units, and further defines the regulon that is dependent on rpoN for expression. The analysis of the NCTC11168 transcriptome was supplemented by additional proteomic analysis using liquid chromatography-MS. The transcriptomic and proteomic datasets represent an important resource for the Campylobacter research community. © 2011 SGM.

"Nicotiana tabacum" L. cv Xanthi como sistema heterólogo para la producción de lactógeno placentario humano (hPL)

152 p. : il., col.

蛋白质芯片用表面改性剂的合成——蛋白质偶联剂与蛋白质非特异吸附抑制剂的合成

以巯丙基三乙氧基硅烷和单取代三聚乙二醇为原料，先把巯基转换成巯钠，把醇羟基转换成对甲苯磺酰基，利用巯钠和对甲苯磺酰基间的亲核取代反应，在温和条件下合成了标题试剂。

Support for Integrated Ecosystem Assessments of NOAA’s National Estuarine Research Reserves System (NERRS), Volume I: The Impacts of Coastal Development on the Ecology and Human Well-being of Tidal Creek Ecosystems of the US Southeast

A study was conducted, in association with the Sapelo Island and North Carolina National Estuarine Research Reserves (NERRs), to evaluate the impacts of coastal development on sentinel habitats (e.g., tidal creek ecosystems), including potential impacts to human health and well-being. Uplands associated with southeastern tidal creeks and the salt marshes they drain are popular locations for building homes, resorts, and recreational facilities because of the high quality of life and mild climate associated with these environments. Tidal creeks form part of the estuarine ecosystem characterized by high biological productivity, great ecological value, complex environmental gradients, and numerous interconnected processes. This research combined a watershed-level study integrating ecological, public health and human dimension attributes with watershed-level land use data. The approach used for this research was based upon a comparative watershed and ecosystem approach that sampled tidal creek networks draining developed watersheds (e.g., suburban, urban, and industrial) as well as undeveloped sites. The primary objective of this work was to clearly define the relationships between coastal development with its concomitant land use changes and non-point source pollution loading and the ecological and human health and well-being status of tidal creek ecosystems. Nineteen tidal creek systems, located along the southeastern United States coast from southern North Carolina to southern Georgia, were sampled during summer (June-August), 2005 and 2006. Within each system, creeks were divided into two primary segments based upon tidal zoning: intertidal (i.e., shallow, narrow headwater sections) and subtidal (i.e., deeper and wider sections), and watersheds were delineated for each segment. In total, we report findings on 24 intertidal and 19 subtidal creeks. Indicators sampled throughout each creek included water quality (e.g., dissolved oxygen concentration, salinity, nutrients, chlorophyll-a levels), sediment quality (e.g., characteristics, contaminants levels including emerging contaminants), pathogen and viral indicators, and abundance and genetic responses of biological resources (e.g., macrobenthic and nektonic communities, shellfish tissue contaminants, oyster microarray responses). For many indicators, the intertidally-dominated or headwater portions of tidal creeks were found to respond differently than the subtidally-dominated or larger and deeper portions of tidal creeks. Study results indicate that the integrity and productivity of headwater tidal creeks were impaired by land use changes and associated non-point source pollution, suggesting these habitats are valuable early warning sentinels of ensuing ecological impacts and potential public health threats. For these headwater creeks, this research has assisted the validation of a previously developed conceptual model for the southeastern US region. This conceptual model identified adverse changes that generally occurred in the physical and chemical environment (e.g., water quality indicators such as indicator bacteria for sewage pollution or sediment chemical contamination) when impervious cover levels in the watershed reach 10-20%. Ecological characteristics responded and were generally impaired when impervious cover levels exceed 20-30%. Estimates of impervious cover levels defining where human uses are impaired are currently being determined, but it appears that shellfish bed closures and the flooding vulnerability of headwater regions become a concern when impervious cover values exceed 10-30%. This information can be used to forecast the impacts of changing land use patterns on tidal creek environmental quality as well as associated human health and well-being. In addition, this study applied tools and technologies that are adaptable, transferable, and repeatable among the high quality NERRS sites as comparable reference entities to other nearby developed coastal watersheds. The findings herein will be of value in addressing local, regional and national needs for understanding multiple stressor (anthropogenic and human impacts) effects upon estuarine ecosystems and response trends in ecosystem condition with changing coastal impacts (i.e., development, climate change). (PDF contaions 88 pages)

Preprocess and data analysis techniques for affymetrix DNA microarrays using bioconductor: a case study in Alzheimer disease

DNA microarray, or DNA chip, is a technology that allows us to obtain the expression level of many genes in a single experiment. The fact that numerical expression values can be easily obtained gives us the possibility to use multiple statistical techniques of data analysis. In this project microarray data is obtained from Gene Expression Omnibus, the repository of National Center for Biotechnology Information (NCBI). Then, the noise is removed and data is normalized, also we use hypothesis tests to find the most relevant genes that may be involved in a disease and use machine learning methods like KNN, Random Forest or Kmeans. For performing the analysis we use Bioconductor, packages in R for the analysis of biological data, and we conduct a case study in Alzheimer disease. The complete code can be found in https://github.com/alberto-poncelas/ bioc-alzheimer