RESUMO
Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders, including reproductive disorders such as polycystic ovarian syndrome, where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mmol/L) of metformin have reported antiproliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20 and 70 µmol/L. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found to be mammalian target of rapamycin (mTOR) dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMP-activated protein kinase (AMPK) signaling. On the contrary, supratherapeutic metformin concentration (10 mmol/L) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation, and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitoTracker™ Red staining and enhanced succinate dehydrogenase expression; however, higher concentration (10 mmol/L) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR-dependent endometrial cell proliferation.
Assuntos
Neoplasias do Endométrio , Metformina , Animais , Feminino , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Proliferação de Células , Células Epiteliais/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Serina-Treonina Quinases TOR/metabolismoRESUMO
In absence of periodic systematic comparisons, biologists/bioinformaticians may be forced to make a subjective selection among the many protein-protein interaction (PPI) databases and tools. We conducted a comprehensive compilation and comparison of such resources. We compiled 375 PPI resources, short-listed 125 important ones (both lists are available at startbioinfo.com), and compared the features and coverage of 16 carefully-selected databases related to human PPIs. We quantitatively compared the coverage of 'experimentally verified' as well as 'total' (experimentally verified and predicted) PPIs for these 16 databases. Coverage was compared in two ways: (a) PPIs obtained in response to gene queries using the web interfaces were compared. As a query set, 108 genes expressed differently across tissues (specific to kidney, testis, and uterus, and ubiquitous - i.e., expressed in 43 human normal tissues) or associated with certain diseases (breast cancer, lung cancer, Alzheimer's, cystic fibrosis, diabetes, and cardiomyopathy) were chosen. The coverage was also compared for the well-studied genes versus the less-studied ones. The coverage of the databases for high-quality interactions was separately assessed using a set of literature curated experimentally-proven PPIs (gold standard PPI-set); (b) the back-end-data from 15 PPI databases was downloaded and compared. Combined results from STRING and UniHI covered around 84% of 'experimentally verified' PPIs. Approximately 94% of the 'total' PPIs available across the databases were retrieved by the combined use of hPRINT, STRING, and IID. Among the experimentally verified PPIs found exclusively in each database, STRING contributed around 71% of the hits. The coverage of certain databases was skewed for some gene-types. Analysis with the gold-standard PPI-set revealed that GPS-Prot, STRING, APID, and HIPPIE, each covered ~70% of the curated interactions. The database usage frequencies did not always correlate with their respective advantages, thereby justifying the need for more frequent studies of this nature.
Assuntos
Mapeamento de Interação de Proteínas , Bases de Dados de Proteínas , HumanosRESUMO
Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 proximal promoter activates transcription in spermatids; and the goal of the present study was to separate the enhancer responsible. Transgenic mice showed that three copies of the -186/-135 fragment (50 bp enhancer) placed upstream of the Acrv1 core promoter (-91/+28) activated reporter expression in testis but not somatic tissues (n = 4). Immunohistochemistry showed that enhancer activity was restricted to the round spermatids. The Acrv1 enhancer failed to activate transcription in the context of a heterologous core promoter (n = 4), indicating a likely requirement for enhancer-core promoter compatibility. Chromatin accessibility assays showed that the Acrv1 enhancer assumes a nucleosome-free state in male germ cells (but not liver), indicating occupancy by transcription factors. Southwestern assays (SWA) identified specific binding of the enhancer to a testis nuclear protein of 47 kDa (TNP47). TNP47 was predominantly nuclear and becomes abundant during the haploid phase of spermatogenesis. Two-dimensional SWA revealed the isoelectric point of TNP47 to be 5.2. Taken together, this study delineated a 50-bp enhancer of the Acrv1 gene for round spermatid-specific transcription and identified a putative cognate factor. The 50-bp enhancer could become useful for delivery of proteins into spermatids.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Proteínas de Membrana/genética , Espermátides/metabolismo , Espermatogênese/genética , Animais , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética , Espermátides/fisiologia , Transcrição Gênica/genéticaRESUMO
Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.
Assuntos
Anexina A1/metabolismo , Invasividade Neoplásica , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/patologia , Proteínas e Peptídeos Salivares/fisiologia , Proteínas de Plasma Seminal/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Proteínas e Peptídeos Salivares/genética , Proteínas de Plasma Seminal/genéticaRESUMO
BACKGROUND: Standard 3' Affymetrix gene expression arrays have contributed a significantly higher volume of existing gene expression data than other microarray platforms. These arrays were designed to identify differentially expressed genes, but not their alternatively spliced transcript forms. No resource can currently identify expression pattern of specific mRNA forms using these microarray data, even though it is possible to do this. RESULTS: We report a web server for expression profiling of alternatively spliced transcripts using microarray data sets from 31 standard 3' Affymetrix arrays for human, mouse and rat species. The tool has been experimentally validated for mRNAs transcribed or not-detected in a human disease condition (non-obstructive azoospermia, a male infertility condition). About 4000 gene expression datasets were downloaded from a public repository. 'Good probes' with complete coverage and identity to latest reference transcript sequences were first identified. Using them, 'Transcript specific probe-clusters' were derived for each platform and used to identify expression status of possible transcripts. The web server can lead the user to datasets corresponding to specific tissues, conditions via identifiers of the microarray studies or hybridizations, keywords, official gene symbols or reference transcript identifiers. It can identify, in the tissues and conditions of interest, about 40% of known transcripts as 'transcribed', 'not-detected' or 'differentially regulated'. Corresponding additional information for probes, genes, transcripts and proteins can be viewed too. We identified the expression of transcripts in a specific clinical condition and validated a few of these transcripts by experiments (using reverse transcription followed by polymerase chain reaction). The experimental observations indicated higher agreements with the web server results, than contradictions. The tool is accessible at http://resource.ibab.ac.in/TIPMaP. CONCLUSION: The newly developed online tool forms a reliable means for identification of alternatively spliced transcript-isoforms that may be differentially expressed in various tissues, cell types or physiological conditions. Thus, by making better use of existing data, TIPMaP avoids the dependence on precious tissue-samples, in experiments with a goal to establish expression profiles of alternative splice forms--at least in some cases.
Assuntos
Processamento Alternativo , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Internet , Animais , Biologia Computacional/métodos , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Controle de Qualidade , Isoformas de RNA , Ratos , Reprodutibilidade dos Testes , SoftwareRESUMO
There is a need to understand the molecular basis of testes under Non-Obstructive Azoospermia (NOA), a state of failed spermatogenesis. There has been a lack of attention to the transcriptome at the level of alternatively spliced mRNAs (iso-mRNAs) and the mechanism of gene expression regulation. Hence, we aimed to establish a reliable iso-mRNA profile of NOA-testes, and explore molecular mechanisms - especially those related to gene expression regulation. We sequenced mRNAs from testicular samples of donors with complete spermatogenesis (control samples) and a failure of spermatogenesis (NOA samples). We identified differentially expressed genes and their iso-mRNAs via standard NGS data analyses. We then listed these iso-mRNAs hierarchically based on the extent of consistency of differential quantities across samples and groups, and validated the lists via RT-qPCRs (for 80 iso-mRNAs). In addition, we performed extensive bioinformatic analysis of the splicing features, domains, interactions, and functions of differentially expressed genes and iso-mRNAs. Many top-ranking down-regulated genes and iso-mRNAs, i.e., those down-regulated more consistently across the NOA samples, are associated with mitosis, replication, meiosis, cilium, RNA regulation, and post-translational modifications such as ubiquitination and phosphorylation. Most down-regulated iso-mRNAs correspond to full-length proteins that include all expected domains. The predominance of alternative promoters and termination sites in these iso-mRNAs indicate their gene expression regulation via promoters and UTRs. We compiled a new, comprehensive list of human transcription factors (TFs) and used it to identify TF-'TF gene' interactions with potential significance in down-regulating genes under the NOA condition. The results indicate that RAD51 suppression by HSF4 prevents SP1-activation, and SP1, in turn, could regulate multiple TF genes. This potential regulatory axis and other TF interactions identified in this study could explain the down-regulation of multiple genes in NOA-testes. Such molecular interactions may also have key regulatory roles during normal human spermatogenesis.
Assuntos
Azoospermia , Testículo , Humanos , Masculino , Testículo/metabolismo , Azoospermia/genética , Transcriptoma , Espermatogênese/genética , Regulação da Expressão GênicaRESUMO
Substantial data posit estrogen receptors (ERs) as promising targets for prostate cancer (PCa) therapeutics. However, the trials on assessing the chemo-preventive or therapeutic potential of ER targeting drugs or selective estrogen receptor modulators (SERMs) have not yet established their clinical benefits. This could be ascribed to a possible modulation in the ER expression during PCa progression. Further it is warranted to test various ER targeting drugs in appropriate preclinical models that simulate human ER expression pattern during PCa progression. The study was undertaken to revisit the existing data on the epithelial ER expression pattern in human cancerous prostates and experimentally determine whether these patterns are replicated in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice, a model for human PCa. Estradiol (E2) binding to the plasma membrane of the epithelial cells and its modulation during the PCa progression in TRAMP were also investigated. A reassessment of the existing data revealed a trend towards downregulation in the epithelial expression of wild-type ESR1 transcripts in high-grade PCa, compared to non-cancerous prostate in humans. Next, epithelial cell-enriched populations from TRAMP prostates (TP) displaying low-grade prostatic intraepithelial neoplasia (LGPIN), high-grade PIN (HGPIN), HGPIN with well-differentiated carcinoma (PIN + WDC), WDC (equivalent to grade 2/3 human PCa), and poorly-differentiated carcinoma (PDC-equivalent to grade 4/5 human PCa) revealed significantly higher Esr1 and Esr2 levels in HGPIN and significantly reduced levels in WDC, compared to respective age-matched control prostates. These patterns for the nuclear ERs were similar to the trend shown by E2 binding to the plasma membrane of the epithelial cells during PCa progression in TRAMP. E2 binding to epithelial cells (EpCAM+), though significantly higher in TPs displaying LGPIN, decreased significantly as the disease progressed to WDC. The study highlights a reduction in the epithelial ESR level with the PCa progression and this pattern was evident in both humans and TRAMP. These observations may have major implications in refining PCa therapeutics targeting ER.
Assuntos
Neoplasia Prostática Intraepitelial , Neoplasias da Próstata , Animais , Progressão da Doença , Células Epiteliais/metabolismo , Estrogênios/metabolismo , Humanos , Masculino , Camundongos , Próstata/metabolismo , Próstata/patologia , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismoRESUMO
HOX proteins are homeodomain-containing transcription factors that play a central role in development. We have applied genome-wide approaches to develop time-dependent profile of differentially expressed genes in early and mature adipocytes. The list of differentially expressed HOX genes were developed by analyzing the microarray datasets of murine adipocyte samples at different time points of development. Since these datasets were obtained from Gene Expression Omnibus (GEO), we were able to find a new HOX gene, HOXC13 in adipogenesis. To investigate whether these members of the homeobox gene family are expressed and regulated in preadipocytes or mature adipocytes, RNA was isolated from 3T3-L1 preadipocyte cells at different time point's through-out the preadipocyte and adipocyte state. A reverse transcriptase-polymerase chain reaction strategy was applied for the analysis of gene expression. We have observed that HOXA5 and HOXC13 were differentially expressed in preadipocytes and HOXD4 and HOXD8 in mature adipocytes. To understand this difference in expression pattern, we have considered to investigate the role of the major regulators of adipogenesis in HOX gene regulation. Since Retinoic acid receptor (RAR) was reported previously as a regulator of Hox genes, we chose the combination of Peroxisome proliferator-activated receptor gamma (PPARγ) and Retinoic X receptor (RXR) which are modulated by the presence of RAR. To provide a detailed analysis of retinoic acid (RA) and/or PPARγ induced transcriptional and epigenetic changes within the homeotic clusters of mouse fibroblast cells (3T3-L1), we have performed a promoter mapping of HOX genes and observed an enriched binding site for PPARγ and RXR in their promoter regions. We further confirmed this PPARγ and RXR binding to HOX gene promoters by re-analyzing the anti-PPARγ/anti-RXR ChIP-Seq data. Based on the results, we modulated the PPARγ expression at the transcriptional and translational levels by using 5 different pharmacological molecules (TSA, GW9662, ATRA, FH535, and Pioglitazone) to elucidate their effect on the HOX gene transcription. These pharmacological molecules had a direct or indirect regulatory effect on the PPARγ activity. We observed that PPARγ suppression alone is enough for the upregulation of HOXA5 and HOXD4 genes. In addition, HOXD8 regulation was mediated by RAR activation in mature adipocytes but the regulation of HOXC13 gene expression was not clear. We suggest that it might be partially mediated through suppressing PPARγ activation. Further insights are required to provide a mechanistic detail about HOX gene regulation through PPARγ. In this study, we have reported a time-dependent expression analysis of HOXA5, HOXD4, HOXD8, and HOXC13 in preadipocytes and mature adipocytes. Also, we have suggested PPARγ/RAR dependent regulation for these genes during adipogenesis.
Assuntos
Adipócitos/fisiologia , Expressão Gênica/genética , Genes Homeobox/genética , Células 3T3-L1 , Adipogenia/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Epigênese Genética/genética , Camundongos , PPAR gama/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Receptores do Ácido Retinoico/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Ativação Transcricional/genética , Tretinoína/metabolismo , Regulação para Cima/genéticaRESUMO
AIMS: Gender disparities exist in smoking-related lung cancer epidemiology, but the molecular basis has not been explored so far. We aimed at identifying genes with gender-bias expression pattern in smoking lung cancer patients for understanding the molecular basis of gender bias in smokers using meta-analysis of microarray gene expression data. MATERIALS AND METHODS: Transcriptome of around 1100 samples from 13 studies were used in the meta-analysis to identify 'Lung Cancer genes specific to Female-Smokers' (LCFS) and 'Lung Cancer genes specific to Male-Smokers' (LCMS). The expression profiles of these genes were validated with an independent microarray report and TCGA-RNA-sequencing data. The molecular interactions, pathway, and other functional annotations were portrayed for the key genes identified. KEY FINDINGS: We identified 1159 gender-biased genes in smoking lung cancer patients. Of these, 400 and 474 genes showed differential expression in cancerous compared to normal lung of women (LCFS) and men (LCMS), respectively. While many up-regulated LCFS were involved in 'immune responses' including T-cell activation, leukocyte cell-cell adhesion, the LCMS were mainly involved in 'positive regulation of gene expression', signaling pathways including RAS, VEGF, insulin-receptor signaling, and 'cell cycle'. SIGNIFICANCE: The strategic-method identified genes, particularly, SNX20, GIMAP6, MTMR2, FAM171B, IDH1, MOBP, FBXO17, LPXN and WIPF1, which were consistently differentially expressed in at least 4 studies, and in agreement with RNA-Seq data. Exploring their functions could be beneficial to the gender-based diagnosis, prognosis, and treatment of lung cancer in smokers. The current meta-analysis supports existing knowledge of sexual-dimorphism of immune responses in cancer.
Assuntos
Fumar Cigarros/genética , Neoplasias Pulmonares/metabolismo , Moléculas de Adesão Celular/genética , Fumar Cigarros/efeitos adversos , Proteínas do Citoesqueleto/genética , Bases de Dados Genéticas , Feminino , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/citologia , Pulmão/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosfoproteínas/genética , Prognóstico , Proteínas Tirosina Fosfatases não Receptoras/genética , RNA/genética , Fatores Sexuais , Nexinas de Classificação/genética , TranscriptomaRESUMO
Angiosperm leaves show extensive shape diversity and are broadly divided into two forms; simple leaves with intact lamina and compound leaves with lamina dissected into leaflets. The mechanistic basis of margin dissection and leaflet initiation has been inferred primarily by analysing compound-leaf architecture, and thus whether the intact lamina of simple leaves has the potential to initiate leaflets upon endogenous gene inactivation remains unclear. Here, we show that the CINCINNATA-like TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS (CIN-TCP) transcription factors activate the class II KNOTTED1-LIKE (KNOX-II) genes and the CIN-TCP and KNOX-II proteins together redundantly suppress leaflet initiation in simple leaves. Simultaneous downregulation of CIN-TCP and KNOX-II in Arabidopsis leads to the reactivation of the stemness genes KNOX-I and CUPSHAPED COTYLEDON (CUC) and triggers ectopic organogenesis, eventually converting the simple lamina to a super-compound form that appears to initiate leaflets indefinitely. Thus, a conserved developmental mechanism promotes simple leaf architecture in which CIN-TCP-KNOX-II forms a strong differentiation module that suppresses the KNOX-I-CUC network and leaflet initiation.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Diferenciação Celular/genética , Regulação para Baixo/genética , Folhas de Planta/anatomia & histologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Plantas Geneticamente ModificadasRESUMO
BACKGROUND: In the recent years, there has been a rise in gene expression profiling reports. Unfortunately, it has not been possible to make maximum use of available gene expression data. Many databases and programs can be used to derive the possible expression patterns of mammalian genes, based on existing data. However, these available resources have limitations. For example, it is not possible to obtain a list of genes that are expressed in certain conditions. To overcome such limitations, we have taken up a new strategy to predict gene expression patterns using available information, for one tissue at a time. RESULTS: The first step of this approach involved manual collection of maximum data derived from large-scale (genome-wide) gene expression studies, pertaining to mammalian testis. These data have been compiled into a Mammalian Gene Expression Testis-database (MGEx-Tdb). This process resulted in a richer collection of gene expression data compared to other databases/resources, for multiple testicular conditions. The gene-lists collected this way in turn were exploited to derive a 'consensus' expression status for each gene, across studies. The expression information obtained from the newly developed database mostly agreed with results from multiple small-scale studies on selected genes. A comparative analysis showed that MGEx-Tdb can retrieve the gene expression information more efficiently than other commonly used databases. It has the ability to provide a clear expression status (transcribed or dormant) for most genes, in the testis tissue, under several specific physiological/experimental conditions and/or cell-types. CONCLUSIONS: Manual compilation of gene expression data, which can be a painstaking process, followed by a consensus expression status determination for specific locations and conditions, can be a reliable way of making use of the existing data to predict gene expression patterns. MGEx-Tdb provides expression information for 14 different combinations of specific locations and conditions in humans (25,158 genes), 79 in mice (22,919 genes) and 23 in rats (14,108 genes). It is also the first system that can predict expression of genes with a 'reliability-score', which is calculated based on the extent of agreements and contradictions across gene-sets/studies. This new platform is publicly available at the following web address: http://resource.ibab.ac.in/MGEx-Tdb/.
Assuntos
Bases de Dados de Ácidos Nucleicos , Expressão Gênica , Testículo/química , Animais , Humanos , Internet , Masculino , Camundongos , Especificidade de Órgãos , Ratos , Reprodutibilidade dos Testes , Testículo/metabolismoRESUMO
Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting tumor heterogeneity. We have performed multi-omic profiling of the kinome in bladder cancer patients with the goal of identify therapeutic targets. Our analyses revealed amplification, overexpression, and elevated kinase activity of P21 (RAC1) activated kinase 4 (PAK4) in a subset of Bladder cancer (BLCA). Using bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, we observed that a PAK4 inhibitor was effective in curtailing growth of BLCA cells. Transcriptomic analyses identified elevated expression of another kinase, protein tyrosine kinase 6 (PTK6), upon treatment with a PAK4 inhibitor and RNA interference of PAK4. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared with either drug alone. Thus, PAK4 may be therapeutically actionable for a subset of MIBC patients with amplified and/or overexpressed PAK4 in their tumors. Our results also indicate that combined inhibition of PAK4 and PTK6 may overcome resistance to PAK4. These observations warrant clinical investigations with selected BLCA patients.
Assuntos
Amplificação de Genes , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias da Bexiga Urinária/enzimologia , Quinases Ativadas por p21/biossíntese , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Quinases Ativadas por p21/genéticaRESUMO
The embryonic stem cell line derivation from nonpermissive mouse strains is a challenging and highly inefficient process. The cellular reprogramming strategy provides an alternative route for generating pluripotent stem cell (PSC) lines from such strains. In this study, we successfully derived an enhanced green fluorescent protein (EGFP)-transgenic "N9" induced pluripotent stem cell (iPS cell, iPSC) line from the FVB/N strain-derived mouse embryonic fibroblasts (MEFs). The exposure of MEFs to human OCT4, SOX2, KLF4, and c-MYC (OSKM) transgenes via lentiviral transduction resulted in complete reprogramming. The N9 iPS cell line demonstrated all the criteria of a typical mouse PSC line, including normal colony morphology and karyotype (40,XY), high replication and propagation efficiencies, expression of the pluripotency-associated genes, spontaneous differentiation to three germ lineage-derived cell types, and robust potential of chimeric blastocyst formation. Taken together, using human OSKM genes for transduction, we report, for the first time, the successful derivation of an EGFP-expressing iPS cell line from a genetically nonpermissive transgenic FVB/N mouse. This cell line could provide opportunities for designing protocols for efficient derivation of PSC lines from other nonpermissive strains and developing mouse models of human diseases.
Assuntos
Embrião de Mamíferos/citologia , Fibroblastos/citologia , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Teratoma/patologia , Animais , Linhagem da Célula , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Teratoma/genética , Teratoma/metabolismoRESUMO
Non Hodgkin lymphoma, predominantly Diffuse Large B-cell Lymphoma (DLBCL) has been reported to have a significant association with Hepatitis B virus (HBV). We investigated the presence of different gene segments of HBV in plasma, B-cells and tumor tissues from DLBCL patients and explored the genetic variability of HBV within and across different compartments in a host using Next Generation Sequencing. Despite all 40 patients being HBV seronegative, 68% showed evidence of occult HBV. Sequencing of these gene segments revealed inter-compartment viral variants in 26% of them, each with at least one non-synonymous mutation. Between compartments, core gene variants revealed Arg94Leu, Glu86Arg and Ser41Thr while X gene variants revealed Phe73Val, Ala44Val, Ser146Ala and Ser147Pro. In tumor compartments per se, several mis-sense mutations were detected, notably the classic T1762A/A1764G mutation in the basal core promoter. In addition, a virus surface antigen mis-sense mutation resulting in M125T was detected in all the samples and could account for surface antigen negativity and occult HBV status. It would be interesting to further explore if a temporal accumulation of viral variants within a favored niche, like patients' lymphocytes, could bestow survival advantage to the virus, and if certain pro-oncogenic HBV variants could drive lymphomagenesis in DLBCL.
Assuntos
Vírus da Hepatite B/classificação , Hepatite B/virologia , Linfoma Difuso de Grandes Células B/virologia , Quase-Espécies , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Viral/genética , Variação Genética , Hepatite B/complicações , Antígenos de Superfície da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Estudos Prospectivos , Adulto JovemRESUMO
Urokinase plasminogen activator receptor (PLAUR) has been implicated in a variety of physiological and pathological conditions. The multi-functionality of PLAUR is due to its capacity to interact with many co-receptors to regulate extracellular proteolysis and intracellular signaling. Recent reports are identifying novel functions of PLAUR which were not evident in the past; however, the molecular mechanisms of PLAUR signaling are not completely understood. Here, we have compared the transcriptomes of silencing control (sicon) and PLAUR silenced (PLAURsi) MDA-MB-231 breast cancer cells on treatment with radiation. We isolated RNA from the cells, synthesized cDNA and measured the gene expression changes by microarray. We identified 24 downregulated and 53 upregulated genes, which were significantly (P-value < 0.005) affected by PLAUR silencing. Our analysis revealed 415 canonical pathways and 743 causal disease networks affected on silencing PLAUR. Transcriptomic changes and predicted pathways supported and consolidated some of the earlier understanding in the context of PLAUR signaling; including our recent observations in DNA damage and repair process. In addition, we have identified several novel pathways where PLAUR is implicated.
RESUMO
Exploring novel computational methods in making sense of biological data has not only been a necessity, but also productive. A part of this trend is the search for more efficient in silico methods/tools for analysis of promoters, which are parts of DNA sequences that are involved in regulation of expression of genes into other functional molecules. Promoter regions vary greatly in their function based on the sequence of nucleotides and the arrangement of protein-binding short-regions called motifs. In fact, the regulatory nature of the promoters seems to be largely driven by the selective presence and/or the arrangement of these motifs. Here, we explore computational classification of promoter sequences based on the pattern of motif distributions, as such classification can pave a new way of functional analysis of promoters and to discover the functionally crucial motifs. We make use of Position Specific Motif Matrix (PSMM) features for exploring the possibility of accurately classifying promoter sequences using some of the popular classification techniques. The classification results on the complete feature set are low, perhaps due to the huge number of features. We propose two ways of reducing features. Our test results show improvement in the classification output after the reduction of features. The results also show that decision trees outperform SVM (Support Vector Machine), KNN (K Nearest Neighbor) and ensemble classifier LibD3C, particularly with reduced features. The proposed feature selection methods outperform some of the popular feature transformation methods such as PCA and SVD. Also, the methods proposed are as accurate as MRMR (feature selection method) but much faster than MRMR. Such methods could be useful to categorize new promoters and explore regulatory mechanisms of gene expressions in complex eukaryotic species.
Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica , Modelos Genéticos , Regiões Promotoras Genéticas , AlgoritmosRESUMO
BACKGROUND: Genome-wide repeat sequences, such as LINEs, SINEs and LTRs share a considerable part of the mammalian nuclear genomes. These repeat elements seem to be important for multiple functions including the regulation of transcription initiation, alternative splicing and DNA methylation. But it is not possible to study all repeats and, hence, it would help to short-list before exploring their potential functional significance via experimental studies and/or detailed in silico analyses. RESULT: We developed the 'Genomic Repeat Element Analyzer for Mammals' (GREAM) for analysis, screening and selection of potentially important mammalian genomic repeats. This web-server offers many novel utilities. For example, this is the only tool that can reveal a categorized list of specific types of transposons, retro-transposons and other genome-wide repetitive elements that are statistically over-/under-represented in regions around a set of genes, such as those expressed differentially in a disease condition. The output displays the position and frequency of identified elements within the specified regions. In addition, GREAM offers two other types of analyses of genomic repeat sequences: a) enrichment within chromosomal region(s) of interest, and b) comparative distribution across the neighborhood of orthologous genes. GREAM successfully short-listed a repeat element (MER20) known to contain functional motifs. In other case studies, we could use GREAM to short-list repetitive elements in the azoospermia factor a (AZFa) region of the human Y chromosome and those around the genes associated with rat liver injury. GREAM could also identify five over-represented repeats around some of the human and mouse transcription factor coding genes that had conserved expression patterns across the two species. CONCLUSION: GREAM has been developed to provide an impetus to research on the role of repetitive sequences in mammalian genomes by offering easy selection of more interesting repeats in various contexts/regions. GREAM is freely available at http://resource.ibab.ac.in/GREAM/.
Assuntos
Mapeamento Cromossômico , Mamíferos/genética , Sequências Repetitivas de Ácido Nucleico , Software , Animais , Computadores , Elementos de DNA Transponíveis , Humanos , Internet , Regiões Promotoras Genéticas/genética , Especificidade da EspécieRESUMO
Spermiogenesis is the terminal differentiation process of the male germ cell during which haploid spermatids acquire unique structures such as the acrosome and flagellum and undergo extensive cellular reorganization. Although well described morphologically, the molecular mechanisms underlying spermiogenesis are not well understood. The SP-10 gene, which codes for the acrosomal protein SP-10, has been well characterized in mice and men. This single copy gene is localized to syntenic regions of chromosomes 9 and 11 in mouse and human, respectively. The SP-10 gene is testis-specific, and is transcribed and translated in round spermatids. The differentiation marker SP-10 serves as a useful model to address questions regarding the regulation of round spermatid-specific gene transcription and acrosome biogenesis. This paper defines the temporal pattern of SP-10 gene expression during spermiogenesis and reviews the work done on analysis of the SP-10 promoter. Transgenic mice demonstrated that either the -408/+28 or the -266/+28 region of the SP-10 promoter could drive round spermatid-specific expression of a GFP reporter gene whereas the -91/+28 region lacked promoter activity. The transgene expression mimicked the spatial and temporal patterns of expression of the endogenous SP-10 gene. Surprisingly, none of the transgenic lines showed expression of GFP in tissues other than testis. Given the complexity of eukaryotic transcriptional regulation, the fact that a short 294-bp promoter is capable of conferring developmental stage- and cell type-specific transcription of a gene is intriguing and paradoxical.
Assuntos
Acrossomo , Antígenos/genética , Hormônios Esteroides Gonadais/genética , Espermatogênese/genética , Animais , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Distribuição Tecidual , Transcrição GênicaRESUMO
Human maltase glucoamylase (MGAM) is a potent molecular target for controlling post prandial glucose surplus in type 2 diabetes. Binding of small molecules from Syzygium sp. with α-glucosidase inhibitory potential in MGAM has been investigated in silico. Our results suggest that myricetin was the most potent inhibitor with high binding affinity for both N- and C-terminals of MGAM. Molecular dynamics revealed that myricetin interacts in its stretched conformation through water-mediated interactions with C-terminal of MGAM and by normal hydrogen bonding with the N-terminal. W1369 of the extended 21 amino acid residue helical loop of C-terminal plays a major role in myricetin binding. Owing to its additional sugar sites, overall binding of small molecules favours C-terminal MGAM.
Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores de Glicosídeo Hidrolases , Hipoglicemiantes/metabolismo , Simulação de Dinâmica Molecular , Syzygium/química , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por SubstratoRESUMO
OBJECTIVE: To determine the status of α-tubulin acetylation and of testis-specific acetylable α-tubulin isoforms in asthenozoospermia. DESIGN: Research study. SETTING: Research institute and an infertility clinic. PATIENT(S): 50 men with normal sperm parameters, and 50 men with asthenozoospermia. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Western blot analyses of α-tubulin, acetylated α-tubulin, and isoforms TUBA3C, TUBA4A, and TUBA8 in Percoll separated sperm and flow cytometry, real-time reverse-transcription polymerase chain reaction, and immunofluorescent localization. RESULT(S): A statistically significant decrease in the expression of acetylated α-tubulin in asthenozoosperm was seen with Western blot analysis, double immunostaining by direct immunofluorescence, and flow cytometric analysis. The transcript and protein of testis-specific acetylable α-tubulin isoform TUBA3C was decreased and TUBA4A was statistically significantly increased in asthenozoosperm as compared with normal spermatozoa. TUBA8 was reduced in asthenozoosperm. Similar observations were noted by indirect immunofluorescent localization. The potential transcription factors involved in the differential expression of TUBA4A and TUBA3C have been identified. CONCLUSION(S): Data suggest an association of α-tubulin acetylation with asthenozoospermia. Ours is the first report to demonstrate α-tubulin isoforms in sperm, implicating their role in motility. The differential expression of TUBA3C and TUBA4A suggests that tubulin acetylation may be governed by the isoform of α-tubulin that is expressed or silenced and that this in turn is transcriptionally controlled.