Integrated QSAR study for inhibitors of hedgehog signal pathway against multiple cell lines:a collaborative filtering method

PubMed Central

2012-01-01

Background The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. Results In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Conclusions Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1. PMID:22849868

Integrated QSAR study for inhibitors of Hedgehog Signal Pathway against multiple cell lines:a collaborative filtering method.

PubMed

Gao, Jun; Che, Dongsheng; Zheng, Vincent W; Zhu, Ruixin; Liu, Qi

2012-07-31

The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1.

The Hippo pathway: regulators and regulations

PubMed Central

Yu, Fa-Xing; Guan, Kun-Liang

2013-01-01

Control of cell number is crucial in animal development and tissue homeostasis, and its dysregulation may result in tumor formation or organ degeneration. The Hippo pathway in both Drosophila and mammals regulates cell number by modulating cell proliferation, cell death, and cell differentiation. Recently, numerous upstream components involved in the Hippo pathway have been identified, such as cell polarity, mechanotransduction, and G-protein-coupled receptor (GPCR) signaling. Actin cytoskeleton or cellular tension appears to be the master mediator that integrates and transmits upstream signals to the core Hippo signaling cascade. Here, we review regulatory mechanisms of the Hippo pathway and discuss potential implications involved in different physiological and pathological conditions. PMID:23431053

Core signaling pathways in ovarian cancer stem cell revealed by integrative analysis of multi-marker genomics data.

PubMed

Zhang, Tianyu; Xu, Jielin; Deng, Siyuan; Zhou, Fengqi; Li, Jin; Zhang, Liwei; Li, Lang; Wang, Qi-En; Li, Fuhai

2018-01-01

Tumor recurrence occurs in more than 70% of ovarian cancer patients, and the majority eventually becomes refractory to treatments. Ovarian Cancer Stem Cells (OCSCs) are believed to be responsible for the tumor relapse and drug resistance. Therefore, eliminating ovarian CSCs is important to improve the prognosis of ovarian cancer patients. However, there is a lack of effective drugs to eliminate OCSCs because the core signaling pathways regulating OCSCs remain unclear. Also it is often hard for biologists to identify a few testable targets and infer driver signaling pathways regulating CSCs from a large number of differentially expression genes in an unbiased manner. In this study, we propose a straightforward and integrative analysis to identify potential core signaling pathways of OCSCs by integrating transcriptome data of OCSCs isolated based on two distinctive markers, ALDH and side population, with regulatory network (Transcription Factor (TF) and Target Interactome) and signaling pathways. We first identify the common activated TFs in two OCSC populations integrating the gene expression and TF-target Interactome; and then uncover up-stream signaling cascades regulating the activated TFs. In specific, 22 activated TFs are identified. Through literature search validation, 15 of them have been reported in association with cancer stem cells. Additionally, 10 TFs are found in the KEGG signaling pathways, and their up-stream signaling cascades are extracted, which also provide potential treatment targets. Moreover, 40 FDA approved drugs are identified to target on the up-stream signaling cascades, and 15 of them have been reported in literatures in cancer stem cell treatment. In conclusion, the proposed approach can uncover the activated up-stream signaling, activated TFs and up-regulated target genes that constitute the potential core signaling pathways of ovarian CSC. Also drugs and drug combinations targeting on the core signaling pathways might be able to eliminate OCSCs. The proposed approach can also be applied for identifying potential activated signaling pathways of other types of cancers.

Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism

PubMed Central

Sychev, Zoi E.; Hu, Alex; Lagunoff, Michael

2017-01-01

Kaposi’s Sarcoma associated Herpesvirus (KSHV), an oncogenic, human gamma-herpesvirus, is the etiological agent of Kaposi’s Sarcoma the most common tumor of AIDS patients world-wide. KSHV is predominantly latent in the main KS tumor cell, the spindle cell, a cell of endothelial origin. KSHV modulates numerous host cell-signaling pathways to activate endothelial cells including major metabolic pathways involved in lipid metabolism. To identify the underlying cellular mechanisms of KSHV alteration of host signaling and endothelial cell activation, we identified changes in the host proteome, phosphoproteome and transcriptome landscape following KSHV infection of endothelial cells. A Steiner forest algorithm was used to integrate the global data sets and, together with transcriptome based predicted transcription factor activity, cellular networks altered by latent KSHV were predicted. Several interesting pathways were identified, including peroxisome biogenesis. To validate the predictions, we showed that KSHV latent infection increases the number of peroxisomes per cell. Additionally, proteins involved in peroxisomal lipid metabolism of very long chain fatty acids, including ABCD3 and ACOX1, are required for the survival of latently infected cells. In summary, novel cellular pathways altered during herpesvirus latency that could not be predicted by a single systems biology platform, were identified by integrated proteomics and transcriptomics data analysis and when correlated with our metabolomics data revealed that peroxisome lipid metabolism is essential for KSHV latent infection of endothelial cells. PMID:28257516

Hippo pathway and protection of genome stability in response to DNA damage.

PubMed

Pefani, Dafni E; O'Neill, Eric

2016-04-01

The integrity of DNA is constantly challenged by exposure to the damaging effects of chemical and physical agents. Elucidating the cellular mechanisms that maintain genomic integrity via DNA repair and cell growth control is vital because errors in these processes lead to genomic damage and the development of cancer. By gaining a deep molecular understanding of the signaling pathways regulating genome integrity it is hoped to uncover new therapeutics and treatment designs to combat cancer. Components of the Hippo pathway, a tumor-suppressor cascade, have recently been defined to limit cancer transformation in response to DNA damage. In this review, we briefly introduce the Hippo signaling cascade in mammals and discuss in detail how the Hippo pathway has been established as part of the DNA damage response, activated by apical signaling kinases that recognize breaks in DNA. We also highlight the significance of the Hippo pathway activator RASSF1A tumor suppressor, a direct target of ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related ATR. Furthermore we discuss how Hippo pathway in response DNA lesions can induce cell death via Yes-associated protein (YAP) (the canonical Hippo pathway effector) or promote maintenance of genome integrity in a YAP-independent manner. © 2015 FEBS.

The Cell Wall Integrity Signaling Pathway and Its Involvement in Secondary Metabolite Production.

PubMed

Valiante, Vito

2017-12-06

The fungal cell wall is the external and first layer that fungi use to interact with the environment. Every stress signal, before being translated into an appropriate stress response, needs to overtake this layer. Many signaling pathways are involved in translating stress signals, but the cell wall integrity (CWI) signaling pathway is the one responsible for the maintenance and biosynthesis of the fungal cell wall. In fungi, the CWI signal is composed of a mitogen-activated protein kinase (MAPK) module. After the start of the phosphorylation cascade, the CWI signal induces the expression of cell-wall-related genes. However, the function of the CWI signal is not merely the activation of cell wall biosynthesis, but also the regulation of expression and production of specific molecules that are used by fungi to better compete in the environment. These molecules are normally defined as secondary metabolites or natural products. This review is focused on secondary metabolites affected by the CWI signal pathway with a special focus on relevant natural products such as melanins, mycotoxins, and antibacterial compounds.

An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

PubMed Central

Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

2012-01-01

During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

Daple coordinates organ-wide and cell-intrinsic polarity to pattern inner-ear hair bundles

PubMed Central

Siletti, Kimberly; Hudspeth, A. J.

2017-01-01

The establishment of planar polarization by mammalian cells necessitates the integration of diverse signaling pathways. In the inner ear, at least two systems regulate the planar polarity of sensory hair bundles. The core planar cell polarity (PCP) proteins coordinate the orientations of hair cells across the epithelial plane. The cell-intrinsic patterning of hair bundles is implemented independently by the G protein complex classically known for orienting the mitotic spindle. Although the primary cilium also participates in each of these pathways, its role and the integration of the two systems are poorly understood. We show that Dishevelled-associating protein with a high frequency of leucine residues (Daple) interacts with PCP and cell-intrinsic signals. Regulated by the cell-intrinsic pathway, Daple is required to maintain the polarized distribution of the core PCP protein Dishevelled and to position the primary cilium at the abneural edge of the apical surface. Our results suggest that the primary cilium or an associated structure influences the domain of cell-intrinsic signals that shape the hair bundle. Daple is therefore essential to orient and pattern sensory hair bundles. PMID:29229865

Cell-Specific Imd-NF-κB Responses Enable Simultaneous Antibacterial Immunity and Intestinal Epithelial Cell Shedding upon Bacterial Infection.

PubMed

Zhai, Zongzhao; Boquete, Jean-Philippe; Lemaitre, Bruno

2018-05-03

Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding. Copyright © 2018 Elsevier Inc. All rights reserved.

Pheromone-Induced Morphogenesis Improves Osmoadaptation Capacity by Activating the HOG MAPK Pathway**

PubMed Central

Baltanás, Rodrigo; Bush, Alan; Couto, Alicia; Durrieu, Lucía; Hohmann, Stefan; Colman-Lerner, Alejandro

2013-01-01

Environmental and internal conditions expose cells to a multiplicity of stimuli whose consequences are difficult to predict. Here, we investigate the response to mating pheromone of yeast cells adapted to high osmolarity. Events downstream of pheromone binding involve two mitogen-activated protein kinase (MAPK) cascades: the pheromone response (PR) and the cell-wall integrity response (CWI). Although these MAPK pathways share components with each and a third MAPK pathway, the high osmolarity response (HOG), they are normally only activated by distinct stimuli, a phenomenon called insulation. We found that in cells adapted to high osmolarity, PR activated the HOG pathway in a pheromone- and osmolarity- dependent manner. Activation of HOG by the PR was not due to loss of insulation, but rather a response to a reduction in internal osmolarity, which resulted from an increase in glycerol release caused by the PR. By analyzing single-cell time courses, we found that stimulation of HOG occurred in discrete bursts that coincided with the “shmooing” morphogenetic process. Activation required the polarisome, the cell wall integrity MAPK Slt2, and the aquaglyceroporin Fps1. HOG activation resulted in high glycerol turnover that improved adaptability to rapid changes in osmolarity. Our work shows how a differentiation signal can recruit a second, unrelated sensory pathway to enable responses to yeast to multiple stimuli. PMID:23612707

ECSIT links TLR and BMP signaling in FOP connective tissue progenitor cells.

PubMed

Wang, Haitao; Behrens, Edward M; Pignolo, Robert J; Kaplan, Frederick S

2018-04-01

Clinical and laboratory observations strongly suggest that the innate immune system induces flare-ups in the setting of dysregulated bone morphogenetic protein (BMP) signaling in fibrodysplasia ossificans progressiva (FOP). In order to investigate the signaling substrates of this hypothesis, we examined toll-like receptor (TLR) activation and bone morphogenetic protein (BMP) signaling in connective tissue progenitor cells (CTPCs) from FOP patients and unaffected individuals. We found that inflammatory stimuli broadly activate TLR expression in FOP CTPCs and that TLR3/TLR4 signaling amplifies BMP pathway signaling through both ligand dependent and independent mechanisms. Importantly, Evolutionarily Conserved Signaling Intermediate in the Toll Pathway (ECSIT) integrates TLR injury signaling with dysregulated BMP pathway signaling in FOP CTPCs. These findings provide novel insight into the cell autonomous integration of injury signals from the innate immune system with dysregulated response signals from the BMP signaling pathway and provide new exploratory targets for therapeutic approaches to blocking the induction and amplification of FOP lesions. Copyright © 2018 Elsevier Inc. All rights reserved.

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways

PubMed Central

Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S.; Allen, Joshua E.; Oster, Wolfgang; Duvic, Madeleine

2017-01-01

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4+ malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V+ cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4+ malignant T cells, not in normal CD4+ T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways. PMID:28977902

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

PubMed

Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S; Allen, Joshua E; Oster, Wolfgang; Duvic, Madeleine

2017-09-22

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4 + malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V + cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4 + malignant T cells, not in normal CD4 + T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation.

PubMed

Gallon, Lorenzo; Mathew, James M; Bontha, Sai Vineela; Dumur, Catherine I; Dalal, Pranav; Nadimpalli, Lakshmi; Maluf, Daniel G; Shetty, Aneesha A; Ildstad, Suzanne T; Leventhal, Joseph R; Mas, Valeria R

2018-02-01

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways. Copyright © 2018 by the American Society of Nephrology.

The role of MAPK signal transduction pathways in the response to oxidative stress in the fungal pathogen Candida albicans: implications in virulence.

PubMed

de Dios, Carmen Herrero; Román, Elvira; Monge, Rebeca Alonso; Pla, Jesús

2010-12-01

In recent years, Mitogen-Activated Protein Kinase (MAPK) pathways have emerged as major regulators of cellular physiology. In the fungal pathogen Candida albicans, three different MAPK pathways have been characterized in the last years. The HOG pathway is mainly a stress response pathway that is activated in response to osmotic and oxidative stress and also participates regulating other pathways. The SVG pathway (or mediated by the Cek1 MAPK) is involved in cell wall formation under vegetative and filamentous growth, while the Mkc1-mediated pathway is involved in cell wall integrity. Oxidative stress is one of the types of stress that every fungal cell has to face during colonization of the host, where the cell encounters both hypoxia niches (i.e. gut) and high concentrations of reactive oxygen species (upon challenge with immune cells). Two pathways have been shown to be activated in response to oxidative stress: the HOG pathway and the MKC1-mediated pathway while the third, the Cek1 pathway is deactivated. The timing, kinetics, stimuli and functional responses generated upon oxidative stress differ among them; however, they have essential functional consequences that severely influence pathogenesis. MAPK pathways are, therefore, valuable targets to be explored in antifungal research.

Hypoxia-elicited impairment of cell wall integrity, glycosylation precursor synthesis, and growth in scaled-up high-cell density fed-batch cultures of Saccharomyces cerevisiae.

PubMed

Aon, Juan C; Sun, Jianxin; Leighton, Julie M; Appelbaum, Edward R

2016-08-15

In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW. We tested the cell wall integrity by probing the cell wall sensitivity to Zymolyase. Also exometabolomics data showed changes in supply of precursors for the glycosylation pathway. The results show a more sensitive cell wall later in the production process at industrial scale, while the sensitivity at early time points was similar at both scales. We also report exometabolomics data, in particular a link with the protein glycosylation pathway. Significantly lower levels of Man6P and progressively higher GDP-mannose indicated partially impaired incorporation of this sugar nucleotide during co- or post-translational protein glycosylation pathways at the 10,000 L compared to the 10 L scale. This impairment in glycosylation would be expected to affect cell wall integrity. Although cell viability from samples obtained at both scales were similar, cells harvested from 10 L bioreactors were able to re-initiate growth faster in fresh shake flask media than those harvested from the industrial scale. The results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.

Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

PubMed

Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic; Jennings, Paul

2015-12-25

Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 μM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

An Integrated Human/Murine Transcriptome and Pathway Approach To Identify Prenatal Treatments For Down Syndrome.

PubMed

Guedj, Faycal; Pennings, Jeroen LA; Massingham, Lauren J; Wick, Heather C; Siegel, Ashley E; Tantravahi, Umadevi; Bianchi, Diana W

2016-09-02

Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls, and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn, and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice.

Modeling of cell signaling pathways in macrophages by semantic networks

PubMed Central

Hsing, Michael; Bellenson, Joel L; Shankey, Conor; Cherkasov, Artem

2004-01-01

Background Substantial amounts of data on cell signaling, metabolic, gene regulatory and other biological pathways have been accumulated in literature and electronic databases. Conventionally, this information is stored in the form of pathway diagrams and can be characterized as highly "compartmental" (i.e. individual pathways are not connected into more general networks). Current approaches for representing pathways are limited in their capacity to model molecular interactions in their spatial and temporal context. Moreover, the critical knowledge of cause-effect relationships among signaling events is not reflected by most conventional approaches for manipulating pathways. Results We have applied a semantic network (SN) approach to develop and implement a model for cell signaling pathways. The semantic model has mapped biological concepts to a set of semantic agents and relationships, and characterized cell signaling events and their participants in the hierarchical and spatial context. In particular, the available information on the behaviors and interactions of the PI3K enzyme family has been integrated into the SN environment and a cell signaling network in human macrophages has been constructed. A SN-application has been developed to manipulate the locations and the states of molecules and to observe their actions under different biological scenarios. The approach allowed qualitative simulation of cell signaling events involving PI3Ks and identified pathways of molecular interactions that led to known cellular responses as well as other potential responses during bacterial invasions in macrophages. Conclusions We concluded from our results that the semantic network is an effective method to model cell signaling pathways. The semantic model allows proper representation and integration of information on biological structures and their interactions at different levels. The reconstruction of the cell signaling network in the macrophage allowed detailed investigation of connections among various essential molecules and reflected the cause-effect relationships among signaling events. The simulation demonstrated the dynamics of the semantic network, where a change of states on a molecule can alter its function and potentially cause a chain-reaction effect in the system. PMID:15494071

A lateral signalling pathway coordinates shape volatility during cell migration

PubMed Central

Zhang, Liang; Luga, Valbona; Armitage, Sarah K.; Musiol, Martin; Won, Amy; Yip, Christopher M.; Plotnikov, Sergey V.; Wrana, Jeffrey L.

2016-01-01

Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23. We show that the Pk1–Arhgap21/23 complex inhibits RhoA, is localized on the non-protrusive lateral membrane cortex and its disruption leads to the disorganization of the actomyosin network and altered focal adhesion dynamics. Pk1-mediated lateral signalling confines protrusive activity and is regulated by Smurf2, an E3 ubiquitin ligase in the PCP pathway. Furthermore, we demonstrate that dynamic interplay between lateral and protrusive signalling generates cyclical fluctuations in cell shape that we quantify here as shape volatility, which strongly correlates with migration speed. These studies uncover a previously unrecognized lateral signalling pathway that coordinates shape volatility during productive cell migration. PMID:27226243

Signaling Molecules Governing Pluripotency and Early Lineage Commitments in Human Pluripotent Stem Cells

PubMed Central

Fathi, Ali; Eisa-Beygi, Shahram; Baharvand, Hossein

2017-01-01

Signaling in pluripotent stem cells is a complex and dynamic process involving multiple mediators, finely tuned to balancing pluripotency and differentiation states. Characterizing and modifying the necessary signaling pathways to attain desired cell types is required for stem-cell applications in various fields of regenerative medicine. These signals may help enhance the differentiation potential of pluripotent cells towards each of the embryonic lineages and enable us to achieve pure in vitro cultures of various cell types. This review provides a timely synthesis of recent advances into how maintenance of pluripotency in hPSCs is regulated by extrinsic cues, such as the fibroblast growth factor (FGF) and ACTIVIN signaling pathways, their interplay with other signaling pathways, namely, wingless- type MMTV integration site family (WNT) and mammalian target of rapamycin (mTOR), and the pathways governing the determination of multiple lineages. PMID:28670512

Differential genomic effects on canonical signaling pathways by two different CeO2 nanoparticles in HepG2 cells

EPA Science Inventory

Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells. Sheau-Fung Thai1, Kathleen A. Wallace1, Carlton P. Jones1, Hongzu Ren2, Benjamin T. Castellon1, James Crooks2, Kirk T. Kitchin1. 1Integrated Systems Toxicology Divison, 2Resea...

Integrative Analysis Reveals an Outcome-associated and Targetable Pattern of p53 and Cell Cycle Deregulation in Diffuse Large B-cell Lymphoma

PubMed Central

Monti, Stefano; Chapuy, Bjoern; Takeyama, Kunihiko; Rodig, Scott J; Hao, Yangsheng; Yeda, Kelly T.; Inguilizian, Haig; Mermel, Craig; Curie, Treeve; Dogan, Ahmed; Kutok, Jeffery L; Beroukim, Rameen; Neuberg, Donna; Habermann, Thomas; Getz, Gad; Kung, Andrew L; Golub, Todd R; Shipp, Margaret A

2013-01-01

Summary Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy and suggest targeted treatment approaches. PMID:22975378

Metabolic pathways recruited in the production of a recombinant enveloped virus: mining targets for process and cell engineering.

PubMed

Rodrigues, A F; Formas-Oliveira, A S; Bandeira, V S; Alves, P M; Hu, W S; Coroadinha, A S

2013-11-01

Biopharmaceuticals derived from enveloped virus comprise an expanding market of vaccines, oncolytic vectors and gene therapy products. Thus, increased attention is given to the development of robust high-titer cell hosts for their manufacture. However, the knowledge on the physiological constraints modulating virus production is still scarce and the use of integrated strategies to improve hosts productivity and upstream bioprocess an under-explored territory. In this work, we conducted a functional genomics study, including the transcriptional profiling and central carbon metabolism analysis, following the metabolic changes in the transition 'parental-to-producer' of two human cell lines producing recombinant retrovirus. Results were gathered into three comprehensive metabolic maps, providing a broad and integrated overview of gene expression changes for both cell lines. Eight pathways were identified to be recruited in the virus production state: amino acid catabolism, carbohydrate catabolism and integration of the energy metabolism, nucleotide metabolism, glutathione metabolism, pentose phosphate pathway, polyamines biosynthesis and lipid metabolism. Their ability to modulate viral titers was experimentally challenged, leading to improved specific productivities of recombinant retrovirus up to 6-fold. Within recruited pathways in the virus production state, we sought for metabolic engineering gene targets in the low producing phenotypes. A mining strategy was used alternative to the traditional approach 'high vs. low producer' clonal comparison. Instead, 'high vs. low producer' from different genetic backgrounds (i.e. cell origins) were compared. Several genes were identified as limiting in the low-production phenotype, including two enzymes from cholesterol biosynthesis, two enzymes from glutathione biosynthesis and the regulatory machinery of polyamines biosynthesis. This is thus a frontier work, bridging fundamentals to technological research and contributing to enlarge our understanding of enveloped virus production dynamics in mammalian cell hosts. © 2013 Published by Elsevier Inc.

Multi-pathway Kinase Signatures of Multipotent Stromal Cells are Predictive for Osteogenic Differentiation

PubMed Central

Platt, Manu O.; Wilder, Catera L.; Wells, Alan; Griffith, Linda G.; Lauffenburger, Douglas A.

2010-01-01

Bone marrow-derived multi-potent stromal cells (MSCs) offer great promise for regenerating tissue. While certain transcription factors have been identified in association with tendency toward particular MSC differentiation phenotypes, the regulatory network of key receptor-mediated signaling pathways activated by extracellular ligands that induce various differentiation responses remain poorly understood. Attempts to predict differentiation fate tendencies from individual pathways in isolation are problematic due to the complex pathway interactions inherent in signaling networks. Accordingly, we have undertaken a multi-variate systems approach integrating experimental measurement of multiple kinase pathway activities and osteogenic differentiation in MSCs, together with computational analysis to elucidate quantitative combinations of kinase signals predictive of cell behavior across diverse contexts. In particular, for culture on polymeric biomaterials surfaces presenting tethered epidermal growth factor (tEGF), type-I collagen, neither, or both, we have found that a partial least-squares regression model yields successful prediction of phenotypic behavior on the basis of two principal components comprising the weighted sums of 8 intracellular phosphoproteins: p-EGFR, p-Akt, p-ERK1/2, p-Hsp27, p-c-jun, p-GSK3α/β, p-p38, and p-STAT3. This combination provides strongest predictive capability for 21-day differentiated phenotype status when calculated from day-7 signal measurements (99%); day-4 (88%) and day-14 (89%) signal measurements are also significantly predictive, indicating a broad time-frame during MSC osteogenesis wherein multiple pathways and states of the kinase signaling network are quantitatively integrated to regulate gene expression, cell processes, and ultimately, cell fate. PMID:19750537

Cellular demise and inflammatory microglial activation during beta-amyloid toxicity are governed by Wnt1 and canonical signaling pathways.

PubMed

Chong, Zhao Zhong; Li, Faqi; Maiese, Kenneth

2007-06-01

Initially described as a modulator of embryogenesis for a number of organ systems, Wnt1 has recently been linked to the development of several neurodegenerative disorders, none being of greater significance than Alzheimer's disease. We therefore examined the ability of Wnt1 to oversee vital pathways responsible for cell survival during beta-amyloid (Abeta1-42) exposure. Here we show that Wnt1 is critical for protection in the SH-SY5Y neuronal cell line against genomic DNA degradation, membrane phosphatidylserine (PS) exposure, and microglial activation, since these neuroprotective attributes of Wnt1 are lost during gene silencing of Wnt1 protein expression. Intimately tied to Wnt1 protection is the presence and activation of Akt1. Pharmacological inhibition of the PI 3-K pathway or gene silencing of Akt1 expression can abrogate the protective capacity of Wnt1. Closely aligned with Wnt1 and Akt1 are the integrated canonical pathways of synthase kinase-3beta (GSK-3beta) and beta-catenin. Through Akt1 dependent pathways, Wnt1 phosphorylates GSK-3beta and maintains beta-catenin integrity to insure its translocation from the cytoplasm to the nucleus to block apoptosis. Our work outlines a highly novel role for Wnt1 and its integration with Akt1, GSK-3beta, and beta-catenin to foster neuronal cell survival and repress inflammatory microglial activation that can identify new avenues of therapy against neurodegenerative disorders.

Tolerant industrial yeast Saccharomyces cerevisiae posses a more robust cell wall integrity signaling pathway against 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde.

PubMed

Liu, Z Lewis; Wang, Xu; Weber, Scott A

2018-06-20

Cell wall integrity signaling pathway in Saccharomyces cerevisiae is a conserved function for detecting and responding to cell stress conditions but less understood for industrial yeast. We examined gene expression dynamics for a tolerant industrial yeast strain NRRL Y-50049 in response to challenges of furfural and HMF through comparative quantitative gene expression analysis using pathway-based qRT-PCR array assays. All tested genes from Y-50049, except for MLP2, demonstrated more resistant and significantly increased gene expression than that from a laboratory strain BY4741. While all five sensor encoding genes WSC1, WSC2, WSC3, MID2 and MTL1 from both strains were activated in response to the furfural-HMF treatment, WSC3 from Y-50049 demonstrated the most increased expression over time compared with any other sensor genes. These results suggested the industrial yeast poses more robust cell wall integrity pathway, and gene WSC3 could have the special capability for signal transmission against furfural and HMF. Among five single nucleotide variations discovered in WSC3 from Y-50049, three were found to be non-synonymous mutations resulting in amino acid alterations of Ser 158  → Tyr 158 , Val 186  → Ile 186 , and Glu 430  → Asp 430 . Our results suggest the industrial yeast as a more desirable delivery vehicle for the next-generation biocatalyst development. Published by Elsevier B.V.

Integrative molecular network analysis identifies emergent enzalutamide resistance mechanisms in prostate cancer

PubMed Central

King, Carly J.; Woodward, Josha; Schwartzman, Jacob; Coleman, Daniel J.; Lisac, Robert; Wang, Nicholas J.; Van Hook, Kathryn; Gao, Lina; Urrutia, Joshua; Dane, Mark A.; Heiser, Laura M.; Alumkal, Joshi J.

2017-01-01

Recent work demonstrates that castration-resistant prostate cancer (CRPC) tumors harbor countless genomic aberrations that control many hallmarks of cancer. While some specific mutations in CRPC may be actionable, many others are not. We hypothesized that genomic aberrations in cancer may operate in concert to promote drug resistance and tumor progression, and that organization of these genomic aberrations into therapeutically targetable pathways may improve our ability to treat CRPC. To identify the molecular underpinnings of enzalutamide-resistant CRPC, we performed transcriptional and copy number profiling studies using paired enzalutamide-sensitive and resistant LNCaP prostate cancer cell lines. Gene networks associated with enzalutamide resistance were revealed by performing an integrative genomic analysis with the PAthway Representation and Analysis by Direct Reference on Graphical Models (PARADIGM) tool. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with MEK, EGFR, RAS, and NFKB. Functional validation studies of 64 genes identified 10 candidate genes whose suppression led to greater effects on cell viability in enzalutamide-resistant cells as compared to sensitive parental cells. Examination of a patient cohort demonstrated that several of our functionally-validated gene hits are deregulated in metastatic CRPC tumor samples, suggesting that they may be clinically relevant therapeutic targets for patients with enzalutamide-resistant CRPC. Altogether, our approach demonstrates the potential of integrative genomic analyses to clarify determinants of drug resistance and rational co-targeting strategies to overcome resistance. PMID:29340039

Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR.

PubMed

Jakočiūnas, Tadas; Jensen, Emil D; Jensen, Michael K; Keasling, Jay D

2018-01-01

Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

Cell-autonomous inactivation of the Reelin pathway impairs adult neurogenesis in the hippocampus

PubMed Central

Teixeira, Catia M.; Kron, Michelle M.; Masachs, Nuria; Zhang, Helen; Lagace, Diane C.; Martinez, Albert; Reillo, Isabel; Duan, Xin; Bosch, Carles; Pujadas, Lluis; Brunso, Lucas; Song, Hongjun; Eisch, Amelia J.; Borrell, Victor; Howell, Brian W.; Parent, Jack M.; Soriano, Eduardo

2012-01-01

Adult hippocampal neurogenesis is thought to be essential for learning and memory and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of disabled-1 (Dab1), an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain- and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adult-generated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders. PMID:22933789

Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway.

PubMed

Alonso-Gutierrez, Jorge; Koma, Daisuke; Hu, Qijun; Yang, Yuchen; Chan, Leanne J G; Petzold, Christopher J; Adams, Paul D; Vickers, Claudia E; Nielsen, Lars K; Keasling, Jay D; Lee, Taek S

2018-04-01

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering. © 2017 Wiley Periodicals, Inc.

Overcoming reprogramming resistance of Fanconi anemia cells

PubMed Central

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans.

PubMed

Gerik, Kimberly J; Bhimireddy, Sujit R; Ryerse, Jan S; Specht, Charles A; Lodge, Jennifer K

2008-10-01

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a "top-down" approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Delta strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans▿ †

PubMed Central

Gerik, Kimberly J.; Bhimireddy, Sujit R.; Ryerse, Jan S.; Specht, Charles A.; Lodge, Jennifer K.

2008-01-01

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism. PMID:18689526

The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

PubMed

Miguel-Rojas, Cristina; Hera, Concepcion

2016-01-01

F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Plasmodesmata in integrated cell signalling: insights from development and environmental signals and stresses

PubMed Central

Sager, Ross; Lee, Jung-Youn

2014-01-01

To survive as sedentary organisms built of immobile cells, plants require an effective intercellular communication system, both locally between neighbouring cells within each tissue and systemically across distantly located organs. Such a system enables cells to coordinate their intracellular activities and produce concerted responses to internal and external stimuli. Plasmodesmata, membrane-lined intercellular channels, are essential for direct cell-to-cell communication involving exchange of diffusible factors, including signalling and information molecules. Recent advances corroborate that plasmodesmata are not passive but rather highly dynamic channels, in that their density in the cell walls and gating activities are tightly linked to developmental and physiological processes. Moreover, it is becoming clear that specific hormonal signalling pathways play crucial roles in relaying primary cellular signals to plasmodesmata. In this review, we examine a number of studies in which plasmodesmal structure, occurrence, and/or permeability responses are found to be altered upon given cellular or environmental signals, and discuss common themes illustrating how plasmodesmal regulation is integrated into specific cellular signalling pathways. PMID:25262225

Predictive toxicology using systemic biology and liver microfluidic “on chip” approaches: Application to acetaminophen injury

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

Prot, Jean-Matthieu; Bunescu, Andrei; Elena-Herrmann, Bénédicte

2012-03-15

We have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treatedmore » biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology. -- Highlights: ► We cultivated liver cells in microfluidic biochips ► We integrated transcriptomic, proteomic and metabolomics profiles ► Pathways reconstructions were proposed in control and acetaminophen treated cultures ► Biomarkers were identified ► Comparisons with in vivo studies were proposed.« less

What’s the Damage? The Impact of Pathogens on Pathways that Maintain Host Genome Integrity

PubMed Central

Weitzman, Matthew D.; Weitzman, Jonathan B.

2014-01-01

Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs. PMID:24629335

Discovery of new candidate genes for rheumatoid arthritis through integration of genetic association data with expression pathway analysis.

PubMed

Shchetynsky, Klementy; Diaz-Gallo, Lina-Marcella; Folkersen, Lasse; Hensvold, Aase Haj; Catrina, Anca Irinel; Berg, Louise; Klareskog, Lars; Padyukov, Leonid

2017-02-02

Here we integrate verified signals from previous genetic association studies with gene expression and pathway analysis for discovery of new candidate genes and signaling networks, relevant for rheumatoid arthritis (RA). RNA-sequencing-(RNA-seq)-based expression analysis of 377 genes from previously verified RA-associated loci was performed in blood cells from 5 newly diagnosed, non-treated patients with RA, 7 patients with treated RA and 12 healthy controls. Differentially expressed genes sharing a similar expression pattern in treated and untreated RA sub-groups were selected for pathway analysis. A set of "connector" genes derived from pathway analysis was tested for differential expression in the initial discovery cohort and validated in blood cells from 73 patients with RA and in 35 healthy controls. There were 11 qualifying genes selected for pathway analysis and these were grouped into two evidence-based functional networks, containing 29 and 27 additional connector molecules. The expression of genes, corresponding to connector molecules was then tested in the initial RNA-seq data. Differences in the expression of ERBB2, TP53 and THOP1 were similar in both treated and non-treated patients with RA and an additional nine genes were differentially expressed in at least one group of patients compared to healthy controls. The ERBB2, TP53. THOP1 expression profile was successfully replicated in RNA-seq data from peripheral blood mononuclear cells from healthy controls and non-treated patients with RA, in an independent collection of samples. Integration of RNA-seq data with findings from association studies, and consequent pathway analysis implicate new candidate genes, ERBB2, TP53 and THOP1 in the pathogenesis of RA.

Challenges in horizontal model integration.

PubMed

Kolczyk, Katrin; Conradi, Carsten

2016-03-11

Systems Biology has motivated dynamic models of important intracellular processes at the pathway level, for example, in signal transduction and cell cycle control. To answer important biomedical questions, however, one has to go beyond the study of isolated pathways towards the joint study of interacting signaling pathways or the joint study of signal transduction and cell cycle control. Thereby the reuse of established models is preferable, as it will generally reduce the modeling effort and increase the acceptance of the combined model in the field. Obtaining a combined model can be challenging, especially if the submodels are large and/or come from different working groups (as is generally the case, when models stored in established repositories are used). To support this task, we describe a semi-automatic workflow based on established software tools. In particular, two frequent challenges are described: identification of the overlap and subsequent (re)parameterization of the integrated model. The reparameterization step is crucial, if the goal is to obtain a model that can reproduce the data explained by the individual models. For demonstration purposes we apply our workflow to integrate two signaling pathways (EGF and NGF) from the BioModels Database.

Pleiotrophin regulates lung epithelial cell proliferation and differentiation during fetal lung development via beta-catenin and Dlk1.

PubMed

Weng, Tingting; Gao, Li; Bhaskaran, Manoj; Guo, Yujie; Gou, Deming; Narayanaperumal, Jeyaparthasarathy; Chintagari, Narendranath Reddy; Zhang, Kexiong; Liu, Lin

2009-10-09

The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.

Paper-based microreactor array for rapid screening of cell signaling cascades.

PubMed

Huang, Chia-Hao; Lei, Kin Fong; Tsang, Ngan-Ming

2016-08-07

Investigation of cell signaling pathways is important for the study of pathogenesis of cancer. However, the related operations used in these studies are time consuming and labor intensive. Thus, the development of effective therapeutic strategies may be hampered. In this work, gel-free cell culture and subsequent immunoassay has been successfully integrated and conducted in a paper-based microreactor array. Study of the activation level of different kinases of cells stimulated by different conditions, i.e., IL-6 stimulation, starvation, and hypoxia, was demonstrated. Moreover, rapid screening of cell signaling cascades after the stimulations of HGF, doxorubicin, and UVB irradiation was respectively conducted to simultaneously screen 40 kinases and transcription factors. Activation of multi-signaling pathways could be identified and the correlation between signaling pathways was discussed to provide further information to investigate the entire signaling network. The present technique integrates most of the tedious operations using a single paper substrate, reduces sample and reagent consumption, and shortens the time required by the entire process. Therefore, it provides a first-tier rapid screening tool for the study of complicated signaling cascades. It is expected that the technique can be developed for routine protocol in conventional biological research laboratories.

Characterization of the role of Fhit in maintenance of genomic integrity following low dose radiation, in vivo and in vitro

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

Ya Wang

2010-05-31

The major goal of this study is to determine the effects of the Fhit pathway on low dose ({le} 0.1 Gy) ionizing radiation (IR)-induced genetic instability. Reduction of Fhit protein expression is observed in most solid tumors particularly in those tumors resulting from exposure to environmental carcinogens. Therefore, characterization of the role of the Fhit-dependent pathway in preventing low dose IR-induced genetic instability will provide useful parameters for evaluating the low dose IR-induced risk of mutagenesis and carcinogenesis. We pursued 3 specific aims to study our hypothesis that the Fhit-dependent pathways maintain genomic integrity through adjusting checkpoint response and repairmore » genes expression following low dose IR. Aim 1: Determine whether Fhit interaction with RPA is necessary for Fhit to affect the cellular response to low dose IR. We combined the approaches of in vitro (GST pull-down and site-directed mutagenesis) and in vivo (observing the co-localization and immunoprecipitation of Fhit and RPA in Fhit knock out mouse cells transfected with mutant Fhit which has lost ability to interact with RPA in vitro). Aim 2: Determine the role of genes whose expression is affected by Fhit in low dose irradiated cells. We analyzed the distinct signature of gene expression in low dose irradiated Fhit-/- cells compared with Fhit+/+ cells by combining microarray, gene transfection and siRNA approaches. Aim 3: Determine the role of Fhit in genetic susceptibility to low dose IR in vivo. We compared the gene mutation frequency and the fragile site stability in the cells isolated from the Fhit+/+ and Fhit-/- mice at 1.5 years following low dose IR. These results determine the role of the Fhit-dependent pathway in maintaining genomic integrity in vitro and in vivo, which provide a basis for choosing surrogate markers in the Fhit-dependent pathway to evaluate low dose IR-induced risk of mutagenesis and carcinogenesis.« less

Integrated analysis of miRNA and mRNA expression data identifies multiple miRNAs regulatory networks for the tumorigenesis of colorectal cancer.

PubMed

Xu, Peng; Wang, Junhua; Sun, Bo; Xiao, Zhongdang

2018-06-15

Investigating the potential biological function of differential changed genes through integrating multiple omics data including miRNA and mRNA expression profiles, is always hot topic. However, how to evaluate the repression effect on target genes integrating miRNA and mRNA expression profiles are not fully solved. In this study, we provide an analyzing method by integrating both miRNAs and mRNAs expression data simultaneously. Difference analysis was adopted based on the repression score, then significantly repressed mRNAs were screened out by DEGseq. Pathway analysis for the significantly repressed mRNAs shows that multiple pathways such as MAPK signaling pathway, TGF-beta signaling pathway and so on, may correlated to the colorectal cancer(CRC). Focusing on the MAPK signaling pathway, a miRNA-mRNA network that centering the cell fate genes was constructed. Finally, the miRNA-mRNAs that potentially important in the CRC carcinogenesis were screened out and scored by impact index. Copyright © 2018 Elsevier B.V. All rights reserved.

An integrated global regulatory network of hematopoietic precursor cell self-renewal and differentiation.

PubMed

You, Yanan; Cuevas-Diaz Duran, Raquel; Jiang, Lihua; Dong, Xiaomin; Zong, Shan; Snyder, Michael; Wu, Jia Qian

2018-06-12

Systematic study of the regulatory mechanisms of Hematopoietic Stem Cell and Progenitor Cell (HSPC) self-renewal is fundamentally important for understanding hematopoiesis and for manipulating HSPCs for therapeutic purposes. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent Hematopoietic Progenitor Cell (HPC) line, EML (Erythroid, Myeloid, and Lymphoid) cells. Herein, we report binding maps for additional TFs (SOX4 and STAT3) by using chromatin immunoprecipitation (ChIP)-Sequencing, to address the underlying mechanisms regulating self-renewal properties of lineage-CD34+ subpopulation (Lin-CD34+ EML cells). Furthermore, we applied the Assay for Transposase Accessible Chromatin (ATAC)-Sequencing to globally identify the open chromatin regions associated with TF binding in the self-renewing Lin-CD34+ EML cells. Mass spectrometry (MS) was also used to quantify protein relative expression levels. Finally, by integrating the protein-protein interaction database, we built an expanded transcriptional regulatory and interaction network. We found that MAPK (Mitogen-activated protein kinase) pathway and TGF-β/SMAD signaling pathway components were highly enriched among the binding targets of these TFs in Lin-CD34+ EML cells. The present study integrates regulatory information at multiple levels to paint a more comprehensive picture of the HSPC self-renewal mechanisms.

Two Forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway

PubMed Central

Ahmad, Shaad M.; Tansey, Terese R.; Busser, Brian W.; Nolte, Michael T.; Jeffries, Neal; Gisselbrecht, Stephen S.; Rusan, Nasser M.; Michelson, Alan M.

2012-01-01

SUMMARY The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types, as well as their proper differentiation and correct positioning relative to each other. During Drosophila cardiogenesis, all three of these processes are controlled by jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), two genes encoding forkhead transcription factors that we discovered utilizing an integrated genetic, genomic and computational strategy for identifying genes expressed in the developing Drosophila heart. Both jumu and CHES-1-like are required during asymmetric cell division for the derivation of two distinct cardiac cell types from their mutual precursor, and in symmetric cell divisions that produce yet a third type of heart cell. jumu and CHES-1-like control the division of cardiac progenitors by regulating the activity of Polo, a kinase involved in multiple steps of mitosis. This pathway demonstrates how transcription factors integrate diverse developmental processes during organogenesis. PMID:22814603

The Regulation of Filamentous Growth in Yeast

PubMed Central

Cullen, Paul J.; Sprague, George F.

2012-01-01

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host–cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways—rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)—also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior. PMID:22219507

PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3.

PubMed

Remy, Ingrid; Montmarquette, Annie; Michnick, Stephen W

2004-04-01

Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.

Radiogenomics: a systems biology approach to understanding genetic risk factors for radiotherapy toxicity ?

PubMed Central

Herskind, Carsten; Talbot, Christopher J.; Kerns, Sarah L.; Veldwijk, Marlon R.; Rosenstein, Barry S.; West, Catharine M. L.

2016-01-01

Adverse reactions in normal tissue after radiotherapy (RT) limit the dose that can be given to tumour cells. Since 80% of individual variation in clinical response is estimated to be caused by patient-related factors, identifying these factors might allow prediction of patients with increased risk of developing severe reactions. While inactivation of cell renewal is considered a major cause of toxicity in early-reacting normal tissues, complex interactions involving multiple cell types, cytokines, and hypoxia seem important for late reactions. Here, we review ‘omics’ approaches such as screening of genetic polymorphisms or gene expression analysis, and assess the potential of epigenetic factors, posttranslational modification, signal transduction, and metabolism. Furthermore, functional assays have suggested possible associations with clinical risk of adverse reaction. Pathway analysis incorporating different ‘omics’ approaches may be more efficient in identifying critical pathways than pathway analysis based on single ‘omics’ data sets. Integrating these pathways with functional assays may be powerful in identifying multiple subgroups of RT patients characterized by different mechanisms. Thus ‘omics’ and functional approaches may synergize if they are integrated into radiogenomics ‘systems biology’ to facilitate the goal of individualised radiotherapy. PMID:26944314

Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa.

PubMed

Bowman, Shaun M; Piwowar, Amy; Al Dabbous, Mash'el; Vierula, John; Free, Stephen J

2006-03-01

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.

The TNF receptor and Ig superfamily members form an integrated signaling circuit controlling dendritic cell homeostasis

PubMed Central

De Trez, Carl; Ware, Carl F.

2008-01-01

Dendritic cells (DC) constitute the most potent antigen presenting cells of the immune system, playing a key role bridging innate and adaptive immune responses. Specialized DC subsets differ depending on their origin, tissue location and the influence of trophic factors, the latter remain to be fully understood. Stromal cell and myeloid-associated Lymphotoxin-β receptor (LTβR) signaling is required for the local proliferation of lymphoid tissue DC. This review focuses the LTβR signaling cascade as a crucial positive trophic signal in the homeostasis of DC subsets. The noncanonical coreceptor pathway comprised of the Immunoglobulin (Ig) superfamily member, B and T lymphocyte attenuator (BTLA) and TNFR superfamily member, Herpesvirus entry mediator (HVEM) counter regulates the trophic signaling by LTβR. Together both pathways form an integrated signaling circuit achieving homeostasis of DC subsets. PMID:18511331

DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

PubMed

Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

2016-06-01

A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pathway modulations and epigenetic alterations in ovarian tumorbiogenesis

PubMed Central

Saldanha, Sabita N.; Tollefsbol, Trygve O.

2013-01-01

Cellular pathways are numerous and are highly integrated in function in the control of cellular systems. They collectively regulate cell division, proliferation, survival and apoptosis of cells and mutagenesis of key genes that control these pathways can initiate neoplastic transformations. Understanding these pathways is crucial to future therapeutic and preventive strategies of the disease. Ovarian cancers are of three major types; epithelial, germ-cell and stromal. However, ovarian cancers of epithelial origin, arising from the mesothelium, are the predominant form. Of the subtypes of ovarian cancer, the high-grade serous tumors are fatal, with low survival rate due to late detection and poor response to treatments. Close examination of preserved ovarian tissues and in vitro studies have provided insights into the mechanistic changes occurring in cells mediated by a few key genes. This review will focus on pathways and key genes of the pathways that are mutated or have aberrant functions in the pathology of ovarian cancer. Non-genetic mechanisms that are gaining prominence in the pathology of ovarian cancer, miRNAs and epigenetics, will also be discussed in the review. PMID:24105793

Systems biology by the rules: hybrid intelligent systems for pathway modeling and discovery.

PubMed

Bosl, William J

2007-02-15

Expert knowledge in journal articles is an important source of data for reconstructing biological pathways and creating new hypotheses. An important need for medical research is to integrate this data with high throughput sources to build useful models that span several scales. Researchers traditionally use mental models of pathways to integrate information and development new hypotheses. Unfortunately, the amount of information is often overwhelming and these are inadequate for predicting the dynamic response of complex pathways. Hierarchical computational models that allow exploration of semi-quantitative dynamics are useful systems biology tools for theoreticians, experimentalists and clinicians and may provide a means for cross-communication. A novel approach for biological pathway modeling based on hybrid intelligent systems or soft computing technologies is presented here. Intelligent hybrid systems, which refers to several related computing methods such as fuzzy logic, neural nets, genetic algorithms, and statistical analysis, has become ubiquitous in engineering applications for complex control system modeling and design. Biological pathways may be considered to be complex control systems, which medicine tries to manipulate to achieve desired results. Thus, hybrid intelligent systems may provide a useful tool for modeling biological system dynamics and computational exploration of new drug targets. A new modeling approach based on these methods is presented in the context of hedgehog regulation of the cell cycle in granule cells. Code and input files can be found at the Bionet website: www.chip.ord/~wbosl/Software/Bionet. This paper presents the algorithmic methods needed for modeling complicated biochemical dynamics using rule-based models to represent expert knowledge in the context of cell cycle regulation and tumor growth. A notable feature of this modeling approach is that it allows biologists to build complex models from their knowledge base without the need to translate that knowledge into mathematical form. Dynamics on several levels, from molecular pathways to tissue growth, are seamlessly integrated. A number of common network motifs are examined and used to build a model of hedgehog regulation of the cell cycle in cerebellar neurons, which is believed to play a key role in the etiology of medulloblastoma, a devastating childhood brain cancer.

Oxidative Stress, Redox Regulation and Diseases of Cellular Differentiation

PubMed Central

Ye, Zhi-Wei; Zhang, Jie; Townsend, Danyelle M.; Tew, Kenneth D.

2015-01-01

Background Within cells, there is a narrow concentration threshold that governs whether reactive oxygen species (ROS) induce toxicity or act as second messengers. Scope of review We discuss current understanding of how ROS arise, facilitate cell signaling, cause toxicities and disease related to abnormal cell differentiation and those (primarily) sulfur based pathways that provide nucleophilicity to offset these effects. Primary conclusions Cellular redox homeostasis mediates a plethora of cellular pathways that determine life and death events. For example, ROS intersect with GSH based enzyme pathways to influence cell differentiation, a process integral to normal hematopoiesis, but also affecting a number of diverse cell differentiation related human diseases. Recent attempts to manage such pathologies have focused on intervening in some of these pathways, with the consequence that differentiation therapy targeting redox homeostasis has provided a platform for drug discovery and development. General Significance The balance between electrophilic oxidative stress and protective biomolecular nucleophiles predisposes the evolution of modern life forms. Imbalances of the two can produce aberrant redox homeostasis with resultant pathologies. Understanding the pathways involved provides opportunities to consider interventional strategies. PMID:25445706

Ibrutinib (PCI-32765) in chronic lymphocytic leukemia.

PubMed

Jain, Nitin; O'Brien, Susan

2013-08-01

B-cell receptor (BCR) signaling is essential for chronic lymphocytic leukemia (CLL) cell survival. Many kinases in the BCR signaling pathway are being studied as potential therapeutic targets. Ibrutinib (PCI-32765) is a novel first-in-class selective inhibitor of Bruton tyrosine kinase. Preclinical evidence suggests that ibrutinib inhibits CLL cell survival and proliferation and affects CLL cell migration and homing. Early clinical data in patients with CLL and non-Hodgkin lymphoma is encouraging. It is likely that ibrutinib and other drugs targeting the BCR pathway will become an integral component of CLL therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

Apoptosis by dietary agents for prevention and treatment of prostate cancer

PubMed Central

Khan, Naghma; Adhami, Vaqar Mustafa; Mukhtar, Hasan

2010-01-01

Accumulating data clearly indicate that induction of apoptosis is an important event for chemoprevention of cancer by naturally occurring dietary agents. In mammalian cells, apoptosis has been divided into two major pathways: the extrinsic pathway, activated by pro-apoptotic receptor signals at the cellular surface; and the intrinsic pathway, which involves the disruption of mitochondrial membrane integrity. This process is strictly controlled in response to integrity of pro-death signaling and plays critical roles in development, maintenance of homeostasis, and host defense in multicellular organisms. For chemoprevention studies, prostate cancer (PCa) represents an ideal disease due to its long latency, its high incidence, tumor marker availability, and identifiable preneoplastic lesions and risk groups. In this article, we highlight the studies of various apoptosis-inducing dietary compounds for prevention of PCa in vitro in cell culture, in preclinical studies in animals, and in human clinical trials. PMID:19926708

Dysregulated cellular functions and cell stress pathways provide critical cues for activating and targeting natural killer cells to transformed and infected cells.

PubMed

Raulet, David H; Marcus, Assaf; Coscoy, Laurent

2017-11-01

Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functional analysis of the MAPK pathways in fungi.

PubMed

Martínez-Soto, Domingo; Ruiz-Herrera, José

The Mitogen-Activated Protein Kinase (MAPK) signaling pathways constitute one of the most important and evolutionarily conserved mechanisms for the perception of extracellular information in all the eukaryotic organisms. The MAPK pathways are involved in the transfer to the cell of the information perceived from extracellular stimuli, with the final outcome of activation of different transcription factors that regulate gene expression in response to them. In all species of fungi, the MAPK pathways have important roles in their physiology and development; e.g. cell cycle control, mating, morphogenesis, response to different stresses, resistance to UV radiation and to temperature changes, cell wall assembly and integrity, degradation of cellular organelles, virulence, cell-cell signaling, fungus-plant interaction, and response to damage-associated molecular patterns (DAMPs). Considering the importance of the phylogenetically conserved MAPK pathways in fungi, an updated review of the knowledge on them is discussed in this article. This information reveals their importance, their distribution in fungal species evolutionarily distant and with different lifestyles, their organization and function, and the interactions occurring between different MAPK pathways, and with other signaling pathways, for the regulation of the most complex cellular processes. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

PubMed

Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

2011-02-17

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics. Copyright © 2011 Elsevier Inc. All rights reserved.

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

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

Lee, Kiwon; Lee, Ah-Young; Kwon, Yunhee Kim

Highlights: {yields} Integration of HPV into host genome critical for activation of E6 and E7 oncogenes. {yields} BAF53 is essential for higher-order chromatin structure. {yields} BAF53 knockdown suppresses E6 and E7 from HPV integrants, but not from episomal HPVs. {yields} BAF53 knockdown decreases H3K9Ac and H4K12Ac on P105 promoter of integrated HPV 18. {yields} BAF53 knockdown restores the p53-dependent signaling pathway in HeLa and SiHa cells. -- Abstract: Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways,more » respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.« less

Porous AgPt@Pt Nanooctahedra as an Efficient Catalyst toward Formic Acid Oxidation with Predominant Dehydrogenation Pathway.

PubMed

Jiang, Xian; Yan, Xiaoxiao; Ren, Wangyu; Jia, Yufeng; Chen, Jianian; Sun, Dongmei; Xu, Lin; Tang, Yawen

2016-11-16

For direct formic acid fuel cells (DFAFCs), the dehydrogenation pathway is a desired reaction pathway, to boost the overall cell efficiency. Elaborate composition tuning and nanostructure engineering provide two promising strategies to design efficient electrocatalysts for DFAFCs. Herein, we present a facile synthesis of porous AgPt bimetallic nanooctahedra with enriched Pt surface (denoted as AgPt@Pt nanooctahedra) by a selective etching strategy. The smart integration of geometric and electronic effect confers a substantial enhancement of desired dehydrogenation pathway as well as electro-oxidation activity for the formic acid oxidation reaction (FAOR). We anticipate that the obtained nanocatalyst may hold great promises in fuel cell devices, and furthermore, the facile synthetic strategy demonstrated here can be extendable for the fabrication of other multicomponent nanoalloys with desirable morphologies and enhanced electrocatalytic performances.

Branches of the NF-κB signaling pathway regulate proliferation of oval cells in rat liver regeneration.

PubMed

Zhao, W M; Qin, Y L; Niu, Z P; Chang, C F; Yang, J; Li, M H; Zhou, Y; Xu, C S

2016-03-24

The NF-kB (nuclear factor kB) pathway is involved in the proliferation of many cell types. To explore the mechanism of the NF-kB signaling pathway underlying the oval cell proliferation during rat liver regeneration, the Rat Genome 230 2.0 Array was used to detect expression changes of NF-kB signaling pathway-related genes in oval cells. The results revealed that the expression levels of many genes in the NF-kB pathway were significantly changed. This included 48 known genes and 16 homologous genes, as well as 370 genes and 85 homologous genes related to cell proliferation. To further understand the biological significance of these changes, an expression profile function was used to analyze the potential biological processes. The results showed that the NF-kB pathway promoted oval cell proliferation mainly through three signaling branches; the tumor necrosis factor alpha branch (TNF-a pathway), the growth factor branch, and the chemokine branch. An integrated statistics method was used to define the key genes in the NF-kB pathway. Seven genes were identified to play vital roles in the NF-kB pathway. To confirm these results, the protein content, including two key genes (TNF and FGF11) and two non-key genes (CCL2 and TNFRSF12A), were analyzed using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. The results were generally consistent with those of the array data. To conclude, three branches and seven key genes were involved in the NF-kB signaling pathway that regulates oval cell proliferation during rat liver regeneration.

The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

PubMed Central

Maerz, Sabine; Ziv, Carmit; Vogt, Nico; Helmstaedt, Kerstin; Cohen, Nourit; Gorovits, Rena; Yarden, Oded; Seiler, Stephan

2008-01-01

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development. PMID:18562669

Mitochondrial AKAP1 supports mTOR pathway and tumor growth.

PubMed

Rinaldi, Laura; Sepe, Maria; Delle Donne, Rossella; Conte, Kristel; Arcella, Antonietta; Borzacchiello, Domenica; Amente, Stefano; De Vita, Fernanda; Porpora, Monia; Garbi, Corrado; Oliva, Maria A; Procaccini, Claudio; Faicchia, Deriggio; Matarese, Giuseppe; Zito Marino, Federica; Rocco, Gaetano; Pignatiello, Sara; Franco, Renato; Insabato, Luigi; Majello, Barbara; Feliciello, Antonio

2017-06-01

Mitochondria are the powerhouses of energy production and the sites where metabolic pathway and survival signals integrate and focus, promoting adaptive responses to hormone stimulation and nutrient availability. Increasing evidence suggests that mitochondrial bioenergetics, metabolism and signaling are linked to tumorigenesis. AKAP1 scaffolding protein integrates cAMP and src signaling on mitochondria, regulating organelle biogenesis, oxidative metabolism and cell survival. Here, we provide evidence that AKAP1 is a transcriptional target of Myc and supports the growth of cancer cells. We identify Sestrin2, a leucine sensor and inhibitor of the mammalian target of rapamycin (mTOR), as a novel component of the complex assembled by AKAP1 on mitochondria. Downregulation of AKAP1 impaired mTOR pathway and inhibited glioblastoma growth. Both effects were reversed by concomitant depletion of AKAP1 and sestrin2. High levels of AKAP1 were found in a wide variety of high-grade cancer tissues. In lung cancer, AKAP1 expression correlates with high levels of Myc, mTOR phosphorylation and reduced patient survival. Collectively, these data disclose a previously unrecognized role of AKAP1 in mTOR pathway regulation and cancer growth. AKAP1/mTOR signal integration on mitochondria may provide a new target for cancer therapy.

Could drugs inhibiting the mevalonate pathway also target cancer stem cells?

PubMed

Likus, Wirginia; Siemianowicz, Krzysztof; Bieńk, Konrad; Pakuła, Małgorzata; Pathak, Himani; Dutta, Chhanda; Wang, Qiong; Shojaei, Shahla; Assaraf, Yehuda G; Ghavami, Saeid; Cieślar-Pobuda, Artur; Łos, Marek J

2016-03-01

Understanding the connection between metabolic pathways and cancer is very important for the development of new therapeutic approaches based on regulatory enzymes in pathways associated with tumorigenesis. The mevalonate cascade and its rate-liming enzyme HMG CoA-reductase has recently drawn the attention of cancer researchers because strong evidences arising mostly from epidemiologic studies, show that it could promote transformation. Hence, these studies pinpoint HMG CoA-reductase as a candidate proto-oncogene. Several recent epidemiological studies, in different populations, have proven that statins are beneficial for the treatment-outcome of various cancers, and may improve common cancer therapy strategies involving alkylating agents, and antimetabolites. Cancer stem cells/cancer initiating cells (CSC) are key to cancer progression and metastasis. Therefore, in the current review we address the different effects of statins on cancer stem cells. The mevalonate cascade is among the most pleiotropic, and highly interconnected signaling pathways. Through G-protein-coupled receptors (GRCP), it integrates extra-, and intracellular signals. The mevalonate pathway is implicated in cell stemness, cell proliferation, and organ size regulation through the Hippo pathway (e.g. Yap/Taz signaling axis). This pathway is a prime preventive target through the administration of statins for the prophylaxis of obesity-related cardiovascular diseases. Its prominent role in regulation of cell growth and stemness also invokes its role in cancer development and progression. The mevalonate pathway affects cancer metastasis in several ways by: (i) affecting epithelial-to-mesenchymal transition (EMT), (ii) affecting remodeling of the cytoskeleton as well as cell motility, (iii) affecting cell polarity (non-canonical Wnt/planar pathway), and (iv) modulation of mesenchymal-to-epithelial transition (MET). Herein we provide an overview of the mevalonate signaling network. We then briefly highlight diverse functions of various elements of this mevalonate pathway. We further discuss in detail the role of elements of the mevalonate cascade in stemness, carcinogenesis, cancer progression, metastasis and maintenance of cancer stem cells. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses.

PubMed

Konstantoulas, Constantine James; Indik, Stanislav

2014-04-30

Mouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and then spreads to T and B lymphocytes and finally to the mammary gland. It is not clear how the prototypic betaretrovirus infects mucosal DCs and naïve lymphocytes as these cells are considered to be non-proliferative. Studies of MMTV biology have been hampered by the difficulty of obtaining sufficient virus/vector titers after transfection of a molecular clone in cultured cells. To surmount this barrier we developed a novel MMTV-based vector system with a split genome design containing potent posttranscriptional regulatory functions. Using this system, vector particles were produced to markedly greater titers (>1000-fold) than those obtained previously. The titers (>106 transduction units /ml) were comparable to those achieved with lentiviral or gammaretroviral vectors. Importantly, the vector transduced the enhanced green fluorescence protein gene into the chromosomes of non-dividing cells, such as cells arrested at the G2/M phase of the cell cycle and unstimulated hematopoietic progenitor cells, at an efficiency similar to that obtained with the HIV-1-based vector. In contrast to HIV-1, MMTV transductions were not affected by knocking down the expression of a factor involved in nuclear import of the HIV-1 pre-integration complexes, TNPO3. In contrast to HIV-1, the MMTV-based vector did not preferentially integrate in transcription units. Additionally, no preference for integration near transcription start sites, the regions preferentially targeted by gammaretroviral vectors, was observed. The vector derived from MMTV exhibits a random integration pattern. Overall, the betaretroviral vector system should facilitate molecular virology studies of the prototypic betaretrovirus as well as studies attempting to elucidate fundamental cellular processes such as nuclear import pathways. Random integration in cycling and non-cycling cells may be applicable in unbiased gene delivery.

Parallel evolution of Nitric Oxide signaling: Diversity of synthesis & memory pathways

PubMed Central

Moroz, Leonid L.; Kohn, Andrea B.

2014-01-01

The origin of NO signaling can be traceable back to the origin of life with the large scale of parallel evolution of NO synthases (NOSs). Inducible-like NOSs may be the most basal prototype of all NOSs and that neuronal-like NOS might have evolved several times from this prototype. Other enzymatic and non-enzymatic pathways for NO synthesis have been discovered using reduction of nitrites, an alternative source of NO. Diverse synthetic mechanisms can co-exist within the same cell providing a complex NO-oxygen microenvironment tightly coupled with cellular energetics. The dissection of multiple sources of NO formation is crucial in analysis of complex biological processes such as neuronal integration and learning mechanisms when NO can act as a volume transmitter within memory-forming circuits. In particular, the molecular analysis of learning mechanisms (most notably in insects and gastropod molluscs) opens conceptually different perspectives to understand the logic of recruiting evolutionarily conserved pathways for novel functions. Giant uniquely identified cells from Aplysia and related species precent unuque opportunities for integrative analysis of NO signaling at the single cell level. PMID:21622160

Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2013-01-01

A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways. PMID:23739767

Integrative Approaches to Enhance Understanding of Plant Metabolic Pathway Structure and Regulation1

PubMed Central

Tohge, Takayuki; Scossa, Federico; Fernie, Alisdair R.

2015-01-01

Huge insight into molecular mechanisms and biological network coordination have been achieved following the application of various profiling technologies. Our knowledge of how the different molecular entities of the cell interact with one another suggests that, nevertheless, integration of data from different techniques could drive a more comprehensive understanding of the data emanating from different techniques. Here, we provide an overview of how such data integration is being used to aid the understanding of metabolic pathway structure and regulation. We choose to focus on the pairwise integration of large-scale metabolite data with that of the transcriptomic, proteomics, whole-genome sequence, growth- and yield-associated phenotypes, and archival functional genomic data sets. In doing so, we attempt to provide an update on approaches that integrate data obtained at different levels to reach a better understanding of either single gene function or metabolic pathway structure and regulation within the context of a broader biological process. PMID:26371234

Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways.

PubMed

Mladenov, Emil; Iliakis, George

2011-06-03

A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis. 2011 Elsevier B.V. All rights reserved.

Autophagy and genomic integrity

PubMed Central

Vessoni, A T; Filippi-Chiela, E C; Menck, C FM; Lenz, G

2013-01-01

DNA lesions, constantly produced by endogenous and exogenous sources, activate the DNA damage response (DDR), which involves detection, signaling and repair of the damage. Autophagy, a lysosome-dependent degradation pathway that is activated by stressful situations such as starvation and oxidative stress, regulates cell fate after DNA damage and also has a pivotal role in the maintenance of nuclear and mitochondrial genomic integrity. Here, we review important evidence regarding the role played by autophagy in preventing genomic instability and tumorigenesis, as well as in micronuclei degradation. Several pathways governing autophagy activation after DNA injury and the influence of autophagy upon the processing of genomic lesions are also discussed herein. In this line, the mechanisms by which several proteins participate in both DDR and autophagy, and the importance of this crosstalk in cancer and neurodegeneration will be presented in an integrated fashion. At last, we present a hypothetical model of the role played by autophagy in dictating cell fate after genotoxic stress. PMID:23933813

Integrative genomic profiling reveals conserved genetic mechanisms for tumorigenesis in common entities of non-Hodgkin's lymphoma.

PubMed

Green, Michael R; Aya-Bonilla, Carlos; Gandhi, Maher K; Lea, Rod A; Wellwood, Jeremy; Wood, Peter; Marlton, Paula; Griffiths, Lyn R

2011-05-01

Recent developments in genomic technologies have resulted in increased understanding of pathogenic mechanisms and emphasized the importance of central survival pathways. Here, we use a novel bioinformatic based integrative genomic profiling approach to elucidate conserved mechanisms of lymphomagenesis in the three commonest non-Hodgkin's lymphoma (NHL) entities: diffuse large B-cell lymphoma, follicular lymphoma, and B-cell chronic lymphocytic leukemia. By integrating genome-wide DNA copy number analysis and transcriptome profiling of tumor cohorts, we identified genetic lesions present in each entity and highlighted their likely target genes. This revealed a significant enrichment of components of both the apoptosis pathway and the mitogen activated protein kinase pathway, including amplification of the MAP3K12 locus in all three entities, within the set of genes targeted by genetic alterations in these diseases. Furthermore, amplification of 12p13.33 was identified in all three entities and found to target the FOXM1 oncogene. Amplification of FOXM1 was subsequently found to be associated with an increased MYC oncogenic signaling signature, and siRNA-mediated knock-down of FOXM1 resulted in decreased MYC expression and induced G2 arrest. Together, these findings underscore genetic alteration of the MAPK and apoptosis pathways, and genetic amplification of FOXM1 as conserved mechanisms of lymphomagenesis in common NHL entities. Integrative genomic profiling identifies common central survival mechanisms and highlights them as attractive targets for directed therapy. 2011 Wiley-Liss, Inc.

Integrative ChIP-seq/Microarray Analysis Identifies a CTNNB1 Target Signature Enriched in Intestinal Stem Cells and Colon Cancer

PubMed Central

Watanabe, Kazuhide; Biesinger, Jacob; Salmans, Michael L.; Roberts, Brian S.; Arthur, William T.; Cleary, Michele; Andersen, Bogi; Xie, Xiaohui; Dai, Xing

2014-01-01

Background Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. Results We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. Conclusion Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells. PMID:24651522

Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

PubMed

Watanabe, Kazuhide; Biesinger, Jacob; Salmans, Michael L; Roberts, Brian S; Arthur, William T; Cleary, Michele; Andersen, Bogi; Xie, Xiaohui; Dai, Xing

2014-01-01

Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair

PubMed Central

He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

2016-01-01

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. PMID:26850641

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

PubMed

He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

2016-05-19

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

PubMed

Hanning, Jennifer E; Saini, Harpreet K; Murray, Matthew J; Caffarel, Maria M; van Dongen, Stijn; Ward, Dawn; Barker, Emily M; Scarpini, Cinzia G; Groves, Ian J; Stanley, Margaret A; Enright, Anton J; Pett, Mark R; Coleman, Nicholas

2013-11-01

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to d-Limonene Show Changes to the Cell Wall but Not to the Plasma Membrane

PubMed Central

Brennan, Timothy C. R.; Nielsen, Lars K.

2013-01-01

Monoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae, has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d-limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes (ROM1, RLM1, PIR3, CTT1, YGP1, MLP1, PST1, and CWP1) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis. PMID:23542628

Osmotic Stress Signaling and Osmoadaptation in Yeasts

PubMed Central

Hohmann, Stefan

2002-01-01

The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects. PMID:12040128

Signaling mechanisms regulating adult neural stem cells and neurogenesis

PubMed Central

Faigle, Roland; Song, Hongjun

2012-01-01

Background Adult neurogenesis occurs throughout life in discrete regions of the mammalian brain and is tightly regulated via both extrinsic environmental influences and intrinsic genetic factors. In recent years, several crucial signaling pathways have been identified in regulating self-renewal, proliferation, and differentiation of neural stem cells, as well as migration and functional integration of developing neurons in the adult brain. Scope of review Here we review our current understanding of signaling mechanisms, including Wnt, notch, sonic hedgehog, growth and neurotrophic factors, bone morphogenetic proteins, neurotransmitters, transcription factors, and epigenetic modulators, and crosstalk between these signaling pathways in the regulation of adult neurogenesis. We also highlight emerging principles in the vastly growing field of adult neural stem cell biology and neural plasticity. Major conclusions Recent methodological advances have enabled the field to identify signaling mechanisms that fine-tune and coordinate neurogenesis in the adult brain, leading to a better characterization of both cell-intrinsic and environmental cues defining the neurogenic niche. Significant questions related to niche cell identity and underlying regulatory mechanisms remain to be fully addressed and will be the focus of future studies. General significance A full understanding of the role and function of individual signaling pathways in regulating neural stem cells and generation and integration of newborn neurons in the adult brain may lead to targeted new therapies for neurological diseases in humans. PMID:22982587

Phospholipase D and the Maintenance of Phosphatidic Acid Levels for Regulation of Mammalian Target of Rapamycin (mTOR)*

PubMed Central

Foster, David A.; Salloum, Darin; Menon, Deepak; Frias, Maria A.

2014-01-01

Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival. PMID:24990952

Phospholipase D and the maintenance of phosphatidic acid levels for regulation of mammalian target of rapamycin (mTOR).

PubMed

Foster, David A; Salloum, Darin; Menon, Deepak; Frias, Maria A

2014-08-15

Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

PubMed

Kim, Sang Hoon; Pajarillo, Edward Alain B; Balolong, Marilen P; Lee, Ji Yoon; Kang, Dae-Kyung

2016-06-28

In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

Comparison of human cell signaling pathway databases—evolution, drawbacks and challenges

PubMed Central

Chowdhury, Saikat; Sarkar, Ram Rup

2015-01-01

Elucidating the complexities of cell signaling pathways is of immense importance to gain understanding about various biological phenomenon, such as dynamics of gene/protein expression regulation, cell fate determination, embryogenesis and disease progression. The successful completion of human genome project has also helped experimental and theoretical biologists to analyze various important pathways. To advance this study, during the past two decades, systematic collections of pathway data from experimental studies have been compiled and distributed freely by several databases, which also integrate various computational tools for further analysis. Despite significant advancements, there exist several drawbacks and challenges, such as pathway data heterogeneity, annotation, regular update and automated image reconstructions, which motivated us to perform a thorough review on popular and actively functioning 24 cell signaling databases. Based on two major characteristics, pathway information and technical details, freely accessible data from commercial and academic databases are examined to understand their evolution and enrichment. This review not only helps to identify some novel and useful features, which are not yet included in any of the databases but also highlights their current limitations and subsequently propose the reasonable solutions for future database development, which could be useful to the whole scientific community. PMID:25632107

A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation

PubMed Central

Kozhemyakina, Elena; Lassar, Andrew B.; Zelzer, Elazar

2015-01-01

Decades of work have identified the signaling pathways that regulate the differentiation of chondrocytes during bone formation, from their initial induction from mesenchymal progenitor cells to their terminal maturation into hypertrophic chondrocytes. Here, we review how multiple signaling molecules, mechanical signals and morphological cell features are integrated to activate a set of key transcription factors that determine and regulate the genetic program that induces chondrogenesis and chondrocyte differentiation. Moreover, we describe recent findings regarding the roles of several signaling pathways in modulating the proliferation and maturation of chondrocytes in the growth plate, which is the ‘engine’ of bone elongation. PMID:25715393

Growth Substrate- and Phase-Specific Expression of Biphenyl, Benzoate, and C1 Metabolic Pathways in Burkholderia xenovorans LB400

PubMed Central

Denef, V. J.; Patrauchan, M. A.; Florizone, C.; Park, J.; Tsoi, T. V.; Verstraete, W.; Tiedje, J. M.; Eltis, L. D.

2005-01-01

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C1 metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were ∼16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (boxC) pathway was also expressed during growth on biphenyl: BoxC proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (boxM) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C1 metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain. PMID:16291673

Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model

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

Fukunaga, Satoki; Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-Naka, Konohana-ku, Osaka 554-8558; Kakehashi, Anna

To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective ofmore » initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis. - Highlights: • We analyzed bleomycin-induced pulmonary fibrosis in the rat. • Integrative analyses of miRNA microarray and proteomics were conducted. • We determined the alterations of miRNAs and their potential target proteins. • The alterations may control biological functions and pathways in pulmonary fibrosis. • Our result may provide new insights of pulmonary fibrosis.« less

BiologicalNetworks 2.0 - an integrative view of genome biology data

PubMed Central

2010-01-01

Background A significant problem in the study of mechanisms of an organism's development is the elucidation of interrelated factors which are making an impact on the different levels of the organism, such as genes, biological molecules, cells, and cell systems. Numerous sources of heterogeneous data which exist for these subsystems are still not integrated sufficiently enough to give researchers a straightforward opportunity to analyze them together in the same frame of study. Systematic application of data integration methods is also hampered by a multitude of such factors as the orthogonal nature of the integrated data and naming problems. Results Here we report on a new version of BiologicalNetworks, a research environment for the integral visualization and analysis of heterogeneous biological data. BiologicalNetworks can be queried for properties of thousands of different types of biological entities (genes/proteins, promoters, COGs, pathways, binding sites, and other) and their relations (interactions, co-expression, co-citations, and other). The system includes the build-pathways infrastructure for molecular interactions/relations and module discovery in high-throughput experiments. Also implemented in BiologicalNetworks are the Integrated Genome Viewer and Comparative Genomics Browser applications, which allow for the search and analysis of gene regulatory regions and their conservation in multiple species in conjunction with molecular pathways/networks, experimental data and functional annotations. Conclusions The new release of BiologicalNetworks together with its back-end database introduces extensive functionality for a more efficient integrated multi-level analysis of microarray, sequence, regulatory, and other data. BiologicalNetworks is freely available at http://www.biologicalnetworks.org. PMID:21190573

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells.

PubMed

Tooze, J; Tooze, S A; Fuller, S D

1987-09-01

Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.

SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae.

PubMed

Vanegas, Katherina García; Lehka, Beata Joanna; Mortensen, Uffe Hasbro

2017-02-08

The yeast Saccharomyces cerevisiae is increasingly used as a cell factory. However, cell factory construction time is a major obstacle towards using yeast for bio-production. Hence, tools to speed up cell factory construction are desirable. In this study, we have developed a new Cas9/dCas9 based system, SWITCH, which allows Saccharomyces cerevisiae strains to iteratively alternate between a genetic engineering state and a pathway control state. Since Cas9 induced recombination events are crucial for SWITCH efficiency, we first developed a technique TAPE, which we have successfully used to address protospacer efficiency. As proof of concept of the use of SWITCH in cell factory construction, we have exploited the genetic engineering state of a SWITCH strain to insert the five genes necessary for naringenin production. Next, the naringenin cell factory was switched to the pathway control state where production was optimized by downregulating an essential gene TSC13, hence, reducing formation of a byproduct. We have successfully integrated two CRISPR tools, one for genetic engineering and one for pathway control, into one system and successfully used it for cell factory construction.

Mutational Analysis of the Glycosylphosphatidylinositol (GPI) Anchor Pathway Demonstrates that GPI-Anchored Proteins Are Required for Cell Wall Biogenesis and Normal Hyphal Growth in Neurospora crassa

PubMed Central

Bowman, Shaun M.; Piwowar, Amy; Al Dabbous, Mash'el; Vierula, John; Free, Stephen J.

2006-01-01

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal “cell-within-a-cell” phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa. PMID:16524913

Outside-in control -Does plant cell wall integrity regulate cell cycle progression?

PubMed

Gigli-Bisceglia, Nora; Hamann, Thorsten

2018-04-13

During recent years it has become accepted that plant cell walls are not inert objects surrounding all plant cells but are instead highly dynamic, plastic structures. They are involved in a large number of cell biological processes and contribute actively to plant growth, development and interaction with environment. Therefore, it is not surprising that cellular processes can control plant cell wall integrity while, simultaneously, cell wall integrity can influence cellular processes. In yeast and animal cells such a bi-directional relationship also exists between the yeast/animal extra-cellular matrices and the cell cycle. In yeast, the cell wall integrity maintenance mechanism and a dedicated plasmamembrane integrity checkpoint are mediating this relationship. Recent research has yielded insights into the mechanism controlling plant cell wall metabolism during cytokinesis. However, knowledge regarding putative regulatory pathways controlling adaptive modifications in plant cell cycle activity in response to changes in the state of the plant cell wall are not yet identified. In this review, we summarize similarities and differences in regulatory mechanisms coordinating extra cellular matrices and cell cycle activity in animal and yeast cells, discuss the available evidence supporting the existence of such a mechanism in plants and suggest that the plant cell wall integrity maintenance mechanism might also control cell cycle activity in plant cells. This article is protected by copyright. All rights reserved.

Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

EPA Science Inventory

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

Role of the ceramide-signaling pathways in ionizing radiation-induced apoptosis.

PubMed

Vit, Jean-Philippe; Rosselli, Filippo

2003-11-27

Ionizing radiations (IR) exposure leads to damage on several cellular targets. How signals from different targets are integrated to determine the cell fate remains a controversial issue. Understanding the pathway(s) responsible(s) for the cell killing effect of the IR exposure is of prime importance in light of using radiations as anticancer agent or as diagnostic tool. In this study, we have established that IR-induced cell damage initiates two independent signaling pathways that lead to a biphasic intracellular ceramide increase. A transitory increase of ceramide is observed within minutes after IR exposure as a consequence of DNA damage-independent acid sphingomyelinase activation. Several hours after irradiation, a second wave of ceramide accumulation is observed depending on the DNA damage-dependent activation of ceramide synthase, which requires a signaling pathway involving ATM. Importantly, we have demonstrated that the late ceramide accumulation is also dependent on the first one and is rate limiting for the apoptotic process induced by IR. In conclusion, our observations suggest that ceramide is a major determinant of the IR-induced apoptotic process at the cross-point of different signal transduction pathways.

Lessons Learned From Trials Targeting Cytokine Pathways in Patients With Inflammatory Bowel Diseases

PubMed Central

Abraham, Clara; Dulai, Parambir S.; Vermeire, Séverine; Sandborn, William J.

2016-01-01

Insights into the pathogenesis of inflammatory bowel diseases (IBD) have provided important information for the development of therapeutics. Levels of interleukin 23 (IL23) and T-helper (Th) 17 cell pathway molecules are elevated in inflamed intestinal tissues of patients with IBD. Loss of function variants of the interleukin 23 receptor gene (IL23R) protect against IBD, and in animals, blocking IL23 reduces severity of colitis. These findings indicated that the IL23 and Th17 cell pathways might be promising targets for treatment of IBD. Clinical trials have investigated the effects of agents designed to target distinct levels of the IL23 and Th17 cell pathways, and the results are providing insights into IBD pathogenesis and additional strategies for modulating these pathways. Strategies to reduce levels of proinflammatory cytokines more broadly and increase anti-inflammatory mechanisms are also emerging for treatment of IBD. The results from trials targeting these immune system pathways have provided important lessons for future trials. Findings indicate the importance of improving approaches to integrate patient features and biomarkers of response with selection of therapeutics. PMID:27780712

The high-osmolarity glycerol- and cell wall integrity-MAP kinase pathways of Saccharomyces cerevisiae are involved in adaptation to the action of killer toxin HM-1.

PubMed

Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

2012-11-01

Fps1p is an aquaglyceroporin important for turgor regulation of Saccharomyces cerevisiae. Previously we reported the involvement of Fps1p in the yeast-killing action of killer toxin HM-1. The fps1 cells showed a high HM-1-resistant phenotype in hypotonic medium and an HM-1-susceptible phenotype in hypertonic medium. This osmotic dependency in HM-1 susceptibility was similar to those observed in Congo red, but different from those observed in other cell wall-disturbing agents. These results indicate that HM-1 exerts fungicidal activity mainly by binding and inserting into the yeast cell wall structure, rather than by inhibiting 1,3-β-glucan synthase. We next determined HM-1-susceptibility and diphospho-MAP kinase inductions in S. cerevisiae. In the wild-type cell, expressions of diphospho-Hog1p and -Slt2p, and mRNA transcription of CWP1 and HOR2, were induced within 1 h after an addition of HM-1. ssk1 and pbs2 cells, but not sho1 and hkr1 cells, showed HM-1-sensitive phenotypes and lacked inductions of phospho-Hog1p in response to HM-1. mid2, rom2 and bck1 cells showed HM-1-sensitive phenotypes and decreased inductions of phospho-Slt2p in response to HM-1. From these results, we postulated that the Sln1-Ypd1-Ssk1 branch of the high-osmolality glycerol (HOG) pathway and plasma membrane sensors of the cell wall integrity (CWI) pathway detect cell wall stresses caused by HM-1. We further suggested that activations of both HOG and CWI pathways have an important role in the adaptive response to HM-1 toxicity. Copyright © 2012 John Wiley & Sons, Ltd.

Corrupting the DNA damage response: a critical role for Rad52 in tumor cell survival.

PubMed

Lieberman, Rachel; You, Ming

2017-07-15

The DNA damage response enables cells to survive, maintain genome integrity, and to safeguard the transmission of high-fidelity genetic information. Upon sensing DNA damage, cells respond by activating this multi-faceted DNA damage response leading to restoration of the cell, senescence, programmed cell death, or genomic instability if the cell survives without proper repair. However, unlike normal cells, cancer cells maintain a marked level of genomic instability. Because of this enhanced propensity to accumulate DNA damage, tumor cells rely on homologous recombination repair as a means of protection from the lethal effect of both spontaneous and therapy-induced double-strand breaks (DSBs) in DNA. Thus, modulation of DNA repair pathways have important consequences for genomic instability within tumor cell biology and viability maintenance under high genotoxic stress. Efforts are underway to manipulate specific components of the DNA damage response in order to selectively induce tumor cell death by augmenting genomic instability past a viable threshold. New evidence suggests that RAD52, a component of the homologous recombination pathway, is important for the maintenance of tumor genome integrity. This review highlights recent reports indicating that reducing homologous recombination through inhibition of RAD52 may represent an important focus for cancer therapy and the specific efforts that are already demonstrating potential.

Simulating cyanobacterial phenotypes by integrating flux balance analysis, kinetics, and a light distribution function

DOE PAGES

He, Lian; Wu, Stephen G.; Wan, Ni; ...

2015-12-24

In this study, genome-scale models (GSMs) are widely used to predict cyanobacterial phenotypes in photobioreactors (PBRs). However, stoichiometric GSMs mainly focus on fluxome that result in maximal yields. Cyanobacterial metabolism is controlled by both intracellular enzymes and photobioreactor conditions. To connect both intracellular and extracellular information and achieve a better understanding of PBRs productivities, this study integrates a genome-scale metabolic model of Synechocystis 6803 with growth kinetics, cell movements, and a light distribution function. The hybrid platform not only maps flux dynamics in cells of sub-populations but also predicts overall production titer and rate in PBRs. Analysis of the integratedmore » GSM demonstrates several results. First, cyanobacteria are capable of reaching high biomass concentration (>20 g/L in 21 days) in PBRs without light and CO 2 mass transfer limitations. Second, fluxome in a single cyanobacterium may show stochastic changes due to random cell movements in PBRs. Third, insufficient light due to cell self-shading can activate the oxidative pentose phosphate pathway in subpopulation cells. Fourth, the model indicates that the removal of glycogen synthesis pathway may not improve cyanobacterial bio-production in large-size PBRs, because glycogen can support cell growth in the dark zones. Based on experimental data, the integrated GSM estimates that Synechocystis 6803 in shake flask conditions has a photosynthesis efficiency of ~2.7 %. Conclusions: The multiple-scale integrated GSM, which examines both intracellular and extracellular domains, can be used to predict production yield/rate/titer in large-size PBRs. More importantly, genetic engineering strategies predicted by a traditional GSM may work well only in optimal growth conditions. In contrast, the integrated GSM may reveal mutant physiologies in diverse bioreactor conditions, leading to the design of robust strains with high chances of success in industrial settings.« less

Regulation of Muscle Stem Cell Functions: A Focus on the p38 MAPK Signaling Pathway

PubMed Central

Segalés, Jessica; Perdiguero, Eusebio; Muñoz-Cánoves, Pura

2016-01-01

Formation of skeletal muscle fibers (myogenesis) during development and after tissue injury in the adult constitutes an excellent paradigm to investigate the mechanisms whereby environmental cues control gene expression programs in muscle stem cells (satellite cells) by acting on transcriptional and epigenetic effectors. Here we will review the molecular mechanisms implicated in the transition of satellite cells throughout the distinct myogenic stages (i.e., activation from quiescence, proliferation, differentiation, and self-renewal). We will also discuss recent findings on the causes underlying satellite cell functional decline with aging. In particular, our review will focus on the epigenetic changes underlying fate decisions and on how the p38 MAPK signaling pathway integrates the environmental signals at the chromatin to build up satellite cell adaptive responses during the process of muscle regeneration, and how these responses are altered in aging. A better comprehension of the signaling pathways connecting external and intrinsic factors will illuminate the path for improving muscle regeneration in the aged. PMID:27626031

Defining Causative Factors Contributing in the Activation of Hedgehog Signaling in Diffuse Large B-Cell Lymphoma

PubMed Central

Ramirez, Elisa; Singh, Rajesh R; Kunkalla, Kranthi; Liu, Yadong; Qu, Changju; Cain, Christine; Multani, Asha S.; Lennon, Patrick A; Jackacky, Jared; Ho, Michael; Dawud, Sity; Gu, Jun; Yang, Su; Hu, Peter C; Vega, Francisco

2012-01-01

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-KB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling. PMID:22809693

Bioreactors to Influence Stem Cell Fate: Augmentation of Mesenchymal Stem Cell Signaling Pathways via Dynamic Culture Systems

PubMed Central

Yeatts, Andrew B.; Choquette, Daniel T.; Fisher, John P.

2012-01-01

Background Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro. Scope of Review This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems. Major Conclusions The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased. General Significance Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. PMID:22705676

Cell signaling heterogeneity is modulated by both cell-intrinsic and -extrinsic mechanisms: An integrated approach to understanding targeted therapy.

PubMed

Kim, Eunjung; Kim, Jae-Young; Smith, Matthew A; Haura, Eric B; Anderson, Alexander R A

2018-03-01

During the last decade, our understanding of cancer cell signaling networks has significantly improved, leading to the development of various targeted therapies that have elicited profound but, unfortunately, short-lived responses. This is, in part, due to the fact that these targeted therapies ignore context and average out heterogeneity. Here, we present a mathematical framework that addresses the impact of signaling heterogeneity on targeted therapy outcomes. We employ a simplified oncogenic rat sarcoma (RAS)-driven mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway in lung cancer as an experimental model system and develop a network model of the pathway. We measure how inhibition of the pathway modulates protein phosphorylation as well as cell viability under different microenvironmental conditions. Training the model on this data using Monte Carlo simulation results in a suite of in silico cells whose relative protein activities and cell viability match experimental observation. The calibrated model predicts distributional responses to kinase inhibitors and suggests drug resistance mechanisms that can be exploited in drug combination strategies. The suggested combination strategies are validated using in vitro experimental data. The validated in silico cells are further interrogated through an unsupervised clustering analysis and then integrated into a mathematical model of tumor growth in a homogeneous and resource-limited microenvironment. We assess posttreatment heterogeneity and predict vast differences across treatments with similar efficacy, further emphasizing that heterogeneity should modulate treatment strategies. The signaling model is also integrated into a hybrid cellular automata (HCA) model of tumor growth in a spatially heterogeneous microenvironment. As a proof of concept, we simulate tumor responses to targeted therapies in a spatially segregated tissue structure containing tumor and stroma (derived from patient tissue) and predict complex cell signaling responses that suggest a novel combination treatment strategy.

Immunoinformatics Features Linked to Leishmania Vaccine Development: Data Integration of Experimental and In Silico Studies

PubMed Central

Brito, Rory C. F.; Guimarães, Frederico G.; Velloso, João P. L.; Corrêa-Oliveira, Rodrigo; Ruiz, Jeronimo C.; Reis, Alexandre B.; Resende, Daniela M.

2017-01-01

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. There is no human vaccine available and it is considered by many studies as apotential effective tool for disease control. To discover novel antigens, computational programs have been used in reverse vaccinology strategies. In this work, we developed a validation antigen approach that integrates prediction of B and T cell epitopes, analysis of Protein-Protein Interaction (PPI) networks and metabolic pathways. We selected twenty candidate proteins from Leishmania tested in murine model, with experimental outcome published in the literature. The predictions for CD4+ and CD8+ T cell epitopes were correlated with protection in experimental outcomes. We also mapped immunogenic proteins on PPI networks in order to find Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with them. Our results suggest that non-protective antigens have lowest frequency of predicted T CD4+ and T CD8+ epitopes, compared with protective ones. T CD4+ and T CD8+ cells are more related to leishmaniasis protection in experimental outcomes than B cell predicted epitopes. Considering KEGG analysis, the proteins considered protective are connected to nodes with few pathways, including those associated with ribosome biosynthesis and purine metabolism. PMID:28208616

Immunoinformatics Features Linked to Leishmania Vaccine Development: Data Integration of Experimental and In Silico Studies.

PubMed

Brito, Rory C F; Guimarães, Frederico G; Velloso, João P L; Corrêa-Oliveira, Rodrigo; Ruiz, Jeronimo C; Reis, Alexandre B; Resende, Daniela M

2017-02-10

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. There is no human vaccine available and it is considered by many studies as apotential effective tool for disease control. To discover novel antigens, computational programs have been used in reverse vaccinology strategies. In this work, we developed a validation antigen approach that integrates prediction of B and T cell epitopes, analysis of Protein-Protein Interaction (PPI) networks and metabolic pathways. We selected twenty candidate proteins from Leishmania tested in murine model, with experimental outcome published in the literature. The predictions for CD4⁺ and CD8⁺ T cell epitopes were correlated with protection in experimental outcomes. We also mapped immunogenic proteins on PPI networks in order to find Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with them. Our results suggest that non-protective antigens have lowest frequency of predicted T CD4⁺ and T CD8⁺ epitopes, compared with protective ones. T CD4⁺ and T CD8⁺ cells are more related to leishmaniasis protection in experimental outcomes than B cell predicted epitopes. Considering KEGG analysis, the proteins considered protective are connected to nodes with few pathways, including those associated with ribosome biosynthesis and purine metabolism.

Distinct Corticostriatal and Intracortical Pathways Mediate Bilateral Sensory Responses in the Striatum.

PubMed

Reig, Ramon; Silberberg, Gilad

2016-12-01

Individual striatal neurons integrate somatosensory information from both sides of the body, however, the afferent pathways mediating these bilateral responses are unclear. Whereas ipsilateral corticostriatal projections are prevalent throughout the neocortex, contralateral projections provide sparse input from primary sensory cortices, in contrast to the dense innervation from motor and frontal regions. There is, therefore, an apparent discrepancy between the observed anatomical pathways and the recorded striatal responses. We used simultaneous in vivo whole-cell and extracellular recordings combined with focal cortical silencing, to dissect the afferent pathways underlying bilateral sensory integration in the mouse striatum. We show that unlike direct corticostriatal projections mediating responses to contralateral whisker deflection, responses to ipsilateral stimuli are mediated mainly by intracortical projections from the contralateral somatosensory cortex (S1). The dominant pathway is the callosal projection from contralateral to ipsilateral S1. Our results suggest a functional difference between the cortico-basal ganglia pathways underlying bilateral sensory and motor processes. © The Author 2016. Published by Oxford University Press.

Commentary on: "An integrated metabolic atlas of clear cell renal cell carcinoma." Hakimi AA, Reznik E, Lee CH, Creighton CJ, Brannon AR, Luna A, Aksoy BA, Liu EM, Shen R, Lee W, Chen Y, Stirdivant SM, Russo P, Chen YB, Tickoo SK, Reuter VE, Cheng EH, Sander C, Hsieh JJ.: Cancer Cell. 2016 Jan 11;29(1):104-16.

PubMed

Lee, Byron H

2017-09-01

Dysregulated metabolism is a hallmark of cancer, manifested through alterations in metabolites. We performed metabolomic profiling on 138 matched clear cell renal cell carcinoma (ccRCC)/normal tissue pairs and found that ccRCC is characterized by broad shifts in central carbon metabolism, one-carbon metabolism, and antioxidant response. Tumor progression and metastasis were associated with metabolite increases in glutathione and cysteine/methionine metabolism pathways. We develop an analytic pipeline and visualization tool (metabolograms) to bridge the gap between TCGA transcriptomic profiling and our metabolomic data, which enables us to assemble an integrated pathway-level metabolic atlas and to demonstrate discordance between transcriptome and metabolome. Lastly, expression profiling was performed on a high-glutathione cluster, which corresponds to a poor-survival subgroup in the ccRCC TCGA cohort. Copyright © 2017 Elsevier Inc. All rights reserved.

Modeling Signaling Networks to Advance New Cancer Therapies.

PubMed

Saez-Rodriguez, Julio; MacNamara, Aidan; Cook, Simon

2015-01-01

Cell signaling pathways control cells' responses to their environment through an intricate network of proteins and small molecules partitioned by intracellular structures, such as the cytoskeleton and nucleus. Our understanding of these pathways has been revised recently with the advent of more advanced experimental techniques; no longer are signaling pathways viewed as linear cascades of information flowing from membrane-bound receptors to the nucleus. Instead, such pathways must be understood in the context of networks, and studying such networks requires an integration of computational and experimental approaches. This understanding is becoming more important in designing novel therapies for diseases such as cancer. Using the MAPK (mitogen-activated protein kinase) and PI3K (class I phosphoinositide-3' kinase) pathways as case studies of cellular signaling, we give an overview of these pathways and their functions. We then describe, using a number of case studies, how computational modeling has aided in understanding these pathways' deregulation in cancer, and how such understanding can be used to optimally tailor current therapies or help design new therapies against cancer.

Penalized differential pathway analysis of integrative oncogenomics studies.

PubMed

van Wieringen, Wessel N; van de Wiel, Mark A

2014-04-01

Through integration of genomic data from multiple sources, we may obtain a more accurate and complete picture of the molecular mechanisms underlying tumorigenesis. We discuss the integration of DNA copy number and mRNA gene expression data from an observational integrative genomics study involving cancer patients. The two molecular levels involved are linked through the central dogma of molecular biology. DNA copy number aberrations abound in the cancer cell. Here we investigate how these aberrations affect gene expression levels within a pathway using observational integrative genomics data of cancer patients. In particular, we aim to identify differential edges between regulatory networks of two groups involving these molecular levels. Motivated by the rate equations, the regulatory mechanism between DNA copy number aberrations and gene expression levels within a pathway is modeled by a simultaneous-equations model, for the one- and two-group case. The latter facilitates the identification of differential interactions between the two groups. Model parameters are estimated by penalized least squares using the lasso (L1) penalty to obtain a sparse pathway topology. Simulations show that the inclusion of DNA copy number data benefits the discovery of gene-gene interactions. In addition, the simulations reveal that cis-effects tend to be over-estimated in a univariate (single gene) analysis. In the application to real data from integrative oncogenomic studies we show that inclusion of prior information on the regulatory network architecture benefits the reproducibility of all edges. Furthermore, analyses of the TP53 and TGFb signaling pathways between ER+ and ER- samples from an integrative genomics breast cancer study identify reproducible differential regulatory patterns that corroborate with existing literature.

Mitochondrial Stress Tests Using Seahorse Respirometry on Intact Dictyostelium discoideum Cells.

PubMed

Lay, Sui; Sanislav, Oana; Annesley, Sarah J; Fisher, Paul R

2016-01-01

Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells. Here, we describe a method to successfully use the Seahorse XF(e)24 Extracellular Flux Analyzer to assess the mitochondrial respirometry of the cellular slime mold Dictyostelium discoideum.

A linear programming computational framework integrates phosphor-proteomics and prior knowledge to predict drug efficacy.

PubMed

Ji, Zhiwei; Wang, Bing; Yan, Ke; Dong, Ligang; Meng, Guanmin; Shi, Lei

2017-12-21

In recent years, the integration of 'omics' technologies, high performance computation, and mathematical modeling of biological processes marks that the systems biology has started to fundamentally impact the way of approaching drug discovery. The LINCS public data warehouse provides detailed information about cell responses with various genetic and environmental stressors. It can be greatly helpful in developing new drugs and therapeutics, as well as improving the situations of lacking effective drugs, drug resistance and relapse in cancer therapies, etc. In this study, we developed a Ternary status based Integer Linear Programming (TILP) method to infer cell-specific signaling pathway network and predict compounds' treatment efficacy. The novelty of our study is that phosphor-proteomic data and prior knowledge are combined for modeling and optimizing the signaling network. To test the power of our approach, a generic pathway network was constructed for a human breast cancer cell line MCF7; and the TILP model was used to infer MCF7-specific pathways with a set of phosphor-proteomic data collected from ten representative small molecule chemical compounds (most of them were studied in breast cancer treatment). Cross-validation indicated that the MCF7-specific pathway network inferred by TILP were reliable predicting a compound's efficacy. Finally, we applied TILP to re-optimize the inferred cell-specific pathways and predict the outcomes of five small compounds (carmustine, doxorubicin, GW-8510, daunorubicin, and verapamil), which were rarely used in clinic for breast cancer. In the simulation, the proposed approach facilitates us to identify a compound's treatment efficacy qualitatively and quantitatively, and the cross validation analysis indicated good accuracy in predicting effects of five compounds. In summary, the TILP model is useful for discovering new drugs for clinic use, and also elucidating the potential mechanisms of a compound to targets.

Large-scale integrative network-based analysis identifies common pathways disrupted by copy number alterations across cancers

PubMed Central

2013-01-01

Background Many large-scale studies analyzed high-throughput genomic data to identify altered pathways essential to the development and progression of specific types of cancer. However, no previous study has been extended to provide a comprehensive analysis of pathways disrupted by copy number alterations across different human cancers. Towards this goal, we propose a network-based method to integrate copy number alteration data with human protein-protein interaction networks and pathway databases to identify pathways that are commonly disrupted in many different types of cancer. Results We applied our approach to a data set of 2,172 cancer patients across 16 different types of cancers, and discovered a set of commonly disrupted pathways, which are likely essential for tumor formation in majority of the cancers. We also identified pathways that are only disrupted in specific cancer types, providing molecular markers for different human cancers. Analysis with independent microarray gene expression datasets confirms that the commonly disrupted pathways can be used to identify patient subgroups with significantly different survival outcomes. We also provide a network view of disrupted pathways to explain how copy number alterations affect pathways that regulate cell growth, cycle, and differentiation for tumorigenesis. Conclusions In this work, we demonstrated that the network-based integrative analysis can help to identify pathways disrupted by copy number alterations across 16 types of human cancers, which are not readily identifiable by conventional overrepresentation-based and other pathway-based methods. All the results and source code are available at http://compbio.cs.umn.edu/NetPathID/. PMID:23822816

Quantitative Analysis of Energy Metabolic Pathways in MCF-7 Breast Cancer Cells by Selected Reaction Monitoring Assay*

PubMed Central

Drabovich, Andrei P.; Pavlou, Maria P.; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P.

2012-01-01

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells. PMID:22535206

Atkinesin-13A Modulates Cell-Wall Synthesis and Cell Expansion in Arabidopsis thaliana via the THESEUS1 Pathway

PubMed Central

Fujikura, Ushio; Elsaesser, Lore; Breuninger, Holger; Sánchez-Rodríguez, Clara; Ivakov, Alexander; Laux, Thomas; Findlay, Kim; Persson, Staffan; Lenhard, Michael

2014-01-01

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion. PMID:25232944

Drug Intervention Response Predictions with PARADIGM (DIRPP) identifies drug resistant cancer cell lines and pathway mechanisms of resistance.

PubMed

Brubaker, Douglas; Difeo, Analisa; Chen, Yanwen; Pearl, Taylor; Zhai, Kaide; Bebek, Gurkan; Chance, Mark; Barnholtz-Sloan, Jill

2014-01-01

The revolution in sequencing techniques in the past decade has provided an extensive picture of the molecular mechanisms behind complex diseases such as cancer. The Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Project (CGP) have provided an unprecedented opportunity to examine copy number, gene expression, and mutational information for over 1000 cell lines of multiple tumor types alongside IC50 values for over 150 different drugs and drug related compounds. We present a novel pipeline called DIRPP, Drug Intervention Response Predictions with PARADIGM7, which predicts a cell line's response to a drug intervention from molecular data. PARADIGM (Pathway Recognition Algorithm using Data Integration on Genomic Models) is a probabilistic graphical model used to infer patient specific genetic activity by integrating copy number and gene expression data into a factor graph model of a cellular network. We evaluated the performance of DIRPP on endometrial, ovarian, and breast cancer related cell lines from the CCLE and CGP for nine drugs. The pipeline is sensitive enough to predict the response of a cell line with accuracy and precision across datasets as high as 80 and 88% respectively. We then classify drugs by the specific pathway mechanisms governing drug response. This classification allows us to compare drugs by cellular response mechanisms rather than simply by their specific gene targets. This pipeline represents a novel approach for predicting clinical drug response and generating novel candidates for drug repurposing and repositioning.

Wires in the soup: quantitative models of cell signaling

PubMed Central

Cheong, Raymond; Levchenko, Andre

2014-01-01

Living cells are capable of extracting information from their environments and mounting appropriate responses to a variety of associated challenges. The underlying signal transduction networks enabling this can be quite complex, necessitating for their unraveling by sophisticated computational modeling coupled with precise experimentation. Although we are still at the beginning of this process, some recent examples of integrative analysis of cell signaling are very encouraging. This review highlights the case of the NF-κB pathway in order to illustrate how a quantitative model of a signaling pathway can be gradually constructed through continuous experimental validation, and what lessons one might learn from such exercises. PMID:18291655

HIV integration sites and implications for maintenance of the reservoir.

PubMed

Symons, Jori; Cameron, Paul U; Lewin, Sharon R

2018-03-01

To provide an overview of recent research of how HIV integration relates to productive and latent infection and implications for cure strategies. How and where HIV integrates provides new insights into how HIV persists on antiretroviral therapy (ART). Clonal expansion of infected cells with the same integration site demonstrates that T-cell proliferation is an important factor in HIV persistence, however, the driver of proliferation remains unclear. Clones with identical integration sites harbouring defective provirus can accumulate in HIV-infected individuals on ART and defective proviruses can express RNA and produce protein. HIV integration sites differ in clonally expanded and nonexpanded cells and in latently and productively infected cells and this influences basal and inducible transcription. There is a growing number of cellular proteins that can alter the pattern of integration to favour latency. Understanding these pathways may identify new interventions to eliminate latently infected cells. Using advances in analysing HIV integration sites, T-cell proliferation of latently infected cells is thought to play a major role in HIV persistence. Clonal expansion has been demonstrated with both defective and intact viruses. Production of viral RNA and protein from defective viruses may play a role in driving chronic immune activation. The site of integration may determine the likelihood of proliferation and the degree of basal and induced transcription. Finally, host factors and gene expression at the time of infection may determine the integration site. Together these new insights may lead to novel approaches to elimination of latently infected cells.

Model Checking of a Diabetes-Cancer Model

NASA Astrophysics Data System (ADS)

Gong, Haijun; Zuliani, Paolo; Clarke, Edmund M.

2011-06-01

Accumulating evidence suggests that cancer incidence might be associated with diabetes mellitus, especially Type II diabetes which is characterized by hyperinsulinaemia, hyperglycaemia, obesity, and overexpression of multiple WNT pathway components. These diabetes risk factors can activate a number of signaling pathways that are important in the development of different cancers. To systematically understand the signaling components that link diabetes and cancer risk, we have constructed a single-cell, Boolean network model by integrating the signaling pathways that are influenced by these risk factors to study insulin resistance, cancer cell proliferation and apoptosis. Then, we introduce and apply the Symbolic Model Verifier (SMV), a formal verification tool, to qualitatively study some temporal logic properties of our diabetes-cancer model. The verification results show that the diabetes risk factors might not increase cancer risk in normal cells, but they will promote cell proliferation if the cell is in a precancerous or cancerous stage characterized by losses of the tumor-suppressor proteins ARF and INK4a.

A fully integrated new paradigm for lithium's mode of action - lithium utilizes latent cellular fail-safe mechanisms.

PubMed

van Woerkom, Arthur Ernst

2017-01-01

It is proposed that lithium's therapeutic effects occur indirectly by augmenting a cascade of protective "fail-safe" pathways pre-configured to activate in response to a dangerous low cell [Mg ++ ] situation, eg, posttraumatic brain injury, alongside relative cell adenosine triphosphate depletion. Lithium activates cell protection, as it neatly mimics a lowered intracellular [Mg ++ ] level.

Integrative systems control approach for reactivating Kaposi's sarcoma-associated herpesvirus (KSHV) with combinatory drugs

PubMed Central

Sun, Chien-Pin; Usui, Takane; Yu, Fuqu; Al-Shyoukh, Ibrahim; Shamma, Jeff; Sun, Ren; Ho, Chih-Ming

2009-01-01

Cells serve as basic units of life and represent intricate biological molecular systems. The vast number of cellular molecules with their signaling and regulatory circuitries forms an intertwined network. In this network, each pathway interacts non-linearly with others through different intermediates. Thus, the challenge of manipulating cellular functions for desired outcomes, such as cancer eradication and controlling viral infection lies within the integrative system of regulatory circuitries. By using a closed-loop system control scheme, we can efficiently analyze biological signaling networks and manipulate their behavior through multiple stimulations on a collection of pathways. Specifically, we aimed to maximize the reactivation of Kaposi's Sarcoma-associated Herpesvirus (KSHV) in a Primary Effusion Lymphoma cell line. The advantage of this approach is that it is well-suited to study complex integrated systems; it circumvents the need for detailed information of individual signaling components; and it investigates the network as a whole by utilizing key systemic outputs as indicators. PMID:19851479

Integrative systems control approach for reactivating Kaposi's sarcoma-associated herpesvirus (KSHV) with combinatory drugs.

PubMed

Sun, Chien-Pin; Usui, Takane; Yu, Fuqu; Al-Shyoukh, Ibrahim; Shamma, Jeff; Sun, Ren; Ho, Chih-Ming

2009-01-01

Cells serve as basic units of life and represent intricate biological molecular systems. The vast number of cellular molecules with their signaling and regulatory circuitries forms an intertwined network. In this network, each pathway interacts non-linearly with others through different intermediates. Thus, the challenge of manipulating cellular functions for desired outcomes, such as cancer eradication and controlling viral infection lies within the integrative system of regulatory circuitries. By using a closed-loop system control scheme, we can efficiently analyze biological signaling networks and manipulate their behavior through multiple stimulations on a collection of pathways. Specifically, we aimed to maximize the reactivation of Kaposi's Sarcoma-associated Herpesvirus (KSHV) in a Primary Effusion Lymphoma cell line. The advantage of this approach is that it is well-suited to study complex integrated systems; it circumvents the need for detailed information of individual signaling components; and it investigates the network as a whole by utilizing key systemic outputs as indicators.

Pathway connectivity and signaling coordination in the yeast stress-activated signaling network

PubMed Central

Chasman, Deborah; Ho, Yi-Hsuan; Berry, David B; Nemec, Corey M; MacGilvray, Matthew E; Hose, James; Merrill, Anna E; Lee, M Violet; Will, Jessica L; Coon, Joshua J; Ansari, Aseem Z; Craven, Mark; Gasch, Audrey P

2014-01-01

Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology. PMID:25411400

Wnt Signaling in Adult Epithelial Stem Cells and Cancer.

PubMed

Tan, Si Hui; Barker, Nick

2018-01-01

Wnt/β-catenin signaling is integral to the homeostasis and regeneration of many epithelial tissues due to its critical role in adult stem cell regulation. It is also implicated in many epithelial cancers, with mutations in core pathway components frequently present in patient tumors. In this chapter, we discuss the roles of Wnt/β-catenin signaling and Wnt-regulated stem cells in homeostatic, regenerative and cancer contexts of the intestines, stomach, skin, and liver. We also examine the sources of Wnt ligands that form part of the stem cell niche. Despite the diversity in characteristics of various tissue stem cells, the role(s) of Wnt/β-catenin signaling is generally coherent in maintaining stem cell fate and/or promoting proliferation. It is also likely to play similar roles in cancer stem cells, making the pathway a salient therapeutic target for cancer. While promising progress is being made in the field, deeper understanding of the functions and signaling mechanisms of the pathway in individual epithelial tissues will expedite efforts to modulate Wnt/β-catenin signaling in cancer treatment and tissue regeneration. Copyright © 2018 Elsevier Inc. All rights reserved.

Linking TGF-beta-mediated Cdc25A inhibition and cytoskeletal regulation through RhoA/p160(ROCK) signaling.

PubMed

Brown, Kimberly; Bhowmick, Neil A

2004-04-01

Transforming growth factor-beta (TGF-beta) can mediate G(1)/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-beta-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160(ROCK) signaling pathway. The activation of TGF-beta-mediated p160(ROCK)rapidly inhibits the Cdc25A phosphatase as a component of the G(1)/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160(ROCK) pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-beta.

A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling.

PubMed

Gilroy, Simon; Suzuki, Nobuhiro; Miller, Gad; Choi, Won-Gyu; Toyota, Masatsugu; Devireddy, Amith R; Mittler, Ron

2014-10-01

Systemic signaling pathways enable multicellular organisms to prepare all of their tissues and cells to an upcoming challenge that may initially only be sensed by a few local cells. They are activated in plants in response to different stimuli including mechanical injury, pathogen infection, and abiotic stresses. Key to the mobilization of systemic signals in higher plants are cell-to-cell communication events that have thus far been mostly unstudied. The recent identification of systemically propagating calcium (Ca(2+)) and reactive oxygen species (ROS) waves in plants has unraveled a new and exciting cell-to-cell communication pathway that, together with electric signals, could provide a working model demonstrating how plant cells transmit long-distance signals via cell-to-cell communication mechanisms. Here, we summarize recent findings on the ROS and Ca(2+) waves and outline a possible model for their integration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in Saccharomyces cerevisiae Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation

PubMed Central

Mensonides, Femke I. C.; Brul, Stanley; Klis, Frans M.; Hellingwerf, Klaas J.; Teixeira de Mattos, M. Joost

2005-01-01

This paper reports on physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a tps1 mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such. PMID:16085846

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

PubMed

Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

2015-09-01

There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. Copyright © 2015 John Wiley & Sons, Inc.

Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors.

PubMed

Taniguchi, Toshiyasu; Tischkowitz, Marc; Ameziane, Najim; Hodgson, Shirley V; Mathew, Christopher G; Joenje, Hans; Mok, Samuel C; D'Andrea, Alan D

2003-05-01

Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia-BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor.

PubMed

Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

2015-05-10

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor

PubMed Central

Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

2015-01-01

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted. PMID:25857298

Dally Proteoglycan Mediates the Autonomous and Nonautonomous Effects on Tissue Growth Caused by Activation of the PI3K and TOR Pathways

PubMed Central

Ferreira, Ana; Milán, Marco

2015-01-01

How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The phosphatidylinositol 3-kinase (PI3K)–phosphatase with tensin homology (PTEN) and tuberous sclerosis complex (TSC)-target of rapamycin (TOR) pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. We utilized the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. Our findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. We present evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size. PMID:26313758

YAP and TAZ: a nexus for Hippo signaling and beyond

PubMed Central

Guan, Kun-Liang

2015-01-01

The Hippo pathway is a potent regulator of cellular proliferation, differentiation, and tissue homeostasis. Here we review the regulatory mechanisms of the Hippo pathway and discuss the function of Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding domain (TAZ), the prime mediators of the Hippo pathway, in stem cell biology and tissue regeneration. We highlight their activities in both the nucleus and the cytoplasm and discuss their role as a signaling nexus and integrator of several other prominent signaling pathways such as the Wnt, G protein-coupled receptor (GPCR), epidermal growth factor (EGF), BMP/transforming growth factor beta (TGFβ), and Notch pathways. PMID:26045258

Not so Fast: Co-Requirements for Sonic Hedgehog Induced Brain Tumorigenesis.

PubMed

Ward, Stacey A; Rubin, Joshua B

2015-08-06

The Sonic hedgehog (Shh) pathway plays an integral role in cellular proliferation during normal brain development and also drives growth in a variety of cancers including brain cancer. Clinical trials of Shh pathway inhibitors for brain tumors have yielded disappointing results, indicating a more nuanced role for Shh signaling. We postulate that Shh signaling does not work alone but requires co-activation of other signaling pathways for tumorigenesis and stem cell maintenance. This review will focus on the interplay between the Shh pathway and these pathways to promote tumor growth in brain tumors, presenting opportunities for the study of combinatorial therapies.

Winding through the WNT pathway during cellular development and demise.

PubMed

Li, F; Chong, Z Z; Maiese, K

2006-01-01

In slightly over a period of twenty years, our comprehension of the cellular and molecular mechanisms that govern the Wnt signaling pathway continue to unfold. The Wnt proteins were initially implicated in viral carcinogenesis experiments associated with mammary tumors, but since this period investigations focusing on the Wnt pathways and their transmembrane receptors termed Frizzled have been advanced to demonstrate the critical nature of Wnt for the development of a variety of cell populations as well as the potential of the Wnt pathway to avert apoptotic injury. In particular, Wnt signaling plays a significant role in both the cardiovascular and nervous systems during embryonic cell patterning, proliferation, differentiation, and orientation. Furthermore, modulation of Wnt signaling under specific cellular influences can either promote or prevent the early and late stages of apoptotic cellular injury in neurons, endothelial cells, vascular smooth muscle cells, and cardiomyocytes. A number of downstream signal transduction pathways can mediate the biological response of the Wnt proteins that include Dishevelled, beta-catenin, intracellular calcium, protein kinase C, Akt, and glycogen synthase kinase-3beta. Interestingly, these cellular cascades of the Wnt-Frizzled pathways can participate in several neurodegenerative, vascular, and cardiac disorders and may be closely integrated with the function of trophic factors. Identification of the critical elements that modulate the Wnt-Frizzled signaling pathway should continue to unlock the potential of Wnt pathway for the development of new therapeutic options against neurodegenerative and vascular diseases.

The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation.

PubMed

Bardhan, Kankana; Anagnostou, Theodora; Boussiotis, Vassiliki A

2016-01-01

The immune system maintains a critically organized network to defend against foreign particles, while evading self-reactivity simultaneously. T lymphocytes function as effectors and play an important regulatory role to orchestrate the immune signals. Although central tolerance mechanism results in the removal of the most of the autoreactive T cells during thymic selection, a fraction of self-reactive lymphocytes escapes to the periphery and pose a threat to cause autoimmunity. The immune system evolved various mechanisms to constrain such autoreactive T cells and maintain peripheral tolerance, including T cell anergy, deletion, and suppression by regulatory T cells (T Regs ). These effects are regulated by a complex network of stimulatory and inhibitory receptors expressed on T cells and their ligands, which deliver cell-to-cell signals that dictate the outcome of T cell encountering with cognate antigens. Among the inhibitory immune mediators, the pathway consisting of the programed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) plays an important role in the induction and maintenance of peripheral tolerance and for the maintenance of the stability and the integrity of T cells. However, the PD-1:PD-L1/L2 pathway also mediates potent inhibitory signals to hinder the proliferation and function of T effector cells and have inimical effects on antiviral and antitumor immunity. Therapeutic targeting of this pathway has resulted in successful enhancement of T cell immunity against viral pathogens and tumors. Here, we will provide a brief overview on the properties of the components of the PD-1 pathway, the signaling events regulated by PD-1 engagement, and their consequences on the function of T effector cells.

The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation

PubMed Central

Bardhan, Kankana; Anagnostou, Theodora; Boussiotis, Vassiliki A.

2016-01-01

The immune system maintains a critically organized network to defend against foreign particles, while evading self-reactivity simultaneously. T lymphocytes function as effectors and play an important regulatory role to orchestrate the immune signals. Although central tolerance mechanism results in the removal of the most of the autoreactive T cells during thymic selection, a fraction of self-reactive lymphocytes escapes to the periphery and pose a threat to cause autoimmunity. The immune system evolved various mechanisms to constrain such autoreactive T cells and maintain peripheral tolerance, including T cell anergy, deletion, and suppression by regulatory T cells (TRegs). These effects are regulated by a complex network of stimulatory and inhibitory receptors expressed on T cells and their ligands, which deliver cell-to-cell signals that dictate the outcome of T cell encountering with cognate antigens. Among the inhibitory immune mediators, the pathway consisting of the programed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) plays an important role in the induction and maintenance of peripheral tolerance and for the maintenance of the stability and the integrity of T cells. However, the PD-1:PD-L1/L2 pathway also mediates potent inhibitory signals to hinder the proliferation and function of T effector cells and have inimical effects on antiviral and antitumor immunity. Therapeutic targeting of this pathway has resulted in successful enhancement of T cell immunity against viral pathogens and tumors. Here, we will provide a brief overview on the properties of the components of the PD-1 pathway, the signaling events regulated by PD-1 engagement, and their consequences on the function of T effector cells. PMID:28018338

Application of Monte Carlo cross-validation to identify pathway cross-talk in neonatal sepsis.

PubMed

Zhang, Yuxia; Liu, Cui; Wang, Jingna; Li, Xingxia

2018-03-01

To explore genetic pathway cross-talk in neonates with sepsis, an integrated approach was used in this paper. To explore the potential relationships between differently expressed genes between normal uninfected neonates and neonates with sepsis and pathways, genetic profiling and biologic signaling pathway were first integrated. For different pathways, the score was obtained based upon the genetic expression by quantitatively analyzing the pathway cross-talk. The paired pathways with high cross-talk were identified by random forest classification. The purpose of the work was to find the best pairs of pathways able to discriminate sepsis samples versus normal samples. The results found 10 pairs of pathways, which were probably able to discriminate neonates with sepsis versus normal uninfected neonates. Among them, the best two paired pathways were identified according to analysis of extensive literature. Impact statement To find the best pairs of pathways able to discriminate sepsis samples versus normal samples, an RF classifier, the DS obtained by DEGs of paired pathways significantly associated, and Monte Carlo cross-validation were applied in this paper. Ten pairs of pathways were probably able to discriminate neonates with sepsis versus normal uninfected neonates. Among them, the best two paired pathways ((7) IL-6 Signaling and Phospholipase C Signaling (PLC); (8) Glucocorticoid Receptor (GR) Signaling and Dendritic Cell Maturation) were identified according to analysis of extensive literature.

Integrative analysis of signaling pathways and diseases associated with the miR-106b/25 cluster and their function study in berberine-induced multiple myeloma cells.

PubMed

Gu, Chunming; Li, Tianfu; Yin, Zhao; Chen, Shengting; Fei, Jia; Shen, Jianping; Zhang, Yuan

2017-05-01

Berberine (BBR), a traditional Chinese herbal medicine compound, has emerged as a novel class of anti-tumor agent. Our previous microRNA (miRNA) microarray demonstrated that miR-106b/25 was significantly down-regulated in BBR-treated multiple myeloma (MM) cells. Here, systematic integration showed that miR-106b/25 cluster is involved in multiple cancer-related signaling pathways and tumorigenesis. MiREnvironment database revealed that multiple environmental factors (drug, ionizing radiation, hypoxia) affected the miR-106b/25 cluster expression. By targeting the seed region in the miRNA, tiny anti-mir106b/25 cluster (t-anti-mir106b/25 cluster) significantly induced suppression in cell viability and colony formation. Western blot validated that t-anti-miR-106b/25 cluster effectively inhibited the expression of P38 MAPK and phospho-P38 MAPK in MM cells. These findings indicated the miR-106b/25 cluster functioned as oncogene and might provide a novel molecular insight into MM.

A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity

PubMed Central

Fagnocchi, Luca; Cherubini, Alessandro; Hatsuda, Hiroshi; Fasciani, Alessandra; Mazzoleni, Stefania; Poli, Vittoria; Berno, Valeria; Rossi, Riccardo L.; Reinbold, Rolland; Endele, Max; Schroeder, Timm; Rocchigiani, Marina; Szkarłat, Żaneta; Oliviero, Salvatore; Dalton, Stephen; Zippo, Alessio

2016-01-01

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity. PMID:27301576

A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity.

PubMed

Fagnocchi, Luca; Cherubini, Alessandro; Hatsuda, Hiroshi; Fasciani, Alessandra; Mazzoleni, Stefania; Poli, Vittoria; Berno, Valeria; Rossi, Riccardo L; Reinbold, Rolland; Endele, Max; Schroeder, Timm; Rocchigiani, Marina; Szkarłat, Żaneta; Oliviero, Salvatore; Dalton, Stephen; Zippo, Alessio

2016-06-15

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.

How might flukes and tapeworms maintain genome integrity without a canonical piRNA pathway?

PubMed Central

Skinner, Danielle E.; Rinaldi, Gabriel; Koziol, Uriel; Brehm, Klaus; Brindley, Paul J.

2014-01-01

Surveillance by RNA interference is central to controlling the mobilization of transposable elements (TEs). In stem cells, Piwi argonaute (Ago) proteins and associated proteins repress mobilization of TEs to maintain genome integrity. This defense mechanism targeting TEs is termed the Piwi-interacting RNA (Piwi-piRNA) pathway. In this Opinion, we draw attention to the situation that the genomes of cestodes and trematodes have lost the piwi and vasa genes that are hallmark characters of the germline multipotency program. This absence of Piwi-like Agos and Vasa helicases prompts the question: how does the germline of these flatworms withstand mobilization of TEs? Here we present an interpretation of mechanisms likely to defend the germline integrity of parasitic flatworms. PMID:24485046

Engineering Robustness of Microbial Cell Factories.

PubMed

Gong, Zhiwei; Nielsen, Jens; Zhou, Yongjin J

2017-10-01

Metabolic engineering and synthetic biology offer great prospects in developing microbial cell factories capable of converting renewable feedstocks into fuels, chemicals, food ingredients, and pharmaceuticals. However, prohibitively low production rate and mass concentration remain the major hurdles in industrial processes even though the biosynthetic pathways are comprehensively optimized. These limitations are caused by a variety of factors unamenable for host cell survival, such as harsh industrial conditions, fermentation inhibitors from biomass hydrolysates, and toxic compounds including metabolic intermediates and valuable target products. Therefore, engineered microbes with robust phenotypes is essential for achieving higher yield and productivity. In this review, the recent advances in engineering robustness and tolerance of cell factories is described to cope with these issues and briefly introduce novel strategies with great potential to enhance the robustness of cell factories, including metabolic pathway balancing, transporter engineering, and adaptive laboratory evolution. This review also highlights the integration of advanced systems and synthetic biology principles toward engineering the harmony of overall cell function, more than the specific pathways or enzymes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1.

PubMed

Herold, Inbal; Yarden, Oded

2017-02-01

Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. Deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency characteristic of the cot-1 parent. gul-1 (a homologue of the yeast SSD1 gene) encodes a translational regulator and has been shown to partially suppress cot-1. We demonstrate that the high expression levels of the cell wall remodeling genes analyzed is curbed, and reaches near wild type levels, when gul-1 is inactivated. This is accompanied by morphological changes that include reduced cell wall thickness and restoration of normal chitin levels. We conclude that gul-1 is a mediator of cell wall remodeling within the cot-1 pathway.

The role of HIV integration in viral persistence: no more whistling past the proviral graveyard

PubMed Central

Maldarelli, Frank

2016-01-01

A substantial research effort has been directed to identifying strategies to eradicate or control HIV infection without a requirement for combination antiretroviral therapy (cART). A number of obstacles prevent HIV eradication, including low-level viral persistence during cART, long-term persistence of HIV-infected cells, and latent infection of resting CD4+ T cells. Mechanisms of persistence remain uncertain, but integration of the provirus into the host genome represents a central event in replication and pathogenesis of all retroviruses, including HIV. Analysis of HIV proviruses in CD4+ lymphocytes from individuals after prolonged cART revealed that a substantial proportion of the infected cells that persist have undergone clonal expansion and frequently have proviruses integrated in genes associated with regulation of cell growth. These data suggest that integration may influence persistence and clonal expansion of HIV-infected cells after cART is introduced, and these processes may represent key mechanisms for HIV persistence. Determining the diversity of host genes with integrants in HIV-infected cells that persist for prolonged periods may yield useful information regarding pathways by which infected cells persist for prolonged periods. Moreover, many integrants are defective, and new studies are required to characterize the role of clonal expansion in the persistence of replication-competent HIV. PMID:26829624

Multiple Signals Regulate PLC beta 3 in Human Myometrial Cells

PubMed Central

Zhong, Miao; Murtazina, Dilyara A.; Phillips, Jennifer; Ku, Chun-Ying; Sanborn, Barbara M.

2008-01-01

Summary The regulation of PLCB3-Serine1105 phosphorylation by both negative feedback and negative crosstalk facilitates the integration of multiple signaling pathways in myometrial cells. Phospholipase CB3 (PLCB3) Serine1105, a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-Serine1105 phosphorylation (P-S1105) was studied in immortalized and primary human myometrial cells. CPT-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S1105. Relaxin also stimulated P-S1105; this effect was partially blocked by the protein kinase A (PRKA) inhibitor Rp-8-CPT-cAMPS. Oxytocin, which stimulates Gαq-mediated pathways, also rapidly increased P-S1105, as did PGF2α and ATP. Oxytocin-stimulated phosphorylation was blocked by the protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S1105, whereas the reverse was the case for the oxytocin-stimulated increase in P-S1105. PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 shRNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. Oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid but enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S1105A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S1105 dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium. PMID:18322273

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

PubMed

Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES.

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

PubMed Central

Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES. PMID:22465851

Identification of Significant Gene Signatures and Prognostic Biomarkers for Patients With Cervical Cancer by Integrated Bioinformatic Methods

PubMed Central

Li, Xiaofang; Tian, Run; Gao, Hugh; Yan, Feng; Ying, Le; Yang, Yongkang; Yang, Pei

2018-01-01

Cervical cancer is the leading cause of death with gynecological malignancies. We aimed to explore the molecular mechanism of carcinogenesis and biomarkers for cervical cancer by integrated bioinformatic analysis. We employed RNA-sequencing details of 254 cervical squamous cell carcinomas and 3 normal samples from The Cancer Genome Atlas. To explore the distinct pathways, messenger RNA expression was submitted to a Gene Set Enrichment Analysis. Kyoto Encyclopedia of Genes and Genomes and protein–protein interaction network analysis of differentially expressed genes were performed. Then, we conducted pathway enrichment analysis for modules acquired in protein–protein interaction analysis and obtained a list of pathways in every module. After intersecting the results from the 3 approaches, we evaluated the survival rates of both mutual pathways and genes in the pathway, and 5 survival-related genes were obtained. Finally, Cox hazards ratio analysis of these 5 genes was performed. DNA replication pathway (P < .001; 12 genes included) was suggested to have the strongest association with the prognosis of cervical squamous cancer. In total, 5 of the 12 genes, namely, minichromosome maintenance 2, minichromosome maintenance 4, minichromosome maintenance 5, proliferating cell nuclear antigen, and ribonuclease H2 subunit A were significantly correlated with survival. Minichromosome maintenance 5 was shown as an independent prognostic biomarker for patients with cervical cancer. This study identified a distinct pathway (DNA replication). Five genes which may be prognostic biomarkers and minichromosome maintenance 5 were identified as independent prognostic biomarkers for patients with cervical cancer. PMID:29642758

The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

PubMed Central

2011-01-01

Background Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of CCW12 results in severe cell wall damage and reduced mating efficiency. Results In order to explore the function of CCW12, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of CCW12. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are PFD1, WHI3, SRN2, PAC10, FEN1 and YDR417C, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant ccw12Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are BCK1, CHS3, EDE1, PFD1, SLT2 and SLA1 that were also identified in the SGA. In contrast, a specific feature of mutant ccw12Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection. Conclusions The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of CCW12. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth. The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned. PMID:21320323

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

Peek, Gregory W.; Tollefsbol, Trygve O., E-mail: trygve@uab.edu; Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmitmore » signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-β pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-β pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression. - Highlights: • PX-866 and raloxifene affect the PI3K/Akt and TGF-β pathways. • PX-866 and raloxifene down-regulate genes up-regulated in cancer. • PX-866 and raloxifene decrease transcription of hTERT and Cyclin D1. • Pathological transcription signatures can identify new defense mechanisms.« less

Whole transcriptome profiling of taste bud cells.

PubMed

Sukumaran, Sunil K; Lewandowski, Brian C; Qin, Yumei; Kotha, Ramana; Bachmanov, Alexander A; Margolskee, Robert F

2017-08-08

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

Spatial Regulation of Root Growth: Placing the Plant TOR Pathway in a Developmental Perspective

PubMed Central

Barrada, Adam; Montané, Marie-Hélène; Robaglia, Christophe; Menand, Benoît

2015-01-01

Plant cells contain specialized structures, such as a cell wall and a large vacuole, which play a major role in cell growth. Roots follow an organized pattern of development, making them the organs of choice for studying the spatio-temporal regulation of cell proliferation and growth in plants. During root growth, cells originate from the initials surrounding the quiescent center, proliferate in the division zone of the meristem, and then increase in length in the elongation zone, reaching their final size and differentiation stage in the mature zone. Phytohormones, especially auxins and cytokinins, control the dynamic balance between cell division and differentiation and therefore organ size. Plant growth is also regulated by metabolites and nutrients, such as the sugars produced by photosynthesis or nitrate assimilated from the soil. Recent literature has shown that the conserved eukaryotic TOR (target of rapamycin) kinase pathway plays an important role in orchestrating plant growth. We will summarize how the regulation of cell proliferation and cell expansion by phytohormones are at the heart of root growth and then discuss recent data indicating that the TOR pathway integrates hormonal and nutritive signals to orchestrate root growth. PMID:26295391

The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

PubMed Central

Rocha, Marina Campos; de Godoy, Krissia Franco; de Castro, Patrícia Alves; Hori, Juliana Issa; Bom, Vinícius Leite Pedro; Brown, Neil Andrew; da Cunha, Anderson Ferreira; Goldman, Gustavo Henrique; Malavazi, Iran

2015-01-01

Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcA G579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcA G579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcA G579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection. PMID:26295576

Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

PubMed

Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

2012-12-01

What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To identify more differentially expressed genes with a lower false discovery rate from a previously published microarray data set, an integrative hypothesis-testing statistical approach was applied. • For validation experiments, expression and phosphorylation levels of select proteins were evaluated by western blotting. • Integration analysis of this transcriptomics data set with our own quantitative proteomics data set identified 10 genes that are potentially regulated by APF in vivo from 4140 differentially expressed genes identified with a false discovery rate of 1%. • Of these, five (i.e. JUP, MAPKSP1, GSPT1, PTGS2/COX-2 and XPOT) were found to be prominent after network modelling of the common genes identified in the proteomics and microarray studies. • This molecular signature reflects the biological processes of cell adhesion, cell proliferation and inflammation, which is consistent with the known physiological effects of APF. • Lastly, we found the mammalian target of rapamycin pathway was down-regulated in response to APF. • This unbiased integration analysis of in vitro quantitative proteomics data with in vivo quantitative transcriptomics data led to the identification of potential downstream mediators of the APF signal transduction pathway. © 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.

Neurotrophic factors switch between two signaling pathways that trigger axonal growth.

PubMed

Paveliev, Mikhail; Lume, Maria; Velthut, Agne; Phillips, Matthew; Arumäe, Urmas; Saarma, Mart

2007-08-01

Integration of multiple inputs from the extracellular environment, such as extracellular matrix molecules and growth factors, is a crucial process for cell function and information processing in multicellular organisms. Here we demonstrate that co-stimulation of dorsal root ganglion neurons with neurotrophic factors (NTFs) - glial-cell-line-derived neurotrophic factor, neurturin or nerve growth factor - and laminin leads to axonal growth that requires activation of Src family kinases (SFKs). A different, SFK-independent signaling pathway evokes axonal growth on laminin in the absence of the NTFs. By contrast, axonal branching is regulated by SFKs both in the presence and in the absence of NGF. We propose and experimentally verify a Boolean model of the signaling network triggered by NTFs and laminin. Our results demonstrate that NTFs provide an environmental cue that triggers a switch between separate pathways in the cell signaling network.

BAD phosphorylation determines ovarian cancer chemo-sensitivity and patient survival

PubMed Central

Marchion, Douglas C.; Cottrill, Hope M.; Xiong, Yin; Chen, Ning; Bicaku, Elona; Fulp, William J.; Bansal, Nisha; Chon, Hye Sook; Stickles, Xiaomang B.; Kamath, Siddharth G.; Hakam, Ardeshir; Li, Lihua; Su, Dan; Moreno, Carolina; Judson, Patricia L.; Berchuck, Andrew; Wenham, Robert M.; Apte, Sachin M.; Gonzalez-Bosquet, Jesus; Bloom, Gregory C.; Eschrich, Steven A.; Sebti, Said; Chen, Dung-Tsa; Lancaster, Johnathan M.

2011-01-01

Purpose Despite initial sensitivity to chemotherapy, ovarian cancers (OVCA) often develop drug-resistance, which limits patient survival. Using specimens and/or genomic data from 289 patients and a panel of cancer cell lines, we explored genome-wide expression changes that underlie the evolution of OVCA chemo-resistance and characterized the BCL2 antagonist of cell death (BAD) apoptosis pathway as a determinant of chemo-sensitivity and patient survival. Experimental Design Serial OVCA cell cisplatin treatments were performed in parallel with measurements of genome-wide expression changes. Pathway analysis was performed on genes associated with increasing cisplatin-resistance (EC50). BAD-pathway expression and BAD-protein phosphorylation were evaluated in patient samples and cell lines as determinants of chemo-sensitivity and/or clinical outcome and as therapeutic targets. Results Induced in vitro OVCA cisplatin-resistance was associated with BAD-pathway expression (P < 0.001). In OVCA cell lines and primary specimens, BAD-protein phosphorylation was associated with platinum-resistance (n = 147, P < 0.0001) and also with overall patient survival (n = 134, P = 0.0007). Targeted modulation of BAD-phosphorylation levels influenced cisplatin sensitivity. A 47-gene BAD-pathway score was associated with in vitro phosphorylated-BAD levels and with survival in 142 patients with advanced-stage (III/IV) serous OVCA. Integration of BAD-phosphorylation or BAD-pathway score with OVCA surgical cytoreductive status was significantly associated with overall survival by log-rank test (P = 0.004 and <0.0001, respectively). Conclusion The BAD apoptosis pathway influences OVCA chemo-sensitivity and overall survival, likely via modulation of BAD-phosphorylation. The pathway has clinical relevance as a biomarker of therapeutic response, patient survival, and as a promising therapeutic target. PMID:21849418

T Lymphocyte Activation Threshold is Increased in Reduced Gravity

NASA Technical Reports Server (NTRS)

Adams, Charley L.; Gonzalez, M.; Sams, C. F.

2000-01-01

There have been substantial advances in molecular and cellular biology that have provided new insight into the biochemical and genetic basis of lymphocyte recognition, activation and expression of distinct functional phenotypes. It has now become evident that for both T and B cells, stimuli delivered through their receptors can result in either clonal expansion or apoptosis. In the case of T cells, clonal expansion of helper cells is accompanied by differentiation into two major functional subsets which regulate the immune response. The pathways between the membrane and the nucleus and their molecular components are an area of very active investigation. This meeting will draw together scientists working on diverse aspects of this problem, including receptor ligand interactions, intracellular pathways that transmit receptor mediated signals and the effect of such signal transduction pathways on gene regulation. The aim of this meeting is to integrate the information from these various experimental approaches into a new synthesis and molecular explanation of T cell activation, differentiation and death.

Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network

PubMed Central

Huang, Guanyi; Ye, Shoudong; Zhou, Xingliang; Liu, Dahai

2016-01-01

Embryonic stem cells (ESCs) can be maintained in culture indefinitely while retaining the capacity to generate any type of cell in the body, and therefore not only hold great promise for tissue repair and regeneration, but also provide a powerful tool for modeling human disease and understanding biological development. In order to fulfill the full potential of ESCs, it is critical to understand how ESC fate, whether to self-renew or to differentiate into specialized cells, is regulated. On the molecular level, ESC fate is controlled by the intracellular transcriptional regulatory networks that respond to various extrinsic signaling stimuli. In this review, we discuss and compare important signaling pathways in the self-renewal and differentiation of mouse, rat, and human ESCs with an emphasis on how these pathways integrate into ESC-specific transcription circuitries. This will be beneficial for understanding the common and conserved mechanisms that govern self-renewal, and for developing novel culture conditions that support ESC derivation and maintenance. PMID:25595304

Targeting interlukin-6 to relieve immunosuppression in tumor microenvironment.

PubMed

Liu, Qian; Yu, Shengnan; Li, Anping; Xu, Hanxiao; Han, Xinwei; Wu, Kongming

2017-06-01

Immunotolerance is one of the hallmarks of malignant tumors. Tumor cells escape from host immune surveillance through various mechanisms resulting in tumor progression and therapeutic resistance. Interlukin-6 is a proinflammatory cytokine involved in many physiological and pathological processes by integrating with multiple intracellular signaling pathways. Aberrant expression of interlukin-6 is associated with the growth, metastasis, and chemotherapeutic resistance in a wide range of cancers. Interlukin-6 exerts immunosuppressive capacity mostly by stimulating the infiltrations of myeloid-derived suppressor cells, tumor-associated neutrophils, and cancer stem-like cells via Janus-activated kinase/signal transducer and activator of transcription 3 pathway in tumor microenvironment. On this foundation, blockage of interlukin-6 signal may provide potential approaches to novel therapies. In this review, we introduced interlukin-6 pathways and summarized molecular mechanisms related to interlukin-6-induced immunosuppression of tumor cell. We also concluded recent clinical studies targeting interlukin-6 as an immune-based therapeutic intervention in patients with cancer.

Regulation of expression, activity and localization of fungal chitin synthases

PubMed Central

Rogg, Luise E.; Fortwendel, Jarrod R.; Juvvadi, Praveen R.; Steinbach, William J.

2013-01-01

The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of β-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets. PMID:21526913

Sensing the Environment Through Sestrins: Implications for Cellular Metabolism.

PubMed

Parmigiani, A; Budanov, A V

2016-01-01

Sestrins are a family of stress-responsive genes that have evolved to attenuate damage induced by stress caused to the cell. By virtue of their antioxidant activity, protein products of Sestrin genes prevent the accumulation of reactive oxygen species within the cell, thereby attenuating the detrimental effects of oxidative stress. In parallel, Sestrins participate in several signaling pathways that control the activity of the target of rapamycin protein kinase (TOR). TOR is a crucial sensor of intracellular and extracellular conditions that promotes cell growth and anabolism when nutrients and growth factors are abundant. In addition to reacting to stress-inducing insults, Sestrins also monitor the changes in the availability of nutrients, which allows them to serve as a key checkpoint for the TOR-regulated signaling pathways. In this review, we will discuss how Sestrins integrate signals from numerous stress- and nutrient-responsive signaling pathways to orchestrate cellular metabolism and support cell viability. Copyright © 2016 Elsevier Inc. All rights reserved.

The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice.

PubMed

Nacerddine, Karim; Lehembre, François; Bhaumik, Mantu; Artus, Jérôme; Cohen-Tannoudji, Michel; Babinet, Charles; Pandolfi, Pier Paolo; Dejean, Anne

2005-12-01

Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals.

Metabolic gene products have evolved to interact with the cell wall integrity pathway in Saccharomyces cerevisiae.

PubMed

Ugbogu, Eziuche A; Wang, Ke; Schweizer, Lilian M; Schweizer, Michael

2016-12-01

Two of the five unlinked genes theoretically capable of encoding 5-phosphoribosyl-1(α)-pyrophosphate (PRPP) synthetase (Prs) in Saccharomyces cerevisiae, PRS1 and PRS5, contain in-frame insertions which separate the cation- and PRPP-binding sites, diagnostic of Prs polypeptides. The impairment of cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) cascade in strains lacking PRS1 and the synthetic lethality associated with loss of PRS1 and PRS5 imply that these insertions are not gratuitous. Coimmunoprecipitation revealed that Prs1 interacts with the CWI MAPK pathway, only when Slt2 has been phosphorylated by Mkk1/2. Three serine residues identified by phosphoproteome analysis (Ficarro et al 2002) are located in one of the insertions of PRS5 thereby defining Prs5 as one of the 11 triply phosphorylated proteins in yeast. Mutation of these phosphosites compromised the transcriptional readout of one endpoint of the CWI pathway, Rlm1, as well as the expression of the gene encoding the stress-activated 1,3 β-glucan synthase, Fks2, regulated by a second endpoint of the CWI pathway, Swi4/Swi6 (SBF transcription factor). Therefore, the unexpected impairment of the CWI phenotype encountered in yeast strains either mutated or deleted for PRS1 or PRS5 can be explained by disruption of the communication between primary cell metabolism and CWI signalling. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Uncoupling of transcription and translation of Fanconi anemia (FANC) complex proteins during spermatogenesis

PubMed Central

Jamsai, Duangporn; O’Connor, Anne E; O’Donnell, Liza; Lo, Jennifer Chi Yi; O’Bryan, Moira K

2015-01-01

Male germ cell genome integrity is critical for spermatogenesis, fertility and normal development of the offspring. Several DNA repair pathways exist in male germ cells. One such important pathway is the Fanconi anemia (FANC) pathway. Unlike in somatic cells, expression profiles and the role of the FANC pathway in germ cells remain largely unknown. In this study, we undertook an extensive expression analyses at both mRNA and protein levels of key components of the FANC pathway during spermatogenesis in the mouse. Herein we show that Fanc mRNAs and proteins displayed developmental enrichment within particular male germ cell types. Spermatogonia and pre-leptotene spermatocytes contained the majority of the FANC components examined i.e. complex I members FANCB, FANCG and FANCM, complex II members FANCD2 and FANCI, and complex III member FANCJ. Leptotene, zygotene and early pachytene spermatocytes contained FANCB, FANCG, FANCM and FANCD2. With the exception of FANCL, all FANC proteins examined were not detected in round spermatids. Elongating and elongated spermatids contained FANCB, FANCG, FANCL and FANCJ. qPCR analysis on isolated spermatocytes and round spermatids showed that Fancg, Fancl, Fancm, Fancd2, Fanci and Fancj mRNAs were expressed in both of these germ cell types, indicating that some degree of translational repression of these FANC proteins occurs during the transition from meiosis to spermiogenesis. Taken together, our findings raise the possibility that the assembly of FANC protein complexes in each of the male germ cell type is unique and may be distinct from the proposed model in mitotic cells. PMID:26413409

CD8 Memory Cells Develop Unique DNA Repair Mechanisms Favoring Productive Division.

PubMed

Galgano, Alessia; Barinov, Aleksandr; Vasseur, Florence; de Villartay, Jean-Pierre; Rocha, Benedita

2015-01-01

Immune responses are efficient because the rare antigen-specific naïve cells are able to proliferate extensively and accumulate upon antigen stimulation. Moreover, differentiation into memory cells actually increases T cell accumulation, indicating improved productive division in secondary immune responses. These properties raise an important paradox: how T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation. We here present the first data addressing the DNA damage responses (DDRs) of CD8 T cells in vivo during exponential expansion in primary and secondary responses in mice. We show that during exponential division CD8 T cells engage unique DDRs, which are not present in other exponentially dividing cells, in T lymphocytes after UV or X irradiation or in non-metastatic tumor cells. While in other cell types a single DDR pathway is affected, all DDR pathways and cell cycle checkpoints are affected in dividing CD8 T cells. All DDR pathways collapse in secondary responses in the absence of CD4 help. CD8 T cells are driven to compulsive suicidal divisions preventing the propagation of DNA lesions. In contrast, in the presence of CD4 help all the DDR pathways are up regulated, resembling those present in metastatic tumors. However, this up regulation is present only during the expansion phase; i.e., their dependence on antigen stimulation prevents CD8 transformation. These results explain how CD8 T cells maintain genome integrity in spite of their extensive division, and highlight the fundamental role of DDRs in the efficiency of CD8 immune responses.

BID links ferroptosis to mitochondrial cell death pathways.

PubMed

Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

2017-08-01

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

A systems biology pipeline identifies new immune and disease related molecular signatures and networks in human cells during microgravity exposure

NASA Astrophysics Data System (ADS)

Mukhopadhyay, Sayak; Saha, Rohini; Palanisamy, Anbarasi; Ghosh, Madhurima; Biswas, Anupriya; Roy, Saheli; Pal, Arijit; Sarkar, Kathakali; Bagh, Sangram

2016-05-01

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions.

A systems biology pipeline identifies new immune and disease related molecular signatures and networks in human cells during microgravity exposure.

PubMed

Mukhopadhyay, Sayak; Saha, Rohini; Palanisamy, Anbarasi; Ghosh, Madhurima; Biswas, Anupriya; Roy, Saheli; Pal, Arijit; Sarkar, Kathakali; Bagh, Sangram

2016-05-17

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

PubMed

Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

2016-01-01

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Tissue Growth by the Mammalian Hippo Signaling Pathway

PubMed Central

Watt, Kevin I.; Harvey, Kieran F.; Gregorevic, Paul

2017-01-01

The integrative control of diverse biological processes such as proliferation, differentiation, apoptosis and metabolism is essential to maintain cellular and tissue homeostasis. Disruption of these underlie the development of many disease states including cancer and diabetes, as well as many of the complications that arise as a consequence of aging. These biological outputs are governed by many cellular signaling networks that function independently, and in concert, to convert changes in hormonal, mechanical and metabolic stimuli into alterations in gene expression. First identified in Drosophila melanogaster as a powerful mediator of cell division and apoptosis, the Hippo signaling pathway is a highly conserved regulator of mammalian organ size and functional capacity in both healthy and diseased tissues. Recent studies have implicated the pathway as an effector of diverse physiological cues demonstrating an essential role for the Hippo pathway as an integrative component of cellular homeostasis. In this review, we will: (a) outline the critical signaling elements that constitute the mammalian Hippo pathway, and how they function to regulate Hippo pathway-dependent gene expression and tissue growth, (b) discuss evidence that shows this pathway functions as an effector of diverse physiological stimuli and (c) highlight key questions in this developing field. PMID:29225579

Cellular and molecular specificity of pituitary gland physiology.

PubMed

Perez-Castro, Carolina; Renner, Ulrich; Haedo, Mariana R; Stalla, Gunter K; Arzt, Eduardo

2012-01-01

The anterior pituitary gland has the ability to respond to complex signals derived from central and peripheral systems. Perception of these signals and their integration are mediated by cell interactions and cross-talk of multiple signaling transduction pathways and transcriptional regulatory networks that cooperate for hormone secretion, cell plasticity, and ultimately specific pituitary responses that are essential for an appropriate physiological response. We discuss the physiopathological and molecular mechanisms related to this integrative regulatory system of the anterior pituitary gland and how it contributes to modulate the gland functions and impacts on body homeostasis.

Understanding D-Ribose and Mitochondrial Function.

PubMed

Mahoney, Diane E; Hiebert, John B; Thimmesch, Amanda; Pierce, John T; Vacek, James L; Clancy, Richard L; Sauer, Andrew J; Pierce, Janet D

2018-01-01

Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production. With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage

PubMed Central

Huang, Ruili; Lin, Ja-An; Sedykh, Alexander; Zhao, Jinghua; Tice, Raymond R.; Paules, Richard S.; Xia, Menghang; Auerbach, Scott S.

2017-01-01

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals. PMID:28531190

Control of stem cell fate and function by engineering physical microenvironments

PubMed Central

Kshitiz; Park, Jinseok; Kim, Peter; Helen, Wilda; Engler, Adam J; Levchenko, Andre; Kim, Deok-Ho

2012-01-01

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell-cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niches environment and regulate stem cell fate and function. PMID:23077731

A fully integrated new paradigm for lithium’s mode of action – lithium utilizes latent cellular fail-safe mechanisms

PubMed Central

van Woerkom, Arthur Ernst

2017-01-01

It is proposed that lithium’s therapeutic effects occur indirectly by augmenting a cascade of protective “fail-safe” pathways pre-configured to activate in response to a dangerous low cell [Mg++] situation, eg, posttraumatic brain injury, alongside relative cell adenosine triphosphate depletion. Lithium activates cell protection, as it neatly mimics a lowered intracellular [Mg++] level. PMID:28203080

Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis

PubMed Central

Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Chris; Wu, Yan; Costa, Mike; de Sauvage, Frederic J.; Klein, Ophir D.

2015-01-01

Summary Proper organ homeostasis requires tight control of adult stem cells and differentiation through integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a de-repression of the Wnt signaling pathway, leading to mis-expression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

PubMed Central

Sasatani, Megumi; Xu, Yanbin; Kawai, Hidehiko; Cao, Lili; Tateishi, Satoshi; Shimura, Tsutomu; Li, Jianxiang; Iizuka, Daisuke; Noda, Asao; Hamasaki, Kanya; Kusunoki, Yoichiro; Kamiya, Kenji

2015-01-01

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure. PMID:25675240

Integrin αv in the mechanical response of osteoblast lineage cells.

PubMed

Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji

2014-05-02

Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src-JNK-YAP/TAZ pathway in response to mechanical stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Integrated molecular portrait of non-small cell lung cancers

PubMed Central

2013-01-01

Background Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. Methods Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. Results At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. Conclusions Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes. PMID:24299561

SCF-KIT signaling induces endothelin-3 synthesis and secretion: Thereby activates and regulates endothelin-B-receptor for generating temporally- and spatially-precise nitric oxide to modulate SCF- and or KIT-expressing cell functions.

PubMed

Chen, Lei L; Zhu, Jing; Schumacher, Jonathan; Wei, Chongjuan; Ramdas, Latha; Prieto, Victor G; Jimenez, Arnie; Velasco, Marco A; Tripp, Sheryl R; Andtbacka, Robert H I; Gouw, Launce; Rodgers, George M; Zhang, Liansheng; Chan, Benjamin K; Cassidy, Pamela B; Benjamin, Robert S; Leachman, Sancy A; Frazier, Marsha L

2017-01-01

We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.

SCF-KIT signaling induces endothelin-3 synthesis and secretion: Thereby activates and regulates endothelin-B-receptor for generating temporally- and spatially-precise nitric oxide to modulate SCF- and or KIT-expressing cell functions

PubMed Central

Zhu, Jing; Schumacher, Jonathan; Wei, Chongjuan; Ramdas, Latha; Prieto, Victor G.; Jimenez, Arnie; Velasco, Marco A.; Tripp, Sheryl R.; Andtbacka, Robert H. I.; Gouw, Launce; Rodgers, George M.; Zhang, Liansheng; Chan, Benjamin K.; Cassidy, Pamela B.; Benjamin, Robert S.; Leachman, Sancy A.; Frazier, Marsha L.

2017-01-01

We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation. PMID:28880927

Defining the gene expression signature of rhabdomyosarcoma by meta-analysis

PubMed Central

Romualdi, Chiara; De Pittà, Cristiano; Tombolan, Lucia; Bortoluzzi, Stefania; Sartori, Francesca; Rosolen, Angelo; Lanfranchi, Gerolamo

2006-01-01

Background Rhabdomyosarcoma is a highly malignant soft tissue sarcoma in childhood and arises as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. The pathogenic pathways involved in this tumor are mostly unknown and therefore a better characterization of RMS gene expression profile would represent a considerable advance. The availability of publicly available gene expression datasets have opened up new challenges especially for the integration of data generated by different research groups and different array platforms with the purpose of obtaining new insights on the biological process investigated. Results In this work we performed a meta-analysis on four microarray and two SAGE datasets of gene expression data on RMS in order to evaluate the degree of agreement of the biological results obtained by these different studies and to identify common regulatory pathways that could be responsible of tumor growth. Regulatory pathways and biological processes significantly enriched has been investigated and a list of differentially meta-profiles have been identified as possible candidate of aggressiveness of RMS. Conclusion Our results point to a general down regulation of the energy production pathways, suggesting a hypoxic physiology for RMS cells. This result agrees with the high malignancy of RMS and with its resistance to most of the therapeutic treatments. In this context, different isoforms of the ANT gene have been consistently identified for the first time as differentially expressed in RMS. This gene is involved in anti-apoptotic processes when cells grow in low oxygen conditions. These new insights in the biological processes responsible of RMS growth and development demonstrate the effective advantage of the use of integrated analysis of gene expression studies. PMID:17090319

COMPREHENSIVE MOLECULAR CHARACTERIZATION OF CLEAR CELL RENAL CELL CARCINOMA

PubMed Central

2013-01-01

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (e.g. VHL) and the maintenance of chromatin states (e.g. PBRM1). We surveyed more than 400 tumors using different genomic platforms and identified 19 significantly mutated genes. The PI3K/Akt pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving down-regulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, up-regulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 and GRB10. Remodeling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumor stage and severity and offers new views on the opportunities for disease treatment. PMID:23792563

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis.

PubMed

Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

2014-07-01

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.

How might flukes and tapeworms maintain genome integrity without a canonical piRNA pathway?

PubMed

Skinner, Danielle E; Rinaldi, Gabriel; Koziol, Uriel; Brehm, Klaus; Brindley, Paul J

2014-03-01

Surveillance by RNA interference is central to controlling the mobilization of transposable elements (TEs). In stem cells, Piwi argonaute (Ago) proteins and associated proteins repress mobilization of TEs to maintain genome integrity. This defense mechanism targeting TEs is termed the Piwi-interacting RNA (piRNA) pathway. In this opinion article, we draw attention to the situation that the genomes of cestodes and trematodes have lost the piwi and vasa genes that are hallmark characters of the germline multipotency program. This absence of Piwi-like Agos and Vasa helicases prompts the question: how does the germline of these flatworms withstand mobilization of TEs? Here, we present an interpretation of mechanisms likely to defend the germline integrity of parasitic flatworms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro.

PubMed

Leung, Ada W Y; Hung, Stacy S; Backstrom, Ian; Ricaurte, Daniel; Kwok, Brian; Poon, Steven; McKinney, Steven; Segovia, Romulo; Rawji, Jenna; Qadir, Mohammed A; Aparicio, Samuel; Stirling, Peter C; Steidl, Christian; Bally, Marcel B

2016-01-01

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry.

PubMed

Shan, Sze Wan; Do, Chi Wai; Lam, Thomas Chuen; Kong, Ricky Pak Wing; Li, King Kit; Chun, Ka Man; Stamer, William Daniel; To, Chi Ho

2017-10-06

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.

The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high-osmolarity glycerol signaling pathways.

PubMed

Yun, Yingzi; Liu, Zunyong; Zhang, Jingze; Shim, Won-Bo; Chen, Yun; Ma, Zhonghua

2014-07-01

Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth and pathogenicity, and in responses to the environment. In this study, we characterized a MAP kinase kinase FgMkk1 in Fusarium graminearum, the causal agent of wheat head blight. Phenotypic analyses of the FgMKK1 mutant (ΔFgMKK1) showed that FgMkk1 is involved in the regulation of hyphal growth, pigmentation, conidiation, deoxynivalenol biosynthesis and virulence of F. graminearum. ΔFgMKK1 also showed increased sensitivity to cell wall-damaging agents, and to osmotic and oxidative stresses, but exhibited decreased sensitivity to the fungicides iprodione and fludioxonil. In addition, the mutant revealed increased sensitivity to a biocontrol agent, Trichoderma atroviride. Western blot assays revealed that FgMkk1 positively regulates phosphorylation of the MAP kinases Mgv1 and FgOs-2, the key component in the cell wall integrity (CWI) and high-osmolarity glycerol (HOG) signalling pathway respectively. Yeast two-hybrid assay indicated that Mgv1 interacts with a transcription factor FgRlm1. The FgRLM1 mutant (ΔFgRLM1) showed increased sensitivity to cell wall-damaging agents and exhibited decreased virulence. Taken together, our data indicated that FgMkk1 is an upstream component of Mgv1, and regulates vegetative differentiation, multiple stress response and virulence via the CWI and HOG signalling pathways. FgRlm1 may be a downstream component of Mgv1 in the CWI pathway in F. graminearum. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

PubMed Central

Cimo, Ann-Marie; Ahmed, Zamal; McIntyre, Bradley W.; Lewis, Dorothy E.; Ladbury, John E.

2013-01-01

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation. PMID:23758320

The balance between Gαi-Cdc42/Rac and Gα12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate

PubMed Central

Reinhard, Nathalie R.; Mastop, Marieke; Yin, Taofei; Wu, Yi; Bosma, Esmeralda K.; Gadella, Theodorus W. J.; Goedhart, Joachim; Hordijk, Peter L.

2017-01-01

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein–coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer–based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathway. PMID:28954861

Adapting the Stress Response: Viral Subversion of the mTOR Signaling Pathway.

PubMed

Le Sage, Valerie; Cinti, Alessandro; Amorim, Raquel; Mouland, Andrew J

2016-05-24

The mammalian target of rapamycin (mTOR) is a central regulator of gene expression, translation and various metabolic processes. Multiple extracellular (growth factors) and intracellular (energy status) molecular signals as well as a variety of stressors are integrated into the mTOR pathway. Viral infection is a significant stress that can activate, reduce or even suppress the mTOR signaling pathway. Consequently, viruses have evolved a plethora of different mechanisms to attack and co-opt the mTOR pathway in order to make the host cell a hospitable environment for replication. A more comprehensive knowledge of different viral interactions may provide fruitful targets for new antiviral drugs.

Aspergillus fumigatus Trehalose-Regulatory Subunit Homolog Moonlights To Mediate Cell Wall Homeostasis through Modulation of Chitin Synthase Activity.

PubMed

Thammahong, Arsa; Caffrey-Card, Alayna K; Dhingra, Sourabh; Obar, Joshua J; Cramer, Robert A

2017-04-25

Trehalose biosynthesis is found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for fungal pathogen virulence in humans and plants through multiple mechanisms. Loss of canonical trehalose biosynthesis genes in the human pathogen Aspergillus fumigatus significantly alters cell wall structure and integrity, though the mechanistic link between these virulence-associated pathways remains enigmatic. Here we characterize genes, called tslA and tslB , which encode proteins that contain domains similar to those corresponding to trehalose-6-phosphate phosphatase but lack critical catalytic residues for phosphatase activity. Loss of tslA reduces trehalose content in both conidia and mycelia, impairs cell wall integrity, and significantly alters cell wall structure. To gain mechanistic insights into the role that TslA plays in cell wall homeostasis, immunoprecipitation assays coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to reveal a direct interaction between TslA and CsmA, a type V chitin synthase enzyme. TslA regulates not only chitin synthase activity but also CsmA sub-cellular localization. Loss of TslA impacts the immunopathogenesis of murine invasive pulmonary aspergillosis through altering cytokine production and immune cell recruitment. In conclusion, our data provide a novel model whereby proteins in the trehalose pathway play a direct role in fungal cell wall homeostasis and consequently impact fungus-host interactions. IMPORTANCE Human fungal infections are increasing globally due to HIV infections and increased use of immunosuppressive therapies for many diseases. Therefore, new antifungal drugs with reduced side effects and increased efficacy are needed to improve treatment outcomes. Trehalose biosynthesis exists in pathogenic fungi and is absent in humans. Components of the trehalose biosynthesis pathway are important for the virulence of human-pathogenic fungi, including Aspergillus fumigatus Consequently, it has been proposed that components of this pathway are potential targets for antifungal drug development. However, how trehalose biosynthesis influences the fungus-host interaction remains enigmatic. One phenotype associated with fungal trehalose biosynthesis mutants that remains enigmatic is cell wall perturbation. Here we discovered a novel moonlighting role for a regulatory-like subunit of the trehalose biosynthesis pathway in A. fumigatus that regulates cell wall homeostasis through modulation of chitin synthase localization and activity. As the cell wall is a current and promising therapeutic target for fungal infections, understanding the role of trehalose biosynthesis in cell wall homeostasis and virulence is expected to help define new therapeutic opportunities. Copyright © 2017 Thammahong et al.

Modulation of C. elegans Touch Sensitivity Is Integrated at Multiple Levels

PubMed Central

Chen, Xiaoyin

2014-01-01

Sensory systems can adapt to different environmental signals. Here we identify four conditions that modulate anterior touch sensitivity in Caenorhabditis elegans after several hours and demonstrate that such sensory modulation is integrated at multiple levels to produce a single output. Prolonged vibration involving integrin signaling directly sensitizes the touch receptor neurons (TRNs). In contrast, hypoxia, the dauer state, and high salt reduce touch sensitivity by preventing the release of long-range neuroregulators, including two insulin-like proteins. Integration of these latter inputs occurs at upstream neurohormonal cells and at the insulin signaling cascade within the TRNs. These signals and those from integrin signaling converge to modulate touch sensitivity by regulating AKT kinases and DAF-16/FOXO. Thus, activation of either the integrin or insulin pathways can compensate for defects in the other pathway. This modulatory system integrates conflicting signals from different modalities, and adapts touch sensitivity to both mechanical and non-mechanical conditions. PMID:24806678

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

NASA Technical Reports Server (NTRS)

Wang, Xiang; Khadpe, Jay; Hu, Baocheng; Iliakis, George; Wang, Ya

2003-01-01

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

Type two innate lymphoid cells; the Janus cells in health and disease

PubMed Central

Maazi, Hadi; Akbari, Omid

2017-01-01

Summary Innate lymphoid cells are functionally diverse subsets of immune cells including the conventional natural killer cells, lymphoid tissue inducers, type 1, 2 and 3 with significant roles in immunity and pathogenesis of inflammatory diseases. Type 2 innate lymphoid cells (ILC2s) resemble type 2 helper (Th2) cells in cytokine production and contribute to anti-helminth immunity, maintaining mucosal tissue integrity and adipose tissue browning. ILC2s play important roles in the pathogenesis of allergic diseases and asthma. Studying the pathways of activation and regulation of ILC2s are currently a priority for giving a better understanding of pathogenesis of diseases with immunological roots. Recently, our laboratory and others have shown several pathways of regulation of ILC2s by costimulatory molecules such as ICOS, regulatory T cells and by compounds such as nicotine. In this review, we summarize the current understanding of the mechanisms of activation and regulation of ILC2s and the role of these cells in health and disease. PMID:28658553

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

PubMed

Jones, D L; Petty, J; Hoyle, D C; Hayes, A; Ragni, E; Popolo, L; Oliver, S G; Stateva, L I

2003-12-16

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Convergent evidence from systematic analysis of GWAS revealed genetic basis of esophageal cancer.

PubMed

Gao, Xue-Xin; Gao, Lei; Wang, Jiu-Qiang; Qu, Su-Su; Qu, Yue; Sun, Hong-Lei; Liu, Si-Dang; Shang, Ying-Li

2016-07-12

Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with risk of esophageal cancer (EC). However, investigation of genetic basis from the perspective of systematic biology and integrative genomics remains scarce.In this study, we explored genetic basis of EC based on GWAS data and implemented a series of bioinformatics methods including functional annotation, expression quantitative trait loci (eQTL) analysis, pathway enrichment analysis and pathway grouped network analysis.Two hundred and thirteen risk SNPs were identified, in which 44 SNPs were found to have significantly differential gene expression in esophageal tissues by eQTL analysis. By pathway enrichment analysis, 170 risk genes mapped by risk SNPs were enriched into 38 significant GO terms and 17 significant KEGG pathways, which were significantly grouped into 9 sub-networks by pathway grouped network analysis. The 9 groups of interconnected pathways were mainly involved with muscle cell proliferation, cellular response to interleukin-6, cell adhesion molecules, and ethanol oxidation, which might participate in the development of EC.Our findings provide genetic evidence and new insight for exploring the molecular mechanisms of EC.

Asymmetry between ON and OFF α ganglion cells of mouse retina: integration of signal and noise from synaptic inputs.

PubMed

Freed, Michael A

2017-11-15

Bipolar and amacrine cells presynaptic to the ON sustained α cell of mouse retina provide currents with a higher signal-to-noise power ratio (SNR) than those presynaptic to the OFF sustained α cell. Yet the ON cell loses proportionately more SNR from synaptic inputs to spike output than the OFF cell does. The higher SNR of ON bipolar cells at the beginning of the ON pathway compensates for losses incurred by the ON ganglion cell, and improves the processing of positive contrasts. ON and OFF pathways in the retina include functional pairs of neurons that, at first glance, appear to have symmetrically similar responses to brightening and darkening, respectively. Upon careful examination, however, functional pairs exhibit asymmetries in receptive field size and response kinetics. Until now, descriptions of how light-adapted retinal circuitry maintains a preponderance of signal over the noise have not distinguished between ON and OFF pathways. Here I present evidence of marked asymmetries between members of a functional pair of sustained α ganglion cells in the mouse retina. The ON cell exhibited a proportionately greater loss of signal-to-noise power ratio (SNR) from its presynaptic arrays to its postsynaptic currents. Thus the ON cell combines signal and noise from its presynaptic arrays of bipolar and amacrine cells less efficiently than the OFF cell does. Yet the inefficiency of the ON cell is compensated by its presynaptic arrays providing a higher SNR than the arrays presynaptic to the OFF cell, apparently to improve visual processing of positive contrasts. Dynamic clamp experiments were performed that introduced synaptic conductances into ON and OFF cells. When the amacrine-modulated conductance was removed, the ON cell's spike train exhibited an increase in SNR. The OFF cell, however, showed the opposite effect of removing amacrine input, which was a decrease in SNR. Thus ON and OFF cells have different modes of synaptic integration with direct effects on the SNR of the spike output. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Ties that bind: the integration of plastid signalling pathways in plant cell metabolism.

PubMed

Brunkard, Jacob O; Burch-Smith, Tessa M

2018-04-13

Plastids are critical organelles in plant cells that perform diverse functions and are central to many metabolic pathways. Beyond their major roles in primary metabolism, of which their role in photosynthesis is perhaps best known, plastids contribute to the biosynthesis of phytohormones and other secondary metabolites, store critical biomolecules, and sense a range of environmental stresses. Accordingly, plastid-derived signals coordinate a host of physiological and developmental processes, often by emitting signalling molecules that regulate the expression of nuclear genes. Several excellent recent reviews have provided broad perspectives on plastid signalling pathways. In this review, we will highlight recent advances in our understanding of chloroplast signalling pathways. Our discussion focuses on new discoveries illuminating how chloroplasts determine life and death decisions in cells and on studies elucidating tetrapyrrole biosynthesis signal transduction networks. We will also examine the role of a plastid RNA helicase, ISE2, in chloroplast signalling, and scrutinize intriguing results investigating the potential role of stromules in conducting signals from the chloroplast to other cellular locations. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Nip the HPV encoded evil in the cancer bud: HPV reshapes TRAILs and signaling landscapes

PubMed Central

2013-01-01

HPV encoded proteins can elicit ectopic protein–protein interactions that re-wire signaling pathways, in a mode that promotes malignancy. Moreover, accumulating data related to HPV is now providing compelling substantiation of a central role played by HPV in escaping immunosurveillance and impairment of apoptotic response. What emerges is an intricate network of Wnt, TGF, Notch signaling cascades that forms higher-order ligand–receptor complexes routing downstream signaling in HPV infected cells. These HPV infected cells are regulated both extracellularly by ligand receptor axis and intracellularly by HPV encoded proteins and impair TRAIL mediated apoptosis. We divide this review into different sections addressing how linear signaling pathways integrate to facilitate carcinogenesis and compounds that directly or indirectly reverse these aberrant interactions offer new possibilities for therapy in cancer. Although HPV encoded proteins mediated misrepresentation of pathways is difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can target dysregulated pathways in HPV infected cervical cancer cells, thus setting the stage for preclinical models and clinical trials. PMID:23773282

The Role of the Mammalian Target of Rapamycin (mTOR) in Pulmonary Fibrosis

PubMed Central

Nho, Richard

2018-01-01

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)-dependent pathway is one of the most integral pathways linked to cell metabolism, proliferation, differentiation, and survival. This pathway is dysregulated in a variety of diseases, including neoplasia, immune-mediated diseases, and fibroproliferative diseases such as pulmonary fibrosis. The mTOR kinase is frequently referred to as the master regulator of this pathway. Alterations in mTOR signaling are closely associated with dysregulation of autophagy, inflammation, and cell growth and survival, leading to the development of lung fibrosis. Inhibitors of mTOR have been widely studied in cancer therapy, as they may sensitize cancer cells to radiation therapy. Studies also suggest that mTOR inhibitors are promising modulators of fibroproliferative diseases such as idiopathic pulmonary fibrosis (IPF) and radiation-induced pulmonary fibrosis (RIPF). Therefore, mTOR represents an attractive and unique therapeutic target in pulmonary fibrosis. In this review, we discuss the pathological role of mTOR kinase in pulmonary fibrosis and examine how mTOR inhibitors may mitigate fibrotic progression. PMID:29518028

The NIH Library of Integrated Network-Based Cellular Signatures (LINCS) Program | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

By generating and making public data that indicates how cells respond to various genetic and environmental stressors, the LINCS project will help us gain a more detailed understanding of cell pathways and aid efforts to develop therapies that might restore perturbed pathways and networks to their normal states. The LINCS website is a source of information for the research community and general public about the LINCS project. This website along with the LINCS Data Portal contains details about the assays, cell types, and perturbagens that are currently part of the library, as well as links to participating sites, data releases from the sites, and software that can be used for analyzing the data.

STAT3 signaling in CD4+ T cells is critical for the pathogenesis of chronic sclerodermatous graft-versus-host disease in a murine model.

PubMed

Radojcic, Vedran; Pletneva, Maria A; Yen, Hung-Rong; Ivcevic, Sanja; Panoskaltsis-Mortari, Angela; Gilliam, Anita C; Drake, Charles G; Blazar, Bruce R; Luznik, Leo

2010-01-15

Donor CD4+ T cells are thought to be essential for inducing delayed host tissue injury in chronic graft-versus-host disease (GVHD). However, the relative contributions of distinct effector CD4+ T cell subpopulations and the molecular pathways influencing their generation are not known. We investigated the role of the STAT3 pathway in a murine model of chronic sclerodermatous GVHD. This pathway integrates multiple signaling events during the differentiation of naive CD4+ T cells and impacts their homeostasis. We report that chimeras receiving an allograft containing STAT3-ablated donor CD4+ T cells do not develop classic clinical and pathological manifestations of alloimmune tissue injury. Analysis of chimeras showed that abrogation of STAT3 signaling reduced the in vivo expansion of donor-derived CD4+ T cells and their accumulation in GVHD target tissues without abolishing antihost alloreactivity. STAT3 ablation did not significantly affect Th1 differentiation while enhancing CD4+CD25+Foxp3+ T cell reconstitution through thymus-dependent and -independent pathways. Transient depletion of CD25+ T cells in chimeras receiving STAT3-deficient T cells resulted in delayed development of alloimmune gut and liver injury. This delayed de novo GVHD was associated with the emergence of donor hematopoietic stem cell-derived Th1 and Th17 cells. These results suggest that STAT3 signaling in graft CD4+ T cells links the alloimmune tissue injury of donor graft T cells and the emergence of donor hematopoietic stem cell-derived pathogenic effector cells and that both populations contribute, albeit in different ways, to the genesis of chronic GVHD after allogenic bone marrow transplantation in a murine model.

In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

PubMed Central

Bordonaro, Michael; Shirasawa, Senji; Lazarova, Darina L.

2016-01-01

Although neoplastic cells exhibit relatively higher sensitivity to hyperthermia than normal cells, hyperthermia has had variable success as an anti-cancer therapy. This variable outcome might be due to the fact that cancer cells themselves have differential degrees of sensitivity to high temperature. We hypothesized that the varying sensitivity of colorectal cancer (CRC) cells to hyperthermia depends upon the differential induction of survival pathways. Screening of such pathways revealed that Extracellular Signal-Regulated Kinase (ERK) signaling is augmented by hyperthermia, and the extent of this modulation correlates with the mutation status of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Through clonal growth assays, apoptotic analyses and transcription reporter assays of CRC cells that differ only in KRAS mutation status we established that mutant KRAS cells are more sensitive to hyperthermia, as they exhibit sustained ERK signaling hyperactivation and increased Wingless/Integrated (WNT)/beta-catenin signaling. We propose that whereas increased levels of WNT and ERK signaling and a positive feedback between the two pathways is a major obstacle in anti-cancer therapy today, under hyperthermia the hyperinduction of the pathways and their positive crosstalk contribute to CRC cell death. Ascertaining the causative association between types of mutations and hyperthermia sensitivity may allow for a mutation profile-guided application of hyperthermia as an anti-cancer therapy. Since KRAS and WNT signaling mutations are prevalent in CRC, our results suggest that hyperthermia-based therapy might benefit a significant number, but not all, CRC patients. PMID:27187477

A structure- and chemical genomics-based approach for repositioning of drugs against VCP/p97 ATPase.

PubMed

Segura-Cabrera, Aldo; Tripathi, Reshmi; Zhang, Xiaoyi; Gui, Lin; Chou, Tsui-Fen; Komurov, Kakajan

2017-03-21

Valosin-containing protein (VCP/p97) ATPase (a.k.a. Cdc48) is a key member of the ER-associated protein degradation (ERAD) pathway. ERAD and VCP/p97 have been implicated in a multitude of human diseases, such as neurodegenerative diseases and cancer. Inhibition of VCP/p97 induces proteotoxic ER stress and cell death in cancer cells, making it an attractive target for cancer treatment. However, no drugs exist against this protein in the market. Repositioning of drugs towards new indications is an attractive alternative to the de novo drug development due to the potential for significantly shorter time to clinical translation. Here, we employed an integrative strategy for the repositioning of drugs as novel inhibitors of the VCP/p97 ATPase. We integrated structure-based virtual screening with the chemical genomics analysis of drug molecular signatures, and identified several candidate inhibitors of VCP/p97 ATPase. Importantly, experimental validation with cell-based and in vitro ATPase assays confirmed three (ebastine, astemizole and clotrimazole) out of seven tested candidates (~40% true hit rate) as direct inhibitors of VCP/p97 and ERAD. This study introduces an effective integrative strategy for drug repositioning, and identified new drugs against the VCP/p97/ERAD pathway in human diseases.

Shared molecular pathways and gene networks for cardiovascular disease and type 2 diabetes mellitus in women across diverse ethnicities.

PubMed

Chan, Kei Hang K; Huang, Yen-Tsung; Meng, Qingying; Wu, Chunyuan; Reiner, Alexander; Sobel, Eric M; Tinker, Lesley; Lusis, Aldons J; Yang, Xia; Liu, Simin

2014-12-01

Although cardiovascular disease (CVD) and type 2 diabetes mellitus (T2D) share many common risk factors, potential molecular mechanisms that may also be shared for these 2 disorders remain unknown. Using an integrative pathway and network analysis, we performed genome-wide association studies in 8155 blacks, 3494 Hispanic American, and 3697 Caucasian American women who participated in the national Women's Health Initiative single-nucleotide polymorphism (SNP) Health Association Resource and the Genomics and Randomized Trials Network. Eight top pathways and gene networks related to cardiomyopathy, calcium signaling, axon guidance, cell adhesion, and extracellular matrix seemed to be commonly shared between CVD and T2D across all 3 ethnic groups. We also identified ethnicity-specific pathways, such as cell cycle (specific for Hispanic American and Caucasian American) and tight junction (CVD and combined CVD and T2D in Hispanic American). In network analysis of gene-gene or protein-protein interactions, we identified key drivers that included COL1A1, COL3A1, and ELN in the shared pathways for both CVD and T2D. These key driver genes were cross-validated in multiple mouse models of diabetes mellitus and atherosclerosis. Our integrative analysis of American women of 3 ethnicities identified multiple shared biological pathways and key regulatory genes for the development of CVD and T2D. These prospective findings also support the notion that ethnicity-specific susceptibility genes and process are involved in the pathogenesis of CVD and T2D. © 2014 American Heart Association, Inc.

RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing

PubMed Central

Li, Yingfu

2017-01-01

The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named “RiboFACSeq”. Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis. PMID:29211762

Identification of Key Transcription Factors Associated with Lung Squamous Cell Carcinoma

PubMed Central

Zhang, Feng; Chen, Xia; Wei, Ke; Liu, Daoming; Xu, Xiaodong; Zhang, Xing; Shi, Hong

2017-01-01

Background Lung squamous cell carcinoma (lung SCC) is a common type of lung cancer, but its mechanism of pathogenesis is unclear. The aim of this study was to identify key transcription factors in lung SCC and elucidate its mechanism. Material/Methods Six published microarray datasets of lung SCC were downloaded from Gene Expression Omnibus (GEO) for integrated bioinformatics analysis. Significance analysis of microarrays was used to identify differentially expressed genes (DEGs) between lung SCC and normal controls. The biological functions and signaling pathways of DEGs were mapped in the Gene Otology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, respectively. A transcription factor gene regulatory network was used to obtain insights into the functions of DEGs. Results A total of 1,011 genes, including 539 upregulated genes and 462 downregulated genes, were filtered as DEGs between lung SCC and normal controls. DEGs were significantly enriched in cell cycle, DNA replication, p53 signaling pathway, pathways in cancer, adherens junction, and cell adhesion molecules signaling pathways. There were 57 transcription factors identified, which were used to construct a regulatory network. The network consisted of 736 interactions between 49 transcription factors and 486 DEGs. NFIC, BRCA1, and NFATC2 were the top 3 transcription factors that had the highest connectivity with DEGs and that regulated 83, 82, and 75 DEGs in the network, respectively. Conclusions NFIC, BRCA1, and NFATC2 might be the key transcription factors in the development of lung SCC by regulating the genes involved in cell cycle and DNA replication pathways. PMID:28081052

VIPER: Chronic Pain after Amputation: Inflammatory Mechanisms, Novel Analgesic Pathways, and Improved Patient Safety

DTIC Science & Technology

2017-10-01

Through analysis of data obtained in the Molecular Signatures of Chronic Pain Subtypes study termed Veterans Integrated Pain Evaluation Research...immune cells (macrophages) to chronic pain while also evaluating novel analgesics in relevant animal models. The current proposal also attempts to...analysis of data obtained in the Molecular Signatures of Chronic Pain Subtypes study termed Veterans Integrated Pain Evaluation Research (VIPER

Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro

PubMed Central

Leung, Ada W. Y.; Hung, Stacy S.; Backstrom, Ian; Ricaurte, Daniel; Kwok, Brian; Poon, Steven; McKinney, Steven; Segovia, Romulo; Rawji, Jenna; Qadir, Mohammed A.; Aparicio, Samuel; Stirling, Peter C.; Steidl, Christian; Bally, Marcel B.

2016-01-01

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell’s ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways. PMID:26938915

Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

PubMed

Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

2015-01-01

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Plant cell wall signalling and receptor-like kinases.

PubMed

Wolf, Sebastian

2017-02-15

Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The capacity of Aspergillus niger to sense and respond to cell wall stress requires at least three transcription factors: RlmA, MsnA and CrzA.

PubMed

Fiedler, Markus Rm; Lorenz, Annett; Nitsche, Benjamin M; van den Hondel, Cees Amjj; Ram, Arthur Fj; Meyer, Vera

2014-01-01

Cell wall integrity, vesicle transport and protein secretion are key factors contributing to the vitality and productivity of filamentous fungal cell factories such as Aspergillus niger . In order to pioneer rational strain improvement programs, fundamental knowledge on the genetic basis of these processes is required. The aim of the present study was thus to unravel survival strategies of A. niger when challenged with compounds interfering directly or indirectly with its cell wall integrity: calcofluor white, caspofungin, aureobasidin A, FK506 and fenpropimorph. Transcriptomics signatures of A. niger and phenotypic analyses of selected null mutant strains were used to predict regulator proteins mediating the survival responses against these stressors. This integrated approach allowed us to reconstruct a model for the cell wall salvage gene network of A. niger that ensures survival of the fungus upon cell surface stress. The model predicts that (i) caspofungin and aureobasidin A induce the cell wall integrity pathway as a main compensatory response via induction of RhoB and RhoD, respectively, eventually activating the mitogen-activated protein kinase kinase MkkA and the transcription factor RlmA. (ii) RlmA is the main transcription factor required for the protection against calcofluor white but it cooperates with MsnA and CrzA to ensure survival of A. niger when challenged with caspofungin and aureobasidin A. (iii) Membrane stress provoked by aureobasidin A via disturbance of sphingolipid synthesis induces cell wall stress, whereas fenpropimorph-induced disturbance of ergosterol synthesis does not. The present work uncovered a sophisticated defence system of A. niger which employs at least three transcription factors - RlmA, MsnA and CrzA - to protect itself against cell wall stress. The transcriptomic data furthermore predicts a fourth transfactor, SrbA, which seems to be specifically important to survive fenpropimorph-induced cell membrane stress. Future studies will disclose how these regulators are interlocked in different signaling pathways to secure survival of A. niger under different cell wall stress conditions.

A review of recent progress in heterogeneous silicon tandem solar cells

NASA Astrophysics Data System (ADS)

Yamaguchi, Masafumi; Lee, Kan-Hua; Araki, Kenji; Kojima, Nobuaki

2018-04-01

Silicon solar cells are the most established solar cell technology and are expected to dominate the market in the near future. As state-of-the-art silicon solar cells are approaching the Shockley-Queisser limit, stacking silicon solar cells with other photovoltaic materials to form multi-junction devices is an obvious pathway to further raise the efficiency. However, many challenges stand in the way of fully realizing the potential of silicon tandem solar cells because heterogeneously integrating silicon with other materials often degrades their qualities. Recently, above or near 30% silicon tandem solar cell has been demonstrated, showing the promise of achieving high-efficiency and low-cost solar cells via silicon tandem. This paper reviews the recent progress of integrating solar cell with other mainstream solar cell materials. The first part of this review focuses on the integration of silicon with III-V semiconductor solar cells, which is a long-researched topic since the emergence of III-V semiconductors. We will describe the main approaches—heteroepitaxy, wafer bonding and mechanical stacking—as well as other novel approaches. The second part introduces the integration of silicon with polycrystalline thin-film solar cells, mainly perovskites on silicon solar cells because of its rapid progress recently. We will also use an analytical model to compare the material qualities of different types of silicon tandem solar cells and project their practical efficiency limits.

Transforming growth factor‐β in liver cancer stem cells and regeneration

PubMed Central

Rao, Shuyun; Zaidi, Sobia; Banerjee, Jaideep; Jogunoori, Wilma; Sebastian, Raul; Mishra, Bibhuti; Nguyen, Bao‐Ngoc; Wu, Ray‐Chang; White, Jon; Deng, Chuxia; Amdur, Richard; Li, Shulin

2017-01-01

Cancer stem cells have established mechanisms that contribute to tumor heterogeneity as well as resistance to therapy. Over 40% of hepatocellular carcinomas (HCCs) are considered to be clonal and arise from a stem‐like/cancer stem cell. Moreover, HCC is the second leading cause of cancer death worldwide, and an improved understanding of cancer stem cells and targeting these in this cancer are urgently needed. Multiple studies have revealed etiological patterns and multiple genes/pathways signifying initiation and progression of HCC; however, unlike the transforming growth factor β (TGF‐β) pathway, loss of p53 and/or activation of β‐catenin do not spontaneously drive HCC in animal models. Despite many advances in cancer genetics that include identifying the dominant role of TGF‐β signaling in gastrointestinal cancers, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support effective targeted therapies for these common and lethal cancers. Moreover, pathways involved in stem cell transformation into gastrointestinal cancers remain largely undefined. Identifying the key mechanisms and developing models that reflect the human disease can lead to effective new treatment strategies. In this review, we dissect the evidence obtained from mouse and human liver regeneration, and mouse genetics, to provide insight into the role of TGF‐β in regulating the cancer stem cell niche. (Hepatology Communications 2017;1:477–493) PMID:29404474

Paper-based microreactor integrating cell culture and subsequent immunoassay for the investigation of cellular phosphorylation.

PubMed

Lei, Kin Fong; Huang, Chia-Hao

2014-12-24

Investigation of cellular phosphorylation and signaling pathway has recently gained much attention for the study of pathogenesis of cancer. Related conventional bioanalytical operations for this study including cell culture and Western blotting are time-consuming and labor-intensive. In this work, a paper-based microreactor has been developed to integrate cell culture and subsequent immunoassay on a single paper. The paper-based microreactor was a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells were directly seeded in the circular zones without hydrogel encapsulation and cultured for 1 day. Subsequently, protein expressions including structural, functional, and phosphorylated proteins of the cells could be detected by their specific antibodies, respectively. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by IL-6 cytokine was demonstrated by the paper-based microreactor. This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathway for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore, it has the potential to be developed for routine protocol for various research areas in conventional bioanalytical laboratories.

Human Cpr (Cell Cycle Progression Restoration) Genes Impart a Far(-) Phenotype on Yeast Cells

PubMed Central

Edwards, M. C.; Liegeois, N.; Horecka, J.; DePinho, R. A.; Sprague-Jr., G. F.; Tyers, M.; Elledge, S. J.

1997-01-01

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G(1) arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G(1) arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles. PMID:9383053

Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target.

PubMed

Dufva, Olli; Kankainen, Matti; Kelkka, Tiina; Sekiguchi, Nodoka; Awad, Shady Adnan; Eldfors, Samuli; Yadav, Bhagwan; Kuusanmäki, Heikki; Malani, Disha; Andersson, Emma I; Pietarinen, Paavo; Saikko, Leena; Kovanen, Panu E; Ojala, Teija; Lee, Dean A; Loughran, Thomas P; Nakazawa, Hideyuki; Suzumiya, Junji; Suzuki, Ritsuro; Ko, Young Hyeh; Kim, Won Seog; Chuang, Shih-Sung; Aittokallio, Tero; Chan, Wing C; Ohshima, Koichi; Ishida, Fumihiro; Mustjoki, Satu

2018-04-19

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.

Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release

PubMed Central

Huber, Heinrich J; Dussmann, Heiko; Kilbride, Seán M; Rehm, Markus; Prehn, Jochen H M

2011-01-01

Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨm from −142 to −88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨm. However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation. PMID:21364572

Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

PubMed

Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

2015-10-01

The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

Integrated High Throughput Analysis Identifies GSK3 as a Crucial Determinant of p53-Mediated Apoptosis in Lung Cancer Cells.

PubMed

Zhang, Yu; Zhu, Chenyang; Sun, Bangyao; Lv, Jiawei; Liu, Zhonghua; Liu, Shengwang; Li, Hai

2017-01-01

p53 dysfunction is frequently observed in lung cancer. Although restoring the tumour suppressor function of p53 is recently approved as a putative strategy for combating cancers, the lack of understanding of the molecular mechanism underlying p53-mediated lung cancer suppression has limited the application of p53-based therapies in lung cancer. Using RNA sequencing, we determined the transcriptional profile of human non-small cell lung carcinoma A549 cells after treatment with two p53-activating chemical compounds, nutlin and RITA, which could induce A549 cell cycle arrest and apoptosis, respectively. Bioinformatics analysis of genome-wide gene expression data showed that distinct transcription profiles were induced by nutlin and RITA and 66 pathways were differentially regulated by these two compounds. However, only two of these pathways, 'Adherens junction' and 'Axon guidance', were found to be synthetic lethal with p53 re-activation, as determined via integrated analysis of genome-wide gene expression profile and short hairpin RNA (shRNA) screening. Further functional protein association analysis of significantly regulated genes associated with these two synthetic lethal pathways indicated that GSK3 played a key role in p53-mediated A549 cell apoptosis, and then gene function study was performed, which revealed that GSK3 inhibition promoted p53-mediated A549 cell apoptosis in a p53 post-translational activity-dependent manner. Our findings provide us with new insights regarding the mechanism by which p53 mediates A549 apoptosis and may cast light on the development of more efficient p53-based strategies for treating lung cancer. © 201 The Author(s). Published by S. Karger AG, Basel.

Evolution of Antibody-Drug Conjugate Tumor Disposition Model to Predict Preclinical Tumor Pharmacokinetics of Trastuzumab-Emtansine (T-DM1).

PubMed

Singh, Aman P; Maass, Katie F; Betts, Alison M; Wittrup, K Dane; Kulkarni, Chethana; King, Lindsay E; Khot, Antari; Shah, Dhaval K

2016-07-01

A mathematical model capable of accurately characterizing intracellular disposition of ADCs is essential for a priori predicting unconjugated drug concentrations inside the tumor. Towards this goal, the objectives of this manuscript were to: (1) evolve previously published cellular disposition model of ADC with more intracellular details to characterize the disposition of T-DM1 in different HER2 expressing cell lines, (2) integrate the improved cellular model with the ADC tumor disposition model to a priori predict DM1 concentrations in a preclinical tumor model, and (3) identify prominent pathways and sensitive parameters associated with intracellular activation of ADCs. The cellular disposition model was augmented by incorporating intracellular ADC degradation and passive diffusion of unconjugated drug across tumor cells. Different biomeasures and chemomeasures for T-DM1, quantified in the companion manuscript, were incorporated into the modified model of ADC to characterize in vitro pharmacokinetics of T-DM1 in three HER2+ cell lines. When the cellular model was integrated with the tumor disposition model, the model was able to a priori predict tumor DM1 concentrations in xenograft mice. Pathway analysis suggested different contribution of antigen-mediated and passive diffusion pathways for intracellular unconjugated drug exposure between in vitro and in vivo systems. Global and local sensitivity analyses revealed that non-specific deconjugation and passive diffusion of the drug across tumor cell membrane are key parameters for drug exposure inside a cell. Finally, a systems pharmacokinetic model for intracellular processing of ADCs has been proposed to highlight our current understanding about the determinants of ADC activation inside a cell.

Stage-specific integration of maternal and embryonic peroxisome proliferator-activated receptor delta signaling is critical to pregnancy success.

PubMed

Wang, Haibin; Xie, Huirong; Sun, Xiaofei; Tranguch, Susanne; Zhang, Hao; Jia, Xiangxu; Wang, Dingzhi; Das, Sanjoy K; Desvergne, Béatrice; Wahli, Walter; DuBois, Raymond N; Dey, Sudhansu K

2007-12-28

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas.

PubMed

Campbell, Joshua D; Yau, Christina; Bowlby, Reanne; Liu, Yuexin; Brennan, Kevin; Fan, Huihui; Taylor, Alison M; Wang, Chen; Walter, Vonn; Akbani, Rehan; Byers, Lauren Averett; Creighton, Chad J; Coarfa, Cristian; Shih, Juliann; Cherniack, Andrew D; Gevaert, Olivier; Prunello, Marcos; Shen, Hui; Anur, Pavana; Chen, Jianhong; Cheng, Hui; Hayes, D Neil; Bullman, Susan; Pedamallu, Chandra Sekhar; Ojesina, Akinyemi I; Sadeghi, Sara; Mungall, Karen L; Robertson, A Gordon; Benz, Christopher; Schultz, Andre; Kanchi, Rupa S; Gay, Carl M; Hegde, Apurva; Diao, Lixia; Wang, Jing; Ma, Wencai; Sumazin, Pavel; Chiu, Hua-Sheng; Chen, Ting-Wen; Gunaratne, Preethi; Donehower, Larry; Rader, Janet S; Zuna, Rosemary; Al-Ahmadie, Hikmat; Lazar, Alexander J; Flores, Elsa R; Tsai, Kenneth Y; Zhou, Jane H; Rustgi, Anil K; Drill, Esther; Shen, Ronglei; Wong, Christopher K; Stuart, Joshua M; Laird, Peter W; Hoadley, Katherine A; Weinstein, John N; Peto, Myron; Pickering, Curtis R; Chen, Zhong; Van Waes, Carter

2018-04-03

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches. Published by Elsevier Inc.

[Fanconi anemia: cellular and molecular features].

PubMed

Macé, G; Briot, D; Guervilly, J-H; Rosselli, F

2007-02-01

Fanconi anemia (FA) is a recessive human cancer prone syndrome featuring bone marrow failure, developmental abnormalities and hypersensitivity to DNA crosslinking agents exposure. 11 among 12 FA gene have been isolated. The biochemical functions of the FANC proteins remain poorly understood. Anyhow, to cope with DNA crosslinks a cell needs a functional FANC pathway. Moreover, the FANC proteins appear to be involved in cell protection against oxidative damage and in the control of TNF-alpha activity. In this review, we describe the current understanding of the FANC pathway and we present how it may be integrated in the complex networks of proteins involved in maintaining the cellular homeostasis.

Pericytes of the neurovascular unit: Key functions and signaling pathways

PubMed Central

Sweeney, Melanie D.; Ayyadurai, Shiva; Zlokovic, Berislav V.

2017-01-01

Pericytes are vascular mural cells embedded in the basement membrane of blood microvessels. They extend their processes along capillaries, pre-capillary arterioles, and post-capillary venules. The central nervous system (CNS) pericytes are uniquely positioned within the neurovascular unit between endothelial cells, astrocytes, and neurons. They integrate, coordinate, and process signals from their neighboring cells to generate diverse functional responses that are critical for CNS functions in health and disease including regulation of the blood-brain barrier permeability, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, neuroinflammation, and stem cell activity. Here, we examine the key signaling pathways between pericytes and their neighboring endothelial cells, astrocytes, and neurons that control neurovascular functions. We also review the role of pericytes in different CNS disorders including rare monogenic diseases and complex neurological disorders such as Alzheimer's disease and brain tumors. Finally, we discuss directions for future studies. PMID:27227366

Metabolic and Epigenetic Coordination of T Cell and Macrophage Immunity.

PubMed

Phan, Anthony T; Goldrath, Ananda W; Glass, Christopher K

2017-05-16

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic and epigenetic coordination of T cell and Macrophage immunity

PubMed Central

Phan, Anthony T.; Goldrath, Ananda W.; Glass, Christopher K.

2017-01-01

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection. PMID:28514673

Emerging Imaging and Genomic Tools for Developmental Systems Biology.

PubMed

Liu, Zhe; Keller, Philipp J

2016-03-21

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Boosting the pentose phosphate pathway restores cardiac progenitor cell availability in diabetes.

PubMed

Katare, Rajesh; Oikawa, Atsuhiko; Cesselli, Daniela; Beltrami, Antonio P; Avolio, Elisa; Muthukrishnan, Deepti; Munasinghe, Pujika Emani; Angelini, Gianni; Emanueli, Costanza; Madeddu, Paolo

2013-01-01

Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway. Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt. We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.

Boosting the pentose phosphate pathway restores cardiac progenitor cell availability in diabetes

PubMed Central

Katare, Rajesh; Oikawa, Atsuhiko; Cesselli, Daniela; Beltrami, Antonio P.; Avolio, Elisa; Muthukrishnan, Deepti; Munasinghe, Pujika Emani; Angelini, Gianni; Emanueli, Costanza; Madeddu, Paolo

2013-01-01

Aims Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway. Methods and results Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1pos CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105pos progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt. Conclusion We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity. PMID:22997160

Reduced risk of apoptosis: mechanisms of stress responses.

PubMed

Milisav, Irina; Poljšak, Borut; Ribarič, Samo

2017-02-01

Apoptosis signaling pathways are integrated into a wider network of interconnected apoptotic and anti-apoptotic pathways that regulate a broad range of cell responses from cell death to growth, development and stress responses. An important trigger for anti- or pro-apoptotic cell responses are different forms of stress including hypoxia, energy deprivation, DNA damage or inflammation. Stress duration and intensity determine whether the cell's response will be improved cell survival, due to stress adaptation, or cell death by apoptosis, necrosis or autophagy. Although the interplay between enhanced stress tolerance and modulation of apoptosis triggering is not yet fully understood, there is a substantial body of experimental evidence demonstrating that apoptosis and anti-apoptosis signaling pathways can be manipulated to trigger or delay apoptosis in vitro or in vivo. Anti-apoptotic strategies cover a broad range of approaches. These interventions include mediators that prevent apoptosis (trophic factors and cytokines), apoptosis inhibition (caspase inhibition, stimulation of anti-apoptotic or inhibition of pro-apoptotic proteins and elimination of apoptotic stimulus), adaptive stress responses (induction of maintenance and repair, caspase inactivation) and cell-cell interactions (blocking engulfment and modified micro environment). There is a consensus that preclinical efficacy and safety evaluations of anti-apoptotic strategies should be performed with protocols that simulate as closely as possible the effects of aging, gender, risk factors, comorbidities and co-medications.

Systems heterogeneity: An integrative way to understand cancer heterogeneity.

PubMed

Wang, Diane Catherine; Wang, Xiangdong

2017-04-01

The concept of systems heterogeneity was firstly coined and explained in the Special Issue, as a new alternative to understand the importance and complexity of heterogeneity in cancer. Systems heterogeneity can offer a full image of heterogeneity at multi-dimensional functions and multi-omics by integrating gene or protein expression, epigenetics, sequencing, phosphorylation, transcription, pathway, or interaction. The Special Issue starts with the roles of epigenetics in the initiation and development of cancer heterogeneity through the interaction between permanent genetic mutations and dynamic epigenetic alterations. Cell heterogeneity was defined as the difference in biological function and phenotypes between cells in the same organ/tissue or in different organs, as well as various challenges, as exampled in telocytes. The single cell heterogeneity has the value of identifying diagnostic biomarkers and therapeutic targets and clinical potential of single cell systems heterogeneity in clinical oncology. A number of signaling pathways and factors contribute to the development of systems heterogeneity. Proteomic heterogeneity can change the strategy and thinking of drug discovery and development by understanding the interactions between proteins or proteins with drugs in order to optimize drug efficacy and safety. The association of cancer heterogeneity with cancer cell evolution and metastasis was also overviewed as a new alternative for diagnostic biomarkers and therapeutic targets in clinical application. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tick-borne encephalitis virus infects human brain microvascular endothelial cells without compromising blood-brain barrier integrity.

PubMed

Palus, Martin; Vancova, Marie; Sirmarova, Jana; Elsterova, Jana; Perner, Jan; Ruzek, Daniel

2017-07-01

Alteration of the blood-brain barrier (BBB) is a hallmark of tick-borne encephalitis (TBE), a life-threating human viral neuroinfection. However, the mechanism of BBB breakdown during TBE, as well as TBE virus (TBEV) entry into the brain is unclear. Here, primary human microvascular endothelial cells (HBMECs) were infected with TBEV to study interactions with the BBB. Although the number of infected cells was relatively low in culture (<5%), the infection was persistent with high TBEV yields (>10 6 pfu/ml). Infection did not induce any significant changes in the expression of key tight junction proteins or upregulate the expression of cell adhesion molecules, and did not alter the highly organized intercellular junctions between HBMECs. In an in vitro BBB model, the virus crossed the BBB via a transcellular pathway without compromising the integrity of the cell monolayer. The results indicate that HBMECs may support TBEV entry into the brain without altering BBB integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

The underlying pathway structure of biochemical reaction networks

PubMed Central

Schilling, Christophe H.; Palsson, Bernhard O.

1998-01-01

Bioinformatics is yielding extensive, and in some cases complete, genetic and biochemical information about individual cell types and cellular processes, providing the composition of living cells and the molecular structure of its components. These components together perform integrated cellular functions that now need to be analyzed. In particular, the functional definition of biochemical pathways and their role in the context of the whole cell is lacking. In this study, we show how the mass balance constraints that govern the function of biochemical reaction networks lead to the translation of this problem into the realm of linear algebra. The functional capabilities of biochemical reaction networks, and thus the choices that cells can make, are reflected in the null space of their stoichiometric matrix. The null space is spanned by a finite number of basis vectors. We present an algorithm for the synthesis of a set of basis vectors for spanning the null space of the stoichiometric matrix, in which these basis vectors represent the underlying biochemical pathways that are fundamental to the corresponding biochemical reaction network. In other words, all possible flux distributions achievable by a defined set of biochemical reactions are represented by a linear combination of these basis pathways. These basis pathways thus represent the underlying pathway structure of the defined biochemical reaction network. This development is significant from a fundamental and conceptual standpoint because it yields a holistic definition of biochemical pathways in contrast to definitions that have arisen from the historical development of our knowledge about biochemical processes. Additionally, this new conceptual framework will be important in defining, characterizing, and studying biochemical pathways from the rapidly growing information on cellular function. PMID:9539712

Coordinate Regulation of Stem Cell Competition by Slit-Robo and JAK-STAT Signaling in the Drosophila Testis

PubMed Central

Stine, Rachel R.; Greenspan, Leah J.; Ramachandran, Kapil V.; Matunis, Erika L.

2014-01-01

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche. PMID:25375180

Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter

PubMed Central

Qu, X; Sandmann, T; Frierson, H; Fu, L; Fuentes, E; Walter, K; Okrah, K; Rumpel, C; Moskaluk, C; Lu, S; Wang, Y; Bourgon, R; Penuel, E; Pirzkall, A; Amler, L; Lackner, M R; Tabernero, J; Hampton, G M; Kabbarah, O

2016-01-01

Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma–carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers. PMID:27270421

Convergence of Cortical and Sensory Driver Inputs on Single Thalamocortical Cells

PubMed Central

Groh, Alexander; Bokor, Hajnalka; Mease, Rebecca A.; Plattner, Viktor M.; Hangya, Balázs; Stroh, Albrecht; Deschenes, Martin; Acsády, László

2014-01-01

Ascending and descending information is relayed through the thalamus via strong, “driver” pathways. According to our current knowledge, different driver pathways are organized in parallel streams and do not interact at the thalamic level. Using an electron microscopic approach combined with optogenetics and in vivo physiology, we examined whether driver inputs arising from different sources can interact at single thalamocortical cells in the rodent somatosensory thalamus (nucleus posterior, POm). Both the anatomical and the physiological data demonstrated that ascending driver inputs from the brainstem and descending driver inputs from cortical layer 5 pyramidal neurons converge and interact on single thalamocortical neurons in POm. Both individual pathways displayed driver properties, but they interacted synergistically in a time-dependent manner and when co-activated, supralinearly increased the output of thalamus. As a consequence, thalamocortical neurons reported the relative timing between sensory events and ongoing cortical activity. We conclude that thalamocortical neurons can receive 2 powerful inputs of different origin, rather than only a single one as previously suggested. This allows thalamocortical neurons to integrate raw sensory information with powerful cortical signals and transfer the integrated activity back to cortical networks. PMID:23825316

Interlocked positive and negative feedback network motifs regulate β-catenin activity in the adherens junction pathway

PubMed Central

Klinke, David J.; Horvath, Nicholas; Cuppett, Vanessa; Wu, Yueting; Deng, Wentao; Kanj, Rania

2015-01-01

The integrity of epithelial tissue architecture is maintained through adherens junctions that are created through extracellular homotypic protein–protein interactions between cadherin molecules. Cadherins also provide an intracellular scaffold for the formation of a multiprotein complex that contains signaling proteins, including β-catenin. Environmental factors and controlled tissue reorganization disrupt adherens junctions by cleaving the extracellular binding domain and initiating a series of transcriptional events that aim to restore tissue homeostasis. However, it remains unclear how alterations in cell adhesion coordinate transcriptional events, including those mediated by β-catenin in this pathway. Here were used quantitative single-cell and population-level in vitro assays to quantify the endogenous pathway dynamics after the proteolytic disruption of the adherens junctions. Using prior knowledge of isolated elements of the overall network, we interpreted these data using in silico model-based inference to identify the topology of the regulatory network. Collectively the data suggest that the regulatory network contains interlocked network motifs consisting of a positive feedback loop, which is used to restore the integrity of adherens junctions, and a negative feedback loop, which is used to limit β-catenin–induced gene expression. PMID:26224311

The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

PubMed Central

Vargas, Diego A.; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A.; Zaman, Muhammad H.

2016-01-01

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability of E-cadherin junctions in response to DPAGT1 inhibition. We show the impact of pathway dysregulation through measurements of cell migration in scratch-wound assays. Collectively, our results highlight the importance of numerical analyses of cellular networks dynamics to gain insights into physiological processes and potential design of therapeutic strategies to prevent epithelial cell invasion in cancer. PMID:27427963

Stromal cells can contribute oncogenic signals

NASA Technical Reports Server (NTRS)

Tlsty, T. D.

2001-01-01

The majority of studies of neoplastic transformation have focused attention on events that occur within transformed cells. These cell autonomous events result in the disruption of molecular pathways that regulate basic activities of the cells such as proliferation, death, movement and genomic integrity. Other studies have addressed the microenvironment of tumor cells and documented its importance in supporting tumor progression. Recent work has begun to expand on these initial studies of tumor microenvironment and now provide novel insights into the possible initiation and progression of malignant cells. This review will address the transforming effect of stromal cells on epithelial components. Copyright 2001 Academic Press.

piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

PubMed Central

Pezic, Dubravka; Manakov, Sergei A.; Sachidanandam, Ravi; Aravin, Alexei A.

2014-01-01

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments. PMID:24939875

Adapting the Stress Response: Viral Subversion of the mTOR Signaling Pathway

PubMed Central

Le Sage, Valerie; Cinti, Alessandro; Amorim, Raquel; Mouland, Andrew J.

2016-01-01

The mammalian target of rapamycin (mTOR) is a central regulator of gene expression, translation and various metabolic processes. Multiple extracellular (growth factors) and intracellular (energy status) molecular signals as well as a variety of stressors are integrated into the mTOR pathway. Viral infection is a significant stress that can activate, reduce or even suppress the mTOR signaling pathway. Consequently, viruses have evolved a plethora of different mechanisms to attack and co-opt the mTOR pathway in order to make the host cell a hospitable environment for replication. A more comprehensive knowledge of different viral interactions may provide fruitful targets for new antiviral drugs. PMID:27231932

CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea

PubMed Central

Marraffini, Luciano A.; Sontheimer, Erik J.

2010-01-01

Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway. PMID:20125085

Genomics of immune response to typhoid and cholera vaccines

PubMed Central

Majumder, Partha P.

2015-01-01

Considerable variation in antibody response (AR) was observed among recipients of an injectable typhoid vaccine and an oral cholera vaccine. We sought to find whether polymorphisms in genes of the immune system, both innate and adaptive, were associated with the observed variation in response. For both vaccines, we were able to discover and validate several polymorphisms that were significantly associated with immune response. For the typhoid vaccines, these polymorphisms were on genes that belonged to pathways of polysaccharide recognition, signal transduction, inhibition of T-cell proliferation, pro-inflammatory signalling and eventual production of antimicrobial peptides. For the cholera vaccine, the pathways included epithelial barrier integrity, intestinal homeostasis and leucocyte recruitment. Even though traditional wisdom indicates that both vaccines should act as T-cell-independent antigens, our findings reveal that the vaccines induce AR using different pathways. PMID:25964454

Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

PubMed

Guo, Jinya; Miao, Yansong; Cai, Yi

2017-01-01

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

Fluorescent nanocolloids for differential labeling of the endocytic pathway and drug delivery applications

NASA Astrophysics Data System (ADS)

Delehanty, James B.; Spillmann, Christopher M.; Naciri, Jawad; Algar, W. Russ; Ratna, Banahalli R.; Medintz, Igor L.

2013-02-01

The demonstration of fine control over nanomaterials within biological systems, particularly in live cells, is integral for the successful implementation of nanoparticles (NPs) in biomedical applications. Here, we show the ability to differentially label the endocytic pathway of mammalian cells in a spatiotemporal manner utilizing fluorescent nanocolloids (NCs) doped with a perylene-based dye. EDC-based conjugation of green- and red-emitting NCs to the iron transport protein transferrin resulted in stable bioconjugates that were efficiently endocytosed by HEK 293T/17 cells. The staggered delivery of the bioconjugates allowed for the time-resolved, differential labeling of distinct vesicular compartments along the endocytic pathway in a nontoxic manner. We further demonstrated the ability of the NCs to be impregnated with the anticancer therapeutic, doxorubicin. Delivery of the drug-doped nanoconjugates resulted in the intracellular release and nuclear accumulation of doxorubicin in a time- and dose-dependent manner. We discuss our results in the context of the utility of such materials for NP-mediated drug delivery applications.

Ephs and Ephrins in Cancer: Ephrin-A1 Signaling

PubMed Central

Beauchamp, Amanda; Debinski, Waldemar

2011-01-01

Ephrin-A1 and its primary receptor, EphA2, are involved in numerous physiological processes and have been intensely studied for their roles in malignancy. Ephrin-Eph signalling is complex on its own and is also cell-type dependent, making elucidation of the exact role of ephrin-A1 in neoplasia challenging. Multiple oncogenic signalling pathways, such as MAP/ERK and PI3K are affected by ephrin-A1, and in some cases evidence suggests the promotion of a specific pathway in one cell or cancer type and inhibition of the same pathway in another type of cell or cancer. EphrinA1 also plays an integral role in angiogenesis and tumor neovascularization. Until recently, studies investigating ephrins focused on the ligands as GPI-anchored proteins that required membrane anchoring or artificial clustering for Eph receptor activation. However, recent studies have demonstrated a functional role for soluble, monomeric ephrin-A1. This review will focus on various forms of ephrin-A1-specific signalling in human malignancy. PMID:22040911

NOVEL ROLES FOR INSULIN RECEPTOR (IR) IN ADIPOCYTES AND SKELETAL MUSCLE CELLS VIA NEW AND UNEXPECTED SUBSTRATES

PubMed Central

Ramalingam, Latha; Oh, Eunjin; Thurmond, Debbie C.

2012-01-01

The insulin signaling pathway regulates whole-body glucose homeostasis by transducing extracellular signals from the insulin receptor (IR) to downstream intracellular targets, thus coordinating a multitude of biological functions. Dysregulation of IR or its signal transduction is associated with insulin resistance, which may culminate in type 2 diabetes (T2D). Following initial stimulation of IR, insulin signaling diverges into different pathways, activating multiple substrates which have roles in various metabolic and cellular processes. The integration of multiple pathways arising from IR activation continues to expand as new IR substrates are identified and characterized. Accordingly, our review will focus on roles for IR substrates as they pertain to three primary areas: Metabolism/glucose uptake, Mitogenesis/growth, and Aging/Longevity. While IR functions in a seemingly pleotropic manner in many cell types, through these three main roles in fat and skeletal muscle cells, IR multi-tasks to regulate whole-body glucose homeostasis to impact healthspan and lifespan. PMID:23052216

Enhanced pathway efficiency of Saccharomyces cerevisiae by introducing thermo-tolerant devices.

PubMed

Liu, Yueqin; Zhang, Genli; Sun, Huan; Sun, Xiangying; Jiang, Nisi; Rasool, Aamir; Lin, Zhanglin; Li, Chun

2014-10-01

In this study, thermo-tolerant devices consisting of heat shock genes from thermophiles were designed and introduced into Saccharomyces cerevisiae for improving its thermo-tolerance. Among ten engineered thermo-tolerant yeasts, T.te-TTE2469, T.te-GroS2 and T.te-IbpA displayed over 25% increased cell density and 1.5-4-fold cell viability compared with the control. Physiological characteristics of thermo-tolerant strains revealed that better cell wall integrity, higher trehalose content and enhanced metabolic energy were preserved by thermo-tolerant devices. Engineered thermo-tolerant strain was used to investigate the impact of thermo-tolerant device on pathway efficiency by introducing β-amyrin synthesis pathway, showed 28.1% increased β-amyrin titer, 28-35°C broadened growth temperature range and 72h shortened fermentation period. The results indicated that implanting heat shock proteins from thermophiles to S. cerevisiae would be an efficient approach to improve its thermo-tolerance. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biological interpretation of genome-wide association studies using predicted gene functions.

PubMed

Pers, Tune H; Karjalainen, Juha M; Chan, Yingleong; Westra, Harm-Jan; Wood, Andrew R; Yang, Jian; Lui, Julian C; Vedantam, Sailaja; Gustafsson, Stefan; Esko, Tonu; Frayling, Tim; Speliotes, Elizabeth K; Boehnke, Michael; Raychaudhuri, Soumya; Fehrmann, Rudolf S N; Hirschhorn, Joel N; Franke, Lude

2015-01-19

The main challenge for gaining biological insights from genetic associations is identifying which genes and pathways explain the associations. Here we present DEPICT, an integrative tool that employs predicted gene functions to systematically prioritize the most likely causal genes at associated loci, highlight enriched pathways and identify tissues/cell types where genes from associated loci are highly expressed. DEPICT is not limited to genes with established functions and prioritizes relevant gene sets for many phenotypes.

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

PubMed

Dolinay, Tamas; Himes, Blanca E; Shumyatcher, Maya; Lawrence, Gladys Gray; Margulies, Susan S

2017-08-01

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

PubMed

Ishibashi, Osamu; Akagi, Ichiro; Ogawa, Yota; Inui, Takashi

2018-05-11

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Integrative Genomic Analysis of In Vivo Muscle Regeneration After Severe Trauma

DTIC Science & Technology

2015-11-30

along with pro-inflammatory cytokines activate downstream target genes such as c-Myc, reported to drive satellite cell proliferation21 and repress...between the US Department of Energy and the US Army Medical Research and Materiel Command. The views, opinions, and/or findings of this report are...pathway member Yap plays a key role in influencing fate decisions in muscle satellite cells. J. Cell Sci. 125, 6009-6019 (2012). 27. McKinsey , T.A

Single-particle tracking and modulation of cell entry pathways of a tetrahedral DNA nanostructure in live cells.

PubMed

Liang, Le; Li, Jiang; Li, Qian; Huang, Qing; Shi, Jiye; Yan, Hao; Fan, Chunhai

2014-07-21

DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single-particle tracking. We found that the TDNs were rapidly internalized by a caveolin-dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule-dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA-nanostructure-based drug delivery nanocarriers for targeted therapy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.

PubMed Central

Cid, V J; Durán, A; del Rey, F; Snyder, M P; Nombela, C; Sánchez, M

1995-01-01

In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle. In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast. Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis. A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified. Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled. Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle. PMID:7565410

On-line metabolic pathway analysis based on metabolic signal flow diagram.

PubMed

Shi, H; Shimizu, K

In this work, an integrated modeling approach based on a metabolic signal flow diagram and cellular energetics was used to model the metabolic pathway analysis for the cultivation of yeast on glucose. This approach enables us to make a clear analysis of the flow direction of the carbon fluxes in the metabolic pathways as well as of the degree of activation of a particular pathway for the synthesis of biomaterials for cell growth. The analyses demonstrate that the main metabolic pathways of Saccharomyces cerevisiae change significantly during batch culture. Carbon flow direction is toward glycolysis to satisfy the increase of requirement for precursors and energy. The enzymatic activation of TCA cycle seems to always be at normal level, which may result in the overflow of ethanol due to its limited capacity. The advantage of this approach is that it adopts both virtues of the metabolic signal flow diagram and the simple network analysis method, focusing on the investigation of the flow directions of carbon fluxes and the degree of activation of a particular pathway or reaction loop. All of the variables used in the model equations were determined on-line; the information obtained from the calculated metabolic coefficients may result in a better understanding of cell physiology and help to evaluate the state of the cell culture process. Copyright 1998 John Wiley & Sons, Inc.

Activation of Wnt signaling pathway by human papillomavirus E6 and E7 oncogenes in HPV16-positive oropharyngeal squamous carcinoma cells.

PubMed

Rampias, Theodore; Boutati, Eleni; Pectasides, Eirini; Sasaki, Clarence; Kountourakis, Panteleimon; Weinberger, Paul; Psyrri, Amanda

2010-03-01

We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear beta-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear beta-catenin levels and the beta-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear beta-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by beta-catenin was lower after silencing. The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of beta-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of beta-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of beta-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous beta-catenin in these cells. Thus, E6 and E7 are involved in beta-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells.

Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells.

PubMed

Shao, Yuxin; Wolf, Patricia G; Guo, Shuangshuang; Guo, Yuming; Gaskins, H Rex; Zhang, Bingkun

2017-05-01

Zinc plays an important role in maintaining intestinal barrier function as well as modulating cellular signaling recognition and protein kinase activities. The phosphatidylinositol 3-kinase (PI3K) cascade has been demonstrated to affect intercellular integrity and tight junction (TJ) proteins. The current study investigated the hypothesis that zinc regulates intestinal intercellular junction integrity through the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. A transwell model of Caco-2 cell was incubated with 0, 50 and 100 μM of zinc at various time points. Transepithelial electrical resistance (TEER), paracellular permeability, TJ proteins, cell proliferation, differentiation and cell damage were measured. Compared with controls, 50 and 100 μM of zinc increased cell growth at 6, 12 and 24 h and the expression of proliferating cell nuclear antigen at 24 h. Zinc (100 μM) significantly elevated TEER at 6-24 h and reduced TJ permeability at 24 h, accompanied by the up-regulation of alkaline phosphatase (AP) activity and zonula occludens (ZO)-1 expression. In addition, zinc (100 μM) affected the PI3K/AKT/mTOR pathway by stimulating phosphorylation of AKT and the downstream target mTOR. Inhibition of PI3K signaling by LY294002 counteracted zinc promotion, as shown by a decrease in AP activity, TEER, the abundance of ZO-1 and phosphorylation of AKT and mTOR. Additionally, TJ permeability and the expression of caspase-3 and LC3II (markers of cell damage) were increased by addition of PI3K inhibitor. In conclusion, the activation of PI3K/AKT/mTOR signaling by zinc is involved in improving intestinal barrier function by enhancing cell differentiation and expression of TJ protein ZO-1. Copyright © 2017 Elsevier Inc. All rights reserved.

An oscillating dynamic model of collective cells in a monolayer

NASA Astrophysics Data System (ADS)

Lin, Shao-Zhen; Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao

2018-03-01

Periodic oscillations of collective cells occur in the morphogenesis and organogenesis of various tissues and organs. In this paper, an oscillating cytodynamic model is presented by integrating the chemomechanical interplay between the RhoA effector signaling pathway and cell deformation. We show that both an isolated cell and a cell aggregate can undergo spontaneous oscillations as a result of Hopf bifurcation, upon which the system evolves into a limit cycle of chemomechanical oscillations. The dynamic characteristics are tailored by the mechanical properties of cells (e.g., elasticity, contractility, and intercellular tension) and the chemical reactions involved in the RhoA effector signaling pathway. External forces are found to modulate the oscillation intensity of collective cells in the monolayer and to polarize their oscillations along the direction of external tension. The proposed cytodynamic model can recapitulate the prominent features of cell oscillations observed in a variety of experiments, including both isolated cells (e.g., spreading mouse embryonic fibroblasts, migrating amoeboid cells, and suspending 3T3 fibroblasts) and multicellular systems (e.g., Drosophila embryogenesis and oogenesis).

Combined-modality treatment of solid tumors using radiotherapy and molecular targeted agents.

PubMed

Ma, Brigette B Y; Bristow, Robert G; Kim, John; Siu, Lillian L

2003-07-15

Molecular targeted agents have been combined with radiotherapy (RT) in recent clinical trials in an effort to optimize the therapeutic index of RT. The appeal of this strategy lies in their potential target specificity and clinically acceptable toxicity. This article integrates the salient, published research findings into the underlying molecular mechanisms, preclinical efficacy, and clinical applicability of combining RT with molecular targeted agents. These agents include inhibitors of intracellular signal transduction molecules, modulators of apoptosis, inhibitors of cell cycle checkpoints control, antiangiogenic agents, and cyclo-oxygenase-2 inhibitors. Molecular targeted agents can have direct effects on the cytoprotective and cytotoxic pathways implicated in the cellular response to ionizing radiation (IR). These pathways involve cellular proliferation, DNA repair, cell cycle progression, nuclear transcription, tumor angiogenesis, and prostanoid-associated inflammation. These pathways can also converge to alter RT-induced apoptosis, terminal growth arrest, and reproductive cell death. Pharmacologic modulation of these pathways may potentially enhance tumor response to RT though inhibition of tumor repopulation, improvement of tumor oxygenation, redistribution during the cell cycle, and alteration of intrinsic tumor radiosensitivity. Combining RT and molecular targeted agents is a rational approach in the treatment of solid tumors. Translation of this approach from promising preclinical data to clinical trials is actively underway.

Integrated metabolomics and proteomics highlight altered nicotinamide and polyamine pathways in lung adenocarcinoma

PubMed Central

Fahrmann, Johannes F.; Grapov, Dmitry; Wanichthanarak, Kwanjeera; DeFelice, Brian C.; Salemi, Michelle R.; Rom, William N.; Gandara, David R.; Phinney, Brett S.; Fiehn, Oliver; Pass, Harvey

2017-01-01

Abstract Lung cancer is the leading cause of cancer mortality in the United States with non-small cell lung cancer adenocarcinoma being the most common histological type. Early perturbations in cellular metabolism are a hallmark of cancer, but the extent of these changes in early stage lung adenocarcinoma remains largely unknown. In the current study, an integrated metabolomics and proteomics approach was utilized to characterize the biochemical and molecular alterations between malignant and matched control tissue from 27 subjects diagnosed with early stage lung adenocarcinoma. Differential analysis identified 71 metabolites and 1102 proteins that delineated tumor from control tissue. Integrated results indicated four major metabolic changes in early stage adenocarcinoma (1): increased glycosylation and glutaminolysis (2); elevated Nrf2 activation (3); increase in nicotinic and nicotinamide salvaging pathways and (4) elevated polyamine biosynthesis linked to differential regulation of the s-adenosylmethionine/nicotinamide methyl-donor pathway. Genomic data from publicly available databases were included to strengthen proteomic findings. Our findings provide insight into the biochemical and molecular biological reprogramming that may accompany early stage lung tumorigenesis and highlight potential therapeutic targets. PMID:28049629

Signaling network of dendritic cells in response to pathogens: a community-input supported knowledgebase.

PubMed

Patil, Sonali; Pincas, Hanna; Seto, Jeremy; Nudelman, German; Nudelman, Irina; Sealfon, Stuart C

2010-10-07

Dendritic cells are antigen-presenting cells that play an essential role in linking the innate and adaptive immune systems. Much research has focused on the signaling pathways triggered upon infection of dendritic cells by various pathogens. The high level of activity in the field makes it desirable to have a pathway-based resource to access the information in the literature. Current pathway diagrams lack either comprehensiveness, or an open-access editorial interface. Hence, there is a need for a dependable, expertly curated knowledgebase that integrates this information into a map of signaling networks. We have built a detailed diagram of the dendritic cell signaling network, with the goal of providing researchers with a valuable resource and a facile method for community input. Network construction has relied on comprehensive review of the literature and regular updates. The diagram includes detailed depictions of pathways activated downstream of different pathogen recognition receptors such as Toll-like receptors, retinoic acid-inducible gene-I-like receptors, C-type lectin receptors and nucleotide-binding oligomerization domain-like receptors. Initially assembled using CellDesigner software, it provides an annotated graphical representation of interactions stored in Systems Biology Mark-up Language. The network, which comprises 249 nodes and 213 edges, has been web-published through the Biological Pathway Publisher software suite. Nodes are annotated with PubMed references and gene-related information, and linked to a public wiki, providing a discussion forum for updates and corrections. To gain more insight into regulatory patterns of dendritic cell signaling, we analyzed the network using graph-theory methods: bifan, feedforward and multi-input convergence motifs were enriched. This emphasis on activating control mechanisms is consonant with a network that subserves persistent and coordinated responses to pathogen detection. This map represents a navigable aid for presenting a consensus view of the current knowledge on dendritic cell signaling that can be continuously improved through contributions of research community experts. Because the map is available in a machine readable format, it can be edited and may assist researchers in data analysis. Furthermore, the availability of a comprehensive knowledgebase might help further research in this area such as vaccine development. The dendritic cell signaling knowledgebase is accessible at http://tsb.mssm.edu/pathwayPublisher/DC_pathway/DC_pathway_index.html.

Encoding of temporal signals by the TGF-β pathway and implications for embryonic patterning

PubMed Central

Sorre, Benoit; Warmflash, Aryeh; Brivanlou, Ali H.; Siggia, Eric D.

2014-01-01

Summary Genetics and biochemistry have defined the components and wiring of the signaling pathways that pattern the embryo. Among them, the TGF-β pathway has the potential to behave as a morphogen: invitro experiments have clearly established that it can dictate cell fate in a concentration dependent manner. How morphogens convey positional information in a developing embryo, where signal levels are changing with time, is less understood. Using integrated microfluidic cell culture and time-lapse microscopy, we demonstrate here that the speed of ligand presentation has a key and previously unexpected influence on TGF-β signaling outcomes. The response to a TGF-β concentration step is transient and adaptive, slowly increasing the ligand concentration diminishes the response and well-spaced pulses of ligand combine additively resulting in greater pathway output than with constant stimulation. Our results suggest that in an embryonic context, the speed of change of ligand concentration is an instructive signal for patterning. PMID:25065773

Red blood cells open promising avenues for longitudinal studies of ageing in laboratory, non-model and wild animals.

PubMed

Stier, Antoine; Reichert, Sophie; Criscuolo, Francois; Bize, Pierre

2015-11-01

Ageing is characterized by a progressive deterioration of multiple physiological and molecular pathways, which impair organismal performance and increase risks of death with advancing age. Hence, ageing studies must identify physiological and molecular pathways that show signs of age-related deterioration, and test their association with the risk of death and longevity. This approach necessitates longitudinal sampling of the same individuals, and therefore requires a minimally invasive sampling technique that provides access to the larger spectrum of physiological and molecular pathways that are putatively associated with ageing. The present paper underlines the interest in using red blood cells (RBCs) as a promising target for longitudinal studies of ageing in vertebrates. RBCs provide valuable information on the following six pathways: cell maintenance and turnover (RBC number, size, and heterogeneity), glucose homeostasis (RBC glycated haemoglobin), oxidative stress parameters, membrane composition and integrity, mitochondrial functioning, and telomere dynamics. The last two pathways are specific to RBCs of non-mammalian species, which possess a nucleus and functional mitochondria. We present the current knowledge about RBCs and age-dependent changes in these pathways in non-model and wild species that are especially suitable to address questions related to ageing using longitudinal studies. We discuss how the different pathways relate with survival and lifespan and give information on their genetic and environmental determinants to appraise their evolutionary potential. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiscale modeling of mucosal immune responses

PubMed Central

2015-01-01

Computational modeling techniques are playing increasingly important roles in advancing a systems-level mechanistic understanding of biological processes. Computer simulations guide and underpin experimental and clinical efforts. This study presents ENteric Immune Simulator (ENISI), a multiscale modeling tool for modeling the mucosal immune responses. ENISI's modeling environment can simulate in silico experiments from molecular signaling pathways to tissue level events such as tissue lesion formation. ENISI's architecture integrates multiple modeling technologies including ABM (agent-based modeling), ODE (ordinary differential equations), SDE (stochastic modeling equations), and PDE (partial differential equations). This paper focuses on the implementation and developmental challenges of ENISI. A multiscale model of mucosal immune responses during colonic inflammation, including CD4+ T cell differentiation and tissue level cell-cell interactions was developed to illustrate the capabilities, power and scope of ENISI MSM. Background Computational techniques are becoming increasingly powerful and modeling tools for biological systems are of greater needs. Biological systems are inherently multiscale, from molecules to tissues and from nano-seconds to a lifespan of several years or decades. ENISI MSM integrates multiple modeling technologies to understand immunological processes from signaling pathways within cells to lesion formation at the tissue level. This paper examines and summarizes the technical details of ENISI, from its initial version to its latest cutting-edge implementation. Implementation Object-oriented programming approach is adopted to develop a suite of tools based on ENISI. Multiple modeling technologies are integrated to visualize tissues, cells as well as proteins; furthermore, performance matching between the scales is addressed. Conclusion We used ENISI MSM for developing predictive multiscale models of the mucosal immune system during gut inflammation. Our modeling predictions dissect the mechanisms by which effector CD4+ T cell responses contribute to tissue damage in the gut mucosa following immune dysregulation. PMID:26329787

Multiscale modeling of mucosal immune responses.

PubMed

Mei, Yongguo; Abedi, Vida; Carbo, Adria; Zhang, Xiaoying; Lu, Pinyi; Philipson, Casandra; Hontecillas, Raquel; Hoops, Stefan; Liles, Nathan; Bassaganya-Riera, Josep

2015-01-01

Computational techniques are becoming increasingly powerful and modeling tools for biological systems are of greater needs. Biological systems are inherently multiscale, from molecules to tissues and from nano-seconds to a lifespan of several years or decades. ENISI MSM integrates multiple modeling technologies to understand immunological processes from signaling pathways within cells to lesion formation at the tissue level. This paper examines and summarizes the technical details of ENISI, from its initial version to its latest cutting-edge implementation. Object-oriented programming approach is adopted to develop a suite of tools based on ENISI. Multiple modeling technologies are integrated to visualize tissues, cells as well as proteins; furthermore, performance matching between the scales is addressed. We used ENISI MSM for developing predictive multiscale models of the mucosal immune system during gut inflammation. Our modeling predictions dissect the mechanisms by which effector CD4+ T cell responses contribute to tissue damage in the gut mucosa following immune dysregulation.Computational modeling techniques are playing increasingly important roles in advancing a systems-level mechanistic understanding of biological processes. Computer simulations guide and underpin experimental and clinical efforts. This study presents ENteric Immune Simulator (ENISI), a multiscale modeling tool for modeling the mucosal immune responses. ENISI's modeling environment can simulate in silico experiments from molecular signaling pathways to tissue level events such as tissue lesion formation. ENISI's architecture integrates multiple modeling technologies including ABM (agent-based modeling), ODE (ordinary differential equations), SDE (stochastic modeling equations), and PDE (partial differential equations). This paper focuses on the implementation and developmental challenges of ENISI. A multiscale model of mucosal immune responses during colonic inflammation, including CD4+ T cell differentiation and tissue level cell-cell interactions was developed to illustrate the capabilities, power and scope of ENISI MSM.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway

PubMed Central

Alam, Maroof; Bouillez, Audrey; Tagde, Ashujit; Ahmad, Rehan; Rajabi, Hasan; Maeda, Takahiro; Hiraki, Masayuki; Suzuki, Yozo; Kufe, Donald

2016-01-01

Apical-basal polarity and epithelial integrity are maintained in part by the Crumbs (CRB) complex. The C-terminal subunit of MUC1 (MUC1-C) is a transmembrane protein that is expressed at the apical border of normal epithelial cells and aberrantly at high levels over the entire surface of their transformed counterparts. However, it is not known if MUC1-C contributes to this loss of polarity that is characteristic of carcinoma cells. Here it is demonstrated that MUC1-C downregulates expression of the Crumbs complex CRB3 protein in triple-negative breast cancer (TNBC) cells. MUC1-C associates with ZEB1 on the CRB3 promoter and represses CRB3 transcription. Notably, CRB3 activates the core kinase cassette of the Hippo pathway, which includes LATS1 and LATS2. In this context, targeting MUC1-C was associated with increased phosphorylation of LATS1, consistent with activation of the Hippo pathway, which is critical for regulating cell contact, tissue repair, proliferation and apoptosis. Also shown is that MUC1-C-mediated suppression of CRB3 and the Hippo pathway is associated with dephosphorylation and activation of the oncogenic YAP protein. In turn, MUC1-C interacts with YAP, promotes formation of YAP/β-catenin complexes and induces the WNT target gene MYC. These data support a previously unrecognized model in which targeting MUC1-C in TNBC cells (i) induces CRB3 expression, (ii) activates the CRB3-driven Hippo pathway, (iii) inactivates YAP, and thereby (iv) suppresses YAP/β-catenin-mediated induction of MYC expression. Implications These findings demonstrate a previously unrecognized role for the MUC1-C oncoprotein in the regulation of polarity and the Hippo pathway in breast cancer. PMID:27658423

Cross-Species Analysis of Nicotine-Induced Proteomic Alterations in Pancreatic Cells

PubMed Central

Paulo, Joao A.; Urrutia, Raul; Kadiyala, Vivek; Banks, Peter

2014-01-01

Background Toxic compounds in tobacco, such as nicotine, may have adversely affect pancreatic function. We aim to determine nicotine-induced protein alterations in pancreatic cells, which may reveal a link between nicotine exposure and pancreatic disease. Methods We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat and human stellate cells and human duct cells) using mass spectrometry-based techniques, specifically GeLC-MS/MS and spectral counting. Results We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine-treated or untreated cells. Inter-species comparisons of stellate cell proteins revealed several differentially-abundant proteins (in nicotine treated versus untreated cells) common among the 3 species. Proteins appearing in all nicotine-treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B,and Toll interacting protein. Conclusions Proteins which were differentially expressed upon nicotine treatment across cell lines, were enriched in certain pathways, including nAChR, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease. PMID:23456891

Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone.

PubMed

Oberhofer, Georg; Grossmann, Daniela; Siemanowski, Janna L; Beissbarth, Tim; Bucher, Gregor

2014-12-01

Wnt/β-catenin and hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. Here, we use the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. We find that Wnt/β-catenin signaling acts upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. We determine the target gene sets of the Wnt/β-catenin and Hh pathways and find that the growth zone signaling center activates a much greater number of genes and that the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist. Furthermore, the Wnt pathway is required for hindgut development and we identify Tc-senseless as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function. © 2014. Published by The Company of Biologists Ltd.

Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone

PubMed Central

Oberhofer, Georg; Grossmann, Daniela; Siemanowski, Janna L.; Beissbarth, Tim; Bucher, Gregor

2014-01-01

Wnt/β-catenin and hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. Here, we use the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. We find that Wnt/β-catenin signaling acts upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. We determine the target gene sets of the Wnt/β-catenin and Hh pathways and find that the growth zone signaling center activates a much greater number of genes and that the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist. Furthermore, the Wnt pathway is required for hindgut development and we identify Tc-senseless as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function. PMID:25395458

Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago truncatula Root Border Cells1[OPEN

PubMed Central

Watson, Bonnie S.; Bedair, Mohamed F.; Urbanczyk-Wochniak, Ewa; Huhman, David V.; Yang, Dong Sik; Allen, Stacy N.; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W.

2015-01-01

Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4′-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4′-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

Radiation induced genome instability: multiscale modelling and data analysis

NASA Astrophysics Data System (ADS)

Andreev, Sergey; Eidelman, Yuri

2012-07-01

Genome instability (GI) is thought to be an important step in cancer induction and progression. Radiation induced GI is usually defined as genome alterations in the progeny of irradiated cells. The aim of this report is to demonstrate an opportunity for integrative analysis of radiation induced GI on the basis of multiscale modelling. Integrative, systems level modelling is necessary to assess different pathways resulting in GI in which a variety of genetic and epigenetic processes are involved. The multilevel modelling includes the Monte Carlo based simulation of several key processes involved in GI: DNA double strand breaks (DSBs) generation in cells initially irradiated as well as in descendants of irradiated cells, damage transmission through mitosis. Taking the cell-cycle-dependent generation of DNA/chromosome breakage into account ensures an advantage in estimating the contribution of different DNA damage response pathways to GI, as to nonhomologous vs homologous recombination repair mechanisms, the role of DSBs at telomeres or interstitial chromosomal sites, etc. The preliminary estimates show that both telomeric and non-telomeric DSB interactions are involved in delayed effects of radiation although differentially for different cell types. The computational experiments provide the data on the wide spectrum of GI endpoints (dicentrics, micronuclei, nonclonal translocations, chromatid exchanges, chromosome fragments) similar to those obtained experimentally for various cell lines under various experimental conditions. The modelling based analysis of experimental data demonstrates that radiation induced GI may be viewed as processes of delayed DSB induction/interaction/transmission being a key for quantification of GI. On the other hand, this conclusion is not sufficient to understand GI as a whole because factors of DNA non-damaging origin can also induce GI. Additionally, new data on induced pluripotent stem cells reveal that GI is acquired in normal mature cells during genome reprogramming by the oncogene c-myc and three additional transcription factors. These and other data reveal the need for generalisation of current model of GI. One can expect that different early events of both DNA damaging and non-damaging origins merge in a single late pathway. To search for a deeper view we propose to redefine GI as genome destabilisation manifested in erosion of genome states and altered transitions between states. This changing view on GI may help to integrate the inducing factors of various origins in the single basic model of GI.

Skeletal muscle wasting with disuse atrophy is multi-dimensional: the response and interaction of myonuclei, satellite cells and signaling pathways

PubMed Central

Brooks, Naomi E.; Myburgh, Kathryn H.

2014-01-01

Maintenance of skeletal muscle is essential for health and survival. There are marked losses of skeletal muscle mass as well as strength and physiological function under conditions of low mechanical load, such as space flight, as well as ground based models such as bed rest, immobilization, disuse, and various animal models. Disuse atrophy is caused by mechanical unloading of muscle and this leads to reduced muscle mass without fiber attrition. Skeletal muscle stem cells (satellite cells) and myonuclei are integrally involved in skeletal muscle responses to environmental changes that induce atrophy. Myonuclear domain size is influenced differently in fast and slow twitch muscle, but also by different models of muscle wasting, a factor that is not yet understood. Although the myonuclear domain is 3-dimensional this is rarely considered. Apoptosis as a mechanism for myonuclear loss with atrophy is controversial, whereas cell death of satellite cells has not been considered. Molecular signals such as myostatin/SMAD pathway, MAFbx, and MuRF1 E3 ligases of the ubiquitin proteasome pathway and IGF1-AKT-mTOR pathway are 3 distinctly different contributors to skeletal muscle protein adaptation to disuse. Molecular signaling pathways activated in muscle fibers by disuse are rarely considered within satellite cells themselves despite similar exposure to unloading or low mechanical load. These molecular pathways interact with each other during atrophy and also when various interventions are applied that could alleviate atrophy. Re-applying mechanical load is an obvious method to restore muscle mass, however how nutrient supplementation (e.g., amino acids) may further enhance recovery (or reduce atrophy despite unloading or ageing) is currently of great interest. Satellite cells are particularly responsive to myostatin and to growth factors. Recently, the hibernating squirrel has been identified as an innovative model to study resistance to atrophy. PMID:24672488

MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells

PubMed Central

Collins, Carol M.; Ellis, Joseph A.

2017-01-01

ABSTRACT Mutations in the gene encoding emerin cause Emery–Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo. PMID:28188262

Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs

PubMed Central

Sharifpoor, Sara; van Dyk, Dewald; Costanzo, Michael; Baryshnikova, Anastasia; Friesen, Helena; Douglas, Alison C.; Youn, Ji-Young; VanderSluis, Benjamin; Myers, Chad L.; Papp, Balázs; Boone, Charles; Andrews, Brenda J.

2012-01-01

A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of synthetic dosage lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs, and literature-curated kinase–substrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale GI networks. PMID:22282571

Combining differential expression, chromosomal and pathway analyses for the molecular characterization of renal cell carcinoma

PubMed Central

Furge, Kyle A; Dykema, Karl; Petillo, David; Westphal, Michael; Zhang, Zhongfa; Kort, Eric J; Teh, Bin Tean

2007-01-01

Using high-throughput gene-expression profiling technology, we can now gain a better understanding of the complex biology that is taking place in cancer cells. This complexity is largely dictated by the abnormal genetic makeup of the cancer cells. This abnormal genetic makeup can have profound effects on cellular activities such as cell growth, cell survival and other regulatory processes. Based on the pattern of gene expression, or molecular signatures of the tumours, we can distinguish or subclassify different types of cancers according to their cell of origin, behaviour, and the way they respond to therapeutic agents and radiation. These approaches will lead to better molecular subclassification of tumours, the basis of personalized medicine. We have, to date, done whole-genome microarray gene-expression profiling on several hundreds of kidney tumours. We adopt a combined bioinformatic approach, based on an integrative analysis of the gene-expression data. These data are used to identify both cytogenetic abnormalities and molecular pathways that are deregulated in renal cell carcinoma (RCC). For example, we have identified the deregulation of the VHL-hypoxia pathway in clear-cell RCC, as previously known, and the c-Myc pathway in aggressive papillary RCC. Besides the more common clear-cell, papillary and chromophobe RCCs, we are currently characterizing the molecular signatures of rarer forms of renal neoplasia such as carcinoma of the collecting ducts, mixed epithelial and stromal tumours, chromosome Xp11 translocations associated with papillary RCC, renal medullary carcinoma, mucinous tubular and spindle-cell carcinoma, and a group of unclassified tumours. Continued development and improvement in the field of molecular profiling will better characterize cancer and provide more accurate diagnosis, prognosis and prediction of drug response. PMID:18542781

Type two innate lymphoid cells: the Janus cells in health and disease.

PubMed

Maazi, Hadi; Akbari, Omid

2017-07-01

Innate lymphoid cells are functionally diverse subsets of immune cells including the conventional natural killer cells, lymphoid tissue inducers, type 1, 2, and 3 with significant roles in immunity and pathogenesis of inflammatory diseases. Type 2 innate lymphoid cells (ILC2s) resemble type 2 helper (Th2) cells in cytokine production and contribute to anti-helminth immunity, maintaining mucosal tissue integrity, and adipose tissue browning. ILC2s play important roles in the pathogenesis of allergic diseases and asthma. Studying the pathways of activation and regulation of ILC2s are currently a priority for giving a better understanding of pathogenesis of diseases with immunological roots. Recently, our laboratory and others have shown several pathways of regulation of ILC2s by co-stimulatory molecules such as ICOS, regulatory T cells and by compounds such as nicotine. In this review, we summarize the current understanding of the mechanisms of activation and regulation of ILC2s and the role of these cells in health and disease. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

PubMed Central

Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

2013-01-01

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

A change in structural integrity of c-Kit mutant D816V causes constitutive signaling.

PubMed

Raghav, Pawan Kumar; Singh, Ajay Kumar; Gangenahalli, Gurudutta

2018-03-01

Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Mitochondrial Redox Signaling and Tumor Progression.

PubMed

Chen, Yuxin; Zhang, Haiqing; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

2016-03-25

Cancer cell can reprogram their energy production by switching mitochondrial oxidative phosphorylation to glycolysis. However, mitochondria play multiple roles in cancer cells, including redox regulation, reactive oxygen species (ROS) generation, and apoptotic signaling. Moreover, these mitochondrial roles are integrated via multiple interconnected metabolic and redox sensitive pathways. Interestingly, mitochondrial redox proteins biphasically regulate tumor progression depending on cellular ROS levels. Low level of ROS functions as signaling messengers promoting cancer cell proliferation and cancer invasion. However, anti-cancer drug-initiated stress signaling could induce excessive ROS, which is detrimental to cancer cells. Mitochondrial redox proteins could scavenger basal ROS and function as "tumor suppressors" or prevent excessive ROS to act as "tumor promoter". Paradoxically, excessive ROS often also induce DNA mutations and/or promotes tumor metastasis at various stages of cancer progression. Targeting redox-sensitive pathways and transcriptional factors in the appropriate context offers great promise for cancer prevention and therapy. However, the therapeutics should be cancer-type and stage-dependent.

AMP kinase promotes glioblastoma bioenergetics and tumour growth.

PubMed

Chhipa, Rishi Raj; Fan, Qiang; Anderson, Jane; Muraleedharan, Ranjithmenon; Huang, Yan; Ciraolo, Georgianne; Chen, Xiaoting; Waclaw, Ronald; Chow, Lionel M; Khuchua, Zaza; Kofron, Matthew; Weirauch, Matthew T; Kendler, Ady; McPherson, Christopher; Ratner, Nancy; Nakano, Ichiro; Dasgupta, Nupur; Komurov, Kakajan; Dasgupta, Biplab

2018-06-18

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.

Host-microbiota interactions: Epigenomic regulation

PubMed Central

Woo, Vivienne; Alenghat, Theresa

2016-01-01

The coevolution of mammalian hosts and their commensal microbiota has led to the development of complex symbiotic relationships between resident microbes and mammalian cells. Epigenomic modifications enable host cells to alter gene expression without modifying the genetic code, and therefore represent potent mechanisms by which mammalian cells can transcriptionally respond, transiently or stably, to environmental cues. Advances in genome-wide approaches are accelerating our appreciation of microbial influences on host physiology, and increasing evidence highlights that epigenomics represent a level of regulation by which the host integrates and responds to microbial signals. In particular, bacterial-derived short chain fatty acids have emerged as one clear link between how the microbiota intersects with host epigenomic pathways. Here we review recent findings describing crosstalk between the microbiota and epigenomic pathways in multiple mammalian cell populations. Further, we discuss interesting links that suggest that the scope of our understanding of epigenomic regulation in the host-microbiota relationship is still in its infancy. PMID:28103497

Ibrutinib (PCI-32765) in Chronic Lymphocytic Leukemia

PubMed Central

Jain, Nitin; O’Brien, Susan

2015-01-01

SYNOPSIS B-cell receptor (BCR) signaling is essential for chronic lymphocytic leukemia (CLL) cell survival. Many kinases in the BCR signaling pathway are currently being studied as potential therapeutic targets. These include Lyn, Syk, PI3 and Bruton tyrosine (BTK). Ibrutinib (PCI-32765) is a novel first-in-class selective inhibitor of BTK. Preclinical evidence suggests that ibrutinib inhibits CLL cell survival and proliferation. In addition, it also affects CLL cell migration and homing. Early clinical data in CLL and non-Hodgkin’s lymphoma patients is very encouraging. In relapsed-refractory patients with CLL, a 67% response rate was observed (420mg dose cohort) with single-agent ibrutinib. Long-term follow-up of these studies and other ongoing/planned studies of ibrutinib either as single-agent or in combination with monoclonal antibodies and chemoimmunotherapy is eagerly awaited. It is likely that ibrutinib and other drugs targeting the BCR pathway will become an integral component of CLL therapy. PMID:23915749

A high throughput screening for TLR3-IRF3 signaling pathway modulators identifies several antipsychotic drugs as TLR inhibitors1

PubMed Central

Zhu, Jianzhong; Smith, Kevin; Hsieh, Paishiun N.; Mburu, Yvonne K.; Chattopadhyay, Saurabh; Sen, Ganes C.; Sarkar, Saumendra N.

2010-01-01

Toll-like Receptor 3 (TLR3) is one of the major innate immune sensors of double stranded RNA (dsRNA). The signal transduction pathway activated by TLR3, upon binding to dsRNA, leads to the activation of two major transcription factors: NF-κB and IRF3. In an effort to identify specific chemical modulators of TLR3-IRF3 signal transduction pathway we developed a cell-based read out system. Using the interferon stimulated gene 56 (ISG56) promoter driven firefly luciferase gene stably integrated in a TLR3 expressing HEK293 cell line, we were able to generate a cell line where treatment with dsRNA resulted in a dose dependent induction of luciferase activity. A screen of two pharmacologically active compound libraries using this system, identified a number of TLR3-IRF3 signaling pathway modulators. Among them we focused on a subset of inhibitors and characterized their mode of action. Several antipsychotic drugs, such as Sertraline, Trifluoperazine and Fluphenazine were found to be direct inhibitors of the innate immune signaling pathway. These inhibitors also showed the ability to inhibit ISG56 induction mediated by TLR4 and TLR7/8 pathways. Interestingly, they did not show significant effect on TLR3, TLR7 and TLR8 mediated NF-κB activation. Detailed analysis of the signaling pathway indicated that these drugs may be exerting their inhibitory effects on IRF3 via PI3K signaling pathway. The data presented here provides mechanistic explanation of possible anti-inflammatory roles of some antipsychotic drugs. PMID:20382888

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival.

PubMed

Davì, Valeria; Tanimoto, Hirokazu; Ershov, Dmitry; Haupt, Armin; De Belly, Henry; Le Borgne, Rémi; Couturier, Etienne; Boudaoud, Arezki; Minc, Nicolas

2018-04-23

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival. Copyright © 2018 Elsevier Inc. All rights reserved.

Systematic reconstruction of TRANSPATH data into Cell System Markup Language

PubMed Central

Nagasaki, Masao; Saito, Ayumu; Li, Chen; Jeong, Euna; Miyano, Satoru

2008-01-01

Background Many biological repositories store information based on experimental study of the biological processes within a cell, such as protein-protein interactions, metabolic pathways, signal transduction pathways, or regulations of transcription factors and miRNA. Unfortunately, it is difficult to directly use such information when generating simulation-based models. Thus, modeling rules for encoding biological knowledge into system-dynamics-oriented standardized formats would be very useful for fully understanding cellular dynamics at the system level. Results We selected the TRANSPATH database, a manually curated high-quality pathway database, which provides a plentiful source of cellular events in humans, mice, and rats, collected from over 31,500 publications. In this work, we have developed 16 modeling rules based on hybrid functional Petri net with extension (HFPNe), which is suitable for graphical representing and simulating biological processes. In the modeling rules, each Petri net element is incorporated with Cell System Ontology to enable semantic interoperability of models. As a formal ontology for biological pathway modeling with dynamics, CSO also defines biological terminology and corresponding icons. By combining HFPNe with the CSO features, it is possible to make TRANSPATH data to simulation-based and semantically valid models. The results are encoded into a biological pathway format, Cell System Markup Language (CSML), which eases the exchange and integration of biological data and models. Conclusion By using the 16 modeling rules, 97% of the reactions in TRANSPATH are converted into simulation-based models represented in CSML. This reconstruction demonstrates that it is possible to use our rules to generate quantitative models from static pathway descriptions. PMID:18570683

Differential transcriptional activation by human T-cell leukemia virus type 1 Tax mutants is mediated by distinct interactions with CREB binding protein and p300.

PubMed

Bex, F; Yin, M J; Burny, A; Gaynor, R B

1998-04-01

The human T-cell leukemia virus type 1 Tax protein transforms human T lymphocytes, which can lead to the development of adult T-cell leukemia. Tax transformation is related to its ability to activate gene expression via the ATF/CREB and the NF-kappaB pathways. Transcriptional activation of these pathways is mediated by the actions of the related coactivators CREB binding protein (CBP) and p300. In this study, immunocytochemistry and confocal microscopy were used to localize CBP and p300 in cells expressing wild-type Tax or Tax mutants that are able to selectively activate gene expression from either the NF-kappaB or ATF/CREB pathway. Wild-type Tax colocalized with both CBP and p300 in nuclear bodies which also contained ATF-1 and the RelA subunit of NF-kappaB. However, a Tax mutant that selectively activates gene expression from only the ATF/CREB pathway colocalized with CBP but not p300, while a Tax mutant that selectively activates gene expression from only the NF-kappaB pathway colocalized with p300 but not CBP. In vitro and in vivo protein interaction studies indicated that the integrity of two independent domains of Tax delineated by these mutants was involved in the direct interaction of Tax with either CBP or p300. These studies are consistent with a model in which activation of either the NF-kappaB or the ATF/CREB pathway by specific Tax mutants is mediated by distinct interactions with related coactivator proteins.

Systematic reconstruction of TRANSPATH data into cell system markup language.

PubMed

Nagasaki, Masao; Saito, Ayumu; Li, Chen; Jeong, Euna; Miyano, Satoru

2008-06-23

Many biological repositories store information based on experimental study of the biological processes within a cell, such as protein-protein interactions, metabolic pathways, signal transduction pathways, or regulations of transcription factors and miRNA. Unfortunately, it is difficult to directly use such information when generating simulation-based models. Thus, modeling rules for encoding biological knowledge into system-dynamics-oriented standardized formats would be very useful for fully understanding cellular dynamics at the system level. We selected the TRANSPATH database, a manually curated high-quality pathway database, which provides a plentiful source of cellular events in humans, mice, and rats, collected from over 31,500 publications. In this work, we have developed 16 modeling rules based on hybrid functional Petri net with extension (HFPNe), which is suitable for graphical representing and simulating biological processes. In the modeling rules, each Petri net element is incorporated with Cell System Ontology to enable semantic interoperability of models. As a formal ontology for biological pathway modeling with dynamics, CSO also defines biological terminology and corresponding icons. By combining HFPNe with the CSO features, it is possible to make TRANSPATH data to simulation-based and semantically valid models. The results are encoded into a biological pathway format, Cell System Markup Language (CSML), which eases the exchange and integration of biological data and models. By using the 16 modeling rules, 97% of the reactions in TRANSPATH are converted into simulation-based models represented in CSML. This reconstruction demonstrates that it is possible to use our rules to generate quantitative models from static pathway descriptions.

Histone-derived piRNA biogenesis depends on the ping-pong partners Piwi5 and Ago3 in Aedes aegypti

PubMed Central

Girardi, Erika; Miesen, Pascal; Pennings, Bas; Frangeul, Lionel; Saleh, Maria-Carla

2017-01-01

Abstract The piRNA pathway is of key importance in controlling transposable elements in most animal species. In the vector mosquito Aedes aegypti, the presence of eight PIWI proteins and the accumulation of viral piRNAs upon arbovirus infection suggest additional functions of the piRNA pathway beyond genome defense. To better understand the regulatory potential of this pathway, we analyzed in detail host-derived piRNAs in A. aegypti Aag2 cells. We show that a large repertoire of protein-coding genes and non-retroviral integrated RNA virus elements are processed into genic piRNAs by different combinations of PIWI proteins. Among these, we identify a class of genes that produces piRNAs from coding sequences in an Ago3- and Piwi5-dependent fashion. We demonstrate that the replication-dependent histone gene family is a genic source of ping-pong dependent piRNAs and that histone-derived piRNAs are dynamically expressed throughout the cell cycle, suggesting a role for the piRNA pathway in the regulation of histone gene expression. Moreover, our results establish the Aag2 cell line as an accessible experimental model to study gene-derived piRNAs. PMID:28115625

CBX7 regulates stem cell-like properties of gastric cancer cells via p16 and AKT-NF-κB-miR-21 pathways.

PubMed

Ni, Su-Jie; Zhao, Li-Qin; Wang, Xiao-Feng; Wu, Zhen-Hua; Hua, Rui-Xi; Wan, Chun-Hua; Zhang, Jie-Yun; Zhang, Xiao-Wei; Huang, Ming-Zhu; Gan, Lu; Sun, Hua-Lin; Dimri, Goberdhan P; Guo, Wei-Jian

2018-02-08

Chromobox protein homolog 7 (CBX7), a member of the polycomb group (PcG) family of proteins, is involved in the regulation of cell proliferation and cancer progression. PcG family members, such as BMI, Mel-18, and EZH2, are integral constituents of the polycomb repressive complexes (PRCs) and have been known to regulate cancer stem cell (CSC) phenotype. However, the role of other PRCs' constituents such as CBX7 in the regulation of CSC phenotype remains largely elusive. This study was to investigate the role of CBX7 in regulating stem cell-like properties of gastric cancer and the underlying mechanisms. Firstly, the role of CBX7 in regulating stem cell-like properties of gastric cancer was investigated using sphere formation, Western blot, and xenograft tumor assays. Next, RNA interference and ectopic CBX7 expression were employed to determine the impact of CBX7 on the expression of CSC marker proteins and CSC characteristics. The expression of CBX7, its downstream targets, and stem cell markers were analyzed in gastric stem cell spheres, common cancer cells, and gastric cancer tissues. Finally, the pathways by which CBX7 regulates stem cell-like properties of gastric cancer were explored. We found that CBX7, a constituent of the polycomb repressive complex 1 (PRC1), plays an important role in maintaining stem cell-like characteristics of gastric cancer cells via the activation of AKT pathway and the downregulation of p16. Spearman rank correlation analysis showed positive correlations among the expression of CBX7 and phospho-AKT (pAKT), stem cell markers OCT-4, and CD133 in gastric cancer tissues. In addition, CBX7 was found to upregulate microRNA-21 (miR-21) via the activation of AKT-NF-κB pathway, and miR-21 contributes to CBX7-mediated CSC characteristics. CBX7 positively regulates stem cell-like characteristics of gastric cancer cells by inhibiting p16 and activating AKT-NF-κB-miR-21 pathway.

Quercetin inhibits prostate cancer by attenuating cell survival and inhibiting anti-apoptotic pathways.

PubMed

Ward, Ashley B; Mir, Hina; Kapur, Neeraj; Gales, Dominique N; Carriere, Patrick P; Singh, Shailesh

2018-06-14

Despite recent advances in diagnosis and treatment, prostate cancer (PCa) remains the leading cause of cancer-related deaths in men. Current treatments offered in the clinics are often toxic and have severe side effects. Hence, to treat and manage PCa, new agents with fewer side effects or having potential to reduce side effects of conventional therapy are needed. In this study, we show anti-cancer effects of quercetin, an abundant bioflavonoid commonly used to treat prostatitis, and defined quercetin-induced cellular and molecular changes leading to PCa cell death. Cell viability was assessed using MTT. Cell death mode, mitochondrial outer membrane potential, and oxidative stress levels were determined by flow cytometry using Annexin V-7 AAD dual staining kit, JC-1 dye, and ROS detection kit, respectively. Antibody microarray and western blot were used to delineate the molecular changes induced by quercetin. PCa cells treated with various concentrations of quercetin showed time- and dose-dependent decrease in cell viability compared to controls, without affecting normal prostate epithelial cells. Quercetin led to apoptotic and necrotic cell death in PCa cells by affecting the mitochondrial integrity and disturbing the ROS homeostasis depending upon the genetic makeup and oxidative status of the cells. LNCaP and PC-3 cells that have an oxidative cellular environment showed ROS quenching after quercetin treatment while DU-145 showed rise in ROS levels despite having a highly reductive environment. Opposing effects of quercetin were also observed on the pro-survival pathways of PCa cells. PCa cells with mutated p53 (DU-145) and increased ROS showed significant reduction in the activation of pro-survival Akt pathway while Raf/MEK were activated in response to quercetin. PC-3 cells lacking p53 and PTEN with reduced ROS levels showed significant activation of Akt and NF-κB pathway. Although some of these changes are commonly associated with oncogenic response, the cumulative effect of these alterations is PCa cell death. Our results demonstrated quercetin exerts its anti-cancer effects by modulating ROS, Akt, and NF-κB pathways. Quercetin could be used as a chemopreventive option as well as in combination with chemotherapeutic drugs to improve clinical outcomes of PCa patients.

Biological interpretation of genome-wide association studies using predicted gene functions

PubMed Central

Pers, Tune H.; Karjalainen, Juha M.; Chan, Yingleong; Westra, Harm-Jan; Wood, Andrew R.; Yang, Jian; Lui, Julian C.; Vedantam, Sailaja; Gustafsson, Stefan; Esko, Tonu; Frayling, Tim; Speliotes, Elizabeth K.; Boehnke, Michael; Raychaudhuri, Soumya; Fehrmann, Rudolf S.N.; Hirschhorn, Joel N.; Franke, Lude

2015-01-01

The main challenge for gaining biological insights from genetic associations is identifying which genes and pathways explain the associations. Here we present DEPICT, an integrative tool that employs predicted gene functions to systematically prioritize the most likely causal genes at associated loci, highlight enriched pathways and identify tissues/cell types where genes from associated loci are highly expressed. DEPICT is not limited to genes with established functions and prioritizes relevant gene sets for many phenotypes. PMID:25597830

Integration of oxygen signaling at the consensus HRE.

PubMed

Wenger, Roland H; Stiehl, Daniel P; Camenisch, Gieri

2005-10-18

The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFalpha subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system.

Calcium as a signal integrator in developing epithelial tissues.

PubMed

Brodskiy, Pavel A; Zartman, Jeremiah J

2018-05-16

Decoding how tissue properties emerge across multiple spatial and temporal scales from the integration of local signals is a grand challenge in quantitative biology. For example, the collective behavior of epithelial cells is critical for shaping developing embryos. Understanding how epithelial cells interpret a diverse range of local signals to coordinate tissue-level processes requires a systems-level understanding of development. Integration of multiple signaling pathways that specify cell signaling information requires second messengers such as calcium ions. Increasingly, specific roles have been uncovered for calcium signaling throughout development. Calcium signaling regulates many processes including division, migration, death, and differentiation. However, the pleiotropic and ubiquitous nature of calcium signaling implies that many additional functions remain to be discovered. Here we review a selection of recent studies to highlight important insights into how multiple signals are transduced by calcium transients in developing epithelial tissues. Quantitative imaging and computational modeling have provided important insights into how calcium signaling integration occurs. Reverse-engineering the conserved features of signal integration mediated by calcium signaling will enable novel approaches in regenerative medicine and synthetic control of morphogenesis.

Connecting Neuronal Cell Protective Pathways and Drug Combinations in a Huntington's Disease Model through the Application of Quantitative Systems Pharmacology.

PubMed

Pei, Fen; Li, Hongchun; Henderson, Mark J; Titus, Steven A; Jadhav, Ajit; Simeonov, Anton; Cobanoglu, Murat Can; Mousavi, Seyed H; Shun, Tongying; McDermott, Lee; Iyer, Prema; Fioravanti, Michael; Carlisle, Diane; Friedlander, Robert M; Bahar, Ivet; Taylor, D Lansing; Lezon, Timothy R; Stern, Andrew M; Schurdak, Mark E

2017-12-19

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington's Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdh Q111 cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism. Several mechanistically diverse protective probes were identified, most of which showed less than 50% efficacy. Specific combinations of these probes were synergistic in enhancing efficacy. Computational analysis of these probes revealed a convergence of pathways indicating activation of PKA. Analysis of phospho-PKA levels showed lower cytoplasmic levels in STHdh Q111 cells compared to wild type STHdh Q7 cells, and these levels were increased by several of the protective compounds. Pharmacological inhibition of PKA activity reduced protection supporting the hypothesis that protection may be working, in part, through activation of the PKA network. The systems-level studies described here can be broadly applied to any discovery strategy involving small molecule modulation of disease phenotype.

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae.

PubMed

Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

2016-10-26

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

RaMP: A Comprehensive Relational Database of Metabolomics Pathways for Pathway Enrichment Analysis of Genes and Metabolites

PubMed Central

Zhang, Bofei; Hu, Senyang; Baskin, Elizabeth; Patt, Andrew; Siddiqui, Jalal K.

2018-01-01

The value of metabolomics in translational research is undeniable, and metabolomics data are increasingly generated in large cohorts. The functional interpretation of disease-associated metabolites though is difficult, and the biological mechanisms that underlie cell type or disease-specific metabolomics profiles are oftentimes unknown. To help fully exploit metabolomics data and to aid in its interpretation, analysis of metabolomics data with other complementary omics data, including transcriptomics, is helpful. To facilitate such analyses at a pathway level, we have developed RaMP (Relational database of Metabolomics Pathways), which combines biological pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, WikiPathways, and the Human Metabolome DataBase (HMDB). To the best of our knowledge, an off-the-shelf, public database that maps genes and metabolites to biochemical/disease pathways and can readily be integrated into other existing software is currently lacking. For consistent and comprehensive analysis, RaMP enables batch and complex queries (e.g., list all metabolites involved in glycolysis and lung cancer), can readily be integrated into pathway analysis tools, and supports pathway overrepresentation analysis given a list of genes and/or metabolites of interest. For usability, we have developed a RaMP R package (https://github.com/Mathelab/RaMP-DB), including a user-friendly RShiny web application, that supports basic simple and batch queries, pathway overrepresentation analysis given a list of genes or metabolites of interest, and network visualization of gene-metabolite relationships. The package also includes the raw database file (mysql dump), thereby providing a stand-alone downloadable framework for public use and integration with other tools. In addition, the Python code needed to recreate the database on another system is also publicly available (https://github.com/Mathelab/RaMP-BackEnd). Updates for databases in RaMP will be checked multiple times a year and RaMP will be updated accordingly. PMID:29470400

RaMP: A Comprehensive Relational Database of Metabolomics Pathways for Pathway Enrichment Analysis of Genes and Metabolites.

PubMed

Zhang, Bofei; Hu, Senyang; Baskin, Elizabeth; Patt, Andrew; Siddiqui, Jalal K; Mathé, Ewy A

2018-02-22

The value of metabolomics in translational research is undeniable, and metabolomics data are increasingly generated in large cohorts. The functional interpretation of disease-associated metabolites though is difficult, and the biological mechanisms that underlie cell type or disease-specific metabolomics profiles are oftentimes unknown. To help fully exploit metabolomics data and to aid in its interpretation, analysis of metabolomics data with other complementary omics data, including transcriptomics, is helpful. To facilitate such analyses at a pathway level, we have developed RaMP (Relational database of Metabolomics Pathways), which combines biological pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, WikiPathways, and the Human Metabolome DataBase (HMDB). To the best of our knowledge, an off-the-shelf, public database that maps genes and metabolites to biochemical/disease pathways and can readily be integrated into other existing software is currently lacking. For consistent and comprehensive analysis, RaMP enables batch and complex queries (e.g., list all metabolites involved in glycolysis and lung cancer), can readily be integrated into pathway analysis tools, and supports pathway overrepresentation analysis given a list of genes and/or metabolites of interest. For usability, we have developed a RaMP R package (https://github.com/Mathelab/RaMP-DB), including a user-friendly RShiny web application, that supports basic simple and batch queries, pathway overrepresentation analysis given a list of genes or metabolites of interest, and network visualization of gene-metabolite relationships. The package also includes the raw database file (mysql dump), thereby providing a stand-alone downloadable framework for public use and integration with other tools. In addition, the Python code needed to recreate the database on another system is also publicly available (https://github.com/Mathelab/RaMP-BackEnd). Updates for databases in RaMP will be checked multiple times a year and RaMP will be updated accordingly.

Integration of cellular ubiquitin and membrane traffic systems: focus on deubiquitylases.

PubMed

Clague, Michael J; Urbé, Sylvie

2017-06-01

The cell is comprised of integrated multilevel protein networks or systems. The ubiquitin, protein homeostasis and membrane trafficking systems are highly integrated. Here, we look at the influence of reversible ubiquitylation on membrane trafficking and organelle dynamics. We review the regulation of endocytic sorting, selective autophagy and the secretory pathway by ubiquitin signals, with a particular focus on detailing the contribution of deubiquitylating enzymes. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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

Perkins, Timothy N.; Dentener, Mieke A.

Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damagingmore » inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT5A signaling. • Microarray reveals WNT as a novel complex signaling network in silica-mediated injury.« less

Local and Long-Range Circuit Connections to Hilar Mossy Cells in the Dentate Gyrus

PubMed Central

Sun, Yanjun; Grieco, Steven F.; Holmes, Todd C.

2017-01-01

Abstract Hilar mossy cells are the prominent glutamatergic cell type in the dentate hilus of the dentate gyrus (DG); they have been proposed to have critical roles in the DG network. To better understand how mossy cells contribute to DG function, we have applied new viral genetic and functional circuit mapping approaches to quantitatively map and compare local and long-range circuit connections of mossy cells and dentate granule cells in the mouse. The great majority of inputs to mossy cells consist of two parallel inputs from within the DG: an excitatory input pathway from dentate granule cells and an inhibitory input pathway from local DG inhibitory neurons. Mossy cells also receive a moderate degree of excitatory and inhibitory CA3 input from proximal CA3 subfields. Long range inputs to mossy cells are numerically sparse, and they are only identified readily from the medial septum and the septofimbrial nucleus. In comparison, dentate granule cells receive most of their inputs from the entorhinal cortex. The granule cells receive significant synaptic inputs from the hilus and the medial septum, and they also receive direct inputs from both distal and proximal CA3 subfields, which has been underdescribed in the existing literature. Our slice-based physiological mapping studies further supported the identified circuit connections of mossy cells and granule cells. Together, our data suggest that hilar mossy cells are major local circuit integrators and they exert modulation of the activity of dentate granule cells as well as the CA3 region through “back-projection” pathways. PMID:28451637

Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

PubMed Central

Denhardt, D T

1996-01-01

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways. PMID:8836113

Uncovering transcription factor and microRNA risk regulatory pathways associated with osteoarthritis by network analysis.

PubMed

Song, Zhenhua; Zhang, Chi; He, Lingxiao; Sui, Yanfang; Lin, Xiafei; Pan, Jingjing

2018-06-12

Osteoarthritis (OA) is the most common form of joint disease. The development of inflammation have been considered to play a key role during the progression of OA. Regulatory pathways are known to play crucial roles in many pathogenic processes. Thus, deciphering these risk regulatory pathways is critical for elucidating the mechanisms underlying OA. We constructed an OA-specific regulatory network by integrating comprehensive curated transcription and post-transcriptional resource involving transcription factor (TF) and microRNA (miRNA). To deepen our understanding of underlying molecular mechanisms of OA, we developed an integrated systems approach to identify OA-specific risk regulatory pathways. In this study, we identified 89 significantly differentially expressed genes between normal and inflamed areas of OA patients. We found the OA-specific regulatory network was a standard scale-free network with small-world properties. It significant enriched many immune response-related functions including leukocyte differentiation, myeloid differentiation and T cell activation. Finally, 141 risk regulatory pathways were identified based on OA-specific regulatory network, which contains some known regulator of OA. The risk regulatory pathways may provide clues for the etiology of OA and be a potential resource for the discovery of novel OA-associated disease genes. Copyright © 2018 Elsevier Inc. All rights reserved.

The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts.

PubMed

Jahanshahi, Maryam; Hsiao, Kuangfu; Jenny, Andreas; Pfleger, Cathie M

2016-08-01

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue "synthetic lethality" phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis.

Next-generation sequencing analysis of gene regulation in the rat model of retinopathy of prematurity.

PubMed

Griffith, Rachel M; Li, Hu; Zhang, Nan; Favazza, Tara L; Fulton, Anne B; Hansen, Ronald M; Akula, James D

2013-08-01

The purpose of this study was to identify the genes, biochemical signaling pathways, and biological themes involved in the pathogenesis of retinopathy of prematurity (ROP). Next-generation sequencing (NGS) was performed on the RNA transcriptome of rats with the Penn et al. (Pediatr Res 36:724-731, 1994) oxygen-induced retinopathy model of ROP at the height of vascular abnormality, postnatal day (P) 19, and normalized to age-matched, room-air-reared littermate controls. Eight custom-developed pathways with potential relevance to known ROP sequelae were evaluated for significant regulation in ROP: The three major Wnt signaling pathways, canonical, planar cell polarity (PCP), and Wnt/Ca(2+); two signaling pathways mediated by the Rho GTPases RhoA and Cdc42, which are, respectively, thought to intersect with canonical and non-canonical Wnt signaling; nitric oxide signaling pathways mediated by two nitric oxide synthase (NOS) enzymes, neuronal (nNOS) and endothelial (eNOS); and the retinoic acid (RA) signaling pathway. Regulation of other biological pathways and themes was detected by gene ontology using the Kyoto Encyclopedia of Genes and Genomes and the NIH's Database for Annotation, Visualization, and Integrated Discovery's GO terms databases. Canonical Wnt signaling was found to be regulated, but the non-canonical PCP and Wnt/Ca(2+) pathways were not. Nitric oxide signaling, as measured by the activation of nNOS and eNOS, was also regulated, as was RA signaling. Biological themes related to protein translation (ribosomes), neural signaling, inflammation and immunity, cell cycle, and cell death were (among others) highly regulated in ROP rats. These several genes and pathways identified by NGS might provide novel targets for intervention in ROP.

Next Generation Sequencing Analysis of Gene Regulation in the Rat Model of Retinopathy of Prematurity

PubMed Central

Griffith, Rachel M.; Li, Hu; Zhang, Nan; Favazza, Tara L.; Fulton, Anne B.; Hansen, Ronald M.; Akula, James D.

2013-01-01

Purpose To identify the genes, biochemical signaling pathways and biological themes involved in the pathogenesis of retinopathy of prematurity (ROP). Methods Next-generation sequencing (NGS) was performed on the RNA transcriptome of rats with the Penn et al. (1994) oxygen-induced retinopathy (OIR) model of ROP at the height of vascular abnormality, postnatal day (P) 19, and normalized to age-matched, room-air-reared littermate controls. Eight custom developed pathways with potential relevance to known ROP sequelae were evaluated for significant regulation in ROP: The three major Wnt signaling pathways, canonical, planar cell polarity (PCP), and Wnt/Ca2+, two signaling pathways mediated by the Rho GTPases RhoA and Cdc42, which are respectively thought to intersect with canonical and noncanonical Wnt signaling, nitric oxide signaling pathways mediated by two nitrox oxide synthase (NOS) enzymes, neuronal (nNOS) and endothelial (eNOS), and the retinoic acid (RA) signaling pathway. Regulation of other biological pathways and themes were detected by gene ontology using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the NIH's Database for Annotation, Visualization and Integrated Discovery (DAVID)'s GO terms databases. Results Canonical Wnt signaling was found to be regulated, but the non-canonical PCP and Wnt/Ca2+ pathways were not. Nitric oxide (NO) signaling, as measured by the activation of nNOS eNOS, was also regulated, as was RA signaling. Biological themes related to protein translation (ribosomes), neural signaling, inflammation and immunity, cell cycle and cell death, were (among others) highly regulated in ROP rats. Conclusions These several genes and pathways identified by NGS might provide novel targets for intervention in ROP. PMID:23775346

The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts

PubMed Central

Jenny, Andreas; Pfleger, Cathie M.

2016-01-01

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue “synthetic lethality” phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis. PMID:27494403

POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation.

PubMed

Ma, Xianjue; Guo, Xiaowei; Richardson, Helena E; Xu, Tian; Xue, Lei

2018-02-27

The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. Here we identify Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in Drosophila POSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. Our findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila .

Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells

PubMed Central

Choi, Paul J.; Mitchison, Timothy J.

2013-01-01

Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse imaging to reveal time-dependent statistics of natural killer (NK) killing at the single-cell level. Most kills occurred during serial killing, and a single NK cell killed up to 10 targets over a 6-h assay. The first kill was slower than subsequent kills, especially on poor targets, or when NK signaling pathways were partially inhibited. Spatial analysis showed that sequential kills were usually adjacent. We propose that NK cells integrate signals from the previous and current target, possibly by simultaneous contact. The resulting burst kinetics and spatial coordination may control the activity of NK cells in tissues. PMID:23576740

A complex regulatory network coordinating cell cycles during C. elegans development is revealed by a genome-wide RNAi screen.

PubMed

Roy, Sarah H; Tobin, David V; Memar, Nadin; Beltz, Eleanor; Holmen, Jenna; Clayton, Joseph E; Chiu, Daniel J; Young, Laura D; Green, Travis H; Lubin, Isabella; Liu, Yuying; Conradt, Barbara; Saito, R Mako

2014-02-28

The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development. Copyright © 2014 Roy et al.

Activity-based protein profiling for biochemical pathway discovery in cancer

PubMed Central

Nomura, Daniel K.; Dix, Melissa M.; Cravatt, Benjamin F.

2011-01-01

Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment. PMID:20703252

Modified Vaccinia Virus Ankara-Infected Dendritic Cells Present CD4+ T-Cell Epitopes by Endogenous Major Histocompatibility Complex Class II Presentation Pathways

PubMed Central

Thiele, Frank; Tao, Sha; Zhang, Yi; Muschaweckh, Andreas; Zollmann, Tina; Protzer, Ulrike; Abele, Rubert

2014-01-01

ABSTRACT CD4+ T lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on major histocompatibility complex II (MHCII) molecules on antigen-presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4+ T cells. Certain epitopes, however, can be processed directly from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways that are possibly involved in the immune response upon vaccination with modified vaccinia virus Ankara (MVA), a promising live viral vaccine vector. We established CD4+ T-cell lines specific for MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4+ T cells. By using knockout mice and chemical inhibitory compounds, we further elucidated the molecular basis, showing that among the various subcellular pathways investigated, proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role, neither TAP nor LAMP-2 was found to be involved in the peptide transport. Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis for improving MVA-based vaccination strategies by aiming for enhanced CD4+ T-cell activation by directing antigens into the responsible pathways. IMPORTANCE This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4+ T cells. We identified autophagosome formation, proteasomal activity, and lysosomal integrity as being crucial for endogenous CD4+ T-cell activation. Since poxvirus vectors such as MVA are already used in clinical trials as recombinant vaccines, the data provide important information for the future design of optimized poxviral vaccines for the study of advanced immunotherapy options. PMID:25520512

Phosphoproteomics analyses show subnetwork systems in T-cell receptor signaling.

PubMed

Hatano, Atsushi; Matsumoto, Masaki; Nakayama, Keiichi I

2016-10-01

A key issue in the study of signal transduction is how multiple signaling pathways are systematically integrated into the cell. We have now performed multiple phosphoproteomics analyses focused on the dynamics of the T-cell receptor (TCR) signaling network and its subsystem mediated by the Ca 2+ signaling pathway. Integration of these phosphoproteomics data sets and extraction of components of the TCR signaling network dependent on Ca 2+ signaling showed unexpected phosphorylation kinetics for candidate substrates of the Ca 2+ -dependent phosphatase calcineurin (CN) during TCR stimulation. Detailed characterization of the TCR-induced phosphorylation of a novel CN substrate, Itpkb, showed that phosphorylation of this protein is regulated by both CN and the mitogen-activated protein kinase Erk in a competitive manner. Phosphorylation of additional CN substrates was also found to be regulated by Erk and CN in a similar manner. The combination of multiple phosphoproteomics approaches thus showed two major subsystems mediated by Erk and CN in the TCR signaling network, with these subsystems regulating the phosphorylation of a group of proteins in a competitive manner. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

PubMed

Azad, Gajendra Kumar; Singh, Vikash; Baranwal, Shivani; Thakare, Mayur Jankiram; Tomar, Raghuvir S

2015-01-02

Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N-terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell-wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

On the origin, evolution, and nature of programmed cell death: a timeline of four billion years.

PubMed

Ameisen, J C

2002-04-01

Programmed cell death is a genetically regulated process of cell suicide that is central to the development, homeostasis and integrity of multicellular organisms. Conversely, the dysregulation of mechanisms controlling cell suicide plays a role in the pathogenesis of a wide range of diseases. While great progress has been achieved in the unveiling of the molecular mechanisms of programmed cell death, a new level of complexity, with important therapeutic implications, has begun to emerge, suggesting (i) that several different self-destruction pathways may exist and operate in parallel in our cells, and (ii) that molecular effectors of cell suicide may also perform other functions unrelated to cell death induction and crucial to cell survival. In this review, I will argue that this new level of complexity, implying that there may be no such thing as a 'bona fide' genetic death program in our cells, might be better understood when considered in an evolutionary context. And a new view of the regulated cell suicide pathways emerges when one attempts to ask the question of when and how they may have become selected during evolution, at the level of ancestral single-celled organisms.

Participation of Candida albicans Transcription Factor RLM1 in Cell Wall Biogenesis and Virulence

PubMed Central

Delgado-Silva, Yolanda; Vaz, Catarina; Carvalho-Pereira, Joana; Carneiro, Catarina; Nogueira, Eugénia; Correia, Alexandra; Carreto, Laura; Silva, Sónia; Faustino, Augusto; Pais, Célia; Oliveira, Rui; Sampaio, Paula

2014-01-01

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources. PMID:24466000

Integrated pathway analysis of nasopharyngeal carcinoma implicates the axonemal dynein complex in the Malaysian cohort.

PubMed

Chin, Yoon-Ming; Tan, Lu Ping; Abdul Aziz, Norazlin; Mushiroda, Taisei; Kubo, Michiaki; Mohd Kornain, Noor Kaslina; Tan, Geok Wee; Khoo, Alan Soo-Beng; Krishnan, Gopala; Pua, Kin-Choo; Yap, Yoke-Yeow; Teo, Soo-Hwang; Lim, Paul Vey-Hong; Nakamura, Yusuke; Lum, Chee Lun; Ng, Ching-Ching

2016-10-15

Nasopharyngeal carcinoma (NPC) is an epithelial squamous cell carcinoma on the mucosal lining of the nasopharynx. The etiology of NPC remains elusive despite many reported studies. Most studies employ a single platform approach, neglecting the cumulative influence of both the genome and transcriptome toward NPC development. We aim to employ an integrated pathway approach to identify dysregulated pathways linked to NPC. Our approach combines imputation NPC GWAS data from a Malaysian cohort as well as published expression data GSE12452 from both NPC and non-NPC nasopharynx tissues. Pathway association for GWAS data was performed using MAGENTA while for expression data, GSA-SNP was used with gene p values derived from differential expression values from GEO2R. Our study identified NPC association in the gene ontology (GO) axonemal dynein complex pathway (pGWAS-GSEA  = 1.98 × 10(-2) ; pExpr-GSEA  = 1.27 × 10(-24) ; pBonf-Combined  = 4.15 × 10(-21) ). This association was replicated in a separate cohort using gene expression data from NPC and non-NPC nasopharynx tissues (pAmpliSeq-GSEA  = 6.56 × 10(-4) ). Loss of function in the axonemal dynein complex causes impaired cilia function, leading to poor mucociliary clearance and subsequently upper or lower respiratory tract infection, the former of which includes the nasopharynx. Our approach illustrates the potential use of integrated pathway analysis in detecting gene sets involved in the development of NPC in the Malaysian cohort. © 2016 UICC.

Characterization of p38 MAPK isoforms for drug resistance study using systems biology approach.

PubMed

Peng, Huiming; Peng, Tao; Wen, Jianguo; Engler, David A; Matsunami, Risë K; Su, Jing; Zhang, Le; Chang, Chung-Che Jeff; Zhou, Xiaobo

2014-07-01

p38 mitogen-activated protein kinase activation plays an important role in resistance to chemotherapeutic cytotoxic drugs in treating multiple myeloma (MM). However, how the p38 mitogen-activated protein kinase signaling pathway is involved in drug resistance, in particular the roles that the various p38 isoforms play, remains largely unknown. To explore the underlying mechanisms, we developed a novel systems biology approach by integrating liquid chromatography-mass spectrometry and reverse phase protein array data from human MM cell lines with computational pathway models in which the unknown parameters were inferred using a proposed novel algorithm called modularized factor graph. New mechanisms predicted by our models suggest that combined activation of various p38 isoforms may result in drug resistance in MM via regulating the related pathways including extracellular signal-regulated kinase (ERK) pathway and NFкB pathway. ERK pathway regulating cell growth is synergistically regulated by p38δ isoform, whereas nuclear factor kappa B (NFкB) pathway regulating cell apoptosis is synergistically regulated by p38α isoform. This finding that p38δ isoform promotes the phosphorylation of ERK1/2 in MM cells treated with bortezomib was validated by western blotting. Based on the predicted mechanisms, we further screened drug combinations in silico and found that a promising drug combination targeting ERK1/2 and NFκB might reduce the effects of drug resistance in MM cells. This study provides a framework of a systems biology approach to studying drug resistance and drug combination selection. RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway.

PubMed

Alam, Maroof; Bouillez, Audrey; Tagde, Ashujit; Ahmad, Rehan; Rajabi, Hasan; Maeda, Takahiro; Hiraki, Masayuki; Suzuki, Yozo; Kufe, Donald

2016-12-01

Apical-basal polarity and epithelial integrity are maintained in part by the Crumbs (CRB) complex. The C--terminal subunit of MUC1 (MUC1-C) is a transmembrane protein that is expressed at the apical border of normal epithelial cells and aberrantly at high levels over the entire surface of their transformed counterparts. However, it is not known whether MUC1-C contributes to this loss of polarity that is characteristic of carcinoma cells. Here it is demonstrated that MUC1-C downregulates expression of the Crumbs complex CRB3 protein in triple-negative breast cancer (TNBC) cells. MUC1-C associates with ZEB1 on the CRB3 promoter and represses CRB3 transcription. Notably, CRB3 activates the core kinase cassette of the Hippo pathway, which includes LATS1 and LATS2. In this context, targeting MUC1-C was associated with increased phosphorylation of LATS1, consistent with activation of the Hippo pathway, which is critical for regulating cell contact, tissue repair, proliferation, and apoptosis. Also shown is that MUC1-C--mediated suppression of CRB3 and the Hippo pathway is associated with dephosphorylation and activation of the oncogenic YAP protein. In turn, MUC1-C interacts with YAP, promotes formation of YAP/β-catenin complexes, and induces the WNT target gene MYC. These data support a previously unrecognized pathway in which targeting MUC1-C in TNBC cells (i) induces CRB3 expression, (ii) activates the CRB3-driven Hippo pathway, (iii) inactivates YAP, and thereby (iv) suppresses YAP/β-catenin-mediated induction of MYC expression. These findings demonstrate a previously unrecognized role for the MUC1-C oncoprotein in the regulation of polarity and the Hippo pathway in breast cancer. Mol Cancer Res; 14(12); 1266-76. ©2016 AACR. ©2016 American Association for Cancer Research.

Simultaneous and Sequential Integration by Cre/loxP Site-Specific Recombination in Saccharomyces cerevisiae.

PubMed

Choi, Ho-Jung; Kim, Yeon-Hee

2018-05-28

A Cre/ loxP -δ-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae . To allow repeated integrations, the reusable Candida glabrata MARKER ( CgMARKER ) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and β-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals

PubMed Central

Wang, Baojun; Barahona, Mauricio; Buck, Martin

2013-01-01

Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas. PMID:22981411

Integrated compensatory network is activated in the absence of NCC phosphorylation.

PubMed

Grimm, P Richard; Lazo-Fernandez, Yoskaly; Delpire, Eric; Wall, Susan M; Dorsey, Susan G; Weinman, Edward J; Coleman, Richard; Wade, James B; Welling, Paul A

2015-05-01

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

Integrated compensatory network is activated in the absence of NCC phosphorylation

PubMed Central

Grimm, P. Richard; Lazo-Fernandez, Yoskaly; Delpire, Eric; Wall, Susan M.; Dorsey, Susan G.; Weinman, Edward J.; Coleman, Richard; Wade, James B.; Welling, Paul A.

2015-01-01

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase–deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H+-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG–activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy. PMID:25893600

Sensational placodes: Neurogenesis in the otic and olfactory systems

PubMed Central

Maier, Esther C.; Saxena, Ankur; Alsina, Berta; Bronner, Marianne E.; Whitfield, Tanya T.

2014-01-01

For both the intricate morphogenetic layout of the sensory cells in the ear and the elegantly radial arrangement of the sensory neurons in the nose, numerous signaling molecules and genetic determinants are required in concert to generate these specialized neuronal populations that help connect us to our environment. In this review, we outline many of the proteins and pathways that play essential roles in the differentiation of otic and olfactory neurons and their integration into their non-neuronal support structures. In both cases, well-known signaling pathways together with region-specific factors transform thickened ectodermal placodes into complex sense organs containing numerous, diverse neuronal subtypes. Olfactory and otic placodes, in combination with migratory neural crest stem cells, generate highly specialized subtypes of neuronal cells that sense sound, position and movement in space, odors and pheromones throughout our lives. PMID:24508480

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Cellular Decision Making by Non-Integrative Processing of TLR Inputs.

PubMed

Kellogg, Ryan A; Tian, Chengzhe; Etzrodt, Martin; Tay, Savaş

2017-04-04

Cells receive a multitude of signals from the environment, but how they process simultaneous signaling inputs is not well understood. Response to infection, for example, involves parallel activation of multiple Toll-like receptors (TLRs) that converge on the nuclear factor κB (NF-κB) pathway. Although we increasingly understand inflammatory responses for isolated signals, it is not clear how cells process multiple signals that co-occur in physiological settings. We therefore examined a bacterial infection scenario involving co-stimulation of TLR4 and TLR2. Independent stimulation of these receptors induced distinct NF-κB dynamic profiles, although surprisingly, under co-stimulation, single cells continued to show ligand-specific dynamic responses characteristic of TLR2 or TLR4 signaling rather than a mixed response, comprising a cellular decision that we term "non-integrative" processing. Iterating modeling and microfluidic experiments revealed that non-integrative processing occurred through interaction of switch-like NF-κB activation, receptor-specific processing timescales, cell-to-cell variability, and TLR cross-tolerance mediated by multilayer negative feedback. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

PubMed

Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

2011-01-01

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

RNA sequencing analysis of human podocytes reveals glucocorticoid regulated gene networks targeting non-immune pathways

PubMed Central

Jiang, Lulu; Hindmarch, Charles C. T.; Rogers, Mark; Campbell, Colin; Waterfall, Christy; Coghill, Jane; Mathieson, Peter W.; Welsh, Gavin I.

2016-01-01

Glucocorticoids are steroids that reduce inflammation and are used as immunosuppressive drugs for many diseases. They are also the mainstay for the treatment of minimal change nephropathy (MCN), which is characterised by an absence of inflammation. Their mechanisms of action remain elusive. Evidence suggests that immunomodulatory drugs can directly act on glomerular epithelial cells or ‘podocytes’, the cell type which is the main target of injury in MCN. To understand the nature of glucocorticoid effects on non-immune cell functions, we generated RNA sequencing data from human podocyte cell lines and identified the genes that are significantly regulated in dexamethasone-treated podocytes compared to vehicle-treated cells. The upregulated genes are of functional relevance to cytoskeleton-related processes, whereas the downregulated genes mostly encode pro-inflammatory cytokines and growth factors. We observed a tendency for dexamethasone-upregulated genes to be downregulated in MCN patients. Integrative analysis revealed gene networks composed of critical signaling pathways that are likely targeted by dexamethasone in podocytes. PMID:27774996

Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

PubMed

Wang, Wei-jia; Wang, Yuan; Chen, Hang-zi; Xing, Yong-zhen; Li, Feng-wei; Zhang, Qian; Zhou, Bo; Zhang, Hong-kui; Zhang, Jie; Bian, Xue-li; Li, Li; Liu, Yuan; Zhao, Bi-xing; Chen, Yan; Wu, Rong; Li, An-zhong; Yao, Lu-ming; Chen, Ping; Zhang, Yi; Tian, Xu-yang; Beermann, Friedrich; Wu, Mian; Han, Jiahuai; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

2014-02-01

Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death.

α7 Nicotinic Acetylcholine Receptor (α7nAChR) Expression in Bone Marrow–Derived Non–T Cells Is Required for the Inflammatory Reflex

PubMed Central

Olofsson, Peder S; Katz, David A; Rosas-Ballina, Mauricio; Levine, Yaakov A; Ochani, Mahendar; Valdés-Ferrer, Sergio I; Pavlov, Valentin A; Tracey, Kevin J; Chavan, Sangeeta S

2012-01-01

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow–derived cells significantly impaired vagus nerve–mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow–derived non–T cells is required for the integrity of the inflammatory reflex. PMID:22183893

A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts

PubMed Central

Sacristán-Reviriego, Almudena; Madrid, Marisa; Cansado, José; Martín, Humberto; Molina, María

2014-01-01

Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi. PMID:24465549

Integration of Plant Metabolomics Data with Metabolic Networks: Progresses and Challenges.

PubMed

Töpfer, Nadine; Seaver, Samuel M D; Aharoni, Asaph

2018-01-01

In the last decade, plant genome-scale modeling has developed rapidly and modeling efforts have advanced from representing metabolic behavior of plant heterotrophic cell suspensions to studying the complex interplay of cell types, tissues, and organs. A crucial driving force for such developments is the availability and integration of "omics" data (e.g., transcriptomics, proteomics, and metabolomics) which enable the reconstruction, extraction, and application of context-specific metabolic networks. In this chapter, we demonstrate a workflow to integrate gas chromatography coupled to mass spectrometry (GC-MS)-based metabolomics data of tomato fruit pericarp (flesh) tissue, at five developmental stages, with a genome-scale reconstruction of tomato metabolism. This method allows for the extraction of context-specific networks reflecting changing activities of metabolic pathways throughout fruit development and maturation.

Signature gene expressions of cell wall integrity pathway concur with tolerance response of industrial yeast Saccharomyces cerevisiae against biomass pretreatment inhibitors

USDA-ARS?s Scientific Manuscript database

Traditional industrial ethanologenic yeast Saccharomyces cerevisiae has a robust performance under various environmental conditions and can be served as a candidate for the next-generation biocatalyst development for advanced biofuels production using lignocellulose mateials. Overcoming toxic compou...

COMPUTATIONAL MODELING OF SIGNALING PATHWAYS MEDIATING CELL CYCLE AND APOPTOTIC RESPONSES TO IONIZING RADIATION MEDIATED DNA DAMAGE

EPA Science Inventory

Demonstrated of the use of a computational systems biology approach to model dose response relationships. Also discussed how the biologically motivated dose response models have only limited reference to the underlying molecular level. Discussed the integration of Computational S...

Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells.

PubMed

Thankamony, Sai P; Sackstein, Robert

2011-02-08

According to the multistep model of cell migration, chemokine receptor engagement (step 2) triggers conversion of rolling interactions (step 1) into firm adhesion (step 3), yielding transendothelial migration. We recently reported that glycosyltransferase-programmed stereosubstitution (GPS) of CD44 on human mesenchymal stem cells (hMSCs) creates the E-selectin ligand HCELL (hematopoietic cell E-selectin/L-selectin ligand) and, despite absence of CXCR4, systemically administered HCELL(+)hMSCs display robust osteotropism visualized by intravital microscopy. Here we performed studies to define the molecular effectors of this process. We observed that engagement of hMSC HCELL with E-selectin triggers VLA-4 adhesiveness, resulting in shear-resistant adhesion to ligand VCAM-1. This VLA-4 activation is mediated via a Rac1/Rap1 GTPase signaling pathway, resulting in transendothelial migration on stimulated human umbilical vein endothelial cells without chemokine input. These findings indicate that hMSCs coordinately integrate CD44 ligation and integrin activation, circumventing chemokine-mediated signaling, yielding a step 2-bypass pathway of the canonical multistep paradigm of cell migration.

Traditional Chinese Medicine CFF-1 induced cell growth inhibition, autophagy, and apoptosis via inhibiting EGFR-related pathways in prostate cancer.

PubMed

Wu, Zhaomeng; Zhu, Qingyi; Yin, Yingying; Kang, Dan; Cao, Runyi; Tian, Qian; Zhang, Yu; Lu, Shan; Liu, Ping

2018-04-01

Traditional Chinese medicine (TCM) has a combined therapeutic result in cancer treatment by integrating holistic and local therapeutical effects, by which TCM can enhance the curative effect and reduce the side effect. In this study, we analyzed the effect of CFF-1 (alcohol extract from an anticancer compound Chinese medicine) on prostate cancer (PCa) cell lines and studied in detail the mechanism of cell death induced by CFF-1 in vitro and in vivo. From our data, we found for the first time that CFF-1 obviously arrested cell cycle in G1 phase, decreased cell viability and then increased nuclear rupture in a dose-dependent manner and finally resulted in apoptosis in prostate cancer cells. In molecular level, our data showed that CFF-1 induced inhibition of EGFR auto-phosphorylation and inactivation of EGFR. Disruption of EGFR activity in turn suppressed downstream PI3K/AKT and Raf/Erk signal pathways, resulted in the decrease of p-FOXO1 (Ser256) and regulated the expression of apoptosis-related and cycle-related genes. Moreover, CFF-1 markedly induced cell autophagy through inhibiting PI3K/AKT/mTOR pathway and then up-regulating Beclin-1 and LC-3II and down-regulating phosphorylation of p70S6K. In vivo, CFF-1-treated group exhibited a significant decrease in tumor volume compared with the negative control group in subcutaneous xenograft tumor in nude mice via inhibiting EGFR-related signal pathways. Thus, bio-functions of Chinese medicine CFF-1 in inducing PCa cell growth inhibition, autophagy, and apoptosis suggested that CFF-1 had the clinical potential to treat patients with prostate cancer. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway

PubMed Central

Robertson, Kevin A.; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J.; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J.; Enright, Anton J.; Yamamoto, Mami; Pradeepa, Madapura M.; Lennox, Kimberly A.; Behlke, Mark A.; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J.; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

2016-01-01

In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway. PMID:26938778

An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.

PubMed

Robertson, Kevin A; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J; Enright, Anton J; Yamamoto, Mami; Pradeepa, Madapura M; Lennox, Kimberly A; Behlke, Mark A; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

2016-03-01

In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway.

Integrated Proteomic and Transcriptomic-Based Approaches to Identifying Signature Biomarkers and Pathways for Elucidation of Daoy and UW228 Subtypes.

PubMed

Higdon, Roger; Kala, Jessie; Wilkins, Devan; Yan, Julia Fangfei; Sethi, Manveen K; Lin, Liang; Liu, Siqi; Montague, Elizabeth; Janko, Imre; Choiniere, John; Kolker, Natali; Hancock, William S; Kolker, Eugene; Fanayan, Susan

2017-02-03

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Patient survival has remained largely the same for the past 20 years, with therapies causing significant health, cognitive, behavioral and developmental complications for those who survive the tumor. In this study, we profiled the total transcriptome and proteome of two established MB cell lines, Daoy and UW228, using high-throughput RNA sequencing (RNA-Seq) and label-free nano-LC-MS/MS-based quantitative proteomics, coupled with advanced pathway analysis. While Daoy has been suggested to belong to the sonic hedgehog (SHH) subtype, the exact UW228 subtype is not yet clearly established. Thus, a goal of this study was to identify protein markers and pathways that would help elucidate their subtype classification. A number of differentially expressed genes and proteins, including a number of adhesion, cytoskeletal and signaling molecules, were observed between the two cell lines. While several cancer-associated genes/proteins exhibited similar expression across the two cell lines, upregulation of a number of signature proteins and enrichment of key components of SHH and WNT signaling pathways were uniquely observed in Daoy and UW228, respectively. The novel information on differentially expressed genes/proteins and enriched pathways provide insights into the biology of MB, which could help elucidate their subtype classification.

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

PubMed Central

Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

2016-01-01

Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

PubMed

Hough, Chris; Radu, Maria; Doré, Jules J E

2012-01-01

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

NADPH Oxidase 1 Modulates WNT and NOTCH1 Signaling To Control the Fate of Proliferative Progenitor Cells in the Colon▿

PubMed Central

Coant, Nicolas; Ben Mkaddem, Sanae; Pedruzzi, Eric; Guichard, Cécile; Tréton, Xavier; Ducroc, Robert; Freund, Jean-Noel; Cazals-Hatem, Dominique; Bouhnik, Yoram; Woerther, Paul-Louis; Skurnik, David; Grodet, Alain; Fay, Michèle; Biard, Denis; Lesuffleur, Thécla; Deffert, Christine; Moreau, Richard; Groyer, André; Krause, Karl-Heinz; Daniel, Fanny; Ogier-Denis, Eric

2010-01-01

The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/β-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/β-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/β-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/β-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector β-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/β-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation. PMID:20351171

Bioinformatic Integration of Molecular Networks and Major Pathways Involved in Mice Cochlear and Vestibular Supporting Cells.

PubMed

Requena, Teresa; Gallego-Martinez, Alvaro; Lopez-Escamez, Jose A

2018-01-01

Background : Cochlear and vestibular epithelial non-hair cells (ENHCs) are the supporting elements of the cellular architecture in the organ of Corti and the vestibular neuroepithelium in the inner ear. Intercellular and cell-extracellular matrix interactions are essential to prevent an abnormal ion redistribution leading to hearing and vestibular loss. The aim of this study is to define the main pathways and molecular networks in the mouse ENHCs. Methods : We retrieved microarray and RNA-seq datasets from mouse epithelial sensory and non-sensory cells from gEAR portal (http://umgear.org/index.html) and obtained gene expression fold-change between ENHCs and non-epithelial cells (NECs) against HCs for each gene. Differentially expressed genes (DEG) with a log2 fold change between 1 and -1 were discarded. The remaining genes were selected to search for interactions using Ingenuity Pathway Analysis and STRING platform. Specific molecular networks for ENHCs in the cochlea and the vestibular organs were generated and significant pathways were identified. Results : Between 1723 and 1559 DEG were found in the mouse cochlear and vestibular tissues, respectively. Six main pathways showed enrichment in the supporting cells in both tissues: (1) "Inhibition of Matrix Metalloproteases"; (2) "Calcium Transport I"; (3) "Calcium Signaling"; (4) "Leukocyte Extravasation Signaling"; (5) "Signaling by Rho Family GTPases"; and (6) "Axonal Guidance Si". In the mouse cochlea, ENHCs showed a significant enrichment in 18 pathways highlighting "axonal guidance signaling (AGS)" ( p = 4.37 × 10 -8 ) and "RhoGDI Signaling" ( p = 3.31 × 10 -8 ). In the vestibular dataset, there were 20 enriched pathways in ENHCs, the most significant being "Leukocyte Extravasation Signaling" ( p = 8.71 × 10 -6 ), "Signaling by Rho Family GTPases" ( p = 1.20 × 10 -5 ) and "Calcium Signaling" ( p = 1.20 × 10 -5 ). Among the top ranked networks, the most biologically significant network contained the "auditory and vestibular system development and function" terms. We also found 108 genes showing tonotopic gene expression in the cochlear ENHCs. Conclusions : We have predicted the main pathways and molecular networks for ENHCs in the organ of Corti and vestibular neuroepithelium. These pathways will facilitate the design of molecular maps to select novel candidate genes for hearing or vestibular loss to conduct functional studies.

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

PubMed Central

Chakrabarti, Sveta; Li, Xiaoxue; Collas, Esther Jeanne; Boquete, Jean-Phillipe; Lemaitre, Bruno

2016-01-01

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival. PMID:27231872

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila.

PubMed

Chakrabarti, Sveta; Dudzic, Jan Paul; Li, Xiaoxue; Collas, Esther Jeanne; Boquete, Jean-Phillipe; Lemaitre, Bruno

2016-05-01

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival.

Mutant p53 Promotes Tumor Cell Malignancy by Both Positive and Negative Regulation of the Transforming Growth Factor β (TGF-β) Pathway*

PubMed Central

Ji, Lei; Xu, Jinjin; Liu, Jian; Amjad, Ali; Zhang, Kun; Liu, Qingwu; Zhou, Lei; Xiao, Jianru; Li, Xiaotao

2015-01-01

Specific p53 mutations abrogate tumor-suppressive functions by gaining new abilities to promote tumorigenesis. Inactivation of p53 is known to distort TGF-β signaling, which paradoxically displays both tumor-suppressive and pro-oncogenic functions. The molecular mechanisms of how mutant p53 simultaneously antagonizes the tumor-suppressive and synergizes the tumor-promoting function of the TGF-β pathway remain elusive. Here we demonstrate that mutant p53 differentially regulates subsets of TGF-β target genes by enhanced binding to the MH2 domain in Smad3 upon the integration of ERK signaling, therefore disrupting Smad3/Smad4 complex formation. Silencing Smad2, inhibition of ERK, or introducing a phosphorylation-defective mutation at Ser-392 in p53 abrogates the R175H mutant p53-dependent regulation of these TGF-β target genes. Our study shows a mechanism to reconcile the seemingly contradictory observations that mutant p53 can both attenuate and cooperate with the TGF-β pathway to promote cancer cell malignancy in the same cell type. PMID:25767119

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme

PubMed Central

Tejada-Romero, Belen; Carter, Jean-Michel; Mihaylova, Yuliana; Neumann, Bjoern; Aboobaker, A. Aziz

2015-01-01

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration. PMID:26062938

Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice

PubMed Central

Tomimatsu, Nozomi; Mukherjee, Bipasha; Hardebeck, Molly Catherine; Ilcheva, Mariya; Camacho, Cristel Vanessa; Harris, Janelle Louise; Porteus, Matthew; Llorente, Bertrand; Khanna, Kum Kum; Burma, Sandeep

2014-01-01

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice. PMID:24705021

Retrograde signals arise from reciprocal crosstalk within plastids.

PubMed

Enami, Kazuhiko; Tanaka, Kan; Hanaoka, Mitsumasa

2012-01-01

In addition to the cell nucleus, plant cells also possess genomic DNA and gene expression machineries within mitochondria and plastids. In higher plants, retrograde transcriptional regulation of several nuclear genes encoding plastid-located proteins has been observed in response to changes in a wide variety of physiological properties in plastids, including organelle gene expression (OGE) and tetrapyrrole metabolism. This regulation is postulated to be accomplished by plastid-to-nucleus signaling, (1,2) although the overall signal transduction pathway(s) are not well characterized. By applying a specific differentiation system in tobacco Bright Yellow-2 (BY-2) cultured cells, (3,4) we recently reported that the regulatory system of nuclear gene expressions modulated by a plastid signal was also observed during differentiation of plastids into amyloplasts. (5) While retrograde signaling from plastids was previously speculated to consist of several independent pathways, we found inhibition of OGE and perturbation in the cellular content of one tetrapyrrole intermediate, heme, seemed to interact to regulate amyloplast differentiation. Our results thus highlight the possibility that several sources of retrograde signaling in plastids could be integrated in an intraorganellar manner.

Novel insights into embryonic stem cell self-renewal revealed through comparative human and mouse systems biology networks.

PubMed

Dowell, Karen G; Simons, Allen K; Bai, Hao; Kell, Braden; Wang, Zack Z; Yun, Kyuson; Hibbs, Matthew A

2014-05-01

Embryonic stem cells (ESCs), characterized by their ability to both self-renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self-renewal. Here, we explore the molecular basis of human ESC self-renewal using Bayesian network machine learning to integrate cell-type-specific, high-throughput data for gene function discovery. We integrated high-throughput ESC data from 83 human studies (~1.8 million data points collected under 1,100 conditions) and 62 mouse studies (~2.4 million data points collected under 1,085 conditions) into separate human and mouse predictive networks focused on ESC self-renewal to analyze shared and distinct functional relationships among protein-coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and reverse transcriptase polymerase chain reaction (RT-PCR) validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self-renewal and pluripotency in both species (e.g., POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g., threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g., leukemia inhibitory factor (LIF)-activated JAK/STAT in mouse; NODAL/ACTIVIN-A-activated fibroblast growth factor in human), and reveal many novel genes and pathways predicted to be functionally associated with self-renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation. © 2013 AlphaMed Press.

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes.

PubMed

Young, Nathan P; Kamireddy, Anwesh; Van Nostrand, Jeanine L; Eichner, Lillian J; Shokhirev, Maxim Nikolaievich; Dayn, Yelena; Shaw, Reuben J

2016-03-01

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. © 2016 Young et al.; Published by Cold Spring Harbor Laboratory Press.

Differential modulation of apoptotic processes by proanthocyanidins as a dietary strategy for delaying chronic pathologies.

PubMed

Puiggròs, Francesc; Salvadó, Maria-Josepa; Bladé, Cinta; Arola, Lluís

2014-01-01

Apoptosis is a biological process necessary for maintaining cellular homeostasis. Several diseases can result if it is deregulated. For example, inhibition of apoptotic signaling pathways is linked to the survival of pathological cells, which contributes to cancer, whereas excessive apoptosis is linked to neurodegenerative diseases, partially via oxidative stress. The activation or restoration of apoptosis via extrinsic or intrinsic pathways combined with cell signaling pathways triggered by reactive oxygen specises (ROS) formation is considered a key strategy by which bioactive foods can exert their health effects. Proanthocyanidins, a class of flavonoids naturally found in fruits, vegetables, and beverages, have attracted a great deal of attention not only because they are strong antioxidants but also because they appear to exert a different modulation of apoptosis, stimulating apoptosis in damaged cells, thus preventing cancer or reducing apoptosis in healthy cells, and as a result, preserving the integrity of normal cells and protecting against neurodegenerative diseases. Therefore, proanthocyanidins could provide a defense against apoptosis induced by oxidative stress or directly inhibit apoptosis, and they could also provide a promising treatment for a variety of diseases. Emerging data suggest that proanthocyanidins, especially those that humans can be persuaded to consume, may be used to prevent and manage cancer and mental disorders.

Pathway to a Phenocopy: Heat Stress Effects in Early Embryogenesis

PubMed Central

Crews, Sarah M.; McCleery, W. Tyler; Hutson, M. Shane

2015-01-01

Background Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants – having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from non-specific heat stress to phenocopied abnormalities is unknown. Results Drosophila embryos subjected to 30-min, 38-°C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 μm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. Conclusions The pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity – i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types. PMID:26498920

Network reconstruction based on proteomic data and prior knowledge of protein connectivity using graph theory.

PubMed

Stavrakas, Vassilis; Melas, Ioannis N; Sakellaropoulos, Theodore; Alexopoulos, Leonidas G

2015-01-01

Modeling of signal transduction pathways is instrumental for understanding cells' function. People have been tackling modeling of signaling pathways in order to accurately represent the signaling events inside cells' biochemical microenvironment in a way meaningful for scientists in a biological field. In this article, we propose a method to interrogate such pathways in order to produce cell-specific signaling models. We integrate available prior knowledge of protein connectivity, in a form of a Prior Knowledge Network (PKN) with phosphoproteomic data to construct predictive models of the protein connectivity of the interrogated cell type. Several computational methodologies focusing on pathways' logic modeling using optimization formulations or machine learning algorithms have been published on this front over the past few years. Here, we introduce a light and fast approach that uses a breadth-first traversal of the graph to identify the shortest pathways and score proteins in the PKN, fitting the dependencies extracted from the experimental design. The pathways are then combined through a heuristic formulation to produce a final topology handling inconsistencies between the PKN and the experimental scenarios. Our results show that the algorithm we developed is efficient and accurate for the construction of medium and large scale signaling networks. We demonstrate the applicability of the proposed approach by interrogating a manually curated interaction graph model of EGF/TNFA stimulation against made up experimental data. To avoid the possibility of erroneous predictions, we performed a cross-validation analysis. Finally, we validate that the introduced approach generates predictive topologies, comparable to the ILP formulation. Overall, an efficient approach based on graph theory is presented herein to interrogate protein-protein interaction networks and to provide meaningful biological insights.

Activated HGF-c-Met Axis in Head and Neck Cancer

PubMed Central

Arnold, Levi; Enders, Jonathan; Thomas, Sufi Mary

2017-01-01

Head and neck squamous cell carcinoma (HNSCC) is a highly morbid disease. Recent developments including Food and Drug Administration (FDA) approved molecular targeted agent’s pembrolizumab and cetuximab show promise but did not improve the five-year survival which is currently less than 40%. The hepatocyte growth factor receptor; also known as mesenchymal–epithelial transition factor (c-Met) and its ligand hepatocyte growth factor (HGF) are overexpressed in head and neck squamous cell carcinoma (HNSCC); and regulates tumor progression and response to therapy. The c-Met pathway has been shown to regulate many cellular processes such as cell proliferation, invasion, and angiogenesis. The c-Met pathway is involved in cross-talk, activation, and perpetuation of other signaling pathways, curbing the cogency of a blockade molecule on a single pathway. The receptor and its ligand act on several downstream effectors including phospholipase C gamma (PLCγ), cellular Src kinase (c-Src), phosphotidylinsitol-3-OH kinase (PI3K) alpha serine/threonine-protein kinase (Akt), mitogen activate protein kinase (MAPK), and wingless-related integration site (Wnt) pathways. They are also known to cross-talk with other receptors; namely epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) and specifically contribute to treatment resistance. Clinical trials targeting the c-Met axis in HNSCC have been undertaken because of significant preclinical work demonstrating a relationship between HGF/c-Met signaling and cancer cell survival. Here we focus on HGF/c-Met impact on cellular signaling in HNSCC to potentiate tumor growth and disrupt therapeutic efficacy. Herein we summarize the current understanding of HGF/c-Met signaling and its effects on HNSCC. The intertwining of c-Met signaling with other signaling pathways provides opportunities for more robust and specific therapies, leading to better clinical outcomes. PMID:29231907

Integrated analysis of breast cancer cell lines reveals unique signaling pathways

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

Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised ofmore » 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.« less

Integrated analysis of breast cancer cell lines reveals unique signaling pathways.

PubMed

Heiser, Laura M; Wang, Nicholas J; Talcott, Carolyn L; Laderoute, Keith R; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L; Laquerre, Sylvie; Jackson, Jeffrey R; Wooster, Richard F; Kuo, Wen Lin; Gray, Joe W; Spellman, Paul T

2009-01-01

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EgfR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EgfR-MAPK signaling. This model was composed of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype-specific subnetworks, including one that suggested Pak1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that Pak1 over-expressing cell lines would have increased sensitivity to Mek inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three Mek inhibitors. We found that Pak1 over-expressing luminal breast cancer cell lines are significantly more sensitive to Mek inhibition compared to those that express Pak1 at low levels. This indicates that Pak1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to Mek inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

Branches of NF-κb signaling pathway regulate hepatocyte proliferation in rat liver regeneration.

PubMed

Chang, C F; Zhao, W M; Mei, J X; Zhou, Y; Pan, C Y; Xu, T T; Xu, C S

2015-07-13

Previous studies have demonstrated that the nuclear factor κB (NF-κB) pathway is involved in promoting cell proliferation. To further explore the regulatory branches and their sequence in the NF-κB pathway in the promotion of hepatocyte proliferation at the transcriptional level during rat liver regeneration, Rat Genome 230 2.0 array was used to detect the expression changes of the isolated hepatocytes. We found that many genes involved in the NF-κB pathway (including 73 known genes and 19 homologous genes) and cell proliferation (including 484 genes and 104 homologous genes) were associated with liver regeneration. Expression profile function (Ep) was used to analyze the biological processes. It was revealed that the NF-κB pathway promoted hepatocyte proliferation through three branches. Several methods of integrated statistics were applied to extract and screen key genes in liver regeneration, and it indicated that eight genes may play a vital role in rat liver regeneration. To confirm the above predicted results, Ccnd1, Jun and Myc were analyzed using qRT-PCR, and the results were generally consistent with that of microarray data. It is concluded that 3 branches and 8 key genes involved in the NF-κB pathway regulate hepatocyte proliferation during rat liver regeneration.

THESEUS 1, FERONIA and relatives: a family of cell wall-sensing receptor kinases?

PubMed

Cheung, Alice Y; Wu, Hen-Ming

2011-12-01

The plant cell wall provides form and integrity to the cell as well as a dynamic interface between a cell and its environment. Therefore mechanisms capable of policing changes in the cell wall, signaling cellular responses including those that would feedback regulate cell wall properties are expected to play important roles in facilitating growth and ensuring survival. Discoveries in the last few years that the Arabidopsis THESEUS 1 receptor-like kinase (RLK) may function as a sensor for cell wall defects to regulate growth and that its relatives FERONIA and ANXURs regulate pollen tube integrity imply strongly that they play key roles in cell wall-related processes. Furthermore, FERONIA acts as a cell surface regulator for RAC/ROP GTPases and activates production of reactive oxygen species which are, respectively, important molecular switches and mediators for diverse processes. These findings position the THESEUS 1/FERONIA family RLKs as surface regulators and potential cell wall sensors capable of broadly and profoundly impacting cellular pathways in response to diverse signals. Copyright © 2011 Elsevier Ltd. All rights reserved.

High-Risk Alphapapillomavirus Oncogenes Impair the Homologous Recombination Pathway

PubMed Central

Khanal, Sujita; Robinson, Kristin L.; Wendel, Sebastian O.; Messer, Joshua J.; Galloway, Denise A.

2017-01-01

ABSTRACT Persistent high-risk genus human Alphapapillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA. IMPORTANCE This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV+ malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea since HPV E6 stimulates the integration of episomes. PMID:28768872

Analysis of expression profiles of selected genes associated with the regenerative property and the receptivity to gene transfer during somatic embryogenesis in Triticum aestivum L.

PubMed

Delporte, Fabienne; Muhovski, Yordan; Pretova, Anna; Watillon, Bernard

2013-10-01

The physiological, biochemical and molecular mechanisms regulating the initiation of a regenerative pathway remain partially unknown. Efforts to identify the biological features that confer transformation ability, or the tendency of some cells to induce transgene silencing, would help to improve plant genetic engineering. The objective of our study was to monitor the evolution of plant cell competencies in relation to both in vitro tissue culture regeneration and the genetic transformation properties. We used a simple wheat regeneration procedure as an experimental model for studying the regenerative capacity of plant cells and their receptivity to direct gene transfer over the successive steps of the regenerative pathway. Target gene profiling studies and biochemical assays were conducted to follow some of the mechanisms triggered during the somatic-to-embryogenic transition (i.e. dedifferentiation, cell division activation, redifferentiation) and affecting the accessibility of plant cells to receive and stably express the exogenous DNA introduced by bombardment. Our results seem to indicate that the control of cell-cycle (S-phase) and host defense strategies can be crucial determinants of genetic transformation efficiency. The results from studies conducted at macro-, micro- and molecular scales are then integrated into a holistic approach that addresses the question of tissue culture and transgenesis competencies more broadly. Through this multilevel analysis we try to establish functional links between both regenerative capacity and transformation receptiveness, and thereby to provide a more global and integrated vision of both processes, at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization.

Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

PubMed Central

Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

2015-01-01

SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

Xenopus egg extract: A powerful tool to study genome maintenance mechanisms.

PubMed

Hoogenboom, Wouter S; Klein Douwel, Daisy; Knipscheer, Puck

2017-08-15

DNA repair pathways are crucial to maintain the integrity of our genome and prevent genetic diseases such as cancer. There are many different types of DNA damage and specific DNA repair mechanisms have evolved to deal with these lesions. In addition to these repair pathways there is an extensive signaling network that regulates processes important for repair, such as cell cycle control and transcription. Despite extensive research, DNA damage repair and signaling are not fully understood. In vitro systems such as the Xenopus egg extract system, have played, and still play, an important role in deciphering the molecular details of these processes. Xenopus laevis egg extracts contain all factors required to efficiently perform DNA repair outside a cell, using mechanisms conserved in humans. These extracts have been used to study several genome maintenance pathways, including mismatch repair, non-homologous end joining, ICL repair, DNA damage checkpoint activation, and replication fork stability. Here we describe how the Xenopus egg extract system, in combination with specifically designed DNA templates, contributed to our detailed understanding of these pathways. Copyright © 2017. Published by Elsevier Inc.

BLM and the FANC proteins collaborate in a common pathway in response to stalled replication forks

PubMed Central

Pichierri, Pietro; Franchitto, Annapaola; Rosselli, Filippo

2004-01-01

Fanconi anaemia (FA) and Bloom syndrome (BS) are autosomal recessive diseases characterised by chromosome fragility and cancer proneness. Here, we report that BLM and the FA pathway are activated in response to both crosslinked DNA and replication fork stall. We provide evidence that BLM and FANCD2 colocalise and co-immunoprecipitate following treatment with either DNA crosslinkers or agents inducing replication arrest. We also find that the FA core complex is necessary for BLM phosphorylation and assembly in nuclear foci in response to crosslinked DNA. Moreover, we show that knock-down of the MRE11 complex, whose function is also under the control of the FA core complex, enhances cellular and chromosomal sensitivity to DNA interstrand crosslinks in BS cells. These findings suggest the existence of a functional link between BLM and the FA pathway and that BLM and the MRE11 complex are in two separated branches of a pathway resulting in S-phase checkpoint activation, chromosome integrity and cell survival in response to crosslinked DNA. PMID:15257300

PI3K pathway inhibitors: potential prospects as adjuncts to vaccine immunotherapy for glioblastoma.

PubMed

Oh, Taemin; Ivan, Michael E; Sun, Matthew Z; Safaee, Michael; Fakurnejad, Shayan; Clark, Aaron J; Sayegh, Eli T; Bloch, Orin; Parsa, Andrew T

2014-01-01

Constitutive activation of the PI3K pathway has been implicated in glioblastoma (GBM) pathogenesis. Pharmacologic inhibition can both inhibit tumor survival and downregulate expression of programmed death ligand-1, a protein highly expressed on glioma cells that strongly contributes to cancer immunosuppression. In that manner, PI3K pathway inhibitors can help optimize GBM vaccine immunotherapy. In this review, we describe and assess the potential integration of various classes of PI3K pathway inhibitors into GBM immunotherapy. While early-generation inhibitors have a wide range of immunosuppressive effects that could negate their antitumor potency, further work should better characterize how contemporary inhibitors affect the immune response. This will help determine if these inhibitors are truly a therapeutic avenue with a strong future in GBM immunotherapy.

Key Transcription Factors in the Differentiation of Mesenchymal Stem Cells

PubMed Central

Almalki, Sami G.; Agrawal, Devendra K.

2016-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that represent a promising source for regenerative medicine. MSCs are capable of osteogenic, chondrogenic, adipogenic and myogenic differentiation. Efficacy of differentiated MSCs to regenerate cells in the injured tissues requires the ability to maintain the differentiation toward the desired cell fate. Since MSCs represent an attractive source for autologous transplantation, cellular and molecular signaling pathways and micro-environmental changes have been studied in order to understand the role of cytokines, chemokines, and transcription factors on the differentiation of MSCs. The differentiation of MSC into a mesenchymal lineage is genetically manipulated and promoted by specific transcription factors associated with a particular cell lineage. Recent studies have explored the integration of transcription factors, including Runx2, Sox9, PPARγ, MyoD, GATA4, and GATA6 in the differentiation of MSCs. Therefore, the overexpression of a single transcription factor in MSCs may promote trans-differentiation into specific cell lineage, which can be used for treatment of some diseases. In this review, we critically discussed and evaluated the role of transcription factors and related signaling pathways that affect the differentiation of MSCs toward adipocytes, chondrocytes, osteocytes, skeletal muscle cells, cardiomyocytes, and smooth muscle cells. PMID:27012163

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

PubMed Central

Hayashi, Yujiro; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Mathison, Angela J.; Kendrick, Michael L.; Shen, K. Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Farrugia, Gianrico; Urrutia, Raul A.; Ordog, Tamas

2013-01-01

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

Tissue Equivalents Based on Cell-Seeded Biodegradable Microfluidic Constructs

PubMed Central

Borenstein, Jeffrey T.; Megley, Katie; Wall, Kimberly; Pritchard, Eleanor M.; Truong, David; Kaplan, David L.; Tao, Sarah L.; Herman, Ira M.

2010-01-01

One of the principal challenges in the field of tissue engineering and regenerative medicine is the formation of functional microvascular networks capable of sustaining tissue constructs. Complex tissues and vital organs require a means to support oxygen and nutrient transport during the development of constructs both prior to and after host integration, and current approaches have not demonstrated robust solutions to this challenge. Here, we present a technology platform encompassing the design, construction, cell seeding and functional evaluation of tissue equivalents for wound healing and other clinical applications. These tissue equivalents are comprised of biodegradable microfluidic scaffolds lined with microvascular cells and designed to replicate microenvironmental cues necessary to generate and sustain cell populations to replace dermal and/or epidermal tissues lost due to trauma or disease. Initial results demonstrate that these biodegradable microfluidic devices promote cell adherence and support basic cell functions. These systems represent a promising pathway towards highly integrated three-dimensional engineered tissue constructs for a wide range of clinical applications.

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

PubMed Central

Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

2012-01-01

Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study. PMID:22655088

Chromatin Immunoprecipitation and DNA Sequencing Identified a LIMS1/ILK Pathway Regulated by LMO1 in Neuroblastoma.

PubMed

Saeki, Norihisa; Saito, Akira; Sugaya, Yuki; Amemiya, Mitsuhiro; Ono, Hiroe; Komatsuzaki, Rie; Yanagihara, Kazuyoshi; Sasaki, Hiroki

2018-01-01

Overall survival for the high-risk group of neuroblastoma (NB) remains at 40-50%. An integrative genomics study revealed that LIM domain only 1 (LMO1) encoding a transcriptional regulator to be an NB-susceptibility gene with a tumor-promoting activity, that needs to be revealed. We conducted chromatin immunoprecipitation and DNA sequencing analyses and cell proliferation assays on two NB cell lines. We identified three genes regulated by LMO1 in the cells, LIM and senescent cell antigen-like domains 1 (LIMS1), Ras suppressor protein 1 (RSU1) and relaxin 2 (RLN2). LIMS1 and RSU1 encode proteins functioning with integrin-linked kinase (ILK), and inhibition of LIMS1, ILK or RLN2 by shRNA reduced cell proliferation of the NB cells, which was also suppressed with an ILK inhibiting compound Cpd 22. The downstream of LMO1-regulatory cascade includes a tumor-promoting LIMS1/ILK pathway, which has a potential to be a novel therapeutic target. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

PubMed

Ortega-Molina, Ana; Boss, Isaac W; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A; Gascoyne, Randy D; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M; Wendel, Hans-Guido

2015-10-01

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development

PubMed Central

Ortega-Molina, Ana; Boss, Isaac W.; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W.; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A.; Gascoyne, Randy D.; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M.; Wendel, Hans-Guido

2015-01-01

The lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center (GC) involution, impedes B cell differentiation and class switch recombination (CSR). Integrative genomic analyses indicate that KMT2D affects H3K4 methylation and expression of a specific set of genes including those in the CD40, JAK-STAT, Toll-like receptor, and B cell receptor pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3, and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell activating pathways. PMID:26366710

The Molecular Ecophysiology of Programmed Cell Death in Marine Phytoplankton

NASA Astrophysics Data System (ADS)

Bidle, Kay D.

2015-01-01

Planktonic, prokaryotic, and eukaryotic photoautotrophs (phytoplankton) share a diverse and ancient evolutionary history, during which time they have played key roles in regulating marine food webs, biogeochemical cycles, and Earth's climate. Because phytoplankton represent the basis of marine ecosystems, the manner in which they die critically determines the flow and fate of photosynthetically fixed organic matter (and associated elements), ultimately constraining upper-ocean biogeochemistry. Programmed cell death (PCD) and associated pathway genes, which are triggered by a variety of nutrient stressors and are employed by parasitic viruses, play an integral role in determining the cell fate of diverse photoautotrophs in the modern ocean. Indeed, these multifaceted death pathways continue to shape the success and evolutionary trajectory of diverse phytoplankton lineages at sea. Research over the past two decades has employed physiological, biochemical, and genetic techniques to provide a novel, comprehensive, mechanistic understanding of the factors controlling this key process. Here, I discuss the current understanding of the genetics, activation, and regulation of PCD pathways in marine model systems; how PCD evolved in unicellular photoautotrophs; how it mechanistically interfaces with viral infection pathways; how stress signals are sensed and transduced into cellular responses; and how novel molecular and biochemical tools are revealing the impact of PCD genes on the fate of natural phytoplankton assemblages.

Regulation of necrotic cell death p53, PARP1 and Cyclophilin D -overlapping pathways of regulated necrosis?

PubMed Central

Ying, Yuan; Padanilam, Babu J.

2017-01-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator (ANT) and the phosphate carrier (PiC) are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury. PMID:27048819

Regulation of necrotic cell death: p53, PARP1 and cyclophilin D-overlapping pathways of regulated necrosis?

PubMed

Ying, Yuan; Padanilam, Babu J

2016-06-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator and the phosphate carrier are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury.

An Integrated Phosphoproteomics Work Flow Reveals Extensive Network Regulation in Early Lysophosphatidic Acid Signaling*

PubMed Central

Schreiber, Thiemo B.; Mäusbacher, Nina; Kéri, György; Cox, Jürgen; Daub, Henrik

2010-01-01

Lysophosphatidic acid (LPA) induces a variety of cellular signaling pathways through the activation of its cognate G protein-coupled receptors. To investigate early LPA responses and assess the contribution of epidermal growth factor (EGF) receptor transactivation in LPA signaling, we performed phosphoproteomics analyses of both total cell lysate and protein kinase-enriched fractions as complementary strategies to monitor phosphorylation changes in A498 kidney carcinoma cells. Our integrated work flow enabled the identification and quantification of more than 5,300 phosphorylation sites of which 224 were consistently regulated by LPA. In addition to induced phosphorylation events, we also obtained evidence for early dephosphorylation reactions due to rapid phosphatase regulation upon LPA treatment. Phosphorylation changes induced by direct heparin-binding EGF-like growth factor-mediated EGF receptor activation were typically weaker and only detected on a subset of LPA-regulated sites, indicating signal integration among EGF receptor transactivation and other LPA-triggered pathways. Our results reveal rapid phosphoregulation of many proteins not yet implicated in G protein-coupled receptor signaling and point to various additional mechanisms by which LPA might regulate cell survival and migration as well as gene transcription on the molecular level. Moreover, our phosphoproteomics analysis of both total lysate and kinase-enriched fractions provided highly complementary parts of the LPA-regulated signaling network and thus represents a useful and generic strategy toward comprehensive signaling studies on a system-wide level. PMID:20071362

Integrating non-coding RNAs in JAK-STAT regulatory networks

PubMed Central

Witte, Steven; Muljo, Stefan A

2014-01-01

Being a well-characterized pathway, JAK-STAT signaling serves as a valuable paradigm for studying the architecture of gene regulatory networks. The discovery of untranslated or non-coding RNAs, namely microRNAs and long non-coding RNAs, provides an opportunity to elucidate their roles in such networks. In principle, these regulatory RNAs can act as downstream effectors of the JAK-STAT pathway and/or affect signaling by regulating the expression of JAK-STAT components. Examples of interactions between signaling pathways and non-coding RNAs have already emerged in basic cell biology and human diseases such as cancer, and can potentially guide the identification of novel biomarkers or drug targets for medicine. PMID:24778925

The Future of Molecular Analysis in Melanoma: Diagnostics to Direct Molecularly Targeted Therapy.

PubMed

Akabane, Hugo; Sullivan, Ryan J

2016-02-01

Melanoma is a malignancy of pigment-producing cells that is driven by a variety of genetic mutations and aberrations. In most cases, this leads to upregulation of the mitogen-activated protein kinase (MAPK) pathway through activating mutations of upstream mediators of the pathway including BRAF and NRAS. With the advent of effective MAPK pathway inhibitors, including the US FDA-approved BRAF inhibitors vemurafenib and dabrafenib and MEK inhibitor trametinib, molecular analysis has become an integral part of the care of patients with metastatic melanoma. In this article, the key molecular targets and strategies to inhibit these targets therapeutically are presented, and the techniques of identifying these targets, in both tissue and blood, are discussed.

The Long Terminal Repeat Retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe.

PubMed

Esnault, Caroline; Levin, Henry L

2015-08-01

The long terminal repeat (LTR) retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe are active mobile elements of the Ty3/gypsy family. The mobilization of these retrotransposons depends on particle formation, reverse transcription and integration, processes typical of other LTR retrotransposons. However, Tf1 and Tf2 are distinct from other LTR elements in that they assemble virus-like particles from a single primary translation product, initiate reverse transcription with an unusual self-priming mechanism, and, in the case of Tf1, integrate with a pattern that favors specific promoters of RNA pol II-transcribed genes. To avoid the chromosome instability and genome damage that results from increased copy number, S. pombe applies a variety of defense mechanisms that restrict Tf1 and Tf2 activity. The mRNA of the Tf elements is eliminated by an exosome-based pathway when cells are in favorable conditions whereas nutrient deprivation triggers an RNA interference-dependent pathway that results in the heterochromatization of the elements. Interestingly, Tf1 integrates into the promoters of stress-induced genes and these insertions are capable of increasing the expression of adjacent genes. These properties of Tf1 transposition raise the possibility that Tf1 benefits cells with specific insertions by providing resistance to environmental stress.

Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

PubMed Central

Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J.; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F.; Emili, Andrew

2011-01-01

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. PMID:22125496

Mechanisms and pathways of Toxoplasma gondii transepithelial migration

PubMed Central

Jones, Emily J.; Carding, Simon R.

2017-01-01

ABSTRACT Toxoplasma gondii is a ubiquitous parasite and a prevalent food-borne parasitic pathogen. Infection of the host occurs principally through oral consumption of contaminated food and water with the gastrointestinal tract being the primary route for entry into the host. To promote infection, T. gondii has evolved highly specialized strategies for rapid traversal of the single cell thick intestinal epithelial barrier. Parasite transmigration via the paracellular pathway between adjacent cells enables parasite dissemination to secondary sites of infection where chronic infection of muscle and brain tissue is established. It has recently been proposed that parasite interactions with the integral tight junction (TJ) protein occludin influences parasite transmigration of the intestinal epithelium. We review here the emerging mechanisms of T. gondii transmigration of the small intestinal epithelium alongside the developing role played in modulating the wider TJ-associated proteome to rewire host cell regulatory systems for the benefit of the parasite. PMID:28452683

Mechanisms and pathways of Toxoplasma gondii transepithelial migration.

PubMed

Jones, Emily J; Korcsmaros, Tamas; Carding, Simon R

2017-01-02

Toxoplasma gondii is a ubiquitous parasite and a prevalent food-borne parasitic pathogen. Infection of the host occurs principally through oral consumption of contaminated food and water with the gastrointestinal tract being the primary route for entry into the host. To promote infection, T. gondii has evolved highly specialized strategies for rapid traversal of the single cell thick intestinal epithelial barrier. Parasite transmigration via the paracellular pathway between adjacent cells enables parasite dissemination to secondary sites of infection where chronic infection of muscle and brain tissue is established. It has recently been proposed that parasite interactions with the integral tight junction (TJ) protein occludin influences parasite transmigration of the intestinal epithelium. We review here the emerging mechanisms of T. gondii transmigration of the small intestinal epithelium alongside the developing role played in modulating the wider TJ-associated proteome to rewire host cell regulatory systems for the benefit of the parasite.

Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

PubMed

Babu, Mohan; Díaz-Mejía, J Javier; Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F; Emili, Andrew

2011-11-01

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.

Engineering plant metabolism into microbes: from systems biology to synthetic biology.

PubMed

Xu, Peng; Bhan, Namita; Koffas, Mattheos A G

2013-04-01

Plant metabolism represents an enormous repository of compounds that are of pharmaceutical and biotechnological importance. Engineering plant metabolism into microbes will provide sustainable solutions to produce pharmaceutical and fuel molecules that could one day replace substantial portions of the current fossil-fuel based economy. Metabolic engineering entails targeted manipulation of biosynthetic pathways to maximize yields of desired products. Recent advances in Systems Biology and the emergence of Synthetic Biology have accelerated our ability to design, construct and optimize cell factories for metabolic engineering applications. Progress in predicting and modeling genome-scale metabolic networks, versatile gene assembly platforms and delicate synthetic pathway optimization strategies has provided us exciting opportunities to exploit the full potential of cell metabolism. In this review, we will discuss how systems and synthetic biology tools can be integrated to create tailor-made cell factories for efficient production of natural products and fuel molecules in microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway.

PubMed

Park, Ji-Hyun; Seo, Young Ho; Jang, Jung-Hee; Jeong, Chul-Ho; Lee, Sooyeun; Park, Byoungduck

2017-12-11

Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway. We used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways. METH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on mitochondrial membrane integrity was also confirmed by flow cytometry and immunofluorescence staining. Based on the literatures and our findings, AA is a promising candidate for an anti-neurotoxic agent, and it can potentially be used for the prevention and treatment of various neurological disorders.

Correction: Kousholt, A.N. et al. Pathways for Genome Integrity in G2 Phase of the Cell Cycle. Biomolecules 2012, 2, 579-607.

PubMed

Kousholt, Arne Nedergaard; Menzel, Tobias; Sørensen, Claus Storgaard

2013-01-15

We have discovered an error in our paper published in Biomolecules [1], in Figure 1 on page 589. The protein names ATR and ATRIP have been swapped. A corrected version of the Figure 1 is provided below. [...].

The role of the Hippo pathway in human disease and tumorigenesis

PubMed Central

2014-01-01

Understanding the molecular nature of human cancer is essential to the development of effective and personalized therapies. Several different molecular signal transduction pathways drive tumorigenesis when deregulated and respond to different types of therapeutic interventions. The Hippo signaling pathway has been demonstrated to play a central role in the regulation of tissue and organ size during development. The deregulation of Hippo signaling leads to a concurrent combination of uncontrolled cellular proliferation and inhibition of apoptosis, two key hallmarks in cancer development. The molecular nature of this pathway was first uncovered in Drosophila melanogaster through genetic screens to identify regulators of cell growth and cell division. The pathway is strongly conserved in humans, rendering Drosophila a suitable and efficient model system to better understand the molecular nature of this pathway. In the present study, we review the current understanding of the molecular mechanism and clinical impact of the Hippo pathway. Current studies have demonstrated that a variety of deregulated molecules can alter Hippo signaling, leading to the constitutive activation of the transcriptional activator YAP or its paralog TAZ. Additionally, the Hippo pathway integrates inputs from a number of growth signaling pathways, positioning the Hippo pathway in a central role in the regulation of tissue size. Importantly, deregulated Hippo signaling is frequently observed in human cancers. YAP is commonly activated in a number of in vitro and in vivo models of tumorigenesis, as well as a number of human cancers. The common activation of YAP in many different tumor types provides an attractive target for potential therapeutic intervention. PMID:25097728

JAK/STAT autocontrol of ligand-producing cell number through apoptosis.

PubMed

Borensztejn, Antoine; Boissoneau, Elisabeth; Fernandez, Guillaume; Agnès, François; Pret, Anne-Marie

2013-01-01

During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. Here we show that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. Our results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila.

The DUF59 Family Gene AE7 Acts in the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Maintain Nuclear Genome Integrity in Arabidopsis[C][W][OA

PubMed Central

Luo, Dexian; Bernard, Delphine G.; Balk, Janneke; Hai, Huang; Cui, Xiaofeng

2012-01-01

Eukaryotic organisms have evolved a set of strategies to safeguard genome integrity, but the underlying mechanisms remain poorly understood. Here, we report that ASYMMETRIC LEAVES1/2 ENHANCER7 (AE7), an Arabidopsis thaliana gene encoding a protein in the evolutionarily conserved Domain of Unknown Function 59 family, participates in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway to maintain genome integrity. The severe ae7-2 allele is embryo lethal, whereas plants with the weak ae7 (ae7-1) allele are viable but exhibit highly accumulated DNA damage that activates the DNA damage response to arrest the cell cycle. AE7 is part of a protein complex with CIA1, NAR1, and MET18, which are highly conserved in eukaryotes and are involved in the biogenesis of cytosolic and nuclear Fe-S proteins. ae7-1 plants have lower activities of the cytosolic [4Fe-4S] enzyme aconitase and the nuclear [4Fe-4S] enzyme DNA glycosylase ROS1. Additionally, mutations in the gene encoding the mitochondrial ATP binding cassette transporter ATM3/ABCB25, which is required for the activity of cytosolic Fe-S enzymes in Arabidopsis, also result in defective genome integrity similar to that of ae7-1. These results indicate that AE7 is a central member of the CIA pathway, linking plant mitochondria to nuclear genome integrity through assembly of Fe-S proteins. PMID:23104832

Transcriptomic meta-analysis identifies gene expression characteristics in various samples of HIV-infected patients with nonprogressive disease.

PubMed

Zhang, Le-Le; Zhang, Zi-Ning; Wu, Xian; Jiang, Yong-Jun; Fu, Ya-Jing; Shang, Hong

2017-09-12

A small proportion of HIV-infected patients remain clinically and/or immunologically stable for years, including elite controllers (ECs) who have undetectable viremia (<50 copies/ml) and long-term nonprogressors (LTNPs) who maintain normal CD4 + T cell counts for prolonged periods (>10 years). However, the mechanism of nonprogression needs to be further resolved. In this study, a transcriptome meta-analysis was performed on nonprogressor and progressor microarray data to identify differential transcriptome pathways and potential biomarkers. Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed genes (DEGs) in nonprogressors and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DEGs identified in the meta-analysis. Five microarray datasets (81 cases and 98 controls in total), including whole blood, CD4 + and CD8 + T cells, were collected for meta-analysis. We determined that nonprogressors have reduced expression of important interferon-stimulated genes (ISGs), CD38, lymphocyte activation gene 3 (LAG-3) in whole blood, CD4 + and CD8 + T cells. Gene ontology (GO) analysis showed a significant enrichment in DEGs that function in the type I interferon signaling pathway. Upregulated pathways, including the PI3K-Akt signaling pathway in whole blood, cytokine-cytokine receptor interaction in CD4 + T cells and the MAPK signaling pathway in CD8 + T cells, were identified in nonprogressors compared with progressors. In each metabolic functional category, the number of downregulated DEGs was more than the upregulated DEGs, and almost all genes were downregulated DEGs in the oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle in the three types of samples. Our transcriptomic meta-analysis provides a comprehensive evaluation of the gene expression profiles in major blood types of nonprogressors, providing new insights in the understanding of HIV pathogenesis and developing strategies to delay HIV disease progression.

PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.

PubMed

Montal, Emily D; Dewi, Ruby; Bhalla, Kavita; Ou, Lihui; Hwang, Bor Jang; Ropell, Ashley E; Gordon, Chris; Liu, Wan-Ju; DeBerardinis, Ralph J; Sudderth, Jessica; Twaddel, William; Boros, Laszlo G; Shroyer, Kenneth R; Duraisamy, Sekhar; Drapkin, Ronny; Powers, R Scott; Rohde, Jason M; Boxer, Matthew B; Wong, Kwok-Kin; Girnun, Geoffrey D

2015-11-19

Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

PubMed Central

Deng, Wen; Zhou, Yuan; Tiwari, Agnes FY; Su, Hang; Yang, Jie; Zhu, Dandan; Lau, Victoria Ming Yi; Hau, Pok Man; Yip, Yim Ling; Cheung, Annie LM; Guan, Xin-Yuan; Tsao, Sai Wah

2015-01-01

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21. PMID:25131797

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

In search of the Golden Fleece: Unraveling principles of morphogenesis by studying the integrative biology of skin appendages

PubMed Central

Hughes, Michael W.; Wu, Ping; Jiang, Ting-Xin; Lin, Sung-Jan; Dong, Chen-Yuan; Li, Ang; Hsieh, Fon-Jou; Widelitz, Randall B.; Choung, Cheng Ming

2013-01-01

Summary The mythological story of the Golden Fleece symbolizes the magical regenerative power of skin appendages. Similar to the adventurous pursuit of the Golden Fleece by the multi-talented Argonauts, today we also need an integrated multi-disciplined approach to understand the cellular and molecular processes during development, regeneration and evolution of skin appendages. To this end, we have explored several aspects of skin appendage biology that contribute to the Turing activator / inhibitor model in feather pattern formation, the topo-biological arrangement of stem cells in organ shape determination, the macro-environmental regulation of stem cells in regenerative hair waves, and potential novel molecular pathways in the morphological evolution of feathers. Here we show our current integrative biology efforts to unravel the complex cellular behavior in patterning stem cells and the control of regional specificity in skin appendages. We use feather / scale tissue recombination to demonstrate the timing control of competence and inducibility. Feathers from different body regions are used to study skin regional specificity. Bioinformatic analyses of transcriptome microarrays show the potential involvement of candidate molecular pathways. We further show Hox genes exhibit some region specific expression patterns. To visualize real time events, we applied time-lapse movies, confocal microscopy and multiphoton microscopy to analyze the morphogenesis of cultured embryonic chicken skin explants. These modern imaging technologies reveal unexpectedly complex cellular flow and organization of extracellular matrix molecules in three dimensions. While these approaches are in preliminary stages, this perspective highlights the challenges we face and new integrative tools we will use. Future work will follow these leads to develop a systems biology view and understanding in the morphogenetic principles that govern the development and regeneration of ectodermal organs. PMID:21437328

Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry, free-energy conservation and redox-cofactor balancing.

PubMed

van Rossum, Harmen M; Kozak, Barbara U; Pronk, Jack T; van Maris, Antonius J A

2016-07-01

Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and novel therapeutic target in acute myeloid leukemia

PubMed Central

Porter, Christopher C.; Kim, Jihye; Fosmire, Susan; Gearheart, Christy M.; van Linden, Annemie; Baturin, Dmitry; Zaberezhnyy, Vadym; Patel, Purvi R.; Gao, Dexiang; Tan, Aik Choon; DeGregori, James

2011-01-01

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide shRNA screens to identify proteins that mediate AML cell fate after cytarabine exposure, gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context, and examination of existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. PMID:22289989

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

PubMed

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

PubMed Central

Acharya, Bipul R.; Peyret, Grégoire; Fardin, Marc-Antoine; Mège, René-Marc; Ladoux, Benoit; Yap, Alpha S.; Fanning, Alan S.

2016-01-01

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions. PMID:27114502

A model-based study delineating the roles of the two signaling branches of Saccharomyces cerevisiae, Sho1 and Sln1, during adaptation to osmotic stress

NASA Astrophysics Data System (ADS)

Parmar, J. H.; Bhartiya, Sharad; Venkatesh, K. V.

2009-09-01

Adaptation to osmotic shock in Saccharomyces cerevisiae is brought about by the activation of two independent signaling pathways, Sho1 and Sln1, which in turn trigger the high osmolarity glycerol (HOG) pathway. The HOG pathway thereby activates the transcription of Gpd1p, an enzyme necessary to synthesize glycerol. The production of glycerol brings about a change in the intracellular osmolarity leading to adaptation. We present a detailed mechanistic model for the response of the yeast to hyperosmotic shock. The model integrates the two branches, Sho1 and Sln1, of the HOG pathway and also includes the mitogen-activated protein kinase cascade, gene regulation and metabolism. Model simulations are consistent with known experimental results for wild-type strain, and Ste11Δ and Ssk1Δ mutant strains subjected to osmotic stress. Simulation results predict that both the branches contribute to the overall wild-type response for moderate osmotic shock, while under severe osmotic shock, the cell responds mainly through the Sln1 branch. The analysis shows that the Sln1 branch helps the cell in preventing cross-talk to other signaling pathways by inhibiting ste11ste50 activation and also by increasing the phosphorylation of Ste50. We show that the negative feedbacks to the Sho1 branch must be faster than those to the Sln1 branch to simultaneously achieve pathway specificity and adaptation during hyperosmotic shock. Sensitivity analysis revealed that the presence of both branches imparts robust behavior to the cell under osmoadaptation to perturbations.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

PubMed

Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

NF-κB signaling mechanisms in HTLV-1-induced adult T-cell leukemia/lymphoma.

PubMed

Harhaj, Edward William; Giam, Chou-Zen

2018-05-03

The human T-cell leukemia virus type 1 (HTLV-1) is a complex deltaretrovirus linked to adult T-cell leukemia/lymphoma (ATLL), a fatal CD4+ malignancy in 3-5% of infected individuals. The HTLV-1 Tax regulatory protein plays indispensable roles in regulating viral gene expression and activating cellular signaling pathways that drive the proliferation and clonal expansion of T cells bearing HTLV-1 proviral integrations. Tax is a potent activator of NF-κB, a key signaling pathway that is essential for the survival and proliferation of HTLV-1 infected T cells. However, constitutive NF-κB activation by Tax also triggers a senescence response, suggesting the possibility that only T cells capable of overcoming NF-κB-induced senescence can selectively undergo clonal expansion after HTLV-1 infection. Tax expression is often silenced in the majority of ATLL due to genetic alterations in the tax gene or DNA hypermethylation of the 5'-LTR. Despite the loss of Tax, NF-κB activation remains persistently activated in ATLL due to somatic mutations in genes in the T/B-cell receptor (T/BCR) and NF-κB signaling pathways. In this review, we focus on the key events driving Tax-dependent and independent mechanisms of NF-κB activation during the multi-step process leading to ATLL. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Neuronal Calcium Signaling in Metabolic Regulation and Adaptation to Nutrient Stress.

PubMed

Jayakumar, Siddharth; Hasan, Gaiti

2018-01-01

All organisms can respond physiologically and behaviorally to environmental fluxes in nutrient levels. Different nutrient sensing pathways exist for specific metabolites, and their inputs ultimately define appropriate nutrient uptake and metabolic homeostasis. Nutrient sensing mechanisms at the cellular level require pathways such as insulin and target of rapamycin (TOR) signaling that integrates information from different organ systems like the fat body and the gut. Such integration is essential for coordinating growth with development. Here we review the role of a newly identified set of integrative interneurons and the role of intracellular calcium signaling within these neurons, in regulating nutrient sensing under conditions of nutrient stress. A comparison of the identified Drosophila circuit and cellular mechanisms employed in this circuit, with vertebrate systems, suggests that the identified cell signaling mechanisms may be conserved for neural circuit function related to nutrient sensing by central neurons. The ideas proposed are potentially relevant for understanding the molecular basis of metabolic disorders, because these are frequently linked to nutritional stress.

Towards integrating extracellular matrix and immunological pathways.

PubMed

Boyd, David F; Thomas, Paul G

2017-10-01

The extracellular matrix (ECM) is a complex and dynamic structure made up of an estimated 300 different proteins. The ECM is also a rich source of cytokines and growth factors in addition to numerous bioactive ECM degradation products that influence cell migration, proliferation, and differentiation. The ECM is constantly being remodeled during homeostasis and in a wide range of pathological contexts. Changes in the ECM modulate immune responses, which in turn regulate repair and regeneration of tissues. Here, we review the many components of the ECM, enzymes involved in ECM remodeling, and the signals that feed into immunological pathways in the context of a dynamic ECM. We highlight studies that have taken an integrative approach to studying immune responses in the context of the ECM and studies that use novel proteomic strategies. Finally, we discuss research challenges relevant to the integration of immune and ECM networks and propose experimental and translational approaches to resolve these issues. Copyright © 2017 Elsevier Ltd. All rights reserved.

Histone H3.3 maintains genome integrity during mammalian development

PubMed Central

Jang, Chuan-Wei; Shibata, Yoichiro; Starmer, Joshua; Yee, Della; Magnuson, Terry

2015-01-01

Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development. PMID:26159997

Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer

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

Zhang, Hui; Liu, Tao; Zhang, Zhen

Ovarian cancer remains the most lethal gynecological malignancy in the developed world, despite recent advances in genomic information and treatment. To better understand this disease, define an integrated proteogenomic landscape, and identify factors associated with homologous repair deficiency (HRD) and overall survival, we performed a comprehensive proteomic characterization of ovarian high-grade serous carcinomas (HGSC) previously characterized by The Cancer Genome Atlas (TCGA). We observed that messenger RNA transcript abundance did not reliably predict abundance for 10,030 proteins across 174 tumors. Clustering of tumors based on protein abundance identified five subtypes, two of which correlated robustly with mesenchymal and proliferative subtypes,more » while tumors characterized as immunoreactive or differentiated at the transcript level were intermixed at the protein level. At the genome level, HGSC is characterized by a complex landscape of somatic copy number alterations (CNA), which individually do not correlate significantly with survival. Correlation of CNAs with protein abundances identified loci with significant trans regulatory effects mapping to pathways associated with proliferation, cell motility/invasion, and immune regulation, three known hallmarks of cancer. Using the trans regulated proteins we also created models significantly correlated with patient survival by multivariate analysis. Integrating protein abundance with specific post-translational modification data identified subnetworks correlated with HRD status; specifically, acetylation of Lys12 and Lys16 on histone H4 was associated with HRD status. Using quantitative phosphoproteomics data covering 4,420 proteins as reflective of pathway activity, we identified the PDGFR and VEGFR signaling pathways as significantly up-regulated in patients with short overall survival, independent of PDGFR and VEGFR protein levels, potentially informing the use of anti-angiogenic therapies. Components of the Rho/Rac/Cdc42 cell motility pathways were also significantly enriched for short survival. Overall, proteomics provided new insights into ovarian cancer not apparent from genomic analysis and enabling a deeper understanding of HGSC with the potential to inform targeted therapeutics.« less

A Mitogen-Activated Protein Kinase Tmk3 Participates in High Osmolarity Resistance, Cell Wall Integrity Maintenance and Cellulase Production Regulation in Trichoderma reesei

PubMed Central

Wang, Mingyu; Zhao, Qiushuang; Yang, Jinghua; Jiang, Baojie; Wang, Fangzhong; Liu, Kuimei; Fang, Xu

2013-01-01

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading. PMID:23991059

Steroid hormone receptors: long- and short-term integrators of the internal milieu and the external environment.

PubMed

Blaustein, J D

2012-07-01

Many of the influences of estrogens and progestins on the brain and behavior are mediated by estrogen receptors and progestin receptors, acting as transcriptional regulators. The homologous and heterologous regulation of the concentrations of these receptors by cognate hormones is well established. However, although they were discovered and characterized based on their binding to cognate hormone and their role in transcriptional regulation, steroid hormone receptors have a more complex role and serve many more functions than originally suspected. First, besides being regulated by steroid hormones, the intracellular concentrations of brain steroid hormone receptors are regulated by neurotransmitters, a pathway by which stimuli from the environment, including from conspecific animals, can modulate the concentration of particular steroid hormone receptors in subsets of cells. Further, besides being activated by cognate steroid hormones, the receptors can be activated by a variety of neurotransmitters and phosphorylation pathways, providing a route through which environmental stimulation can activate steroid-receptor-dependent functions in specific cells. In addition, the transcription factor, estrogen receptor-α, produced from the estrogen receptor-α gene, can be modified to be targeted to membranes, where it can signal via kinase pathways. Finally, developmental experiences, such as particular stressors during the pubertal period, can permanently remodel the brain's response to ovarian hormones, most likely by long-term changes in regulation of the receptors mediating those responses. In addition to their function in responding to cognate ligand, it is now more appropriate to think of steroid hormone receptors as integrators of a wide variety of signaling pathways. © Georg Thieme Verlag KG Stuttgart · New York.

(Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin*

PubMed Central

Peruchetti, Diogo B.; Pinheiro, Ana Acacia S.; Landgraf, Sharon S.; Wengert, Mira; Takiya, Christina M.; Guggino, William B.; Caruso-Neves, Celso

2011-01-01

In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion. PMID:22057272

Integrative Genomic Analysis Identifies Isoleucine and CodY as Regulators of Listeria monocytogenes Virulence

PubMed Central

Lobel, Lior; Sigal, Nadejda; Borovok, Ilya; Ruppin, Eytan; Herskovits, Anat A.

2012-01-01

Intracellular bacterial pathogens are metabolically adapted to grow within mammalian cells. While these adaptations are fundamental to the ability to cause disease, we know little about the relationship between the pathogen's metabolism and virulence. Here we used an integrative Metabolic Analysis Tool that combines transcriptome data with genome-scale metabolic models to define the metabolic requirements of Listeria monocytogenes during infection. Twelve metabolic pathways were identified as differentially active during L. monocytogenes growth in macrophage cells. Intracellular replication requires de novo synthesis of histidine, arginine, purine, and branch chain amino acids (BCAAs), as well as catabolism of L-rhamnose and glycerol. The importance of each metabolic pathway during infection was confirmed by generation of gene knockout mutants in the respective pathways. Next, we investigated the association of these metabolic requirements in the regulation of L. monocytogenes virulence. Here we show that limiting BCAA concentrations, primarily isoleucine, results in robust induction of the master virulence activator gene, prfA, and the PrfA-regulated genes. This response was specific and required the nutrient responsive regulator CodY, which is known to bind isoleucine. Further analysis demonstrated that CodY is involved in prfA regulation, playing a role in prfA activation under limiting conditions of BCAAs. This study evidences an additional regulatory mechanism underlying L. monocytogenes virulence, placing CodY at the crossroads of metabolism and virulence. PMID:22969433

RhoA orchestrates glycolysis for Th2 cell differentiation and allergic airway inflammation

PubMed Central

Yang, Jun-Qi; Kalim, Khalid W.; Li, Yuan; Zhang, Shuangmin; Hinge, Ashwini; Filippi, Marie-Dominique; Zheng, Yi; Guo, Fukun

2015-01-01

Background Mitochondrial metabolism is known to be important for T cell activation. However, its involvement in effector T cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T cell activation and effector cell differentiation and function remains largely unknown. Objective We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for Th2 cell differentiation and Th2-mediated allergic airway inflammation. Methods Conditional RhoA-deficient mice were generated by crossing RhoAflox/flox mice with CD2-Cre transgenic mice. Effects of RhoA on Th2 differentiation were evaluated by in vitro Th2-polarized culture conditions, and in vivo in ovalbumin (OVA)-induced allergic airway inflammation. Cytokines were measured by intracellular staining and ELISA. T cell metabolism was measured by Seahorse XF24 Analyzer and flow cytometry. Results Disruption of RhoA inhibited T cell activation and Th2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo, with no effect on Th1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to Th2 cell differentiation and allergic airway inflammation via regulating IL-4 receptor mRNA expression and Th2-specific signaling events. Finally, inhibition of Rho-associated protein kinase (ROCK), an immediate downstream effector of RhoA, blocked Th2 differentiation and allergic airway inflammation. Conclusion RhoA is a key component of the signaling cascades leading to Th2-differentiation and allergic airway inflammation, at least in part, through the control of T cell metabolism and via ROCK pathway. PMID:26100081

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

PubMed Central

Logemann, Elke; Tavernaro, Annette; Schulz, Wolfgang; Somssich, Imre E.; Hahlbrock, Klaus

2000-01-01

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells. PMID:10677554

Cell-cycle control in the face of damage--a matter of life or death.

PubMed

Clarke, Paul R; Allan, Lindsey A

2009-03-01

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

PubMed Central

Shackelford, R E; Kaufmann, W K; Paules, R S

1999-01-01

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling. Images Figure 3 PMID:10229703

E-cadherin-mediated force transduction signals regulate global cell mechanics

PubMed Central

Muhamed, Ismaeel; Wu, Jun; Sehgal, Poonam; Kong, Xinyu; Tajik, Arash; Wang, Ning

2016-01-01

ABSTRACT This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors. PMID:26966187

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia

PubMed Central

Wang, Lili; Fan, Jean; Francis, Joshua M.; Georghiou, George; Hergert, Sarah; Li, Shuqiang; Gambe, Rutendo; Zhou, Chensheng W.; Yang, Chunxiao; Xiao, Sheng; Cin, Paola Dal; Bowden, Michaela; Kotliar, Dylan; Shukla, Sachet A.; Brown, Jennifer R.; Neuberg, Donna; Alessi, Dario R.; Zhang, Cheng-Zhong; Kharchenko, Peter V.; Livak, Kenneth J.; Wu, Catherine J.

2017-01-01

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype–phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy. PMID:28679620

Reconstructing targetable pathways in lung cancer by integrating diverse omics data

PubMed Central

Balbin, O. Alejandro; Prensner, John R.; Sahu, Anirban; Yocum, Anastasia; Shankar, Sunita; Malik, Rohit; Fermin, Damian; Dhanasekaran, Saravana M.; Chandler, Benjamin; Thomas, Dafydd; Beer, David G.; Cao, Xuhong; Nesvizhskii, Alexey I.; Chinnaiyan, Arul M.

2014-01-01

Global ‘multi-omics’ profiling of cancer cells harbours the potential for characterizing the signaling networks associated with specific oncogenes. Here we profile the transcriptome, proteome and phosphoproteome in a panel of non-small cell lung cancer (NSCLC) cell lines in order to reconstruct targetable networks associated with KRAS dependency. We develop a two-step bioinformatics strategy addressing the challenge of integrating these disparate data sets. We first define an ‘abundance-score’ combining transcript, protein and phospho-protein abundances to nominate differentially abundant proteins and then use the Prize Collecting Steiner Tree algorithm to identify functional sub-networks. We identify three modules centered on KRAS and MET, LCK and PAK1 and b-Catenin. We validate activation of these proteins in KRAS-dependent (KRAS-Dep) cells and perform functional studies defining LCK as a critical gene for cell proliferation in KRAS-Dep but not KRAS-independent NSCLCs. These results suggest that LCK is a potential druggable target protein in KRAS-Dep lung cancers. PMID:24135919

Computational Reconstruction of NFκB Pathway Interaction Mechanisms during Prostate Cancer

PubMed Central

Börnigen, Daniela; Tyekucheva, Svitlana; Wang, Xiaodong; Rider, Jennifer R.; Lee, Gwo-Shu; Mucci, Lorelei A.; Sweeney, Christopher; Huttenhower, Curtis

2016-01-01

Molecular research in cancer is one of the largest areas of bioinformatic investigation, but it remains a challenge to understand biomolecular mechanisms in cancer-related pathways from high-throughput genomic data. This includes the Nuclear-factor-kappa-B (NFκB) pathway, which is central to the inflammatory response and cell proliferation in prostate cancer development and progression. Despite close scrutiny and a deep understanding of many of its members’ biomolecular activities, the current list of pathway members and a systems-level understanding of their interactions remains incomplete. Here, we provide the first steps toward computational reconstruction of interaction mechanisms of the NFκB pathway in prostate cancer. We identified novel roles for ATF3, CXCL2, DUSP5, JUNB, NEDD9, SELE, TRIB1, and ZFP36 in this pathway, in addition to new mechanistic interactions between these genes and 10 known NFκB pathway members. A newly predicted interaction between NEDD9 and ZFP36 in particular was validated by co-immunoprecipitation, as was NEDD9's potential biological role in prostate cancer cell growth regulation. We combined 651 gene expression datasets with 1.4M gene product interactions to predict the inclusion of 40 additional genes in the pathway. Molecular mechanisms of interaction among pathway members were inferred using recent advances in Bayesian data integration to simultaneously provide information specific to biological contexts and individual biomolecular activities, resulting in a total of 112 interactions in the fully reconstructed NFκB pathway: 13 (11%) previously known, 29 (26%) supported by existing literature, and 70 (63%) novel. This method is generalizable to other tissue types, cancers, and organisms, and this new information about the NFκB pathway will allow us to further understand prostate cancer and to develop more effective prevention and treatment strategies. PMID:27078000

Immunosenescence-Related Transcriptomic and Immunologic Changes in Older Individuals Following Influenza Vaccination

PubMed Central

Kennedy, Richard B.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Oberg, Ann L.; Zimmermann, Michael T.; Grill, Diane E.; Poland, Gregory A.

2016-01-01

The goal of annual influenza vaccination is to reduce mortality and morbidity associated with this disease through the generation of protective immune responses. The objective of the current study was to examine markers of immunosenescence and identify immunosenescence-related differences in gene expression, gene regulation, cytokine secretion, and immunologic changes in an older study population receiving seasonal influenza A/H1N1 vaccination. Surprisingly, prior studies in this cohort revealed weak correlations between immunosenescence markers and humoral immune response to vaccination. In this report, we further examined the relationship of each immunosenescence marker (age, T cell receptor excision circle frequency, telomerase expression, percentage of CD28− CD4+ T cells, percentage of CD28− CD8+ T cells, and the CD4/CD8 T cell ratio) with additional markers of immune response (serum cytokine and chemokine expression) and measures of gene expression and/or regulation. Many of the immunosenescence markers indeed correlated with distinct sets of individual DNA methylation sites, miRNA expression levels, mRNA expression levels, serum cytokines, and leukocyte subsets. However, when the individual immunosenescence markers were grouped by pathways or functional terms, several shared biological functions were identified: antigen processing and presentation pathways, MAPK, mTOR, TCR, BCR, and calcium signaling pathways, as well as key cellular metabolic, proliferation and survival activities. Furthermore, the percent of CD4+ and/or CD8+ T cells lacking CD28 expression also correlated with miRNAs regulating clusters of genes known to be involved in viral infection. Integrated (DNA methylation, mRNA, miRNA, and protein levels) network biology analysis of immunosenescence-related pathways and genesets identified both known pathways (e.g., chemokine signaling, CTL, and NK cell activity), as well as a gene expression module not previously annotated with a known function. These results may improve our ability to predict immune responses to influenza and aid in new vaccine development, and highlight the need for additional studies to better define and characterize immunosenescence. PMID:27853459

Luminance and chromatic signals interact differently with melanopsin activation to control the pupil light response.

PubMed

Barrionuevo, Pablo A; Cao, Dingcai

2016-09-01

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin. These cells receive afferent inputs from rods and cones, which provide inputs to the postreceptoral visual pathways. It is unknown, however, how melanopsin activation is integrated with postreceptoral signals to control the pupillary light reflex. This study reports human flicker pupillary responses measured using stimuli generated with a five-primary photostimulator that selectively modulated melanopsin, rod, S-, M-, and L-cone excitations in isolation, or in combination to produce postreceptoral signals. We first analyzed the light adaptation behavior of melanopsin activation and rod and cones signals. Second, we determined how melanopsin is integrated with postreceptoral signals by testing with cone luminance, chromatic blue-yellow, and chromatic red-green stimuli that were processed by magnocellular (MC), koniocellular (KC), and parvocellular (PC) pathways, respectively. A combined rod and melanopsin response was also measured. The relative phase of the postreceptoral signals was varied with respect to the melanopsin phase. The results showed that light adaptation behavior for all conditions was weaker than typical Weber adaptation. Melanopsin activation combined linearly with luminance, S-cone, and rod inputs, suggesting the locus of integration with MC and KC signals was retinal. The melanopsin contribution to phasic pupil responses was lower than luminance contributions, but much higher than S-cone contributions. Chromatic red-green modulation interacted with melanopsin activation nonlinearly as described by a "winner-takes-all" process, suggesting the integration with PC signals might be mediated by a postretinal site.

The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

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

Hwang, Hyun Sook; Kim, Soung Soo

Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATPmore » pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 deg C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.« less

Mapping biological process relationships and disease perturbations within a pathway network.

PubMed

Stoney, Ruth; Robertson, David L; Nenadic, Goran; Schwartz, Jean-Marc

2018-01-01

Molecular interaction networks are routinely used to map the organization of cellular function. Edges represent interactions between genes, proteins, or metabolites. However, in living cells, molecular interactions are dynamic, necessitating context-dependent models. Contextual information can be integrated into molecular interaction networks through the inclusion of additional molecular data, but there are concerns about completeness and relevance of this data. We developed an approach for representing the organization of human cellular processes using pathways as the nodes in a network. Pathways represent spatial and temporal sets of context-dependent interactions, generating a high-level network when linked together, which incorporates contextual information without the need for molecular interaction data. Analysis of the pathway network revealed linked communities representing functional relationships, comparable to those found in molecular networks, including metabolism, signaling, immunity, and the cell cycle. We mapped a range of diseases onto this network and find that pathways associated with diseases tend to be functionally connected, highlighting the perturbed functions that result in disease phenotypes. We demonstrated that disease pathways cluster within the network. We then examined the distribution of cancer pathways and showed that cancer pathways tend to localize within the signaling, DNA processes and immune modules, although some cancer-associated nodes are found in other network regions. Altogether, we generated a high-confidence functional network, which avoids some of the shortcomings faced by conventional molecular models. Our representation provides an intuitive functional interpretation of cellular organization, which relies only on high-quality pathway and Gene Ontology data. The network is available at https://data.mendeley.com/datasets/3pbwkxjxg9/1.

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond.

PubMed

Bakshi, Anshika; Chaudhary, Sandeep C; Rana, Mehtab; Elmets, Craig A; Athar, Mohammad

2017-12-01

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10-100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs. © 2017 Wiley Periodicals, Inc.

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond

PubMed Central

Bakshi, Anshika; Chaudhary, Sandeep C.; Rana, Mehtab; Elmets, Craig A.; Athar, Mohammad

2018-01-01

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10–100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs. PMID:28574612

Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

PubMed

Renault, L

2016-01-01

β-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/β-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling. © 2016 Elsevier Inc. All rights reserved.

The effect of tributyltin chloride on Caenorhabditis elegans germline is mediated by a conserved DNA damage checkpoint pathway.

PubMed

Cheng, Zhe; Tian, Huimin; Chu, Hongran; Wu, Jianjian; Li, Yingying; Wang, Yanhai

2014-03-21

Tributyltin (TBT), one of the environmental pollutants, has been shown to impact the reproduction of animals. However, due to the lack of appropriate animal model, analysis of the affected molecular pathways in germ cells is lagging and has been particularly challenging. In the present study, we investigated the effects of tributyltin chloride (TBTCL) on the nematode Caenorhabditis elegans germline. We show that exposure of C. elegans to TBTCL causes significantly elevated level of sterility and embryonic lethality. TBTCL exposure results in an increased number of meiotic DNA double-strand breaks in germ cells, subsequently leading to activated DNA damage checkpoint. Exposing C. elegans to TBTCL causes dose- and time-dependent germline apoptosis. This apoptotic response was blocked in loss-of-function mutants of hus-1 (op241), mrt-2 (e2663) and p53/cep-1 (gk138), indicating that checkpoints and p53 are essential for mediating TBTCL-induced germ cell apoptosis. Moreover, TBTCL exposure can inhibit germ cell proliferation, which is also mediated by the conserved checkpoint pathway. We thereby propose that TBT exhibits its effects on the germline by inducing DNA damage and impaired maintenance of genomic integrity. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Floral pathway integrator gene expression mediates gradual transmission of environmental and endogenous cues to flowering time.

PubMed

van Dijk, Aalt D J; Molenaar, Jaap

2017-01-01

The appropriate timing of flowering is crucial for the reproductive success of plants. Hence, intricate genetic networks integrate various environmental and endogenous cues such as temperature or hormonal statues. These signals integrate into a network of floral pathway integrator genes. At a quantitative level, it is currently unclear how the impact of genetic variation in signaling pathways on flowering time is mediated by floral pathway integrator genes. Here, using datasets available from literature, we connect Arabidopsis thaliana flowering time in genetic backgrounds varying in upstream signalling components with the expression levels of floral pathway integrator genes in these genetic backgrounds. Our modelling results indicate that flowering time depends in a quite linear way on expression levels of floral pathway integrator genes. This gradual, proportional response of flowering time to upstream changes enables a gradual adaptation to changing environmental factors such as temperature and light.

IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway.

PubMed

Kim, Chanyang; Park, Seungjoon

2018-03-01

Insulin-like growth factor (IGF)-1 is a well-known anti-apoptotic pro-survival factor and phosphatidylinositol-3-kinase (PI3K)/Akt pathway is linked to cell survival induced by IGF-1. It is also reported that Akt signaling is modulated by 3-phosphoinositide-dependent kinase-1 (PDK1). In the current study, we investigated whether the anti-apoptotic effect of IGF-1 in SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP + ) is associated with the activity of PI3K/PDK1/Akt pathway. Treatment of cells with IGF-1 inhibited MPP + -induced apoptotic cell death. IGF-1-induced activation of Akt and the protective effect of IGF-1 on MPP + -induced apoptosis were abolished by chemical inhibition of PDK1 (GSK2334470) or PI3K (LY294002). The phosphorylated levels of Akt and PDK1 were significantly suppressed after MPP + exposure, while IGF-1 treatment completely restored MPP+-induced reductions in phosphorylation. IGF-1 protected cells from MPP + insult by suppressing intracellular reactive oxygen species (ROS) production and malondialdehyde levels and increasing superoxide dismutase activity. Mitochondrial ROS levels were also increased during MPP + exposure, which were attenuated by IGF-1 treatment. In addition, IGF-1-treated cells showed increased activities of succinate dehydrogenase and citrate synthase, stabilization of mitochondrial transmembrane potential, increased ratio of Bcl-2 to Bax, prevention of cytochrome c release and inhibition of caspase-3 activation with PARP cleavage. Furthermore, the protective effects of IGF-1 on oxidative stress and mitochondrial dysfunction were attenuated when cells were preincubated with GSK2334470 or LY294002. Our data suggest that IGF-1 protects SH-SY5Y cells against MPP + -associated oxidative stress by preserving mitochondrial integrity and inhibiting mitochondrial apoptotic cascades via the activation of PI3K/PDK1/Akt pathway. © 2018 The authors.

IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway

PubMed Central

Kim, Chanyang; Park, Seungjoon

2018-01-01

Insulin-like growth factor (IGF)-1 is a well-known anti-apoptotic pro-survival factor and phosphatidylinositol-3-kinase (PI3K)/Akt pathway is linked to cell survival induced by IGF-1. It is also reported that Akt signaling is modulated by 3-phosphoinositide-dependent kinase-1 (PDK1). In the current study, we investigated whether the anti-apoptotic effect of IGF-1 in SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP+) is associated with the activity of PI3K/PDK1/Akt pathway. Treatment of cells with IGF-1 inhibited MPP+-induced apoptotic cell death. IGF-1-induced activation of Akt and the protective effect of IGF-1 on MPP+-induced apoptosis were abolished by chemical inhibition of PDK1 (GSK2334470) or PI3K (LY294002). The phosphorylated levels of Akt and PDK1 were significantly suppressed after MPP+ exposure, while IGF-1 treatment completely restored MPP+-induced reductions in phosphorylation. IGF-1 protected cells from MPP+ insult by suppressing intracellular reactive oxygen species (ROS) production and malondialdehyde levels and increasing superoxide dismutase activity. Mitochondrial ROS levels were also increased during MPP+ exposure, which were attenuated by IGF-1 treatment. In addition, IGF-1-treated cells showed increased activities of succinate dehydrogenase and citrate synthase, stabilization of mitochondrial transmembrane potential, increased ratio of Bcl-2 to Bax, prevention of cytochrome c release and inhibition of caspase-3 activation with PARP cleavage. Furthermore, the protective effects of IGF-1 on oxidative stress and mitochondrial dysfunction were attenuated when cells were preincubated with GSK2334470 or LY294002. Our data suggest that IGF-1 protects SH-SY5Y cells against MPP+-associated oxidative stress by preserving mitochondrial integrity and inhibiting mitochondrial apoptotic cascades via the activation of PI3K/PDK1/Akt pathway. PMID:29459421

Nanobodies and recombinant binders in cell biology

PubMed Central

Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge

2015-01-01

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

PubMed

Li, Congfen; Takahashi, Chikara; Zhang, Liangxuan; Huseni, Mahrukh; Stankovich, Basha; Mashhedi, Haider; Lee, Joanna; French, Dorothy; Anderson, Jeff Eastham; Kim, Doris; Howell, Kathy; Brauer, Matthew J; Kowanetz, Marcin; Yan, Yibing; Humke, Eric; Ebens, Allen; Hampton, Garret; Lackner, Mark R; Hegde, Priti; Jia, Shidong

2013-03-23

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

PubMed Central

2013-01-01

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. Results The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. Conclusions We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials. PMID:23522020

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

PubMed

Lee, Eun Jeong; Yun, Un-Jung; Koo, Kyung Hee; Sung, Jee Young; Shim, Jaegal; Ye, Sang-Kyu; Hong, Kyeong-Man; Kim, Yong-Nyun

2014-01-01

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. © 2013.

Activation of YUCCA5 by the Transcription Factor TCP4 Integrates Developmental and Environmental Signals to Promote Hypocotyl Elongation in Arabidopsis.

PubMed

Challa, Krishna Reddy; Aggarwal, Pooja; Nath, Utpal

2016-09-05

Cell expansion is an essential process in plant morphogenesis and is regulated by the coordinated action of environmental stimuli and endogenous factors, such as the phytohormones auxin and brassinosteroid. Although the biosynthetic pathways that generate these hormones and their downstream signaling mechanisms have been extensively studied, the upstream transcriptional network that modulates their levels and connects their action to cell morphogenesis is less clear. Here we show that the miR319-regulated TCP (TEOSINTE BRANCHED 1, CYCLODEA, PROLIFERATING CELL FACTORS) transcription factors, notably TCP4, directly activate YUCCA5 transcription and integrate the auxin response to a brassinosteroid-dependent molecular circuit that promotes cell elongation in Arabidopsis hypocotyls. Further, TCP4 modulates the common transcriptional network downstream to auxin-BR signaling, which is also triggered by environmental cues, such as light, to promote cell expansion. Our study links TCP function with the hormone response during cell morphogenesis and shows that developmental and environmental signals converge on a common transcriptional network to promote cell elongation. {copyright, serif} 2016 American Society of Plant Biologists. All rights reserved.

Unraveling Interfaces between Energy Metabolism and Cell Cycle in Plants.

PubMed

Siqueira, João Antonio; Hardoim, Pablo; Ferreira, Paulo C G; Nunes-Nesi, Adriano; Hemerly, Adriana S

2018-06-19

Oscillation in energy levels is widely variable in dividing and differentiated cells. To synchronize cell proliferation and energy fluctuations, cell cycle-related proteins have been implicated in the regulation of mitochondrial energy-generating pathways in yeasts and animals. Plants have chloroplasts and mitochondria, coordinating the cell energy flow. Recent findings suggest an integrated regulation of these organelles and the nuclear cell cycle. Furthermore, reports indicate a set of interactions between the cell cycle and energy metabolism, coordinating the turnover of proteins in plants. Here, we discuss how cell cycle-related proteins directly interact with energy metabolism-related proteins to modulate energy homeostasis and cell cycle progression. We provide interfaces between cell cycle and energy metabolism-related proteins that could be explored to maximize plant yield. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow.

PubMed

El-Ali, Jamil; Gaudet, Suzanne; Günther, Axel; Sorger, Peter K; Jensen, Klavs F

2005-06-01

We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using alpha-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.

Integration of Nuclear- and Extranuclear-Initiated Estrogen Receptor Signaling in Breast Cancer Cells

ERIC Educational Resources Information Center

Madak Erdogan, Zeynep

2009-01-01

Estrogenic hormones exert their effects through binding to Estrogen Receptors (ERs), which work in concert with coregulators and extranuclear signaling pathways to control gene expression in normal as well as cancerous states, including breast tumors. In this thesis, we have used multiple genome-wide analysis tools to elucidate various ways that…

Reconstructing the regulatory circuit of cell fate determination in yeast mating response.

PubMed

Shao, Bin; Yuan, Haiyu; Zhang, Rongfei; Wang, Xuan; Zhang, Shuwen; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

2017-07-01

Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our integrated experimental and modeling strategies could be widely applicable to other biological systems.

Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses.

PubMed

Bacete, Laura; Mélida, Hugo; Miedes, Eva; Molina, Antonio

2018-02-01

Plants have evolved a repertoire of monitoring systems to sense plant morphogenesis and to face environmental changes and threats caused by different attackers. These systems integrate different signals into overreaching triggering pathways which coordinate developmental and defence-associated responses. The plant cell wall, a dynamic and complex structure surrounding every plant cell, has emerged recently as an essential component of plant monitoring systems, thus expanding its function as a passive defensive barrier. Plants have a dedicated mechanism for maintaining cell wall integrity (CWI) which comprises a diverse set of plasma membrane-resident sensors and pattern recognition receptors (PRRs). The PRRs perceive plant-derived ligands, such as peptides or wall glycans, known as damage-associated molecular patterns (DAMPs). These DAMPs function as 'danger' alert signals activating DAMP-triggered immunity (DTI), which shares signalling components and responses with the immune pathways triggered by non-self microbe-associated molecular patterns that mediate disease resistance. Alteration of CWI by impairment of the expression or activity of proteins involved in cell wall biosynthesis and/or remodelling, as occurs in some plant cell wall mutants, or by wall damage due to colonization by pathogens/pests, activates specific defensive and growth responses. Our current understanding of how these alterations of CWI are perceived by the wall monitoring systems is scarce and few plant sensors/PRRs and DAMPs have been characterized. The identification of these CWI sensors and PRR-DAMP pairs will help us to understand the immune functions of the wall monitoring system, and might allow the breeding of crop varieties and the design of agricultural strategies that would enhance crop disease resistance. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway.

PubMed

Serrano, Raquel; Martín, Humberto; Casamayor, Antonio; Ariño, Joaquín

2006-12-29

Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.

Knowledge-guided fuzzy logic modeling to infer cellular signaling networks from proteomic data

PubMed Central

Liu, Hui; Zhang, Fan; Mishra, Shital Kumar; Zhou, Shuigeng; Zheng, Jie

2016-01-01

Modeling of signaling pathways is crucial for understanding and predicting cellular responses to drug treatments. However, canonical signaling pathways curated from literature are seldom context-specific and thus can hardly predict cell type-specific response to external perturbations; purely data-driven methods also have drawbacks such as limited biological interpretability. Therefore, hybrid methods that can integrate prior knowledge and real data for network inference are highly desirable. In this paper, we propose a knowledge-guided fuzzy logic network model to infer signaling pathways by exploiting both prior knowledge and time-series data. In particular, the dynamic time warping algorithm is employed to measure the goodness of fit between experimental and predicted data, so that our method can model temporally-ordered experimental observations. We evaluated the proposed method on a synthetic dataset and two real phosphoproteomic datasets. The experimental results demonstrate that our model can uncover drug-induced alterations in signaling pathways in cancer cells. Compared with existing hybrid models, our method can model feedback loops so that the dynamical mechanisms of signaling networks can be uncovered from time-series data. By calibrating generic models of signaling pathways against real data, our method supports precise predictions of context-specific anticancer drug effects, which is an important step towards precision medicine. PMID:27774993

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in A. niger.

PubMed

Park, Joohae; Tefsen, Boris; Arentshorst, Mark; Lagendijk, Ellen; van den Hondel, Cees Amjj; van Die, Irma; Ram, Arthur Fj

2014-01-01

Galactofuranose (Gal f )-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Gal f -containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in Aspergillus niger is characterized by the specific induction of the agsA gene, which encodes a cell wall α-glucan synthase. In this study, we screened a collection of cell wall mutants with an induced expression of agsA for defects in Gal f biosynthesis using a with anti-Gal f antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene ( ugmA ). Here, we have identified six additional UDP-galactopyranose mutase ( ugmA ) mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Gal f -levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of ugeA resulted in an even more severe reduction of Gal f in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The ugeA gene is also required for growth on galactose despite the presence of two UgeA homologs in the A. niger genome. By using a classical mutant screen and whole genome sequencing of a new Gal f -deficient mutant, the UDP-glucose-4-epimerase gene ( ugeA ) has been identified. UgeA is required for the biosynthesis of Gal f as well as for galactose metabolism in Aspergillus niger .

Identification of Biological Targets of Therapeutic Intervention for Hepatocellular Carcinoma by Integrated Bioinformatical Analysis.

PubMed

Hu, Wei Qi; Wang, Wei; Fang, Di Long; Yin, Xue Feng

2018-05-24

BACKGROUND We screened the potential molecular targets and investigated the molecular mechanisms of hepatocellular carcinoma (HCC). MATERIAL AND METHODS Microarray data of GSE47786, including the 40 μM berberine-treated HepG2 human hepatoma cell line and 0.08% DMSO-treated as control cells samples, was downloaded from the GEO database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed; the protein-protein interaction (PPI) networks were constructed using STRING database and Cytoscape; the genetic alteration, neighboring genes networks, and survival analysis of hub genes were explored by cBio portal; and the expression of mRNA level of hub genes was obtained from the Oncomine databases. RESULTS A total of 56 upregulated and 8 downregulated DEGs were identified. The GO analysis results were significantly enriched in cell-cycle arrest, regulation of transcription, DNA-dependent, protein amino acid phosphorylation, cell cycle, and apoptosis. The KEGG pathway analysis showed that DEGs were enriched in MAPK signaling pathway, ErbB signaling pathway, and p53 signaling pathway. JUN, EGR1, MYC, and CDKN1A were identified as hub genes in PPI networks. The genetic alteration of hub genes was mainly concentrated in amplification. TP53, NDRG1, and MAPK15 were found in neighboring genes networks. Altered genes had worse overall survival and disease-free survival than unaltered genes. The expressions of EGR1, MYC, and CDKN1A were significantly increased, but expression of JUN was not, in the Roessler Liver datasets. CONCLUSIONS We found that JUN, EGR1, MYC, and CDKN1A might be used as diagnostic and therapeutic molecular biomarkers and broaden our understanding of the molecular mechanisms of HCC.

Dynamic Fluctuations in Subcellular Localization of the Hippo Pathway Effector Yorkie In Vivo.

PubMed

Manning, Samuel A; Dent, Lucas G; Kondo, Shu; Zhao, Ziqing W; Plachta, Nicolas; Harvey, Kieran F

2018-05-21

The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors [1-8]. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins [9-12]. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, we have little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, we used live multiphoton microscopy to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. We found that the majority of Yorkie rapidly traffics between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, we found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking. Copyright © 2018 Elsevier Ltd. All rights reserved.

RAS oncogene-mediated deregulation of the transcriptome: from molecular signature to function.

PubMed

Schäfer, Reinhold; Sers, Christine

2011-01-01

Transcriptome analysis of cancer cells has developed into a standard procedure to elucidate multiple features of the malignant process and to link gene expression to clinical properties. Gene expression profiling based on microarrays provides essentially correlative information and needs to be transferred to the functional level in order to understand the activity and contribution of individual genes or sets of genes as elements of the gene signature. To date, there exist significant gaps in the functional understanding of gene expression profiles. Moreover, the processes that drive the profound transcriptional alterations that characterize cancer cells remain mainly elusive. We have used pathway-restricted gene expression profiles derived from RAS oncogene-transformed cells and from RAS-expressing cancer cells to identify regulators downstream of the MAPK pathway.We describe the role of epigenetic regulation exemplified by the control of several immune genes in generic cell lines and colorectal cancer cells, particularly the functional interaction between signaling and DNA methylation. Moreover, we assess the role of the architectural transcription factor high mobility AT-hook 2 (HMGA2) as a regulator of the RAS-responsive transcriptome in ovarian epithelial cells. Finally, we describe an integrated approach combining pathway interference in colorectal cancer cells, gene expression profiling and computational analysis of regulatory elements of deregulated target genes. This strategy resulted in the identification of Y-box binding protein 1 (YBX1) as a regulator of MAPK-dependent proliferation and gene expression. The implications for a therapeutic application of HMGA2 gene silencing and the role of YBX1 as a prognostic factor are discussed.

Integration of nodal and BMP signals in the heart requires FoxH1 to create left-right differences in cell migration rates that direct cardiac asymmetry.

PubMed

Lenhart, Kari F; Holtzman, Nathalia G; Williams, Jessica R; Burdine, Rebecca D

2013-01-01

Failure to properly establish the left-right (L/R) axis is a major cause of congenital heart defects in humans, but how L/R patterning of the embryo leads to asymmetric cardiac morphogenesis is still unclear. We find that asymmetric Nodal signaling on the left and Bmp signaling act in parallel to establish zebrafish cardiac laterality by modulating cell migration velocities across the L/R axis. Moreover, we demonstrate that Nodal plays the crucial role in generating asymmetry in the heart and that Bmp signaling via Bmp4 is dispensable in the presence of asymmetric Nodal signaling. In addition, we identify a previously unappreciated role for the Nodal-transcription factor FoxH1 in mediating cell responsiveness to Bmp, further linking the control of these two pathways in the heart. The interplay between these TGFβ pathways is complex, with Nodal signaling potentially acting to limit the response to Bmp pathway activation and the dosage of Bmp signals being critical to limit migration rates. These findings have implications for understanding the complex genetic interactions that lead to congenital heart disease in humans.

Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway.

PubMed

Lallemand-Breitenbach, Valérie; Jeanne, Marion; Benhenda, Shirine; Nasr, Rihab; Lei, Ming; Peres, Laurent; Zhou, Jun; Zhu, Jun; Raught, Brian; de Thé, Hugues

2008-05-01

In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.

Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic.

PubMed

Marat, Andrea L; Haucke, Volker

2016-03-15

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network. © 2016 The Authors.

Clonal selection versus clonal cooperation: the integrated perception of immune objects

PubMed Central

Nataf, Serge

2016-01-01

Analogies between the immune and nervous systems were first envisioned by the immunologist Niels Jerne who introduced the concepts of antigen "recognition" and immune "memory". However, since then, it appears that only the cognitive immunology paradigm proposed by Irun Cohen, attempted to further theorize the immune system functions through the prism of neurosciences. The present paper is aimed at revisiting this analogy-based reasoning. In particular, a parallel is drawn between the brain pathways of visual perception and the processes allowing the global perception of an "immune object". Thus, in the visual system, distinct features of a visual object (shape, color, motion) are perceived separately by distinct neuronal populations during a primary perception task. The output signals generated during this first step instruct then an integrated perception task performed by other neuronal networks. Such a higher order perception step is by essence a cooperative task that is mandatory for the global perception of visual objects. Based on a re-interpretation of recent experimental data, it is suggested that similar general principles drive the integrated perception of immune objects in secondary lymphoid organs (SLOs). In this scheme, the four main categories of signals characterizing an immune object (antigenic, contextual, temporal and localization signals) are first perceived separately by distinct networks of immunocompetent cells.  Then, in a multitude of SLO niches, the output signals generated during this primary perception step are integrated by TH-cells at the single cell level. This process eventually generates a multitude of T-cell and B-cell clones that perform, at the scale of SLOs, an integrated perception of immune objects. Overall, this new framework proposes that integrated immune perception and, consequently, integrated immune responses, rely essentially on clonal cooperation rather than clonal selection. PMID:27830060

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

Identification of miRNA-Mediated Core Gene Module for Glioma Patient Prediction by Integrating High-Throughput miRNA, mRNA Expression and Pathway Structure

PubMed Central

Han, Junwei; Shang, Desi; Zhang, Yunpeng; Zhang, Wei; Yao, Qianlan; Han, Lei; Xu, Yanjun; Yan, Wei; Bao, Zhaoshi; You, Gan; Jiang, Tao; Kang, Chunsheng; Li, Xia

2014-01-01

The prognosis of glioma patients is usually poor, especially in patients with glioblastoma (World Health Organization (WHO) grade IV). The regulatory functions of microRNA (miRNA) on genes have important implications in glioma cell survival. However, there are not many studies that have investigated glioma survival by integrating miRNAs and genes while also considering pathway structure. In this study, we performed sample-matched miRNA and mRNA expression profilings to systematically analyze glioma patient survival. During this analytical process, we developed pathway-based random walk to identify a glioma core miRNA-gene module, simultaneously considering pathway structure information and multi-level involvement of miRNAs and genes. The core miRNA-gene module we identified was comprised of four apparent sub-modules; all four sub-modules displayed a significant correlation with patient survival in the testing set (P-values≤0.001). Notably, one sub-module that consisted of 6 miRNAs and 26 genes also correlated with survival time in the high-grade subgroup (WHO grade III and IV), P-value = 0.0062. Furthermore, the 26-gene expression signature from this sub-module had robust predictive power in four independent, publicly available glioma datasets. Our findings suggested that the expression signatures, which were identified by integration of miRNA and gene level, were closely associated with overall survival among the glioma patients with various grades. PMID:24809850

The integrity of the plant Golgi apparatus depends on cell growth-controlled activity of GNL1.

PubMed

Du, Wenyan; Tamura, Kentaro; Stefano, Giovanni; Brandizzi, Federica

2013-05-01

Membrane traffic and organelle integrity in the plant secretory pathway depend on ARF-GTPases, which are activated by guanine-nucleotide exchange factors (ARF-GEFs). While maintenance of conserved roles, evolution of unique functions as well as tissue-specific roles have been shown for a handful of plant ARF-GEFs, a fundamental yet unanswered question concerns the extent to which their function overlaps during cell growth. To address this, we have characterized pao, a novel allele of GNOM-like 1 (GNL1), a brefeldin A (BFA)-insensitive ARF-GEF, isolated through a confocal microscopy-based forward genetics screen of the Golgi in Arabidopsis thaliana. Specifically, we have analyzed the dependence of the integrity of trafficking routes and secretory organelles on GNL1 availability during expansion stages of cotyledon epidermal cells, an exquisite model system for vegetative cell growth analyses in intact tissues. We show that Golgi traffic is influenced largely by GNL1 availability at early stages of cotyledon cell expansion but by BFA-sensitive GEFs when cell growth terminates. These data reveal an unanticipated level of complexity in the biology of GNL1 by showing that its cellular roles are correlated with cell growth. These results also indicate that the cell growth stage is an important element weighting into functional analyses of the cellular roles of ARF-GEFs.

Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior

PubMed Central

Li, Suyan; Kumar T, Peeyush; Joshee, Sampada; Kirschstein, Timo; Subburaju, Sivan; Khalili, Jahan S; Kloepper, Jonas; Du, Chuang; Elkhal, Abdallah; Szabó, Gábor; Jain, Rakesh K; Köhling, Rüdiger; Vasudevan, Anju

2018-01-01

The cerebral cortex is essential for integration and processing of information that is required for most behaviors. The exquisitely precise laminar organization of the cerebral cortex arises during embryonic development when neurons migrate successively from ventricular zones to coalesce into specific cortical layers. While radial glia act as guide rails for projection neuron migration, pre-formed vascular networks provide support and guidance cues for GABAergic interneuron migration. This study provides novel conceptual and mechanistic insights into this paradigm of vascular-neuronal interactions, revealing new mechanisms of GABA and its receptor-mediated signaling via embryonic forebrain endothelial cells. With the use of two new endothelial cell specific conditional mouse models of the GABA pathway (Gabrb3ΔTie2-Cre and VgatΔTie2-Cre), we show that partial or complete loss of GABA release from endothelial cells during embryogenesis results in vascular defects and impairs long-distance migration and positioning of cortical interneurons. The downstream effects of perturbed endothelial cell-derived GABA signaling are critical, leading to lasting changes to cortical circuits and persistent behavioral deficits. Furthermore, we illustrate new mechanisms of activation of GABA signaling in forebrain endothelial cells that promotes their migration, angiogenesis and acquisition of blood-brain barrier properties. Our findings uncover and elucidate a novel endothelial GABA signaling pathway in the CNS that is distinct from the classical neuronal GABA signaling pathway and shed new light on the etiology and pathophysiology of neuropsychiatric diseases, such as autism spectrum disorders, epilepsy, anxiety, depression and schizophrenia. PMID:29086765

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

PubMed

Pardossi-Piquard, Raphaëlle; Dunys, Julie; Giaime, Emilie; Guillot-Sestier, Marie-Victoire; St George-Hyslop, Peter; Checler, Frédéric; Alves da Costa, Cristine

2009-04-01

Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.

PubMed

Fraguas, Susanna; Barberán, Sara; Cebrià, Francesc

2011-06-01

Similarly to development, the process of regeneration requires that cells accurately sense and respond to their external environment. Thus, intrinsic cues must be integrated with signals from the surrounding environment to ensure appropriate temporal and spatial regulation of tissue regeneration. Identifying the signaling pathways that control these events will not only provide insights into a fascinating biological phenomenon but may also yield new molecular targets for use in regenerative medicine. Among classical models to study regeneration, freshwater planarians represent an attractive system in which to investigate the signals that regulate cell proliferation and differentiation, as well as the proper patterning of the structures being regenerated. Recent studies in planarians have begun to define the role of conserved signaling pathways during regeneration. Here, we extend these analyses to the epidermal growth factor (EGF) receptor pathway. We report the characterization of three epidermal growth factor (EGF) receptors in the planarian Schmidtea mediterranea. Silencing of these genes by RNA interference (RNAi) yielded multiple defects in intact and regenerating planarians. Smed-egfr-1(RNAi) resulted in decreased differentiation of eye pigment cells, abnormal pharynx regeneration and maintenance, and the development of dorsal outgrowths. In contrast, Smed-egfr-3(RNAi) animals produced smaller blastemas associated with abnormal differentiation of certain cell types. Our results suggest important roles for the EGFR signaling in controlling cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis. Copyright © 2011 Elsevier Inc. All rights reserved.

Intracellular signaling by phospholipase D as a therapeutic target.

PubMed

Steed, P M; Chow, A H

2001-09-01

The pharmaceutical industry has recently focused on intracellular signaling as a means to integrate the multiple facets of complex disease states, such as inflammation, because these pathways respond to numerous extracellular signals and coordinate a collection of cell responses contributing to pathology. One critical aspect of intracellular signaling is regulation of key cell functions by lipid mediators, in particular the generation of a key mediator, phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine by phospholipase D (PLD). Research in this field has intensified, due in part to the recent cloning and partial characterization of the two PLD isoforms in mammalian cells, and this work has contributed significantly to our understanding of events downstream of PA generation. It is these effector functions of PLD activity that make this pathway attractive as a therapeutic target while the biochemical properties of the PLD isozymes make them amenable to small molecule intervention. Recent studies indicate that PA, and its immediate metabolites diacylglycerol and lyso-PA, affect numerous cellular pathways including ligand-mediated secretion, cytoskeletal reorganisations, respiratory burst, prostaglandin release, cell migration, cytokine release, and mitogenesis. This review summarises the data implicating signaling via PLD in these cell functions, obtained from: (i) molecular analyses of PLD/effector interactions, (ii) correlation between PA production and cell responses, (iii) experimental manipulation of PA levels, (iv) inhibition of PLD regulators, and (v) direct inhibition of PA production. The utility of targeting PLD signaling for the treatment of acute/chronic inflammation and other indications is discussed in light of these data.

Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation

PubMed Central

Mittra, Indraneel; Samant, Urmila; Sharma, Suvarna; Raghuram, Gorantla V; Saha, Tannistha; Tidke, Pritishkumar; Pancholi, Namrata; Gupta, Deepika; Prasannan, Preeti; Gaikwad, Ashwini; Gardi, Nilesh; Chaubal, Rohan; Upadhyay, Pawan; Pal, Kavita; Rane, Bhagyeshri; Shaikh, Alfina; Salunkhe, Sameer; Dutt, Shilpee; Mishra, Pradyumna K; Khare, Naveen K; Nair, Naveen K; Dutt, Amit

2017-01-01

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell–cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities. PMID:28580170

Vectorial signalling mechanism required for cell-cell communication during sporulation in Bacillus subtilis.

PubMed

Diez, Veronica; Schujman, Gustavo E; Gueiros-Filho, Frederico J; de Mendoza, Diego

2012-01-01

Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cell-cell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor σ(E) to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of σ(E) activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cell-cell communication during development. © 2011 Blackwell Publishing Ltd.

ROP GTPase-mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana.

PubMed

Lin, Deshu; Ren, Huibo; Fu, Ying

2015-01-01

In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell-to-cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP-mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin-ROP signaling networks have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes. Here, we review findings related to the discovery of this novel auxin-signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well-known Transport Inhibitor Response (TIR)-Auxin Signaling F-Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane-localized, transmembrane kinase (TMK) receptor-like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self-organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis. © 2014 Institute of Botany, Chinese Academy of Sciences.

Metabologenomics of Phaeochromocytoma and Paraganglioma: An Integrated Approach for Personalised Biochemical and Genetic Testing

PubMed Central

Eisenhofer, Graeme; Klink, Barbara; Richter, Susan; Lenders, Jacques WM; Robledo, Mercedes

2017-01-01

The tremendous advances over the past two decades in both clinical genetics and biochemical testing of chromaffin cell tumours have led to new considerations about how these aspects of laboratory medicine can be integrated to improve diagnosis and management of affected patients. With germline mutations in 15 genes now identified to be responsible for over a third of all cases of phaeochromocytomas and paragangliomas, these tumours are recognised to have one of the richest hereditary backgrounds among all neoplasms. Depending on the mutation, tumours show distinct differences in metabolic pathways that relate to or even directly impact clinical presentation. At the same time, there has been improved understanding about how catecholamines are synthesised, stored, secreted and metabolised by chromaffin cell tumours. Although the tumours may not always secrete catecholamines it has become clear that almost all continuously produce and metabolise catecholamines. This has not only fuelled changes in laboratory medicine, but has also assisted in recognition of genotype-biochemical phenotype relationships important for diagnostics and clinical care. In particular, differences in catecholamine and energy pathway metabolomes can guide genetic testing, assist with test interpretation and provide predictions about the nature, behaviour and imaging characteristics of the tumours. Conversely, results of genetic testing are important for guiding how routine biochemical testing should be employed and interpreted in surveillance programmes for at-risk patients. In these ways there are emerging needs for modern laboratory medicine to seamlessly integrate biochemical and genetic testing into the diagnosis and management of patients with chromaffin cell tumours. PMID:29332973

Differentiation of a Highly Tumorigenic Basal Cell Compartment in Urothelial Carcinoma

PubMed Central

He, Xiaobing; Marchionni, Luigi; Hansel, Donna E.; Yu, Wayne; Sood, Akshay; Yang, Jie; Parmigiani, Giovanni; Matsui, William; Berman, David M.

2011-01-01

Highly tumorigenic cancer cell (HTC) populations have been identified for a variety of solid tumors and assigned stem cell properties. Strategies for identifying HTCs in solid tumors have been primarily empirical rather than rational, particularly in epithelial tumors, which are responsible for 80% of cancer deaths. We report evidence for a spatially restricted bladder epithelial (urothelial) differentiation program in primary urothelial cancers (UCs) and in UC xenografts. We identified a highly tumorigenic UC cell compartment that resembles benign urothelial stem cells (basal cells), co-expresses the 67-kDa laminin receptor and the basal cell-specific cytokeratin CK17, and lacks the carcinoembryonic antigen family member CEACAM6 (CD66c). This multipotent compartment resides at the tumor-stroma interface, is easily identified on histologic sections, and possesses most, if not all, of the engraftable tumor-forming ability in the parental xenograft. We analyzed differential expression of genes and pathways in basal-like cells versus more differentiated cells. Among these, we found significant enrichment of pathways comprising “hallmarks” of cancer, and pharmacologically targetable signaling pathways, including Janus kinase-signal transducer and activator of transcription, Notch, focal adhesion, mammalian target of rapamycin, epidermal growth factor receptor (erythroblastic leukemia viral oncogene homolog [ErbB]), and wingless-type MMTV integration site family (Wnt). The basal/HTC gene expression signature was essentially invisible within the context of nontumorigenic cell gene expression and overlapped significantly with genes driving progression and death in primary human UC. The spatially restricted epithelial differentiation program described here represents a conceptual advance in understanding cellular heterogeneity of carcinomas and identifies basal-like HTCs as attractive targets for cancer therapy. PMID:19544456

FAP-α (Fibroblast activation protein-α) is involved in the control of human breast cancer cell line growth and motility via the FAK pathway

PubMed Central

2014-01-01

Background Fibroblast Activation Protein alpha (FAP-α) or seprase is an integral membrane serine peptidase. Previous work has not satisfactorily explained both the suppression and promotion effects that have been observed in cancer. The purpose of this work was to investigate the role of FAP-α in human breast cancer. Expression of FAP-α was characterized in primary tumour samples and in cell lines, along with the effects of FAP-α expression on in vitro growth, invasion, attachment and migration. Furthermore the potential interaction of FAP-α with other signalling pathways was investigated. Results FAP-α was significantly increased in patients with poor outcome and survival. In vitro results showed that breast cancer cells over expressing FAP-α had increased growth ability and impaired migratory ability. The growth of MDA-MB-231 cells and the adhesion and invasion ability of both MCF-7 cells and MDA-MB-231 cells were not dramatically influenced by FAP-α expression. Over-expression of FAP-α resulted in a reduction of phosphorylated focal adhesion kinase (FAK) level in both cells cultured in normal media and serum-free media. An inhibitor to FAK restored the reduced motility ability of both MCF-7exp cells and MDA-MB-231exp cells and prevented the change in phosphorylated FAK levels. However, inhibitors to PI3K, ERK, PLCϒ, NWASP, ARP2/3, and ROCK had no influence this. Conclusions FAP-α in significantly associated with poor outcome in patients with breast cancer. In vitro, FAP-α promotes proliferation and inhibits migration of breast cancer cells, potentially by regulating the FAK pathway. These results suggest FAP-α could be a target for future therapies. PMID:24885257

FAP-α (Fibroblast activation protein-α) is involved in the control of human breast cancer cell line growth and motility via the FAK pathway.

PubMed

Jia, Jun; Martin, Tracey Amanda; Ye, Lin; Jiang, Wen Guo

2014-05-21

Fibroblast Activation Protein alpha (FAP-α) or seprase is an integral membrane serine peptidase. Previous work has not satisfactorily explained both the suppression and promotion effects that have been observed in cancer. The purpose of this work was to investigate the role of FAP-α in human breast cancer. Expression of FAP-α was characterized in primary tumour samples and in cell lines, along with the effects of FAP-α expression on in vitro growth, invasion, attachment and migration. Furthermore the potential interaction of FAP-α with other signalling pathways was investigated. FAP-α was significantly increased in patients with poor outcome and survival. In vitro results showed that breast cancer cells over expressing FAP-α had increased growth ability and impaired migratory ability. The growth of MDA-MB-231 cells and the adhesion and invasion ability of both MCF-7 cells and MDA-MB-231 cells were not dramatically influenced by FAP-α expression. Over-expression of FAP-α resulted in a reduction of phosphorylated focal adhesion kinase (FAK) level in both cells cultured in normal media and serum-free media. An inhibitor to FAK restored the reduced motility ability of both MCF-7exp cells and MDA-MB-231exp cells and prevented the change in phosphorylated FAK levels. However, inhibitors to PI3K, ERK, PLCΥ, NWASP, ARP2/3, and ROCK had no influence this. FAP-α in significantly associated with poor outcome in patients with breast cancer. In vitro, FAP-α promotes proliferation and inhibits migration of breast cancer cells, potentially by regulating the FAK pathway. These results suggest FAP-α could be a target for future therapies.

PANDA: pathway and annotation explorer for visualizing and interpreting gene-centric data.

PubMed

Hart, Steven N; Moore, Raymond M; Zimmermann, Michael T; Oliver, Gavin R; Egan, Jan B; Bryce, Alan H; Kocher, Jean-Pierre A

2015-01-01

Objective. Bringing together genomics, transcriptomics, proteomics, and other -omics technologies is an important step towards developing highly personalized medicine. However, instrumentation has advances far beyond expectations and now we are able to generate data faster than it can be interpreted. Materials and Methods. We have developed PANDA (Pathway AND Annotation) Explorer, a visualization tool that integrates gene-level annotation in the context of biological pathways to help interpret complex data from disparate sources. PANDA is a web-based application that displays data in the context of well-studied pathways like KEGG, BioCarta, and PharmGKB. PANDA represents data/annotations as icons in the graph while maintaining the other data elements (i.e., other columns for the table of annotations). Custom pathways from underrepresented diseases can be imported when existing data sources are inadequate. PANDA also allows sharing annotations among collaborators. Results. In our first use case, we show how easy it is to view supplemental data from a manuscript in the context of a user's own data. Another use-case is provided describing how PANDA was leveraged to design a treatment strategy from the somatic variants found in the tumor of a patient with metastatic sarcomatoid renal cell carcinoma. Conclusion. PANDA facilitates the interpretation of gene-centric annotations by visually integrating this information with context of biological pathways. The application can be downloaded or used directly from our website: http://bioinformaticstools.mayo.edu/research/panda-viewer/.

Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells

PubMed Central

Gandhi, Nishant; Wild, Aaron T.; Chettiar, Sivarajan T.; Aziz, Khaled; Kato, Yoshinori; Gajula, Rajendra P.; Williams, Russell D.; Cades, Jessica A.; Annadanam, Anvesh; Song, Danny; Zhang, Yonggang; Hales, Russell K.; Herman, Joseph M.; Armour, Elwood; DeWeese, Theodore L.; Schaeffer, Edward M.; Tran, Phuoc T.

2013-01-01

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies. PMID:23358469

Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells.

PubMed

Gandhi, Nishant; Wild, Aaron T; Chettiar, Sivarajan T; Aziz, Khaled; Kato, Yoshinori; Gajula, Rajendra P; Williams, Russell D; Cades, Jessica A; Annadanam, Anvesh; Song, Danny; Zhang, Yonggang; Hales, Russell K; Herman, Joseph M; Armour, Elwood; DeWeese, Theodore L; Schaeffer, Edward M; Tran, Phuoc T

2013-04-01

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the "non-oncogene" addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded "client" proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4-1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G 2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.

Biphasic effects of FGF2 on odontoblast differentiation involve changes in the BMP and Wnt signaling pathways.

PubMed

Sagomonyants, Karen; Mina, Mina

2014-08-01

Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study, we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of the components of the BMP (Bmp2, Dlx5, Msx2, Osx) and Wnt (Wnt10a, Wisp2) pathways, and decreased expression of an inhibitor of the Wnt signaling, Nkd2. Further addition of FGF2 during the differentiation/mineralization phase of growth resulted in decreased expression of components of the BMP signaling (Bmp2, Runx2, Osx) and increased expression of inhibitors of the Wnt signaling (Nkd2, Dkk3). This suggests that both BMP and Wnt pathways may be involved in mediating the effects of FGF2 on dental pulp cells.

Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data.

PubMed

Singh, Nitesh Kumar; Ernst, Mathias; Liebscher, Volkmar; Fuellen, Georg; Taher, Leila

2016-10-20

The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.

Data-Driven Discovery of Extravasation Pathway in Circulating Tumor Cells

PubMed Central

Yadavalli, S.; Jayaram, S.; Manda, S. S.; Madugundu, A. K.; Nayakanti, D. S.; Tan, T. Z.; Bhat, R.; Rangarajan, A.; Chatterjee, A.; Gowda, H.; Thiery, J. P.; Kumar, P.

2017-01-01

Circulating tumor cells (CTCs) play a crucial role in cancer dissemination and provide a promising source of blood-based markers. Understanding the spectrum of transcriptional profiles of CTCs and their corresponding regulatory mechanisms will allow for a more robust analysis of CTC phenotypes. The current challenge in CTC research is the acquisition of useful clinical information from the multitude of high-throughput studies. To gain a deeper understanding of CTC heterogeneity and identify genes, pathways and processes that are consistently affected across tumors, we mined the literature for gene expression profiles in CTCs. Through in silico analysis and the integration of CTC-specific genes, we found highly significant biological mechanisms and regulatory processes acting in CTCs across various cancers, with a particular enrichment of the leukocyte extravasation pathway. This pathway appears to play a pivotal role in the migration of CTCs to distant metastatic sites. We find that CTCs from multiple cancers express both epithelial and mesenchymal markers in varying amounts, which is suggestive of dynamic and hybrid states along the epithelial-mesenchymal transition (EMT) spectrum. Targeting the specific molecular nodes to monitor disease and therapeutic control of CTCs in real time will likely improve the clinical management of cancer progression and metastases. PMID:28262832

The inhibitory HVEM-BTLA pathway counter regulates lymphotoxin receptor signaling to achieve homeostasis of dendritic cells.

PubMed

De Trez, Carl; Schneider, Kirsten; Potter, Karen; Droin, Nathalie; Fulton, James; Norris, Paula S; Ha, Suk-won; Fu, Yang-Xin; Murphy, Theresa; Murphy, Kenneth M; Pfeffer, Klaus; Benedict, Chris A; Ware, Carl F

2008-01-01

Proliferation of dendritic cells (DC) in the spleen is regulated by positive growth signals through the lymphotoxin (LT)-beta receptor; however, the countering inhibitory signals that achieve homeostatic control are unresolved. Mice deficient in LTalpha, LTbeta, LTbetaR, and the NFkappaB inducing kinase show a specific loss of CD8- DC subsets. In contrast, the CD8alpha- DC subsets were overpopulated in mice deficient in the herpesvirus entry mediator (HVEM) or B and T lymphocyte attenuator (BTLA). HVEM- and BTLA-deficient DC subsets displayed a specific growth advantage in repopulating the spleen in competitive replacement bone marrow chimeric mice. Expression of HVEM and BTLA were required in DC and in the surrounding microenvironment, although DC expression of LTbetaR was necessary to maintain homeostasis. Moreover, enforced activation of the LTbetaR with an agonist Ab drove expansion of CD8alpha- DC subsets, overriding regulation by the HVEM-BTLA pathway. These results indicate the HVEM-BTLA pathway provides an inhibitory checkpoint for DC homeostasis in lymphoid tissue. Together, the LTbetaR and HVEM-BTLA pathways form an integrated signaling network regulating DC homeostasis.

Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering.

PubMed

Chen, Yan; Xiao, Wenhai; Wang, Ying; Liu, Hong; Li, Xia; Yuan, Yingjin

2016-06-21

Microbial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered. In this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of BtCrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets (YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts. Saccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products.

Improvement of Blood-Brain Barrier Integrity in Traumatic Brain Injury and Hemorrhagic Shock Following Treatment With Valproic Acid and Fresh Frozen Plasma.

PubMed

Nikolian, Vahagn C; Dekker, Simone E; Bambakidis, Ted; Higgins, Gerald A; Dennahy, Isabel S; Georgoff, Patrick E; Williams, Aaron M; Andjelkovic, Anuska V; Alam, Hasan B

2018-01-01

Combined traumatic brain injury and hemorrhagic shock are highly lethal. Following injuries, the integrity of the blood-brain barrier can be impaired, contributing to secondary brain insults. The status of the blood-brain barrier represents a potential factor impacting long-term neurologic outcomes in combined injuries. Treatment strategies involving plasma-based resuscitation and valproic acid therapy have shown efficacy in this setting. We hypothesize that a component of this beneficial effect is related to blood-brain barrier preservation. Following controlled traumatic brain injury, hemorrhagic shock, various resuscitation and treatment strategies were evaluated for their association with blood-brain barrier integrity. Analysis of gene expression profiles was performed using Porcine Gene ST 1.1 microarray. Pathway analysis was completed using network analysis tools (Gene Ontology, Ingenuity Pathway Analysis, and Parametric Gene Set Enrichment Analysis). Female Yorkshire swine were subjected to controlled traumatic brain injury and 2 hours of hemorrhagic shock (40% blood volume, mean arterial pressure 30-35 mmHg). Subjects were resuscitated with 1) normal saline, 2) fresh frozen plasma, 3) hetastarch, 4) fresh frozen plasma + valproic acid, or 5) hetastarch + valproic acid (n = 5 per group). After 6 hours of observation, brains were harvested for evaluation. Immunofluoroscopic evaluation of the traumatic brain injury site revealed significantly increased expression of tight-junction associated proteins (zona occludin-1, claudin-5) following combination therapy (fresh frozen plasma + valproic acid and hetastarch + valproic acid). The extracellular matrix protein laminin was found to have significantly improved expression with combination therapies. Pathway analysis indicated that valproic acid significantly modulated pathways involved in endothelial barrier function and cell signaling. Resuscitation with fresh frozen plasma results in improved expression of proteins essential for blood-brain barrier integrity. The addition of valproic acid provides significant improvement to these protein expression profiles. This is likely secondary to activation of key pathways related to endothelial functions.

Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis

PubMed Central

Vanhollebeke, Benoit; Stone, Oliver A; Bostaille, Naguissa; Cho, Chris; Zhou, Yulian; Maquet, Emilie; Gauquier, Anne; Cabochette, Pauline; Fukuhara, Shigetomo; Mochizuki, Naoki; Nathans, Jeremy; Stainier, Didier YR

2015-01-01

Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function. DOI: http://dx.doi.org/10.7554/eLife.06489.001 PMID:26051822

On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

NASA Astrophysics Data System (ADS)

Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

2004-08-01

The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

Network Analysis of Epidermal Growth Factor Signaling Using Integrated Genomic, Proteomic and Phosphorylation Data

PubMed Central

Waters, Katrina M.; Liu, Tao; Quesenberry, Ryan D.; Willse, Alan R.; Bandyopadhyay, Somnath; Kathmann, Loel E.; Weber, Thomas J.; Smith, Richard D.; Wiley, H. Steven; Thrall, Brian D.

2012-01-01

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response. PMID:22479638