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Differentiation of skeletal muscle cells, like most other cell types, requires a permanent exit from the cell cycle. The epigenetic programming underlying these distinct cellular states is not fully understood. In this study, we provide evidence that the lysine methyltransferase G9a functions as a central axis to regulate proliferation and differentiation of skeletal muscle cells. Transcriptome analysis of G9a knockdown cells revealed deregulation of many cell cycle regulatory genes. We demonstrate that G9a enhances cellular proliferation by two distinct mechanisms. G9a blocks cell cycle exit via methylation-dependent transcriptional repression of the MyoD target genes p21(Cip/Waf1) and Rb1. In addition, it activates E2F1-target genes in a methyltransferase activity-independent manner. We show that G9a is present in the E2F1/PCAF complex, and enhances PCAF occupancy and histone acetylation marks at E2F1-target promoters. Interestingly, G9a preferentially associates with E2F1 at the G1/S phase and with MyoD at the G2/M phase. Our results provide evidence that G9a functions both as a co-activator and a co-repressor to enhance cellular proliferation and inhibit myogenic differentiation.

Anaphylaxis is a sudden immune reaction against an allergen that can potentially lead to Anaphylactic Shock (AS). This immune reaction is characterized by an increase in Immunoglobulin-E (IgE) type of antibodies that bind with FceRI receptors on mast cells to release inflammatory mediators. Various intracellular signaling molecules downstream of IgE/ FceRI axis play a potential role in cytokine, chemokine and eicosanoid secretion as well as degranulation of immune cells causing vasodilation, vascular permeability, and reduction of intravascular volume leading to cardiovascular collapse. Here, we discuss the cellular machinery of anaphylaxis and the de novo paradigm shift in the cellular aspects of AS.

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.

MicroRNAs (miRNAs) are known to be dysregulated and play a key role in cancer progression. The present study aims to identify the miRNAs associated with colorectal adenoma and carcinoma to evaluate their role in tumor progression and metastasis using microarray. In silico analysis of miRNAs was performed on five different microarray data sets that represented the genes and miRNAs expressed in colorectal adenoma and carcinoma. We identified 10 different miRNAs that were common to both colorectal adenoma and carcinoma, namely, miR9, miR96, miR135b, miR137, miR147, miR182, miR183, miR196b, miR224, and miR503. Of these, miR135b and miR147 were significantly downregulated in colorectal adenoma but upregulated in carcinoma. In addition, we studied the gene expression profile associated with colorectal adenocarcinoma and identified three genes, namely, ZBED3, SLC10A3, and FOXQ1, that were significantly downregulated in colorectal adenoma compared to carcinoma. Interestingly, of all the miRNAs and genes associated with colorectal adenocarcinoma, the myoglobin (MB) gene was identified to be under the direct influence of miR135b, showing an inverse relationship between them in adenoma and carcinoma. Most of the identified miRNAs and associated genes are involved in signaling pathways of cell proliferation, angiogenesis, and metastasis. The present study has identified putative miRNA targets and their associated gene networks which could be used as potential biomarkers of colon adenocarcinoma. Moreover, the association of miR135b and MB gene is very unique and can be considered as a lead candidate for novel cancer therapeutics.

The relationship between the two age-related diseases namely, Alzheimer's disease (AD) and type II diabetes mellitus (T2DM), is gaining much attention in research because of the alarming forecast on both increasing incidence and economic burden. Recent research studies have identified some of the existing links, between AD and T2DM, such as the dysfunctional glucose metabolism and insulin signaling, stress and inflammation, defective protein processing and the role of advanced glycation end products. It is, therefore, crucial to understand the cellular and molecular mechanisms to identify the common linking mechanisms involved in the pathogenesis of both AD and T2DM. Genome wide association studies may lead to identification of novel targets and provide clues for possible interventional strategies to limit the progression of these two age-related diseases. Hence, the purpose of the present review is to provide an update, on the various possible linking cellular and molecular mechanisms, including our experience on the use of high throughput applications to investigate the molecular mechanisms underneath the neurodegeneration in animal models. Besides, using this knowledge-driven approach, we discuss how the current technological advancements can effectively be used to identify possible associations between these age-related diseases.

Traditional medicine plays a vital role for primary health care in India, where it is widely practiced to treat various ailments. Among those obtained from the healers, 78 medicinal plants were scientifically evaluated for antibacterial activity. Methanol extract of plants (100 μg of residue) was tested against the multidrug resistant (MDR) Gram-negative and Gram-positive bacteria. Forty-seven plants showed strong activity againstBurkholderia pseudomallei(strain TES and KHW) andStaphylococcus aureus, of whichTragia involucrataL.,Citrus acidaRoxb. Hook.f., andAegle marmelos(L.) Correa ex Roxb. showed powerful inhibition of bacteria. Eighteen plants displayed only a moderate effect, while six plants failed to provide any evidence of inhibition against the tested bacteria. Purified compounds showed higher antimicrobial activity than crude extracts. The compounds showed less toxic effect to the human skin fibroblasts (HEPK) cells than their corresponding aromatic fractions. Phytochemical screening indicates that the presence of various secondary metabolites may be responsible for this activity. Most of the plant extracts contained high levels of phenolic or polyphenolic compounds and exhibited activity against MDR pathogens. In conclusion, plants are promising agents that deserve further exploration. Lead molecules available from such extracts may serve as potential antimicrobial agents for future drug development to combat diseases caused by the MDR bacterial strains as reported in this study.

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ±1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8–24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.

Allergy and anaphylaxis are inflammatory disorders caused by immune reactions mainly induced by immunoglobulin-E that signal through the high-affinity FcεRI receptor to release the inflammatory mediators from innate immune cells. The FcεRI/mast cell axis is potently involved in triggering various intracellular signaling molecules to induce calcium release from the internal stores, induction of transcription factors such as NF-kB, secretion of various cytokines as well as lipid mediators, and degranulation, resulting in the induction of allergy and anaphylaxis. In this review, we discuss various cellular and molecular mechanisms triggered through FcεRI/mast cell axis in allergy and anaphylaxis with a special emphasis on the functional genomics paradigm.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease of unidentified aetiology, chiefly affecting the synovial membranes of joints, cartilage, bone, bursa and tendon sheath. Osteoarthritis (OA) is a degenerative disorder and encompass different sets of pathogenic pathways than RA; however, it shows a medley of clinical manifestations or symptoms resembling RA. Hence, we intend to identify more disease specific biomarkers through the meta-analysis of microarray datasets that can be crucial in the differential diagnosis, disease specific treatment as well as management of both RA and OA in a typical clinical setting.

Recruitment of leukocytes circulating in our blood to the sites of infection or tissue damage is the key phenomenon in the acute inflammatory response(s). Among the leukocytes, neutrophils are primarily recruited into the areas of acute inflammation. When neutrophils interact with activated endothelium of the blood vessels, they become migratory and cross the endothelial layer of the blood vessel wall in a process called as leukocyte extravasation. Identifying and understanding the gene regulation of this extravasation phenomenon is one of the key objective of biomedical research aimed at ameliorating or alleviating the symptoms of various diseases, such as rheumatoid arthritis, asthma, anaphylaxis, atherosclerosis, ulcerative colitis etc., that are exacerbated by inappropriate inflammatory stimuli. Here, we decipher and discuss the key genes implicated in the leukocyte transmigration using the acute inflammation model called as the Dextran Sulphate Sodium (DSS) induced Colitis in mice as a classic paradigm.

Transient cerebral ischemia often results in secondary ischemic/reperfusion injury, the pathogenesis of which remains unclear. This study provides a comprehensive, temporal description of the molecular events contributing to neuronal injury after transient cerebral ischemia. Intraluminal middle cerebral artery occlusion (MCAO) was performed to induce a 2-h ischemia with reperfusion. Microarray analysis was then performed on the infarct cortex of wild-type (WT) and glutathione peroxidase-1 (a major antioxidant enzyme) knockout (Gpx1(-/-)) mice at 8 and 24h postreperfusion to identify differential gene expression profile patterns and potential alternative injury cascades in the absence of Gpx1, a crucial antioxidant enzyme, in cerebral ischemia. Genes with at least ±1.5-fold change in expression at either time point were considered significant. Global transcriptomic analyses demonstrated that 70% of the WT-MCAO profile overlapped with that of Gpx1(-/-)-MCAO, and 28% vice versa. Critical analysis of the 1034 gene probes specific to the Gpx1(-/-)-MCAO profile revealed regulation of additional novel pathways, including the p53-mediated proapoptotic pathway and Fas ligand (CD95/Apo1)-mediated pathways; downplay of the Nrf2 antioxidative cascade; and ubiquitin-proteasome system dysfunction. Therefore, this comparative study forms the foundation for the establishment of screening platforms for target definition in acute cerebral ischemia intervention.

Biodistribution of gold nanoparticles (AuNPs) in more than 25 organs were examined on 1 day, 1 week, 1 month and 2 months after a single intravenous (i.v.) injection in rats. Au was rapidly and consistently accumulated in liver (49.4+/-50.4-72.2+/-40.5 ng/g) and spleen (8.4+/-5.0-9.5+/-6.4 ng/g) throughout the entire timeframe of the study (2 months). Significant accumulation of Au in kidney (up to 5.5+/-2.5 ng/g) and testis (up to 0.6+/-0.1 ng/g) occurred from 1 month post-injection when Au level in urine and feces decreased. Significant increase of Au in blood occurred 2 months after injection, coincident with the delayed accumulation in kidney. Au accumulation in lungs was found at 1 day post-injection but decreased within a week. No accumulation of Au was found in the brain. Microarray results of liver and spleen point to significant effects on genes related to detoxification, lipid metabolism, cell cycle, defense response, and circadian rhythm. These results demonstrate that significant biodistribution of Au occurs in the body over 2 months after a single i.v. injection of AuNPs, accompanied by gene expression changes in target organs.

Deletion of mouse preprotachykinin-A (PPTA), which encodes mainly for neuropeptide substance P, has been shown to protect against lung injury and mortality in sepsis. This study explored microarray-based differential gene expression profiles in mouse lung tissue 8 h after inducing microbial sepsis and the effect of PPTA gene deletion. A range of genes differentially expressed (more than two-fold) in microarray analysis was assessed, comparing wild-type and PPTA-knockout septic mice with their respective sham controls, and the data were further validated. Genetic deletion of substance P resulted in a significantly different expression profile of genes involved in inflammation and immunomodulation after the induction of sepsis, compared with wild-type mice. Interestingly, apart from the various proinflammatory mediators, the antiinflammatory cytokine interleukin-1 receptor antagonist gene (IL1RN) was also elevated much more in PPTA(-/-) septic mice. In addition, semiquantitative RT-PCR analysis supported the microarray data. The microarray data imply that the elevated levels of inflammatory gene expression in the early stages of sepsis in PPTA-knockout mice are possibly aimed to resolve the infection without excessive immunosuppression. As scientists are divided over the effects of pro- and antiinflammatory mediators in sepsis, it seems prudent to define the status depending on a complete genome profile. This is the first report exploring pulmonary gene expression profiles using microarray analysis in PPTA-knockout mice subjected to cecal ligation and puncture-induced sepsis and providing additional biological insight into the protection received against lung injury and mortality.

The mechanistic understanding of low-level sarin-induced neurotoxicity after single or repeated doses has yet to be explored at a cellular level. Using the microarray (Affymetrix-GeneChips) transcription profiling approach, the present study examined gene expression in human SH-SY5Y cells exposed to single (3 and 24 h) or repeated (2 x 24 h) doses of sarin (5 microg/mL) to delineate the possible mechanism. Two hundred twenty-four genes whose expression was significantly (P0.01) altered by at least 3-fold were selected by GeneSpringGX analysis. The comparative gene expression data confirmed the transcriptional changes to be related to dose and exposure time of sarin. The effect of a single noncytotoxic sarin dose on gene transcription was variable, whereas repeated doses over 48 h persistently down-regulated genes linked to neurodegenerative mechanisms. Thirty persistently altered genes were validated using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Similar qRT-PCR profiles obtained in sarin-treated SH-SY5Y and HCN-1A cells confirmed the cell-independent alterations in expression levels. Genes (ETS2, APOE, PSEN1, DDC, and CD9) implicated mainly in the regulation of sarin-induced neuropathogenesis were further confirmed by Western blot and double-immunofluorescence assays. The regulome pathway suggests a new feasible mechanism by which sarin increases ETS2 expression and takes control over other genes involved in the neurodegenerative pathway. The overall data delineate an in vitro experimental model suitable for studying the neuropathology of cells and may provide novel insights into therapeutic interventions.

Maternal diabetes causes congenital malformations in various organs including the neural tube in fetuses. In this study, we have analyzed the differential gene expression profiling in the cranial neural tube of embryos from diabetic and control mice by using the oligonucleotide microarray. Expression patterns of genes and proteins that are differentially expressed in the cranial neural tube were further examined by the real-time reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. Proliferation index and apoptosis were examined by BrdU (5-bromo-2-deoxyuridine) labeling and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, respectively. Embryos (E11.5) of diabetic pregnancies displayed distortion in neuroepithelia of the cranial neural tube. Microarray analysis revealed that a total of 390 genes exhibited more than twofold changes in expression level in the cranial neural tube of embryos from diabetic mice. Several genes involving apoptosis, proliferation, migration, and differentiation of neurons in the cranial neural tube were differentially expressed in embryos of diabetic pregnancy. In addition, maternal diabetes perturbed the development of choroid plexus and ventricular systems and reduced the production of proteins such as Ttr and Igf2 in the developing brain, indicating that these changes could impair the survival and proliferation of neuroepithelial cells and neurogenesis in embryos of diabetic mice. It is concluded that altered expression of a variety of genes involved in brain development is associated with cranial neural tube dysmorphogenesis that may subsequently contribute to intellectual impairment of the offspring of a diabetic mother.

MicroRNAs (miRNAs) are members of a family of non-coding RNAs of 8-24 nucleotide RNA molecules that regulate target mRNAs. The first miRNAs, lin-4 and let-7, were first discovered in the year 1993 by Ambros, Ruvkun, and co-workers while studying development in Caenorhabditis elegans. miRNAs can play vital functions form C. elegans to higher vertebrates by typical Watson-Crick base pairing to specific mRNAs to regulate the expression of a specific gene. It has been well established that multicellular eukaryotes utilize miRNAs to regulate many biological processes such as embryonic development, proliferation, differentiation, and cell death. Recent studies have shown that miRNAs may provide new insight in cancer research. A recent study demonstrated that more than 50% of miRNA genes are located in fragile sites and cancer-associated genomic regions, suggesting that miRNAs may play a more important role in the pathogenesis of human cancers. Exploiting the emerging knowledge of miRNAs for the development of new human therapeutic applications will be important. Recent studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer. In this review, we focus on how miRNAs regulate tumorigenesis by acting as oncogenes and anti-oncogenes in higher eukaryotes.

Programmed cell death (PCD), is a highly regulated and sophisticated cellular mechanism that commits cell to isolated death fate. PCD has been implicated in the pathogenesis of numerous neurodegenerative disorders. Countless molecular events underlie this phenomenon, with each playing a crucial role in death commitment. A precedent event, apoptotic volume decrease (AVD), is ubiquitously observed in various forms of PCD induced by different cellular insults. Under physiological conditions, cells when subjected to osmotic fluctuations will undergo regulatory volume increase/decrease (RVI/RVD) to achieve homeostatic balance with neurons in the brain being additionally protected by the blood-brain-barrier. However, during AVD following apoptotic trigger, cell undergoes anistonic shrinkage that involves the loss of water and ions, particularly monovalent ions e.g. K(+), Na(+) and Cl(-). It is worthwhile to concentrate on the molecular implications underlying the loss of these cellular components which posed to be significant and crucial in the successful propagation of the apoptotic signals. Microarray and real-time PCR analyses demonstrated several ion and water channel genes are regulated upon the onset of lactacystin (a proteosomal inhibitor)-mediated apoptosis. A time course study revealed that gene expressions of water and ion channels are being modulated just prior to apoptosis, some of which are aquaporin 4 and 9, potassium channels and chloride channels. In this review, we shall looked into the molecular protein machineries involved in the execution of AVD in the central nervous system (CNS), and focus on the significance of movements of each cellular component in affecting PCD commitment, thus provide some pharmacological advantages in the global apoptotic cell death.

Studies have shown that the lipid peroxidation by-product, 4-hydroxynonenal (HNE), is involved in many pathological events in several neurodegenerative diseases. A number of signaling pathways mediating HNE-induced cell death in the brain have been proposed. However, the exact mechanism remains unknown. In the present study, we have examined the effects of HNE on cultured primary cortical neurons and found that HNE treatment leads to cell death via apoptosis. Both the caspase and calpain proteolytic systems were activated. There were also increased levels of phospho-p53 and cell cycle-related proteins. Gene transcription was further studied using microarray analysis. Results showed that majority of the genes associated with cell cycle regulation, response to stress, and signal transduction were differentially expressed. The various categories of differentially-expressed genes suggested that there are other parallel pathways regulating HNE-induced neuronal apoptosis. Collectively, these might help to elucidate similar molecular mechanisms involved during cell death in neurodegenerative diseases.

The present study was designed to investigate the hypoglycemic and hypolipidemic properties of an ethanolic extract of Cichorium intybus (CIE) which is widely used in India as a traditional treatment for diabetes mellitus. Male Sprague-Dawley rats aged 9 weeks (160-200 g) were administered with streptozotocin (STZ, 50mg/kg) intraperitoneally to induce experimental diabetes. The Cichorium intybus whole plant was exhaustively extracted with 80% ethanol, concentrated at 40 degrees C using a rotavapor and freeze dried to get powder. Hypoglycemic effects of CIE were observed in an oral glucose tolerance test (OGTT) in which, a dose of 125 mg of plant extract/kg body weight exhibited the most potent hypoglycemic effect. Moreover, daily administration of CIE (125 mg/kg) for 14 days to diabetic rats attenuated serum glucose by 20%, triglycerides by 91% and total cholesterol by 16%. However, there was no change in serum insulin levels, which ruled out the possibility that CIE induces insulin secretion from pancreatic beta-cells. In addition, hepatic glucose-6-phosphatase activity (Glc-6-Pase) was markedly reduced by CIE when compared to the control group. The reduction in the hepatic Glc-6-Pase activity could decrease hepatic glucose production, which in turn results in lower concentration of blood glucose in CIE-treated diabetic rats. In conclusion, our results support the traditional belief that Cichorium intybus could ameliorate diabetic state.

Previously, we demonstrated that undifferentiated human embryonic stem cells (hESC) displayed higher resistance to oxidative and genotoxic stress compared to somatic cells, but did not further probe the underlying mechanisms. Using H₂O₂-induced genotoxicity as a model, this study investigated whether higher resistance of hESC to oxidative and genotoxic stress could be due to lower innate basal intracellular levels of reactive oxygen species (ROS), as compared to their differentiated fibroblastic progenies (H1F) and two other somatic cell types - human embryonic palatal mesenchymal (HEPM) cells and peripheral blood lymphocytes (PBL). Comet assay demonstrated that undifferentiated hESC consistently sustained lower levels of DNA damage upon acute exposure to H₂O₂ for 30 min, compared to somatic cells. DCFDA and HE staining with flow cytometry showed that undifferentiated hESC had lower innate basal intracellular levels of reactive oxygen species compared to somatic cells, which could lead to their higher resistance to genotoxic stress upon acute exposure to H₂O₂.

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.

Congenital heart defects (CHD) are one of the most common defects in offspring of diabetic mothers. There is a clear association between maternal diabetes and CHD; however the underlying molecular mechanism remains unknown. We hypothesized that maternal diabetes affects with the expression of early developmental genes that regulate the essential developmental processes of the heart, thereby resulting in the pathogenesis of CHD. We analyzed genome-wide expression profiling in the developing heart of embryos from diabetic and control mice by using the oligonucleotide microarray. Microarray analysis revealed that a total of 878 genes exhibited more than 1.5 fold changes in expression level in the hearts of experimental embryos in either E13.5 or E15.5 compared with their respective controls. Expression pattern of genes that is differentially expressed in the developing heart was further examined by the real-time reverse transcriptase-polymerase chain reaction. Several genes involved in a number of molecular signaling pathways such as apoptosis, proliferation, migration and differentiation in the developing heart were differentially expressed in embryos of diabetic pregnancy. It is concluded that altered expression of several genes involved in heart development may contribute to CHD in offspring of diabetic mothers.

Mitochondrial dysfunction and oxidative stress are currently considered two key mechanisms contributing to pathobiology in neurodegenerative conditions. The current study investigated the temporal molecular events contributing to programmed cell death after treatment with the mitochondrial complex I inhibitor rotenone. Microarray analysis was performed using cultured neocortical neurons treated with 10nM rotenone for 8, 15, and 24h. Genes showing at least ±1.2-fold change in expression at one time point were considered significant. Transcriptomic analysis of the 4178 genes probes revealed major changes to nine biological processes, including those eliciting mitochondrial dysfunction, activation of calcium signaling, increased expression of apoptotic genes, and downplay of chaperones/co-chaperones, ubiquitin-proteasome system and autophagy. These data define targets for intervention where mitochondrial complex I dysfunction plays a substantial role, most notably Parkinson's disease.

Excitotoxicity, induced by the aberrant rise in cytosolic Ca(2+) level, is a major neuropathological process in numerous neurodegenerative disorders. It is triggered when extracellular glutamate (Glu) concentration reaches neuropathological levels resulting in dysregulation and hyper-activation of ionotropic glutamate receptor subtype (iGluRs). Even though all three members of the iGluRs, namely N-methyl-d-aspartate (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and kainate (KAR) receptors are implicated in excitotoxicity, their individual contributions to downstream signaling transduction have not been explored. In this study, we report a comprehensive description of the recruitment of cellular processes in neurons upon iGluR activation during excitotoxicity through temporal (5h, 15h, and 24h) global gene profiling of AMPA, KA, NMDA, and Glu excitotoxic models. DNA microarray analyses of mouse primary cortical neurons treated with these four pharmacological agonists are further validated via real-time PCR. Bi-model analyses against Glu model demonstrate that NMDARs and KARs play a more pivotal role in Glu-mediated excitotoxicity, with a higher degree of global gene profiling overlaps, as compared to that of AMPARs. Comparison of global transcriptomic profiles reveals aberrant calcium ion binding and homeostasis, organellar (lysosomal and endoplasmic reticulum) stress, oxidative stress, cell cycle re-entry and activation of cell death processes as the main pathways that are significantly modulated across all excitotoxicity models. Singular profile analyses demonstrate substantial transcriptional regulation of numerous cell cycle proteins. For the first time, we show that iGluR activation forms the basis of cell cycle re-activation, and together with oxidative stress fulfill the "two-hit" hypothesis that accelerates neurodegeneration.

Recently the role of hydrogen sulphide (H(2) S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H(2) S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H(2) S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H(2) S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5-24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H(2) S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H(2) S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H(2) S neuropathologies where NMDAR-activated signaling cascades played a substantial role.

Palladin is a scaffold protein involved in the formation of actin-associated protein complexes. Gene expression array analysis on the poorly metastatic HCT116 colon cancer cell line and a metastatic derivative cell line (E1) with EMT (epithelial-mesenchymal transition) features showed a down-regulation of palladin gene expression in the latter. Knockdown of palladin expression in the HCT116 cells suppressed junctional localization of E-cadherin, reduced intercellular adhesion and collective cell migration, showing that palladin plays an important role in maintaining the integrity of adherens junctions. The acquisition of the EMT features by the E1 cell line was dependent on the Erk pathway. Inhibition of this pathway by U0126 treatment in E1 cells resulted in the re-expression of palladin, relocalization of E-cadherin to the adherens junctions and a reversal of EMT features. The re-establishment of intercellular adhesion was dependent on palladin expression. The down-regulation of palladin was also observed in poorly-differentiated tumor tubules and dissociated tumor cells that have undergone de-differentiation in human primary colon tumors. Our data show that palladin is an integral component of adherens junctions and plays a role in the localization of E-cadherin to the junctions. The loss of palladin may be an integral part of EMT, an early step in the metastatic spread of colon carcinoma.

Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. We and others demonstrated that treatment of cortical neurons with the proteasomal inhibitor lactacystin leads to apoptosis. We discovered by microarray analysis that lactacystin treatment modulates the expression of both potentially neuroprotective as well as pro-apoptotic genes in neurons. However, the significance of the genes which upon transcriptional modulation contributed to proteasomal inhibition-induced apoptosis, remained unidentified. By employing microarray analysis to decipher the time-dependent changes in transcription of these genes in cultured cortical neurons, we discovered different groups of genes were transcriptionally regulated in the early and late phase of lactacystin-induced cell death. In the early phase, several neuroprotective genes such as those encoding the proteasome subunits and ubiquitin-associated enzymes, as well as the heat-shock proteins (HSP) were up-regulated. However, the pro-apoptotic endoplasmic reticulum (ER) stress-associated genes were also up-regulated at the early phase of lactacystin-induced neuronal cell death. In the late phase, genes encoding antioxidants and calcium-binding proteins were up-regulated while those associated with cholesterol biosynthesis were down-regulated. The data suggest that ER stress may participate in mediating the apoptotic responses induced by proteasomal inhibition. The up-regulation of the neuroprotective antioxidant genes and calcium-binding protein genes and down-regulation of the cholesterol biosynthesis genes in the later phase are likely consequences of stimulation of the pro-apoptotic signaling pathways in the early phase of lactacystin treatment.

Background: The telomerase enzyme is a viable target for anti-cancer therapy given the innate differences in telomerase activity between tumour cells and normal somatic cells. However, the time lag between telomerase inhibition and telomeres becoming critically short to trigger cell death, allows cancer cells to acquire drug resistance. Inhibition of DNA repair pathways along with telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. Poly (ADP-Ribose) Polymerase-1 (PARP-1), an important DNA damage sensor and a DNA repair factor, has important roles in maintaining telomeres and chromosomal stability. In this study, the effects of combined inhibition of PARP-1 and telomerase in mouse embryonic fibroblasts (MEFs) following sodium arsenite exposure (a carcinogen and potent DNA damaging agent), were evaluated. Results: Inhibition of PARP in telomerase deficient MEFs induced an increase in arsenite-induced DNA damage as compared to control cells. Combined inhibition also resulted in enhanced genomic instability, demonstrated by elevated micronuclei induction and chromosomal aberrations with decreased cell survival. In addition, telomerase inhibition in PARP-1 deficient MEFs led to greater telomere shortening and increased genomic instability. Conclusions: Our study demonstrated that the co-inhibition of PARP-1 and telomerase in MEFs rendered cells more susceptible to DNA damaging agents. Hence, these results offer support for the use of combined inhibition of PARP-1 and telomerase as a strategy to minimise the problems associated with long-term telomerase inhibition in cancer therapeutics.

Aims With the identification of hypochlorous acid (HOCl) as a biomarker in diseased brains and endogenous detection of its modified proteins, HOCl might be implicated in the development of neurodegenerative disorders. However, its effect on neuronal cell death has not yet been investigated at gene expression level. Main methods Therefore, DNA microarray was performed for screening of HOCl-responsive genes in primary mouse cortical neurons. Neurotoxicity caused by physiological relevant HOCl (250 μM) exhibited several biochemical markers of apoptosis. Key findings The biological processes affected during HOCl-mediated apoptosis included cell death, response to stress, cellular metabolism, and cell cycle. Among them, mRNAs level of cell death and stress response genes were up-regulated while expression of metabolism and cell cycle genes were down-regulated. Significance Our results showed, for the first time, that HOCl induces apoptosis in cortical neurons by upregulating apoptotic genes and gene expression of stress response such as heat shock proteins and antioxidant proteins were enhanced to provide protection. These data form a foundation for the development of screening platforms and define targets for intervention in HOCl neuropathologies where HOCl-mediated injury is causative.

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcεRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcεRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcεRI stimulation including: Ca2+ signals, NFκB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1−/− and SPHK2−/− mice, which showed that SphK2 was required for FcεRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1−/− and SPHK2−/− mice and show that the calcium response and degranulation, triggered by FcεRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

RNA interference (RNAi), an accurate and potent gene-silencing method, was first experimentally documented in 1998 in Caenorhabditis elegans by Fire et al., who subsequently were awarded the 2006 Nobel Prize in Physiology/Medicine. Subsequent RNAi studies have demonstrated the clinical potential of synthetic small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) in dental diseases, eye diseases, cancer, metabolic diseases, neurodegenerative disorders, and other illnesses. siRNAs are generally from 21 to 25 base-pairs (bp) in length and have sequence-homology-driven gene-knockdown capability. RNAi offers researchers an effortless tool for investigating biological systems by selectively silencing genes. Key technical aspects—such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety—need to be investigated before RNAi can become a successful therapeutic strategy. Nevertheless, this area shows a huge potential for the pharmaceutical industry around the globe. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs, could effectively activate a particular gene in a sequence-specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed ‘RNA activation’ (RNAa). In this review, we analyze these research findings and discussed the in vivo applications of siRNAs, miRNAs, and shRNAs.

The present study was carried out to examine global gene expression in the brainstem, in a mouse facial carrageenan injection model of orofacial pain. Mice that received facial carrageenan injection showed increased mechanical allodynia, demonstrated by increased responses to von Frey hair stimulation of the face. The brainstem was harvested at 3 days post-injection, corresponding to the time of peak responses, and analyzed by Affymetrix Mouse Genome 430 2.0 microarrays. We sought to identify common genes that are changed in the respective sides of the brainstem after either right- or left-sided facial carrageenan injection. The result is a relatively small list of genes (22 genes), which were then classified using DAVID software. Many of them fell into the categories of "response to stress", "defence response", "response to biotic stimulus", "cell adhesion" and "leukocyte adhesion". Of these, increased expression of P-selectin, ICAM-1 and CCL12 after carrageenan injection could be verified by real-time RT-PCR on both the right and left sides, and increased in P-selectin and ICAM-1 further verified by Western blot analysis. P-selectin and ICAM-1 were immunolocalized to endothelial cells, and were double labelled with von Willebrand factor. Intraperitoneal injection of the P-selectin inhibitor KF38789 significantly reduced mechanical allodynia in the facial carrageenan-injected mice. P-selectin mediates the capturing of leukocytes from the bloodstream and rolling of leukocytes along the endothelial surface. We hypothesize that increased nociceptive input to the brainstem could attract circulating macrophages into the brain, resulting in neuroinflammation and pain.

RNA interference (RNAi) is a powerful technology with huge applications for functional genomics, target identification in drug discovery and elucidation of molecular signaling pathways. Current RNAi studies have demonstrated the clinical potential of small interfering RNAs (siRNAs) in metabolic diseases, cancer, AIDS, malaria, neurodegenerative disorders, dental diseases and other illnesses. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs could effectively activate a particular gene in a sequence specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed as RNA activation (RNAa). The paradoxical concept of Yin and Yang, which describe two primal opposing but complementary principles, can potentially be applied to elucidate the complex phenomenon of RNAa/RNAi in the RNAome. This warrants a proper understanding of the RNAi/RNAa molecular pathways in living organisms before any of the small dsRNAs can potentially be exploited for therapeutics in human beings.

Receptors for the constant (Fc) region of immunoglobulin G, play an important role in linking the humoral and cellular arms of immune response. Three different classes of receptors have been defined, FcγRI (CD64), FcγRII (CD 32), and FcγRIII (CD16). We have previously shown that in a human monocyte model (IFNγ-primed U937 cells), FcγRI and FcγRII stimulation trigger two very distinct intracellular signalling pathways. Here we show for the first time, the use of oligonucleotide microarrays to investigate the overall gene-expression profile of human monocytes triggered with FcγRI or FcγRII. Our data indicate that several interesting genes are differentially expressed in response to the different receptors, pointing out to a complete different genetic response of human monocytes regulated by the different signal transduction cascade triggered by FcγRI or FcγRIIa. Thus, providing new insights into the different mechanisms utilized by the different IgG receptors to effect their functions.

High-density oligonucleotide microarray is a promising approach for high throughput analysis. It has been extensively used in many areas of biomedical research. Immunoglobulin E (IgE) mediated allergic response (type-1 hypersensitivity) is one of the most powerful reactions of the immune system. Tissue Mast Cells (MCs) and circulating basophils are the major effector cells in these reactions. By dissecting the regulatory circuitry of mast cells by analyzing the genome wide effects of antigen stimulation triggered by FcεRI, offers a potential for finding novel genes as ‘targets’ for therapeutic intervention. In this work, we tried to study the gene expression pattern in IgE sensitized and FcεRI cross linked cord blood derived MCs using one of the latest techniques, high density oligonucleotide expression probe array (HG-Focus array, Gene Chip, Affymetrix, Santa Clara, CA). Microarray hybridization of RNA from cord blood derived MCs revealed coordinated changes in gene expression in response to IgE stimulation and receptor cross linking at different time points. Among the most prominent findings, we observed 2 to 32-fold increased expression of different transcripts. Real-time PCR confirmed reliability of microarray data. This enabled us to classify and cluster genes by functional families as well as to understand known genes in signaling pathways. These results defined a list of primary candidates for finding novel genes as ‘targets’ for therapeutic intervention.