Network assessment of demethylation treatment in melanoma: Differential transcriptome-methylome and antigen profile signatures.

BACKGROUND: In melanoma, like in other cancers, both genetic alterations and epigenetic underlie the metastatic process. These effects are usually measured by changes in both methylome and transcriptome profiles, whose cross-correlation remains uncertain. We aimed to assess at systems scale the significance of epigenetic treatment in melanoma cells with different metastatic potential. METHODS AND FINDINGS: Treatment by DAC demethylation with 5-Aza-2'-deoxycytidine of two melanoma cell lines endowed with different metastatic potential, SKMEL-2 and HS294T, was performed and high-throughput coupled RNA-Seq and RRBS-Seq experiments delivered differential profiles (DiP) of both transcriptomes and methylomes. Methylation levels measured at both TSS and gene body were studied to inspect correlated patterns with wide-spectrum transcript abundance levels quantified in both protein coding and non-coding RNA (ncRNA) regions. The DiP were then mapped onto standard bio-annotation sources (pathways, biological processes) and network configurations were obtained. The prioritized associations for target identification purposes were expected to elucidate the reprogramming dynamics induced by the epigenetic therapy. The interactomic connectivity maps of each cell line were formed to support the analysis of epigenetically re-activated genes. i.e. those supposedly silenced by melanoma. In particular, modular protein interaction networks (PIN) were used, evidencing a limited number of shared annotations, with an example being MAPK13 (cascade of cellular responses evoked by extracellular stimuli). This gene is also a target associated to the PANDAR ncRNA, therapeutically relevant because of its aberrant expression observed in various cancers. Overall, the non-metastatic SKMEL-2 map reveals post-treatment re-activation of a richer pathway landscape, involving cadherins and integrins as signatures of cell adhesion and proliferation. Relatively more lncRNAs were also annotated, indicating more complex regulation patterns in view of target identification. Finally, the antigen maps matched to DiP display other differential signatures with respect to the metastatic potential of the cell lines. In particular, as demethylated melanomas show connected targets that grow with the increased metastatic potential, also the potential target actionability seems to depend to some degree on the metastatic state. However, caution is required when assessing the direct influence of re-activated genes over the identified targets. In light of the stronger treatment effects observed in non-metastatic conditions, some limitations likely refer to in silico data integration tools and resources available for the analysis of tumor antigens. CONCLUSION: Demethylation treatment strongly affects early melanoma progression by re-activating many genes. This evidence suggests that the efficacy of this type of therapeutic intervention is potentially high at the pre-metastatic stages. The biomarkers that can be assessed through antigens seem informative depending on the metastatic conditions, and networks help to elucidate the assessment of possible targets actionability.

Simulated Microgravity Impairs Cardiac Autonomic Neurogenesis from Neural Crest Cells.

Microgravity-induced alterations in the autonomic nervous system (ANS) contribute to derangements in both the mechanical and electrophysiological function of the cardiovascular system, leading to severe symptoms in humans following space travel. Because the ANS forms embryonically from neural crest (NC) progenitors, we hypothesized that microgravity can impair NC-derived cardiac structures. Accordingly, we conducted in vitro simulated microgravity experiments employing NC genetic lineage tracing in mice with cKitCreERT2/+, Isl1nLacZ, and Wnt1-Cre reporter alleles. Inducible fate mapping in adult mouse hearts and pluripotent stem cells (iPSCs) demonstrated reduced cKitCreERT2/+-mediated labeling of both NC-derived cardiomyocytes and autonomic neurons (P < 0.0005 vs. controls). Whole transcriptome analysis, suggested that this effect was associated with repressed cardiac NC- and upregulated mesoderm-related gene expression profiles, coupled with abnormal bone morphogenetic protein (BMP)/transforming growth factor beta (TGF-ß) and Wnt/ß-catenin signaling. To separate the manifestations of simulated microgravity on NC versus mesodermal-cardiac derivatives, we conducted Isl1nLacZ lineage analyses, which indicated an approximately 3-fold expansion (P < 0.05) in mesoderm-derived Isl-1+ pacemaker sinoatrial nodal cells; and an approximately 3-fold reduction (P < 0.05) in cardiac NC-derived ANS cells, including sympathetic nerves and Isl-1+ cardiac ganglia. Finally, NC-specific fate mapping with a Wnt1-Cre reporter iPSC model of murine NC development confirmed that simulated microgravity directly impacted the in vitro development of cardiac NC progenitors and their contribution to the sympathetic and parasympathetic innervation of the iPSC-derived myocardium. Altogether, these findings reveal an important role for gravity in the development of NCs and their postnatal derivatives, and have important therapeutic implications for human space exploration, providing insights into cellular and molecular mechanisms of microgravity-induced cardiomyopathies/channelopathies.

Induction of endogenous retroelements as a potential mechanism for mouse-specific drug-induced carcinogenicity.

A number of chemical compounds have been shown to induce liver tumors in mice but not in other species. While several mechanisms for this species-specific tumorigenicity have been proposed, no definitive mechanism has been established. We examined the effects of the nongenotoxic rodent hepatic carcinogen, WY-14,643, in male mice from a high liver tumor susceptible strain (C3H/HeJ), and from a low tumor susceptible strain (C57BL/6). WY-14,643, a PPARα activator induced widespread increases in the expression of some endogenous retroelements, namely members of LTR and LINE elements in both strains. The expression of a number of known retroviral defense genes was also elevated. We also demonstrated that basal immune-mediated viral defense was elevated in C57BL/6 mice (the resistant strain) and that WY-14,643 further activated those immuno-defense processes. We propose that the previously reported >100X activity of retroelements in mice drives mouse-specific tumorigenicity. We also propose that C57BL/6's competent immune to retroviral activation allows it to remove cells before the activation of these elements can result in significant chromosomal insertions and mutation. Finally, we showed that WY-14,643 treatment induced gene signatures of DNA recombination in the sensitive C3H/HeJ strain.

The landscape of BRAF transcript and protein variants in human cancer.

BACKGROUND: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood. RESULTS: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation. CONCLUSIONS: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data.

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein-protein interaction networks, other connectivity paths were identified between proteins already investigated in studies on disorders, such as Parkinson, Down syndrome, Huntington disease, and Alzheimer. Our findings suggest the importance of reusing gene expression data by meta-analysis approaches.

Non-coding RNAs profiling in head and neck cancers.

The majority of studies on human cancers published to date focus on coding genes. More recently, however, non-coding RNAs (ncRNAs) are gaining growing recognition as important regulatory components. Here we characterise the ncRNA landscape in 442 head and neck squamous cell carcinomas (HNSCs) from the cancer genome atlas (TCGA). HNSCs represent an intriguing case to study the potential role of ncRNA as a function of viral presence, especially as HPV is potentially oncogenic. Thus, we identify HPV16-positive (HPV16+) and HPV-negative (HPV-) tumours and study the expression of ncRNAs on both groups. Overall, the ncRNAs comprise 36% of all differentially expressed genes, with antisense RNAs being the most represented ncRNA type (12.6%). Protein-coding genes appear to be more frequently downregulated in tumours compared with controls, whereas ncRNAs show significant upregulation in tumours, especially in HPV16+ tumours. Overall, expression of pseudogenes, antisense and short RNAs is elevated in HPV16+ tumours, while the remaining long non-coding RNA types are more active in all HNSC tumours independent of HPV status. In addition, we identify putative regulatory targets of differentially expressed ncRNAs. Among these 'targets' we find several well-established oncogenes, tumour suppressors, cytokines, growth factors and cell differentiation genes, which indicates the potential involvement of ncRNA in the control of these key regulators as a direct consequence of HPV oncogenic activity. In conclusion, our findings establish the ncRNAs as crucial transcriptional components in HNSCs. Our results display the great potential for the study of ncRNAs and the role they have in human cancers.

Integrative analysis of cancer imaging readouts by networks.

Cancer is a multifactorial and heterogeneous disease. The corresponding complexity appears at multiple levels: from the molecular and the cellular constitution to the macroscopic phenotype, and at the diagnostic and therapeutic management stages. The overall complexity can be approximated to a certain extent, e.g. characterized by a set of quantitative phenotypic observables recorded in time-space resolved dimensions by using multimodal imaging approaches. The transition from measures to data can be made effective through various computational inference methods, including networks, which are inherently capable of mapping variables and data to node- and/or edge-valued topological properties, dynamic modularity configurations, and functional motifs. We illustrate how networks can integrate imaging data to explain cancer complexity, and assess potential pre-clinical and clinical impact.

Identification of potential therapeutic targets in a model of neuropathic pain.

Neuropathic pain (NP) is caused by damage to the nervous system, resulting in dysfunction and aberrant pain. The cellular functions (e.g., peripheral neuron spinal cord innervation, neuronal excitability) associated with NP often develop over time and are likely associated with gene expression changes. Gene expression studies on the cells involved in NP (e.g., sensory dorsal root ganglion neurons) are publically available; the mining of these studies may enable the identification of novel targets and the subsequent development of therapies that are essential for improving quality of life for the millions of individuals suffering with NP. Here we analyzed a publically available microarray dataset (GSE30165) in order to identify new RNAs (e.g., messenger RNA (mRNA) isoforms and non-coding RNAs) underlying NP. GSE30165 profiled gene expression in dorsal root ganglion neurons (DRG) and in sciatic nerve (SN) after resection, a NP model. Gene ontological analysis shows enrichment for sensory and neuronal processes. Protein network analysis demonstrates DRG upregulated genes typical to an injury and NP response. Of the top changing genes, 34 and 36% are associated with more than one protein coding isoform in the DRG and SN, respectively. The majority of genes are receptor and enzymes. We identified 15 long non-coding RNAs (lncRNAs) targeting these genes in LNCipedia.org, an online comprehensive lncRNA database. These RNAs represent new therapeutic targets for preventing NP development and this approach demonstrates the feasibility of data reanalysis for their identification.

Comparative analysis of genome sequences from four strains of the Buchnera aphidicola Mp endosymbion of the green peach aphid, Myzus persicae.

BACKGROUND: Myzus persicae, the green peach aphid, is a polyphagous herbivore that feeds from hundreds of species of mostly dicot crop plants. Like other phloem-feeding aphids, M. persicae rely on the endosymbiotic bacterium, Buchnera aphidicola (Buchnera Mp), for biosynthesis of essential amino acids and other nutrients that are not sufficiently abundant in their phloem sap diet. Tobacco-specialized M. persicae are typically red and somewhat distinct from other lineages of this species. To determine whether the endosymbiotic bacteria of M. persicae could play a role in tobacco adaptation, we sequenced the Buchnera Mp genomes from two tobacco-adapted and two non-tobacco M. persicae lineages. RESULTS: With a genome size of 643.5 kb and 579 predicted genes, Buchnera Mp is the largest Buchnera genome sequenced to date. No differences in gene content were found between the four sequenced Buchnera Mp strains. Compared to Buchnera APS from the well-studied pea aphid, Acyrthosiphon pisum, Buchnera Mp has 21 additional genes. These include genes encoding five enzymes required for biosynthesis of the modified nucleoside queosine, the heme pathway enzyme uroporphyrinogen III synthase, and asparaginase. Asparaginase, which is also encoded by the genome of the aphid host, may allow Buchnera Mp to synthesize essential amino acids from asparagine, a relatively abundant phloem amino acid. CONCLUSIONS: Together our results indicate that the obligate intracellular symbiont Buchnera aphidicola does not contribute to the adaptation of Myzus persicae to feeding on tobacco.

MicroRNA-20a constrains p300-driven myocardial angiogenic transcription by direct targeting of p300.

OBJECTIVE: To characterize downstream effectors of p300 acetyltransferase in the myocardium. BACKGROUND: Acetyltransferase p300 is a central driver of the hypertrophic response to increased workload, but its biological targets and downstream effectors are incompletely known. METHODS AND RESULTS: Mice expressing a myocyte-restricted transgene encoding acetyltransferase p300, previously shown to develop spontaneous hypertrophy, were observed to undergo robust compensatory blood vessel growth together with increased angiogenic gene expression. Chromatin immunoprecipitation demonstrated binding of p300 to the enhancers of the angiogenic regulators Angpt1 and Egln3. Interestingly, p300 overexpression in vivo was also associated with relative upregulation of several members of the anti-angiogenic miR-17∼92 cluster in vivo. Confirming this finding, both miR-17-3p and miR-20a were upregulated in neonatal rat ventricular myocytes following adenoviral transduction of p300. Relative expression of most members of the 17∼92 cluster was similar in all 4 cardiac chambers and in other organs, however, significant downregulation of miR-17-3p and miR-20a occurred between 1 and 8 months of age in both wt and tg mice. The decline in expression of these microRNAs was associated with increased expression of VEGFA, a validated miR-20a target. In addition, miR-20a was demonstrated to directly repress p300 expression through a consensus binding site in the p300 3'UTR. In vivo transduction of p300 resulted in repression both of p300 and of p300-induced angiogenic transcripts. CONCLUSION: p300 drives an angiogenic transcription program during hypertrophy that is fine-tuned in part through direct repression of p300 by miR-20a.

Viral expression associated with gastrointestinal adenocarcinomas in TCGA high-throughput sequencing data.

BACKGROUND: Up to 20% of cancers worldwide are thought to be associated with microbial pathogens, including bacteria and viruses. The widely used methods of viral infection detection are usually limited to a few a priori suspected viruses in one cancer type. To our knowledge, there have not been many broad screening approaches to address this problem more comprehensively. METHODS: In this study, we performed a comprehensive screening for viruses in nine common cancers using a multistep computational approach. Tumor transcriptome and genome sequencing data were available from The Cancer Genome Atlas (TCGA). Nine hundred fifty eight primary tumors in nine common cancers with poor prognosis were screened against a non-redundant database of virus sequences. DNA sequences from normal matched tissue specimens were used as controls to test whether each virus is associated with tumors. RESULTS: We identified human papilloma virus type 18 (HPV-18) and four human herpes viruses (HHV) types 4, 5, 6B, and 8, also known as EBV, CMV, roseola virus, and KSHV, in colon, rectal, and stomach adenocarcinomas. In total, 59% of screened gastrointestinal adenocarcinomas (GIA) were positive for at least one virus: 26% for EBV, 21% for CMV, 7% for HHV-6B, and 20% for HPV-18. Over 20% of tumors were co-infected with multiple viruses. Two viruses (EBV and CMV) were statistically significantly associated with colorectal cancers when compared to the matched healthy tissues from the same individuals (p = 0.02 and 0.03, respectively). HPV-18 was not detected in DNA, and thus, no association testing was possible. Nevertheless, HPV-18 expression patterns suggest viral integration in the host genome, consistent with the potentially oncogenic nature of HPV-18 in colorectal adenocarcinomas. The estimated counts of viral copies were below one per cell for all identified viruses and approached the detection limit. CONCLUSIONS: Our comprehensive screening for viruses in multiple cancer types using next-generation sequencing data clearly demonstrates the presence of viral sequences in GIA. EBV, CMV, and HPV-18 are potentially causal for GIA, although their oncogenic role is yet to be established.

Characteristics of cross-hybridization and cross-alignment of expression in pseudo-xenograft samples by RNA-Seq and microarrays.

BACKGROUND: Exploring stromal changes associated with tumor growth and development is a growing area of oncologic research. In order to study molecular changes in the stroma it is recommended to separate tumor tissue from stromal tissue. This is relevant to xenograft models where tumors can be small and difficult to separate from host tissue. We introduce a novel definition of cross-alignment/cross-hybridization to compare qualitatively the ability of high-throughput mRNA sequencing, RNA-Seq, and microarrays to detect tumor and stromal expression from mixed 'pseudo-xenograft' samples vis-à-vis genes and pathways in cross-alignment (RNA-Seq) and cross-hybridization (microarrays). Samples consisted of normal mouse lung and human breast cancer cells; these were combined in fixed proportions to create a titration series of 25% steps. Our definition identifies genes in a given species (human or mouse) with undetectable expression in same-species RNA but detectable expression in cross-species RNA. We demonstrate the comparative value of this method and discuss its potential contribution in cancer research. RESULTS: Our method can identify genes from either species that demonstrate cross-hybridization and/or cross-alignment properties. Surprisingly, the set of genes identified using a simpler and more common approach (using a 'pure' cross-species sample and calling all detected genes as 'crossers') is not a superset of the genes identified using our technique. The observed levels of cross-hybridization are relatively low: 5.3% of human genes detected in mouse, and 3.5% of mouse genes detected in human. Observed levels of cross-alignment are practically comparable to the levels of cross-hybridization: 6.5% of human genes detected in mouse, and 2.3% of mouse genes detected in human. We also observed a relatively high percentage of orthologs: 40.3% of cross-hybridizing genes, and 32.2% of cross-aligning genes.Normalizing the gene catalog to use Consensus Coding Sequence (CCDS) IDs (Genome Res 19:1316-1323, 2009), our results show that the observed levels of cross-hybridization are low: 2.7% of human CCDS IDs are detected in mouse, and 2.4% of mouse CCDS IDs are detected in human. Levels of cross-alignment using the RNA-Seq data are comparable for the mouse, 2.2% of mouse CCDS IDs detected in human, and 9.9% of human CCDS IDs detected in mouse. However, the lists of cross-aligning/cross-hybridizing genes contain many that are of specific interest to oncologic researchers. CONCLUSIONS: The conservative definition that we propose identifies genes in mouse whose expression can be attributed to human RNA, and vice versa, as well as revealing genes with cross-alignment/cross-hybridization properties which could not be identified using a simpler but more established approach. The overall percentage of genes affected by cross-hybridization/cross-alignment is small, but includes genes that are of interest to oncologic researchers. Which platform to use with mixed xenograft samples, microarrays or RNA-Seq, appears to be primarily a question of cost and whether the detection and measurement of expression of specific genes of interest are likely to be affected by cross-hybridization or cross-alignment.

MicroRNA expression in alpha and beta cells of human pancreatic islets.

microRNAs (miRNAs) play an important role in pancreatic development and adult ß-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and ß-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and ß-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in ß-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in ß-cells (ß-miRNAs). Bioinformatic analysis identified potential targets of ß-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by ß-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and ß-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

A clinical evaluation tool for SNP arrays, especially for autosomal recessive conditions in offspring of consanguineous parents.

PURPOSE: This report describes a fast online tool to accelerate and improve clinical interpretation of single nucleotide polymorphism array results for diagnostic purposes, when consanguinity or inbreeding is identified. METHODS: We developed a web-based program that permits entry of regions of homozygosity and, using OMIM, UCSC, and NCBI databases, retrieves genes within these regions as well as their associated autosomal recessive disorders. Relevant OMIM Clinical Synopses can be searched, using key clinical terms permitting further filtering for candidate genes and disorders. RESULTS: The tool aids the clinician by arriving at a short list of relevant candidate disorders, guiding the continued diagnostic work-up. Its efficacy is illustrated by presenting seven patients who were diagnosed using this tool. CONCLUSION: The online single nucleotide polymorphism array evaluation tool rapidly and systematically identifies relevant genes and associated conditions mapping to identified regions of homozygosity. The built-in OMIM clinical feature search allows the user to further filter to reach a short list of candidate conditions relevant for the diagnosis, making it possible to strategize more focused diagnostic testing. The tabulated results can be downloaded and saved to the desktop in an Excel format. Its efficacy is illustrated by providing a few clinical examples.Genet Med 2013:15(5):354-360.

Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets.

Nonspecific inflammation in the transplant microenvironment results in ß-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of ß-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca(2+) sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions.

GenSensor Suite: A Web-Based Tool for the Analysis of Gene and Protein Interactions, Pathways, and Regulation.

The GenSensor Suite consists of four web tools for elucidating relationships among genes and proteins. GenPath results show which biochemical, regulatory, or other gene set categories are over- or under-represented in an input list compared to a background list. All common gene sets are available for searching in GenPath, plus some specialized sets. Users can add custom background lists. GenInteract builds an interaction gene list from a single gene input and then analyzes this in GenPath. GenPubMed uses a PubMed query to identify a list of PubMed IDs, from which a gene list is extracted and queried in GenPath. GenViewer allows the user to query one gene set against another in GenPath. GenPath results are presented with relevant P- and q-values in an uncluttered, fully linked, and integrated table. Users can easily copy this table and paste it directly into a spreadsheet or document.

Heterogeneity in SDF-1 expression defines the vasculogenic potential of adult cardiac progenitor cells.

RATIONALE: The adult myocardium has been reported to harbor several classes of multipotent progenitor cells (CPCs) with tri-lineage differentiation potential. It is not clear whether c-kit+CPCs represent a uniform precursor population or a more complex mixture of cell types. OBJECTIVE: To characterize and understand vasculogenic heterogeneity within c-kit+presumptive cardiac progenitor cell populations. METHODS AND RESULTS: c-kit+, sca-1+ CPCs obtained from adult mouse left ventricle expressed stem cell-associated genes, including Oct-4 and Myc, and were self-renewing, pluripotent and clonogenic. Detailed single cell clonal analysis of 17 clones revealed that most (14/17) exhibited trilineage differentiation potential. However, striking morphological differences were observed among clones that were heritable and stable in long-term culture. 3 major groups were identified: round (7/17), flat or spindle-shaped (5/17) and stellate (5/17). Stellate morphology was predictive of vasculogenic differentiation in Matrigel. Genome-wide expression studies and bioinformatic analysis revealed clonally stable, heritable differences in stromal cell-derived factor-1 (SDF-1) expression that correlated strongly with stellate morphology and vasculogenic capacity. Endogenous SDF-1 production contributed directly to vasculogenic differentiation: both shRNA-mediated knockdown of SDF-1 and AMD3100, an antagonist of the SDF-1 receptor CXC chemokine Receptor-4 (CXCR4), reduced tube-forming capacity, while exogenous SDF-1 induced tube formation by 2 non-vasculogenic clones. CPCs producing SDF-1 were able to vascularize Matrigel dermal implants in vivo, while CPCs with low SDF-1 production were not. CONCLUSIONS: Clonogenic c-kit+, sca-1+ CPCs are heterogeneous in morphology, gene expression patterns and differentiation potential. Clone-specific levels of SDF-1 expression both predict and promote development of a vasculogenic phenotype via a previously unreported autocrine mechanism.

Identifying consensus disease pathways in Parkinson's disease using an integrative systems biology approach.

Parkinson's disease (PD) has had six genome-wide association studies (GWAS) conducted as well as several gene expression studies. However, only variants in MAPT and SNCA have been consistently replicated. To improve the utility of these approaches, we applied pathway analyses integrating both GWAS and gene expression. The top 5000 SNPs (p<0.01) from a joint analysis of three existing PD GWAS were identified and each assigned to a gene. For gene expression, rather than the traditional comparison of one anatomical region between sets of patients and controls, we identified differentially expressed genes between adjacent Braak regions in each individual and adjusted using average control expression profiles. Over-represented pathways were calculated using a hyper-geometric statistical comparison. An integrated, systems meta-analysis of the over-represented pathways combined the expression and GWAS results using a Fisher's combined probability test. Four of the top seven pathways from each approach were identical. The top three pathways in the meta-analysis, with their corrected p-values, were axonal guidance (p = 2.8E-07), focal adhesion (p = 7.7E-06) and calcium signaling (p = 2.9E-05). These results support that a systems biology (pathway) approach will provide additional insight into the genetic etiology of PD and that these pathways have both biological and statistical support to be important in PD.

MicroRNA signature of the human developing pancreas.

BACKGROUND: MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. RESULTS: The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase algorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. CONCLUSIONS: We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.