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The human prolyl-4-hydroxylase domain (PHD) proteins 1–3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways. ISSN:1362-4962 ISSN:0301-5610

The timing of daily circadian behavior can be highly variable among different individuals, and twin studies have suggested that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment, for example, 22-h instead of 24-h, which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. We found that transient exposure of mice to such lighting stably altered global transcription in the suprachiasmatic nucleus (SCN) of the hypothalamus (the master clock tissue regulating circadian behavior in mammals). In parallel, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN that correlated with these changes. Behavioral, transcriptional and DNA methylation changes were reversible after prolonged re-entrainment to 24-h d. Notably, infusion of a methyltransferase inhibitor to the SCN suppressed period changes. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity.

Tissue-resident macrophages constitute heterogeneous populations with unique functions and distinct gene-expression signatures. While it has been established that they originate mostly from embryonic progenitor cells, the signals that induce a characteristic tissue-specific differentiation program remain unknown. We found that the nuclear receptor PPAR-γ determined the perinatal differentiation and identity of alveolar macrophages (AMs). In contrast, PPAR-γ was dispensable for the development of macrophages located in the peritoneum, liver, brain, heart, kidneys, intestine and fat. Transcriptome analysis of the precursors of AMs from newborn mice showed that PPAR-γ conferred a unique signature, including several transcription factors and genes associated with the differentiation and function of AMs. Expression of PPAR-γ in fetal lung monocytes was dependent on the cytokine GM-CSF. Therefore, GM-CSF has a lung-specific role in the perinatal development of AMs through the induction of PPAR-γ in fetal monocytes.

The protozoan Giardia lamblia has a minimized organelle repertoire, and most strikingly lacks a classical stacked Golgi apparatus. Nevertheless, Giardia trophozoites constitutively secrete variant surface proteins, and dramatically increase the volume of protein secretion during differentiation to cysts. Eukaryotic cells have evolved an elaborate system for quality control (QC) of protein folding and capacity in the endoplasmic reticulum (ER). Upon ER-overload, an unfolded protein response (UPR) is triggered on transcriptional/translational level aiming at alleviating ER stress. In Giardia, a minimized secretory machinery and absence of glycan-dependent QC suggests that a genetically conserved UPR (or functional equivalent) to cope with insults to the secretory system has been eliminated. We tested this hypothesis of UPR elimination by profiling the transcriptional response during induced ER-folding stress. We show that on the contrary, ER-folding stress triggers a stressor-specific, ER-directed response with upregulation of only ~ 30 genes, with different kinetics and scope compared with the UPR of other eukaryotes. Computational genomics revealed conserved cis-acting motifs in upstream regions of responder genes capable of stressor-specific gene regulation in transfected cells. Interestingly, the sensors/transducers of folding stress, well conserved in model eukaryotes, are absent in Giardia suggesting the presence of a novel version of this essential eukaryotic function.

Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients' miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-β and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states.

As a revolutionary technology for life sciences, RNA-seq has many applications and the computation pipeline has also many variations. Here, we describe a protocol to perform RNA-seq data analysis where the aim is to identify differentially expressed genes in comparisons of two conditions. The protocol follows the recently published RNA-seq data analysis best practice and applies quality checkpoints throughout the analysis to ensure reliable data interpretation. It is written to help new RNA-seq users to understand the basic steps necessary to analyze an RNA-seq dataset properly. An extension of the protocol has been implemented as automated workflows in the R package ezRun, available also in the data analysis framework SUSHI, for reliable, repeatable, and easily interpretable analysis results.

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is fundamental for cell proliferation and survival and is frequently altered and activated in neoplasia, including carcinomas of the lung. In this study, we investigated the potential of targeting the catalytic class IA PI3K isoforms in small cell lung cancer (SCLC), which is the most aggressive of all lung cancer types. Experimental Design: The expression of PI3K isoforms in patient specimens was analyzed. The effects on SCLC cell survival and downstream signaling were determined following PI3K isoform inhibition by selective inhibitors or downregulation by siRNA. Results: Overexpression of the PI3K isoforms p110-α and p110-β and the antiapoptotic protein Bcl-2 was shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K p110-α with RNA interference or selective pharmacologic inhibitors resulted in strongly affected cell proliferation of SCLC cells in vitro and in vivo, whereas targeting p110-β was less effective. Inhibition of p110-α also resulted in increased apoptosis and autophagy, which was accompanied by decreased phosphorylation of Akt and components of the mTOR pathway, such as the ribosomal S6 protein, and the eukaryotic translation initiation factor 4E-binding protein 1. A DNA microarray analysis revealed that p110-α inhibition profoundly affected the balance of pro- and antiapoptotic Bcl-2 family proteins. Finally, p110-α inhibition led to impaired SCLC tumor formation and vascularization in vivo. Conclusion: Together our data show the key involvement of the PI3K isoform p110-α in the regulation of multiple tumor-promoting processes in SCLC. Clin Cancer Res; 19(1); 96–105. ©2012 AACR.

Post-translational chromatin modifications are an important regulatory mechanism in light signalling and circadian clock function. The regulation of diurnal transcript level changes requires fine-tuning of the expression of generally active genes depending on the prevailing environmental conditions. We investigated the association of histone modifications H3K4me3, H3K9ac, H3K9me2, H3S10p, H3K27ac, H3K27me3 and H3S28p with diurnal changes in transcript expression using chromatin immunoprecipitations followed by sequencing (ChIP-Seq) in fully expanded leaves 6 of Arabidopsis thaliana grown in short-day optimal and water-deficit conditions. We identified a differential H3K9ac, H3K27ac and H3S28p signature between end-of-day and end-of-night that is correlated with changes in diurnal transcript levels. Genes with this signature have particular over-represented promoter elements and encode proteins that are significantly enriched for transcription factors, circadian clock and starch catabolic process. Additional activating modifications were prevalent in optimally watered (H3S10p) and in water-deficit (H3K4me3) plants. The data suggest a mechanism for diurnal transcript level regulation in which reduced binding of repressive transcription factors facilitates activating H3K9ac, H3K27ac and H3S28p chromatin modifications. The presence of activating chromatin modification patterns on genes only at times of the day when their expression is required can explain why some genes are differentially inducible during the diurnal cycle., Plant, Cell & Environment, 39 (11), ISSN:0140-7791, ISSN:1365-3040

Post-translational chromatin modifications are an important regulatory mechanism in light signalling and circadian clock function. The regulation of diurnal transcript level changes requires fine-tuning of the expression of generally active genes depending on the prevailing environmental conditions. We investigated the association of histone modifications H3K4me3, H3K9ac, H3K9me2, H3S10p, H3K27ac, H3K27me3 and H3S28p with diurnal changes in transcript expression using chromatin immunoprecipitations followed by sequencing (ChIP-Seq) in fully expanded leaves 6 of Arabidopsis thaliana grown in short-day optimal and water-deficit conditions. We identified a differential H3K9ac, H3K27ac and H3S28p signature between end-of-day and end-of-night that is correlated with changes in diurnal transcript levels. Genes with this signature have particular over-represented promoter elements and encode proteins that are significantly enriched for transcription factors, circadian clock and starch catabolic process. Additional activating modifications were prevalent in optimally watered (H3S10p) and in water-deficit (H3K4me3) plants. The data suggest a mechanism for diurnal transcript level regulation in which reduced binding of repressive transcription factors facilitates activating H3K9ac, H3K27ac and H3S28p chromatin modifications. The presence of activating chromatin modification patterns on genes only at times of the day when their expression is required can explain why some genes are differentially inducible during the diurnal cycle.

We have shown recently that the hybridization of short oligonucleotides can be studied in a zero-mode waveguide nanostructure (ZMW) chip using a modified DNA sequencer.[1] Here we present an extension of this method enabling the parallel measurement of kinetic constants of a large number of hybridization reactions on a single chip. This can be achieved by immobilization of a mixture of oligonucleotides, which leads to a statistical and random distribution of single molecules in the 150’000 ZMWs of a SMRT™ cell. This setup is comparable to a classical microarray with ZMWs in place of spots but unknown allocation of probes. The probe surface density is reduced by a factor of ~1010 allowing the study of hybridization in the absence of interactions with neighboring probes. Hybridization with a dye labelled oligonucleotide results in trains of fluorescence pulses from which interpulse durations (IPDs) and pulse widths (PWs) can be extracted. Since the identity of a probe in a ZMW is unknown, the immobilized oligonucleotide is sequenced in a subsequent step. After mapping the fluorescence traces to the sequence, the association and dissociation rate constant for each oligonucleotide can be calculated. By selecting suitable probes, the method can be used to determine rate constants of hybridization for a large number of mismatch oligonucleotides in a single measurement and at single-molecule level. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Purpose: The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM). Experimental Design: The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice. Results: A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors. Conclusions: An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach. Clin Cancer Res; 18(17); 4646–56. ©2012 AACR.

Glacier forefield environments are exposed to extreme and fluctuating climatic and nutritional conditions. The high diversity of free-living diazotrophic communities found in these environments indicates that nitrogen fixers are able to efficiently cope with such conditions. In this study, a nifH microarray was used to monitor changes in diazotrophic populations in the field over a season, in the presence or absence of plants and in 2 glacier forefields characterized by a different bedrock type (siliceous or calcareous), as well as at different temperatures (10 °C, 15 °C) and under different nitrogen fertilization regimes (0, 10, 40 kg N·ha–1·year–1) in laboratory systems. Population structures responded highly dynamically to environmental changes. Plant presence had the strongest impact, which decreased toward the end of the season and with high amounts of nitrogen fertilization. Temperature and nitrogen fertilization increases indirectly affected diazotrophic communities through their positive impact on plant growth. These results indicate strong carbon limitation in young glacier forefield soils. Phylotypes related to the genus Methylocystis strongly responded to environmental variations. These methanotrophic microorganisms, which are able to retrieve nitrogen and carbon from the atmospheric pool, are particularly adapted to the extreme nutritional conditions found in glacier forefields.

Rampant activity of the hypoxia-inducible factor (HIF)-1 in cancer is frequently associated with the malignant progression into a harder-to-treat, increasingly aggressive phenotype. Clearly, anti-HIF strategies in cancer cells are of considerable clinical interest. One way to fine-tune, or inhibit, HIF's transcriptional outflow independently of hydroxylase activities could be through competing transcription factors. A CACGTG-binding activity in human hepatoma cells was previously found to restrict HIF's access to hypoxia response cis-elements (HRE) in a Daphnia globin gene promoter construct (phb2). The CACGTG factor, and its impact on hypoxia-responsive human genes, was analyzed in this study by genome-wide computational scans as well as gene-specific quantitative PCR, reporter and DNA-binding assays in hepatoma (Hep3B), cervical carcinoma (HeLa), and breast carcinoma (MCF7) cells. Among six basic helix-loop-helix transcription factors known to target CACGTG palindromes, we identified upstream stimulatory factor (USF)-1/2 as predominant phb2 CACGTG constituents in Hep3B, HeLa, and MCF7 cells. Human genes with adjacent or overlapping HRE and CACGTG motifs included with lactate dehydrogenase A (LDHA) and Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3) hypoxia-induced HIF-1 targets. Parallel recruitment of HIF-1α and USF1/2a to the respective promoter chromatin was verified for all cell lines investigated. Mutual complementing (LDHA) or moderating (BNIP3) cross-talk was seen upon overexpression or silencing of HIF-1α and USF1/2a. Distinct (LDHA) or overlapping (BNIP3) promoter-binding sites for HIF-1 and USFs were subsequently characterized. We propose that, depending on abundance or activity of its protein constituents, O2-independent USF signaling can function to fine-tune or interfere with HIF-mediated transcription in cancer cells. Mol Cancer Res; 9(11); 1520–36. ©2011 AACR.

International audience; Summary Histone modification is an important mechanism regulating both gene expression and the establishment and maintenance of cellular phenotypes during development. Regulation of histone acetylation via histone acetylases and deacetylases (HDACs) appears to be particularly crucial in determining gene expression patterns. In this study we explored the effect of HDAC inhibition on the life cycle of the human pathogen Giardia lamblia, a highly reduced parasitic protozoan characterized by minimized cellular processes. We found that the HDAC inhibitor FR235222 increased the level of histone acetylation and induced transcriptional regulation of approximately 2% of genes in proliferating and encysting parasites. In addition, our analyses showed that the levels of histone acetylation decreased during differentiation into cysts, the infective stage of the parasite. Importantly, FR235222 treatment during encystation reversed this histone hypo-acetylation and potently blocked the formation of cysts. These results provide the first direct evidence for epigenetic regulation of gene expression in this simple eukaryote. This suggests that regulation of histone acetylation is involved in the control of Giardia stage differentiation, and identifies epigenetic mechanisms as a promising target to prevent Giardia transmission.

Functional microarrays are powerful tools that allow the parallel detection of multiple strains at the species level and therefore to rapidly obtain information on microbial communities in the environment. However, the design of suitable probes is prone to uncertainties, as it is based so far on in silico predictions including weighted mismatch number and Gibbs free-energy values. This study describes the experimental selection of probes targeting subsequences of the nifH gene to study the community structure of diazotrophic populations present in Damma glacier (Swiss Central Alps) forefield soils. Using the Geniom One in situ synthesis technology (Febit, Germany), 2727 in silico designed candidate probes were tested. A total of 946 specific probes were selected and validated. This probe set covered a large diversity of the NifH phylotypes (35 out of the 45) found in the forefield. Hybridization predictors were tested statistically. Gibbs free-energy value for probe-target binding gave the best prediction for hybridization efficiency, while the weighted mismatch number was not significantly associated to probe specificity. In this study, we demonstrate that extensive experimental tests of probe-hybridization behaviour against sequences present in the studied environment remain a prerequisite for meaningful probe selection.

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia, an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia, Pandoraea, Ralstonia and Limnobacter. Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool Phylo- Detect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmiumcontaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.

Nuclear factor kappaB (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65-/- cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor alpha was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFalpha identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.

Production and excretion of acids are balanced to maintain systemic acid-base homeostasis. During metabolic acidosis (MA) excess acid accumulates and is removed from the body, a process achieved, at least in part, by increasing renal acid excretion. This acid-secretory process requires the concerted regulation of metabolic and transport pathways, which are only partially understood. Chronic MA causes also morphological remodeling of the kidney. Therefore, we characterized transcriptional changes in mammalian kidney during MA to gain insights into adaptive pathways. Total kidney RNA from control and 2- and 7-days NH4Cl treated mice was subjected to microarray gene profiling. We identified 4,075 transcripts significantly ( P < 0.05) regulated after 2 and/or 7 days of treatment. Microarray results were confirmed by qRT-PCR. Analysis of candidate genes revealed that a large group of regulated transcripts was represented by different solute carrier transporters, genes involved in cell growth, proliferation, apoptosis, water homeostasis, and ammoniagenesis. Pathway analysis revealed that oxidative phosphorylation was the most affected pathway. Interestingly, the majority of acutely regulated genes after 2 days, returned to normal values after 7 days suggesting that adaptation had occurred. Besides these temporal changes, we detected also differential regulation of selected genes (SNAT3, PEPCK, PDG) between early and late proximal tubule. In conclusion, the mammalian kidney responds to MA by temporally and spatially altering the expression of a large number of genes. Our analysis suggests that many of these genes may participate in various processes leading to adaptation and restoration of normal systemic acid-base and electrolyte homeostasis.

Background Kidney renal clear cell carcinoma (KIRC) is a type of cancer that is resistant to chemotherapy and radiotherapy and has limited treatment possibilities. Large-scale molecular profiling of KIRC tumors offers a great potential to uncover the genetic and epigenetic changes underlying this disease and to improve the clinical management of KIRC patients. However, in practice the clinicians and researchers typically focus on single-platform molecular data or on a small set of genes. Using molecular and clinical data of over 500 patients, we have systematically studied which type of molecular data is the most informative in predicting the clinical outcome of KIRC patients, as a standalone platform and integrated with clinical data. Results We applied different computational approaches to preselect on survival-predictive genomic markers and evaluated the usability of mRNA/miRNA/protein expression data, copy number variation (CNV) data and DNA methylation data in predicting survival of KIRC patients. Our analyses show that expression and methylation data have statistically significant predictive powers compared to a random guess, but do not perform better than predictions on clinical data alone. However, the integration of molecular data with clinical variables resulted in improved predictions. We present a set of survival associated genomic loci that could potentially be employed as clinically useful biomarkers. Conclusions Our study evaluates the survival prediction of different large-scale molecular data of KIRC patients and describes the prognostic relevance of such data over clinical-variable-only models. It also demonstrates the survival prognostic importance of methylation alterations in KIRC tumors and points to the potential of epigenetic modulators in KIRC treatment. Reviewers An extended abstract of this research paper was selected for the CAMDA Satellite Meeting to ISMB 2015 by the CAMDA Programme Committee. The full research paper then underwent one round of Open Peer Review under a responsible CAMDA Programme Committee member, Djork-Arné Clevert, PhD (Bayer AG, Germany). Open Peer Review was provided by Martin Otava, PhD (Janssen Pharmaceutica, Belgium) and Hendrik Luuk, PhD (The Centre for Disease Models and Biomedical Imaging, University of Tartu, Estonia). The Reviewer comments section shows the full reviews and author responses., Biology Direct, 11, ISSN:1745-6150

Current methods for the determination of molecular interactions are widely used in the analytical sciences. To identify new methods, we investigated as a model system the hybridisation of a short 7 nt oligonucleotide labelled with, structurally, very similar cyanine dyes CY3 and DY-547, respectively, to a 34 nt oligonucleotide probe immobilised in a zero-mode waveguide (ZMW) nanostructure. Using a modified commercial off-the-shelf DNA sequencer, we established the principles to measure biomolecular interactions at the single-molecule level. Kinetic data were obtained from trains of fluorescence pulses, allowing the calculation of association and dissociation rate constants (k on, k off). For the 7mer labelled with the positively charged CY3 dye, k on and k off are ~3 larger and ~2 times smaller, respectively, compared with the oligonucleotide labelled with negatively charged DY-547 dye. The effect of neighbouring molecules lacking the 7nt binding sequence on single-molecule rate constants is small. The association rate constants is reduced by only 20–35%. Hybrid dissociation is not affected, since as a consequence of the experimental design, rebinding cannot take place. Results of single-molecule experiments were compared with data obtained from surface plasmon resonance (SPR) performed under comparable conditions. A good correlation for the association rate constants within a factor of 1.5 was found. Dissociation rate constants are smaller by a factor of 2–3 which we interpreted as a result of rebinding to neighbouring probes. Results of SPR measurements tend to systematically underestimate dissociation rate constants. The amount of this deviation depends on the association rate constant and the surface probe density. As a consequence, it is recommended to work at low probe densities to keep this effect small.

The web application MAGMA provides a simple and intuitive interface to identify differentially expressed genes from two-channel microarray data. While the underlying algorithms are not superior to those of similar web applications, MAGMA is particularly user friendly and can be used without prior training. The user interface guides the novice user through the most typical microarray analysis workflow consisting of data upload, annotation, normalization and statistical analysis. It automatically generates R-scripts that document MAGMA's entire data processing steps, thereby allowing the user to regenerate all results in his local R installation. The implementation of MAGMA follows the model-view-controller design pattern that strictly separates the R-based statistical data processing, the web-representation and the application logic. This modular design makes the application flexible and easily extendible by experts in one of the fields: statistical microarray analysis, web design or software development. State-of-the-art Java Server Faces technology was used to generate the web interface and to perform user input processing. MAGMA's object-oriented modular framework makes it easily extendible and applicable to other fields and demonstrates that modern Java technology is also suitable for rather small and concise academic projects. MAGMA is freely available at www.magma-fgcz.uzh.ch.

MicroRNA miR-199a-5p impairs tight junction formation, leading to increased urothelial permeability in bladder pain syndrome. Now, using transcriptome analysis in urothelial TEU-2 cells, we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF, and WNT signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder, and we altered its levels in bladder smooth muscle cells (SMCs) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size, and up-regulated miR-199a-5p targets, including WNT2. Overexpression of WNT2 protein or treating SMCs with recombinant WNT2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of WNT2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM α-actin, and SM myosin heavy chain mRNA and protein levels. These changes as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of the smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor A downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of WNT-dependent inhibitory Krüppel-like transcription factor 4 in miR-199a-5p-overexpressing cells. In contrast, Krüppel-like transcription factor 4 was induced in antimiR-expressing cells following the activation of WNT2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the WNT2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, which is relevant for organ remodeling., Journal of Biological Chemistry, 290 (11), ISSN:0021-9258, ISSN:1083-351X