GPR15 in colon cancer development and anti-tumor immune responses.

Introduction: The chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies. Methods: In human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry. Results: Our analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to 'tumor-free' surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15 +-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden. Discussion: Our findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.

Integrating inflammatory biomarker analysis and artificial-intelligence-enabled image-based profiling to identify drug targets for intestinal fibrosis.

Intestinal fibrosis, often caused by inflammatory bowel disease, can lead to intestinal stenosis and obstruction, but there are no approved treatments. Drug discovery has been hindered by the lack of screenable cellular phenotypes. To address this, we used a scalable image-based morphology assay called Cell Painting, augmented with machine learning algorithms, to identify small molecules that could reverse the activated fibrotic phenotype of intestinal myofibroblasts. We then conducted a high-throughput small molecule chemogenomics screen of approximately 5,000 compounds with known targets or mechanisms, which have achieved clinical stage or approval by the FDA. By integrating morphological analyses and AI using pathologically relevant cells and disease-relevant stimuli, we identified several compounds and target classes that are potentially able to treat intestinal fibrosis. This phenotypic screening platform offers significant improvements over conventional methods for identifying a wide range of drug targets.

Characterizing miRNA-lncRNA Interplay.

Long noncoding RNAs (lncRNAs) are noncoding transcripts, usually longer than 200 nt, that constitute one of the largest and significantly heterogeneous RNA families. The annotation of lncRNAs and the characterization of their function is a constantly evolving field. LncRNA interplay with microRNAs (miRNAs) is thoroughly studied in several physiological and disease states. miRNAs are small noncoding RNAs (~22 nt) that posttranscriptionally regulate the expression of protein coding genes, through mRNA target cleavage, degradation or direct translational suppression. miRNAs can affect lncRNA half-life by promoting their degradation, or lncRNAs can act as miRNA "sponges," reducing miRNA regulatory effect on target mRNAs. This chapter outlines the miRNA-lncRNA interplay and provides hands-on methodologies for experimentally supported and in silico-guided analyses. The proposed techniques are a valuable asset to further understand lncRNA functions and can be appropriately adapted to become the backbone for further downstream analyses.

AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing.

AKT-phosphorylated IWS1 regulates alternative RNA splicing via a pathway that is active in lung cancer. RNA-seq studies in lung adenocarcinoma cells lacking phosphorylated IWS1, identified a exon 2-deficient U2AF2 splice variant. Here, we show that exon 2 inclusion in the U2AF2 mRNA is a cell cycle-dependent process that is regulated by LEDGF/SRSF1 splicing complexes, whose assembly is controlled by the IWS1 phosphorylation-dependent deposition of histone H3K36me3 marks in the body of target genes. The exon 2-deficient U2AF2 mRNA encodes a Serine-Arginine-Rich (RS) domain-deficient U2AF65, which is defective in CDCA5 pre-mRNA processing. This results in downregulation of the CDCA5-encoded protein Sororin, a phosphorylation target and regulator of ERK, G2/M arrest and impaired cell proliferation and tumor growth. Analysis of human lung adenocarcinomas, confirmed activation of the pathway in EGFR-mutant tumors and showed that pathway activity correlates with tumor stage, histologic grade, metastasis, relapse after treatment, and poor prognosis.

A Novel Microphysiological Colon Platform to Decipher Mechanisms Driving Human Intestinal Permeability.

BACKGROUND & AIMS: The limited availability of organoid systems that mimic the molecular signatures and architecture of human intestinal epithelium has been an impediment to allowing them to be harnessed for the development of therapeutics as well as physiological insights. We developed a microphysiological Organ-on-Chip (Emulate, Inc, Boston, MA) platform designed to mimic properties of human intestinal epithelium leading to insights into barrier integrity. METHODS: We combined the human biopsy-derived leucine-rich repeat-containing G-protein-coupled receptor 5-positive organoids and Organ-on-Chip technologies to establish a micro-engineered human Colon Intestine-Chip (Emulate, Inc, Boston, MA). We characterized the proximity of the model to human tissue and organoids maintained in suspension by RNA sequencing analysis, and their differentiation to intestinal epithelial cells on the Colon Intestine-Chip under variable conditions. Furthermore, organoids from different donors were evaluated to understand variability in the system. Our system was applied to understanding the epithelial barrier and characterizing mechanisms driving the cytokine-induced barrier disruption. RESULTS: Our data highlight the importance of the endothelium and the in vivo tissue-relevant dynamic microenvironment in the Colon Intestine-Chip in the establishment of a tight monolayer of differentiated, polarized, organoid-derived intestinal epithelial cells. We confirmed the effect of interferon-γ on the colonic barrier and identified reorganization of apical junctional complexes, and induction of apoptosis in the intestinal epithelial cells as mediating mechanisms. We show that in the human Colon Intestine-Chip exposure to interleukin 22 induces disruption of the barrier, unlike its described protective role in experimental colitis in mice. CONCLUSIONS: We developed a human Colon Intestine-Chip platform and showed its value in the characterization of the mechanism of action of interleukin 22 in the human epithelial barrier. This system can be used to elucidate, in a time- and challenge-dependent manner, the mechanism driving the development of leaky gut in human beings and to identify associated biomarkers.

DIANA-LncBase v3: indexing experimentally supported miRNA targets on non-coding transcripts.

DIANA-LncBase v3.0 (www.microrna.gr/LncBase) is a reference repository with experimentally supported miRNA targets on non-coding transcripts. Its third version provides approximately half a million entries, corresponding to ∼240 000 unique tissue and cell type specific miRNA-lncRNA pairs. This compilation of interactions is derived from the manual curation of publications and the analysis of >300 high-throughput datasets. miRNA targets are supported by 14 experimental methodologies, applied to 243 distinct cell types and tissues in human and mouse. The largest part of the database is highly confident, AGO-CLIP-derived miRNA-binding events. LncBase v3.0 is the first relevant database to employ a robust CLIP-Seq-guided algorithm, microCLIP framework, to analyze 236 AGO-CLIP-Seq libraries and catalogue ∼370 000 miRNA binding events. The database was redesigned from the ground up, providing new functionalities. Known short variant information, on >67,000 experimentally supported target sites and lncRNA expression profiles in different cellular compartments are catered to users. Interactive visualization plots, portraying correlations of miRNA-lncRNA pairs, as well as lncRNA expression profiles in a wide range of cell types and tissues, are presented for the first time through a dedicated page. LncBase v3.0 constitutes a valuable asset for ncRNA research, providing new insights to the understanding of the still widely unexplored lncRNA functions.

The combination of TPL2 knockdown and TNFα causes synthetic lethality via caspase-8 activation in human carcinoma cell lines.

Most normal and tumor cells are protected from tumor necrosis factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 kinase tumor progression locus-2 (TPL2) as a player contributing to the protection of a subset of tumor cell lines. The combination of TPL2 knockdown and TNFα gives rise to a synthetic lethality phenotype via receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent and -independent mechanisms. Whereas wild-type TPL2 rescues the phenotype, its kinase-dead mutant does not. Comparison of the molecular events initiated by small interfering RNA for TPL2 (siTPL2) ± TNFα in treatment-sensitive and -resistant lines revealed that the activation of caspase-8, downstream of miR-21-5p and cFLIP, is the dominant TPL2-dependent event. More important, comparison of the gene expression profiles of all of the tested cell lines results in the clustering of sensitive and resistant lines into distinct groups, providing proof of principle for the feasibility of generating a predictive tool for treatment sensitivity.

microCLIP super learning framework uncovers functional transcriptome-wide miRNA interactions.

Argonaute crosslinking and immunoprecipitation (CLIP) experiments are the most widely used high-throughput methodologies for miRNA targetome characterization. The analysis of Photoactivatable Ribonucleoside-Enhanced (PAR) CLIP methodology focuses on sequence clusters containing T-to-C conversions. Here, we demonstrate for the first time that the non-T-to-C clusters, frequently observed in PAR-CLIP experiments, exhibit functional miRNA-binding events and strong RNA accessibility. This discovery is based on the analysis of an extensive compendium of bona fide miRNA-binding events, and is further supported by numerous miRNA perturbation experiments and structural sequencing data. The incorporation of these previously neglected clusters yields an average of 14% increase in miRNA-target interactions per PAR-CLIP library. Our findings are integrated in microCLIP ( www.microrna.gr/microCLIP ), a cutting-edge framework that combines deep learning classifiers under a super learning scheme. The increased performance of microCLIP in CLIP-Seq-guided detection of miRNA interactions, uncovers previously elusive regulatory events and miRNA-controlled pathways.

DIANA-TarBase v8: a decade-long collection of experimentally supported miRNA-gene interactions.

DIANA-TarBase v8 (http://www.microrna.gr/tarbase) is a reference database devoted to the indexing of experimentally supported microRNA (miRNA) targets. Its eighth version is the first database indexing >1 million entries, corresponding to ∼670 000 unique miRNA-target pairs. The interactions are supported by >33 experimental methodologies, applied to ∼600 cell types/tissues under ∼451 experimental conditions. It integrates information on cell-type specific miRNA-gene regulation, while hundreds of thousands of miRNA-binding locations are reported. TarBase is coming of age, with more than a decade of continuous support in the non-coding RNA field. A new module has been implemented that enables the browsing of interactions through different filtering combinations. It permits easy retrieval of positive and negative miRNA targets per species, methodology, cell type and tissue. An incorporated ranking system is utilized for the display of interactions based on the robustness of their supporting methodologies. Statistics, pie-charts and interactive bar-plots depicting the database content are available through a dedicated result page. An intuitive interface is introduced, providing a user-friendly application with flexible options to different queries.

BUFET: boosting the unbiased miRNA functional enrichment analysis using bitsets.

BACKGROUND: A group of miRNAs can regulate a biological process by targeting genes involved in the process. The unbiased miRNA functional enrichment analysis is the most precise in silico approach to predict the biological processes that may be regulated by a given miRNA group. However, it is computationally intensive and significantly more expensive than its alternatives. RESULTS: We introduce BUFET, a new approach to significantly reduce the time required for the execution of the unbiased miRNA functional enrichment analysis. It derives its strength from the utilization of efficient bitset-based methods and parallel computation techniques. CONCLUSIONS: BUFET outperforms the state-of-the-art implementation, in regard to computational efficiency, in all scenarios (both single- and multi-core), being, in some cases, more than one order of magnitude faster.

A perspective on AKT 25-plus years after its discovery.

Since its discovery more than 25 years ago, the kinase AKT has become a central figure in cell signaling. We highlight some of the landmark findings in those 25 years that contributed to our understanding of the regulation and function of AKT in directing cellular processes and behavior. Future progress toward fully understanding the roles of AKT in cell, tissue, and organismal biology will depend on technological innovations and the combination of in-depth reductionist analyses with systems-based strategies.

Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP.

Aß peptide that accumulates in Alzheimer's disease brain, derives from proteolytic processing of the amyloid precursor protein (APP) that exists in three main isoforms derived by alternative splicing. The isoform APP695, lacking exons 7 and 8, is predominately expressed in neurons and abnormal neuronal splicing of APP has been observed in the brain of patients with Alzheimer's disease. Herein, we demonstrate that expression of the neuronal members of the ELAVL protein family (nELAVLs) correlate with APP695 levels in vitro and in vivo. Moreover, we provide evidence that nELAVLs regulate the production of APP695; by using a series of reporters we show that concurrent binding of nELAVLs to sequences located both upstream and downstream of exon 7 is required for its skipping, whereas nELAVL-binding to a highly conserved U-rich sequence upstream of exon 8, is sufficient for its exclusion. Finally, we report that nELAVLs block APP exon 7 or 8 definition by reducing the binding of the essential splicing factor U2AF65, an effect facilitated by the concurrent binding of AUF-1. Our study provides new insights into the regulation of APP pre-mRNA processing, supports the role for nELAVLs as neuron-specific splicing regulators and reveals a novel function of AUF1 in alternative splicing.

RNAcentral: a comprehensive database of non-coding RNA sequences.

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.

DIANA-TarBase and DIANA Suite Tools: Studying Experimentally Supported microRNA Targets.

microRNAs (miRNAs) are short non-coding RNAs (∼22 nts) present in animals, plants, and viruses. They are considered central post-transcriptional regulators of gene expression and are key components in a great number of physiological and pathological conditions. The accurate characterization of their targets is considered essential to a series of applications and basic or applied research settings. DIANA-TarBase (http://www.microrna.gr/tarbase) was initially launched in 2006. It is a reference repository indexing experimentally derived miRNA-gene interactions in different cell types, tissues, and conditions across numerous species. This unit focuses on the study of experimentally supported miRNA-gene interactions, as well as their functional interpretation through the use of available tools in the DIANA suite (http://www.microrna.gr). The proposed use-case scenarios are presented in protocols, describing how to utilize the DIANA-TarBase database and DIANA-microT-CDS server and perform miRNA-targeted pathway analysis with DIANA-miRPath-v3. All analyses are directly invoked or initiated from DIANA-TarBase. © 2016 by John Wiley & Sons, Inc.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. RESULTS: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. CONCLUSIONS: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.

DIANA-mirExTra v2.0: Uncovering microRNAs and transcription factors with crucial roles in NGS expression data.

Differential expression analysis (DEA) is one of the main instruments utilized for revealing molecular mechanisms in pathological and physiological conditions. DIANA-mirExTra v2.0 (http://www.microrna.gr/mirextrav2) performs a combined DEA of mRNAs and microRNAs (miRNAs) to uncover miRNAs and transcription factors (TFs) playing important regulatory roles between two investigated states. The web server uses as input miRNA/RNA-Seq read count data sets that can be uploaded for analysis. Users can combine their data with 350 small-RNA-Seq and 65 RNA-Seq in-house analyzed libraries which are provided by DIANA-mirExTra v2.0.The web server utilizes miRNA:mRNA, TF:mRNA and TF:miRNA interactions derived from extensive experimental data sets. More than 450 000 miRNA interactions and 2 000 000 TF binding sites from specific or high-throughput techniques have been incorporated, while accurate miRNA TSS annotation is obtained from microTSS experimental/in silico framework. These comprehensive data sets enable users to perform analyses based solely on experimentally supported information and to uncover central regulators within sequencing data: miRNAs controlling mRNAs and TFs regulating mRNA or miRNA expression. The server also supports predicted miRNA:gene interactions from DIANA-microT-CDS for 4 species (human, mouse, nematode and fruit fly). DIANA-mirExTra v2.0 has an intuitive user interface and is freely available to all users without any login requirement.

Analyzing MiRNA-LncRNA Interactions.

Long noncoding RNAs (lncRNAs) are noncoding transcripts usually longer than 200 nts that have recently emerged as one of the largest and significantly diverse RNA families. The biological role and functions of lncRNAs are still mostly uncharacterized. Their target-mimetic, sponge/decoy function on microRNAs was recently uncovered. miRNAs are a class of noncoding RNA species (~22 nts) that play a central role in posttranscriptional regulation of protein coding genes by mRNA cleavage, direct translational repression and/or mRNA destabilization. LncRNAs can act as miRNA sponges, reducing their regulatory effect on mRNAs. This function introduces an extra layer of complexity in the miRNA-target interaction network. This chapter focuses on the study of miRNA-lncRNA interactions with either in silico or experimentally supported analyses. The proposed methodologies can be appropriately adapted in order to become the backbone of advanced multistep functional miRNA analyses.

DIANA-miRGen v3.0: accurate characterization of microRNA promoters and their regulators.

microRNAs (miRNAs) are small non-coding RNAs that actively fine-tune gene expression. The accurate characterization of the mechanisms underlying miRNA transcription regulation will further expand our knowledge regarding their implication in homeostatic and pathobiological networks. Aim of DIANA-miRGen v3.0 (http://www.microrna.gr/mirgen) is to provide for the first time accurate cell-line-specific miRNA gene transcription start sites (TSSs), coupled with genome-wide maps of transcription factor (TF) binding sites in order to unveil the mechanisms of miRNA transcription regulation. To this end, more than 7.3 billion RNA-, ChIP- and DNase-Seq next generation sequencing reads were analyzed/assembled and combined with state-of-the-art miRNA TSS prediction and TF binding site identification algorithms. The new database schema and web interface facilitates user interaction, provides advanced queries and innate connection with other DIANA resources for miRNA target identification and pathway analysis. The database currently supports 276 miRNA TSSs that correspond to 428 precursors and >19M binding sites of 202 TFs on a genome-wide scale in nine cell-lines and six tissues of Homo sapiens and Mus musculus.