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Objective: Pancreatic beta-cells exposed to proinflammatory cytokines display alterations in gene expression resulting in defective insulin secretion and apoptosis. MicroRNAs are small noncoding RNAs emerging as key regulators of gene expression. Here, we evaluated the contribution of microRNAs to cytokine-mediated beta-cell cytotoxicity.Research Design and Methods: We used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in beta-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated beta-cell failure by modifying their expression in insulin-secreting MIN6 cells.Results: We found that IL-1beta and TNF-alpha induce the expression of miR-21, miR-34a, and miR-146a both in MIN6 cells and human pancreatic islets. We further show an increase of these microRNAs in islets of NOD mice during development of pre-diabetic insulitis. Blocking miR-21, miR-34a, or miR-146a function using antisense molecules did not restore insulin-promoter activity but prevented the reduction in glucose-induced insulin secretion observed upon IL-1beta exposure. Moreover, anti-miR-34a and anti-miR-146a treatment protected MIN6 cells from cytokine-triggered cell death.Conclusions: Our data identify miR-21, miR-34a, and miR-146a as novel players in beta-cell failure elicited in vitro and in vivo by proinflammatory cytokines, notably during the development of peri-insulitis that precedes overt diabetes in NOD mice. [ABSTRACT FROM AUTHOR]

Pleomorphic hyalinizing angiectatic tumor (PHAT) is a rare mesenchymal tumor of intermediate malignancy. PHAT, and the related hemosiderotic fibrolipomatous tumor, show a recurrent t(1;10)(p22;q24). Fluorescence in situ hybridization (FISH) and BAC (bacterial artificial chromosome) clones have previously identified TGFBR3 and MGEA5 as fusion partners. However, targeted RNA-sequencing allowed for the correct identification of FBXW4 and not MGEA5 as the fusion partner of TGFBR3 in a subcutaneous PHAT, a finding further confirmed by RT-PCR. FBXW4 and MGEA5 share a common cytogenetic location at 10q24.32, thereby suggesting that the use of less precise technology may have led to inaccurate gene identification. The study of additional cases is however required.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8 + T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8 + T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8 + T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8 + T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Liver stem cell therapy (SCT) has been suggested as a promising means to improve liver regeneration in advanced liver disease. However, data from trials are heterogeneous, with no systematic histological evaluation. The aim of this study is to specifically analyze the effect of autologous SCT on liver regeneration and on gene expression changes., Methods: Individuals in the randomized controlled trial of SCT in alcoholic hepatitis with paired liver biopsies were included (n = 58). Immunohistochemistry (Ki67, K7, and CD68), in situ hybridization (SPINK1), and global gene expression analysis were performed on liver biopsies (30 control patients and 28 patients with transarterial administration of bone marrow-derived stem cells) both at baseline and after 4 weeks of follow-up., Results: No difference between the two groups could be observed regarding the proliferative hepatocyte number, proliferative K7-positive cells, or total K7-positive cells at the 4-week follow-up liver biopsy. However, patients who received SCT showed a more important liver macrophagic expansion as compared to standard treatment. Transcriptome data revealed changes in genes linked with inflammation (CD68 and SAA), regeneration (SPINK1 and HGF), fibrosis (COL1A1), and stem cells (CD45). No changes in gene pathways involved in liver growth and cell cycle proteins were evident. SPINK1 mRNA was present by in situ hybridization at week 4 in SCT patients in the liver parenchyma areas adjacent to macrophage recruitment and liver cell proliferation., Conclusions: The analysis of liver tissue after SCT demonstrated an expansion of macrophages concurrent with an upregulated expression of genes involved in inflammatory and regenerative pathways. With the negative results from the clinical trial, the impact of the SCT has to be interpreted as weak, and it is not able to modify the clinical course of this severe liver disease.

Background and Aims: Dilated peribiliary glands (PBG) in patients with cirrhosis are often an incidental finding although their significance and physiopathology remain unclear. We aimed to identify clinical factors associated with dilated PBG and to perform a detailed morphometric assessment of dilated PBG in cirrhotic patients undergoing liver transplantation (LT)., Methods: All consecutive cirrhotic patients undergoing LT at our institution between October 2006 and October 2011 were assessed for inclusion. Ten non-cirrhotic patients were included as controls. We performed morphometrical assessment of PBG, assessed baseline clinical factors associated with dilated PBG, immunohistochemistry staining with CK-19, MiB-1 and EpCAM, and radiological assessment of all available cases., Results: Seventy-one patients met the inclusion criteria, 24% had PBG dilatation of >1000 µm. On multivariable analysis, MELD (OR 1.11 per unit increase in MELD, p = 0.004) was the only significant factor associated with dilated PBG. Compared to PBG < 1000 µm, large PBG had a higher proportion of EpCAM-positive (69 vs. 28%, p < 0.001) and MiB-1-positive lining cells (2.8 vs. 0.55%, p = 0.036). Computed tomography and magnetic resonance imaging had high specificity but low sensitivity for the diagnosis of dilated PBG > 1000 µm (specificity 90-100%, sensitivity 25-29%)., Conclusions: Dilated PBGs are a common finding in explants of cirrhotic subjects undergoing LT and are associated with liver failure although diagnostic performance of cross-sectional imaging is inconstant. The high number of proliferative and EpCAM-positive cells lining the PBG may suggest a role of PBG in organ repair during liver failure.

Background & Aims: The role of liver progenitor cell (LPC) expansion, known as a marker of disease severity, as well as the impact of macrophage activation on liver regeneration remains unclear in humans. We aimed to characterize the LPC and macrophage compartments in alcoholic hepatitis (AH), as well as gene expression patterns to identify predictors of a good prognosis in this setting., Methods: Immunohistochemical studies for macrophages, proliferative hepatocytes, total and proliferative LPC, as well as whole liver microarray gene expression were performed on baseline liver biopsies of 58 AH patients early after admission. Abstinent cirrhotic patients were used as controls. Patients were qualified as "improvers" or "non-improvers" based on the change in MELD score three months after baseline., Results: Compared to controls, AH patients demonstrated a significant expansion of macrophages, invasion of LPC and a higher number of proliferating hepatocytes and LPC. In AH patients, total LPC expansion (total Keratin7(+) cells) was associated with liver disease severity. The group of improvers (n=34) was characterized at baseline by a higher number of proliferating hepatocytes, proliferative LPC (double Keratin7(+)Ki67(+) cells) and liver macrophages as compared to non-improvers (n=24), despite similar clinical and biological variables. Upregulated genes in improvers were associated with cell cycle mitosis together with a major expression of SPINK1., Conclusions: Higher liver macrophage expansion, increased proliferative hepatocyte but also LPC number, as well as an upregulation of cell proliferation-related genes are associated with a favourable outcome. These new findings open novel therapeutic targets in AH., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

We report a fatal case of acute lower respiratory tract disease with human rhinovirus C (HRV-C) as the unique cause in a 19-month-old girl with a history of repeated episodes of bronchiolitis. HRV-C type 8 nucleic acids were observed in respiratory, stool, and cerebrospinal fluid samples, and infectious virions were isolated from patient serum after inoculation onto reconstituted airway epithelia., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease., Competing Interests: The authors have declared that no competing interests exist.

Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.

Down syndrome (DS) is the most common cause of mental retardation. Many neural phenotypes are shared between DS individuals and DS mouse models; however, the common underlying molecular pathogenetic mechanisms remain unclear. Using a transchromosomic model of DS, we show that a 30%-60% reduced expression of Nrsf/Rest (a key regulator of pluripotency and neuronal differentiation) is an alteration that persists in trisomy 21 from undifferentiated embryonic stem (ES) cells to adult brain and is reproducible across several DS models. Using partially trisomic ES cells, we map this effect to a three-gene segment of HSA21, containing DYRK1A. We independently identify the same locus as the most significant eQTL controlling REST expression in the human genome. We show that specifically silencing the third copy of DYRK1A rescues Rest levels, and we demonstrate altered Rest expression in response to inhibition of DYRK1A expression or kinase activity, and in a transgenic Dyrk1A mouse. We reveal that undifferentiated trisomy 21 ES cells show DYRK1A-dose-sensitive reductions in levels of some pluripotency regulators, causing premature expression of transcription factors driving early endodermal and mesodermal differentiation, partially overlapping recently reported downstream effects of Rest +/-. They produce embryoid bodies with elevated levels of the primitive endoderm progenitor marker Gata4 and a strongly reduced neuroectodermal progenitor compartment. Our results suggest that DYRK1A-mediated deregulation of REST is a very early pathological consequence of trisomy 21 with potential to disturb the development of all embryonic lineages, warranting closer research into its contribution to DS pathology and new rationales for therapeutic approaches.

As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix. Four cytokines were overexpressed on 804G-ECM compared with poly-L-lysine: C-X-C motif ligand 1 (CXCL1), CXCL2, interferon-inducible protein-10, and IL-1beta. A time-course experiment indicated that maximal induction by 804G-ECM of CXCL1/2 and interferon-inducible protein-10 occurred at 4 h. Stimulation of CXCL1 release by beta-cells on 804G-ECM was confirmed at the protein level. Moreover, secreted CXCL1 was shown to be functionally active by attracting rat granulocytes. Preventing the interaction of beta1 integrins and laminin-5 (a major component of 804G-ECM) with specific antibodies resulted in a 40-50% inhibition of CXCL1 expression. Using the nuclear factor-kappaB pathway inhibitor Bay 11-7082 it is demonstrated that CXCL1 expression and secretion are dependent on nuclear factor-kappaB activation. IL-1 secreted by beta-cells plated on 804G-ECM was found to be a key soluble mediator because treatment of cells with the IL-1 receptor antagonist significantly reduced both CXCL1 gene expression and secretion. It is concluded that ECM induces expression of cytokines including CXCL1 with amplification by IL-1 acting via a positive autocrine feedback loop.

Germline mutations of the LKB1 (STK11) tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer. LKB1 encodes a serine/threonine kinase generally inactivated in PJS patients. We identified the dual phosphatase and tumor suppressor protein PTEN as an LKB1-interacting protein. Several LKB1 point mutations associated with PJS disrupt the interaction with PTEN suggesting that the loss of this interaction might contribute to PJS. Although PTEN and LKB1 are predominantly cytoplasmic and nuclear, respectively, their interaction leads to a cytoplasmic relocalization of LKB1. In addition, we show that PTEN is a substrate of the kinase LKB1 in vitro. As PTEN is a dual phosphatase mutated in autosomal inherited disorders with phenotypes similar to those of PJS (Bannayan-Riley-Ruvalcaba syndrome and Cowden disease), our study suggests a functional link between the proteins involved in different hamartomatous polyposis syndromes and emphasizes the central role played by LKB1 as a tumor suppressor in the small intestine.

Peutz-Jeghers syndrome (PJS) is caused by germline mutations in the LKB1 gene, which encodes a serine-threonine kinase that regulates cell proliferation and polarity. This autosomal dominant disorder is characterized by mucocutaneous melanin pigmentation, multiple gastrointestinal hamartomatous polyposis and an increased risk of developing various neoplasms. To understand the molecular pathogenesis of PJS phenotypes, we used microarrays to analyze gene expression profiles in proliferating HeLa cells transduced with lentiviral vectors expressing wild type or mutant LKB1 proteins. We show that gene expression is differentially affected by mutations that impair the kinase activity (K78I) or alter the cellular localization of the LKB1 protein. However, both mutations abrogate the ability of LKB1 to up-regulate the transcription of several genes involved in Wnt signaling, including DKK3, WNT5B and FZD2. In addition-and in contrast to the wild type protein-these LKB1 mutants fail to activate the GSK-3beta kinase, which otherwise phosphorylates beta-catenin. The increase in beta-catenin phosphorylation that occurs upon expression of wild-type LKB1 results in transcriptional inhibition of a canonical Wnt reporter gene. This suggests that pathogenic LKB1 mutations that lead to activation of the Wnt/beta-catenin pathway could contribute to the cancer predisposition of PJS patients.

The hepatitis B virus X protein (HBx) is essential for viral infection and strongly interferes with cell growth and viability in culture. These activities involve interaction of HBx with the DDB1 subunit of UV-damaged DNA-binding factor UV-DDB. UV-DDB consists of DDB1 and a DDB2 subunit that mediates nuclear import and has recognized functions in DNA repair and E2F1-mediated transcription. Here we show that HBx retains DDB1-binding-dependent cytotoxic activities when engineered to accumulate in the nucleus but not when excluded from the nucleus. Nuclear localization of HBx does not require binding to DDB1 and remains unaffected by ectopically expressed UV-DDB subunits, indicating that HBx reaches the nuclear compartment independently of UV-DDB. Unexpectedly, HBx appears to largely exist in association with DDB1 and is in direct competition with DDB2 for binding to DDB1. Hence, HBx-mediated cell death can be relieved by increased levels of DDB2, an effect that is not observed with a naturally occurring mutant of DDB2 that lacks DDB1-binding activity. These findings indicate that HBx acts through a pathway that involves a DDB2-independent nuclear function of DDB1 and that this activity will depend on the relative concentration of DDB1 and DDB2 in cells.

The hepatitis B virus X protein (HBx) is essential for establishing natural viral infection and has been implicated in the development of liver cancer associated with chronic infection. The basis for HBx function in either process is not understood. In cell culture, HBx exhibits pleiotropic activities affecting transcription, DNA repair, cell growth, and apoptotic cell death. Numerous cellular proteins including the p127-kDa subunit of UV-damaged DNA-binding activity have been reported to interact with HBx but the functional significance of these interactions remains unclear. Here we show that the binding of HBx to p127 interferes with cell viability. Mutational analysis reveals that HBx contacts p127 via a region to which no function has been assigned previously. An HBx variant bearing a single-charge reversal substitution within this region loses p127 binding and concomitant cytotoxicity. This mutant regains activity when directly fused to p127. These studies confirm that p127 is an important cellular target of HBx, and they indicate that HBx does not exert its effect by sequestering p127, and thereby preventing its normal function, but instead by conferring to p127 a deleterious activity., (Copyright 2001 Academic Press.)

La proteins are conserved, abundant and predominantly nuclear phosphoproteins which bind to the 3'-U termini of newly synthesized RNA polymerase III transcripts. The human La protein has been implicated in the synthesis, termination and release of such transcripts. Here we examine the potential transcriptional properties of La in Xenopus laevis, using a homologous tRNA gene as template. Immunodepletion of La from cell-free extracts leads to the formation of tRNA precursors lacking 3'-U residues. This shortening can be uncoupled from RNA polymerase III transcription, indicating that it results from nuclease degradation rather than incomplete synthesis. Extracts containing <1% of the normal La protein content synthesize tRNA precursors just as well as complete extracts, with no change in termination efficiency, and the vast majority of these full-length transcripts are not associated with the template or with residual La protein. Hence, Xenopus La seems not to function as an initiation, termination or release factor for RNA polymerase III. Consistent with the recently discovered role of La in yeast tRNA maturation in vivo, recombinant Xenopus La prevents 3'-exonucleolytic degradation of tRNA precursors in vitro. A conserved RNA chaperone function may best explain the abundance of La in eukaryotic nuclei.

In Xenopus and other vertebrates, ribosomal protein mRNAs share a common sequence in the 5' untranslated region (5' UTR), in particular a pyrimidine tract at the 5' end, which has been demonstrated to be involved in the translational regulation of this class of mRNAs. In previous studies, carried out in the Xenopus system, we demonstrated the specific binding of two proteins (57 kDa and 47 kDa) to the pyrimidine tract of the mRNAs for three different ribosomal proteins. Here, we show that the two binding proteins are in fact one; one being the cleavage product of the other. By immunoprecipitation and protein purification, this binding protein has been identified as the Xenopus homologue of the human La autoantigen, an RNA-binding protein previously reported to be implicated in RNA polymerase III transcription termination and in translation initiation of poliovirus and immunodeficiency virus type 1 RNAs. We show that the specific interaction of La with the 5' pyrimidine tract of ribosomal protein mRNA is mediated by a protease-sensitive factor, which, after assisting La-RNA binding, dissociates from the complex and becomes again available to promote further binding. We show that mutations in the 5' UTR pyrimidine tract, known to disrupt the translational control of ribosomal protein mRNA, severely impair La binding. Although a direct relationship between ribosomal protein mRNA translation and La binding is not yet available, the properties of the interaction suggest that La protein, possibly together with other components, might be involved in translational regulation.

When HA epitope-tagged and untagged Sendai virus (SeV) P proteins are coexpressed and the products reacted with anti-HA, the untagged P protein is also selected because this protein is found as an oligomer. The oligomer was determined to be a homotrimer by coselection studies in which increasing amounts of untagged versus tagged protein were coexpressed, and these findings were extended to mumps virus, a member of the rubulavirus genus. The region of the SeV protein responsible for the oligomerization was localized to residues 344-411. Computer analysis of the 13 Paramyxovirus P proteins in the database revealed that all but one are predicted to form coiled coils in this region, the first of only two regions that can be aligned throughout the entire virus subfamily. The predicted coiled-coil region of the measles virus P protein, when grafted onto the C-terminus of the normally monomeric La protein, led to the efficient oligomerization of this reporter protein. The predicted coiled-coil region of these P proteins thus appears to be sufficient for oligomerization.

The La protein is a 47 kDa polypeptide that frequently acts as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome patients. A key property of this protein is its association with the U-rich termini of newly synthesized RNA polymerase III transcripts. Here we characterize a 32 kDa protein from Saccharomyces cerevisiae that shows sequence similarity to the N termini of vertebrate La proteins. This yeast protein also functionally resembles La in that it binds preferentially in vitro to RNAs ending with a series of U residues, and at least 53 amino acids can be deleted from the C terminus without impeding this activity. Such RNA binding activity can be detected in crude yeast extracts by immunoprecipitation of ribonucleoprotein particles by an antibody to frog La protein. However, the same antibody fails to react with the 32 kDa protein. In addition, the gene encoding this protein is not essential for viability. Together, these results suggest that additional La homologue(s) exist in yeast.

In mammalian nuclei, newly-synthesized RNA polymerase III transcripts are transiently associated with a phosphorylated polypeptide of approximately 50 kDa called the La protein. Here we provide evidence that the frog Xenopus laevis contains mRNAs for two highly related La proteins, each apparently encoded by a single gene. Both forms of the La protein contain the RNP-80 motif previously identified in many RNA binding proteins. The steady state levels of La mRNAs and protein are approximately constant in oocytes, eggs and embryos. This implies a progressive and severe decrease in these levels on a per cell basis during early development. In particular, neither the La mRNA nor protein level increases at the mid-blastula transition, the time when RNA polymerase III transcription first occurs during embryogenesis.