• Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights Its Zoonotic Potential.

      Kindler, Eveline; Jónsdóttir, Hulda R; Muth, Doreen; Hamming, Ole J; Hartmann, Rune; Rodriguez, Regulo; Geffers, Robert; Fouchier, Ron A M; Drosten, Christian; Müller, Marcel A; et al. (2013)
      ABSTRACT The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.
    • Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate.

      Chaturvedi, A; Araujo Cruz, M M; Jyotsana, N; Sharma, A; Goparaju, R; Schwarzer, A; Görlich, K; Schottmann, R; Struys, E A; Jansen, E E; et al. (2016-08)
      Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.
    • Evaluation of latent tuberculosis infection in patients with inflammatory arthropathies before treatment with TNF-alpha blocking drugs using a novel flow-cytometric interferon-gamma release assay.

      Dinser, R; Fousse, M; Sester, U; Albrecht, K; Singh, M; Köhler, H; Müller-Ladner, U; Sester, M; Department of Internal Medicine and Rheumatology, Justus-Liebig University of Giessen, Kerckhoff Clinic, Benekestrasse 2-8, D-61231 Bad Nauheim, Germany. r.dinser@kerckhoff-klinik.de (2008-02)
      OBJECTIVE: To compare the efficacy of the conventional skin test and a novel flow cytometric whole blood assay in the diagnosis of latent tuberculosis infection (LTBI) in patients with rheumatological diseases evaluated for treatment with TNF-alpha-blocking agents. METHODS: Prospective study of 97 consecutively enrolled patients, who were assessed for the presence of LTBI through clinical history, Mendel-Mantoux skin testing and chest X-ray. In addition, T-cell reactivity towards tuberculin (PPD, purified protein derivative) and the Mycobacterium tuberculosis-specific proteins ESAT-6 and CFP-10 was determined ex vivo using a flow cytometric whole blood assay. RESULTS: After standard screening, 15% of patients receiving TNF-alpha-blocking therapy were pretreated with isoniazide (INH), another 5% of patients did not receive TNF-alpha-blocking therapy because of LTBI. PPD-reactivity in the skin was observed in 14% of patients compared with 39% with the whole blood test. Analysis of the M. tuberculosis-specific response to ESAT-6 and CFP-10 revealed positive results in 16% of patients. Using a decision tree incorporating history, chest X-ray and either skin-test or ESAT-6/CFP-10 results, 18 or 22% of the patients, respectively, were classified as latently infected with M. tuberculosis. Four patients treated with INH because of a positive skin reaction did not show reactivity to ESAT-6/CFP-10 in the whole blood assays. Another six patients not pretreated with INH because of negative skin tests would have received INH, had the results of the whole blood assay been taken into account. CONCLUSION: The Mendel-Mantoux skin test has a low sensitivity and specificity for the diagnosis of LTBI in this cohort of patients, potentially resulting in both over- and under-treatment with prophylactic INH when compared with the flow cytometric analysis of whole blood T-cell reactivity to proteins specific to M. tuberculosis. Use of T-cell based in vitro tests may help to refine diagnostic testing for LTBI.
    • Exome sequencing and case-control analyses identify RCC1 as a candidate breast cancer susceptibility gene.

      Riahi, Aouatef; Radmanesh, Hoda; Schürmann, Peter; Bogdanova, Natalia; Geffers, Robert; Meddeb, Rym; Kharrat, Maher; Dörk, Thilo; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-15)
      Breast cancer is a genetic disease but the known genes explain a minority of cases. To elucidate the molecular basis of breast cancer in the Tunisian population, we performed exome sequencing on six BRCA1/BRCA2 mutation-negative patients with familial breast cancer and identified a novel frameshift mutation in RCC1, encoding the Regulator of Chromosome Condensation 1. Subsequent genotyping detected the 19-bp deletion in additional 5 out of 153 (3%) breast cancer patients but in none of 400 female controls (p = 0.0015). The deletion was enriched in patients with a positive family history (5%, p = 0.0009) and co-segregated with breast cancer in the initial pedigree. The mutant allele was lost in 4/6 breast tumors from mutation carriers which may be consistent with the hypothesis that RCC1 dysfunction provides a selective disadvantage at the stage of tumor progression. In summary, we propose RCC1 as a likely breast cancer susceptibility gene in the Tunisian population.
    • Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms.

      Heinemeyer, T; Chen, X; Karas, H; Kel, A E; Kel, O V; Liebich, I; Meinhardt, T; Reuter, I; Schacherer, F; Wingender, E (1999-01-01)
    • An extended transcriptional regulatory network of Escherichia coli and analysis of its hierarchical structure and network motifs

      Ma, Hong-Wu; Kumar, Bharani; Ditges, Uta; Gunzer, Florian; Buer, Jan; Zeng, An-Ping (Oxford University Press, 2004)
    • FMNL2 drives actin-based protrusion and migration downstream of Cdc42.

      Block, Jennifer; Breitsprecher, Dennis; Kühn, Sonja; Winterhoff, Moritz; Kage, Frieda; Geffers, Robert; Duwe, Patrick; Rohn, Jennifer L; Baum, Buzz; Brakebusch, Cord; et al. (2012-06-05)
      Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.
    • From Human Monocytes to Genome-Wide Binding Sites - A Protocol for Small Amounts of Blood: Monocyte Isolation/ChIP-Protocol/Library Amplification/Genome Wide Computational Data Analysis.

      Weiterer, Sebastian; Uhle, Florian; Bhuju, Sabin; Jarek, Michael; Weigand, Markus A; Bartkuhn, Marek (2014)
      Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner. Conclusion: The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.
    • A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.

      Bohla, Dorte; Herold, Martin; Panzer, Imke; Buxa, Melanie K; Ali, Tamer; Demmers, Jeroen; Krüger, Marcus; Scharfe, Maren; Jarek, Michael; Bartkuhn, Marek; et al. (2014)
      Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.
    • Gene expression profiling of macrophages: implications for an immunosuppressive effect of dissolucytotic gold ions

      Seifert, Oliver; Matussek, Andreas; Sjögren, Florence; Geffers, Robert; Anderson, Chris D (2012-11-09)
      Abstract Background Gold salts has previously been used in the treatment of rheumatoid arthritis but have been replaced by biologicals such as TNF-α inhibitors. The mechanisms behind the anti-inflammatory effect of metallic gold ions are still unknown, however, recent data showed that charged gold atoms are released from pure metallic gold implants by macrophages via a dissolucytosis membrane, and that gold ions are taken up by local macrophages, mast cells and to some extent fibroblasts. These findings open the question of possible immunomodulatory effects of metallic gold and motivate efforts on a deeper understanding of the effect of metallic gold on key inflammatory cells as macrophages. Methods Human macrophage cells (cell line THP-1) were grown on gold foils and intracellular uptake was analysed by autometallography. The impact of phagocytised gold ions on viability of THP-1 cells was investigated by trypan blue staining and TUNEL assay. The global gene expression profile of THP-1 cells after incorporation of gold ions was studied using microarray analysis comprising approximately 20,000 genes. The gene expression data was confirmed by measurement of secreted proteins. Results Autometallography showed intracellular uptake of gold ions into THP-1 cells. No significant effect on viability of THP-1 cells was demonstrated. Our data revealed a unique gene expression signature of dissolucytotic THP-1 cells that had taken up gold ions. A large number of regulated genes were functionally related to immunomodulation. Gold ion uptake induced downregulation of genes involved in rheumatoid arthritis such as hepatocyte growth factor, tenascin-C, inhibitor of DNA binding 1 and 3 and matrix metalloproteinase 13. Conclusion The data obtained in this study offer new insights into the mode of action of gold ions and suggest for the investigation of effects on other key cells and a possible future role of metallic gold as implants in rheumatoid arthritis or other inflammatory conditions.
    • Genome Sequence of Strain MOLA814, a Proteorhodopsin-Containing Representative of the Betaproteobacteria Common in the Ocean.

      Courties, Alicia; Riedel, Thomas; Jarek, Michael; Intertaglia, Laurent; Lebaron, Philippe; Suzuki, Marcelino T; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2013-12-19)
      Strain MOLA814 is a marine betaproteobacterium that was isolated from seawater in the Beaufort Sea. Here, we present its genome sequence and annotation. Genome analysis revealed the presence of a proteorhodopsin-encoding sequence together with its retinal-producing pathway, indicating that this strain might generate energy by using light.
    • Genome sequence of the thermophilic strain Bacillus coagulans 2-6, an efficient producer of high-optical-purity L-lactic acid.

      Su, Fei; Yu, Bo; Sun, Jibin; Ou, Hong-Yu; Zhao, Bo; Wang, Limin; Qin, Jiayang; Tang, Hongzhi; Tao, Fei; Jarek, Michael; et al. (2011-09)
      Bacillus coagulans 2-6 is an efficient producer of lactic acid. The genome of B. coagulans 2-6 has the smallest genome among the members of the genus Bacillus known to date. The frameshift mutation at the start of the d-lactate dehydrogenase sequence might be responsible for the production of high-optical-purity l-lactic acid.
    • Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes.

      Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram; et al. (2012-09)
      The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.
    • Genome-Wide Sequencing Reveals MicroRNAs Downregulated in Cerebral Cavernous Malformations.

      Kar, Souvik; Bali, Kiran Kumar; Baisantry, Arpita; Geffers, Robert; Samii, Amir; Bertalanffy, Helmut; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-02)
      Cerebral cavernous malformations (CCM) are vascular lesions associated with loss-of-function mutations in one of the three genes encoding KRIT1 (CCM1), CCM2, and PDCD10. Recent understanding of the molecular mechanisms that lead to CCM development is limited. The role of microRNAs (miRNAs) has been demonstrated in vascular pathologies resulting in loss of tight junction proteins, increased vascular permeability and endothelial cell dysfunction. Since the relevance of miRNAs in CCM pathophysiology has not been elucidated, the primary aim of the study was to identify the miRNA-mRNA expression network associated with CCM. Using small RNA sequencing, we identified a total of 764 matured miRNAs expressed in CCM patients compared to the healthy brains. The expression of the selected miRNAs was validated by qRT-PCR, and the results were found to be consistent with the sequencing data. Upon application of additional statistical stringency, five miRNAs (let-7b-5p, miR-361-5p, miR-370-3p, miR-181a-2-3p, and miR-95-3p) were prioritized to be top CCM-relevant miRNAs. Further in silico analyses revealed that the prioritized miRNAs have a direct functional relation with mRNAs, such as MIB1, HIF1A, PDCD10, TJP1, OCLN, HES1, MAPK1, VEGFA, EGFL7, NF1, and ENG, which are previously characterized as key regulators of CCM pathology. To date, this is the first study to investigate the role of miRNAs in CCM pathology. By employing cutting edge molecular and in silico analyses on clinical samples, the current study reports global miRNA expression changes in CCM patients and provides a rich source of data set to understand detailed molecular machinery involved in CCM pathophysiology.
    • Genomewide analyses define different modes of transcriptional regulation by peroxisome proliferator-activated receptor-β/δ (PPARβ/δ).

      Adhikary, Till; Kaddatz, Kerstin; Finkernagel, Florian; Schönbauer, Anne; Meissner, Wolfgang; Scharfe, Maren; Jarek, Michael; Blöcker, Helmut; Müller-Brüsselbach, Sabine; Müller, Rolf; et al. (2011)
      Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with essential functions in lipid, glucose and energy homeostasis, cell differentiation, inflammation and metabolic disorders, and represent important drug targets. PPARs heterodimerize with retinoid X receptors (RXRs) and can form transcriptional activator or repressor complexes at specific DNA elements (PPREs). It is believed that the decision between repression and activation is generally governed by a ligand-mediated switch. We have performed genomewide analyses of agonist-treated and PPARβ/δ-depleted human myofibroblasts to test this hypothesis and to identify global principles of PPARβ/δ-mediated gene regulation. Chromatin immunoprecipitation sequencing (ChIP-Seq) of PPARβ/δ, H3K4me3 and RNA polymerase II enrichment sites combined with transcriptional profiling enabled the definition of 112 bona fide PPARβ/δ target genes showing either of three distinct types of transcriptional response: (I) ligand-independent repression by PPARβ/δ; (II) ligand-induced activation and/or derepression by PPARβ/δ; and (III) ligand-independent activation by PPARβ/δ. These data identify PPRE-mediated repression as a major mechanism of transcriptional regulation by PPARβ/δ, but, unexpectedly, also show that only a subset of repressed genes are activated by a ligand-mediated switch. Our results also suggest that the type of transcriptional response by a given target gene is connected to the structure of its associated PPRE(s) and the biological function of its encoded protein. These observations have important implications for understanding the regulatory PPAR network and PPARβ/δ ligand-based drugs.
    • A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer.

      Eddyani, Miriam; Vandelannoote, Koen; Meehan, Conor J; Bhuju, Sabin; Porter, Jessica L; Aguiar, Julia; Seemann, Torsten; Jarek, Michael; Singh, Mahavir; Portaels, Françoise; et al. (2015-11)
      Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes.
    • Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

      Dai, Haiping; Ehrentraut, Stefan; Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; et al. (2015)
      Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets.
    • Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

      Mertens, Gabriele; Hoffmann, Andrea; Blöcker, Helmut; Frank, Ronald; Kahmann, Regine (1984-10)
    • Global micro RNA expression profiling in the liver biopsies of Hepatitis B Virus infected patients suggests specific miRNA signatures for viral persistence and hepatocellular injury.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Bhardwaj, Ankit; Venugopal, Senthil Kumar; Trehanpati, Nirupama; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; et al. (2017-11-30)
      Hepatitis B virus (HBV) can manipulate the miRNA regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury and liver fibrosis. We investigated differentially expressed miRNAs by microarray in the liver biopsy samples from different stages of HBV infection and liver disease [immune tolerant (IT; n= 8); acute viral hepatitis (AVH; n=8); no fibrosis (n=16); early (F1+F2) (n=19) or late fibrosis (F3+F4) (n=14) and healthy controls (n=7)]. The miRNA expression levels were analyzed by the unsupervised principal component analysis (PCA) and hierarchical clustering. Analysis of miRNA-mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes each representing a stage-specific gene regulation during disease progression. The IT group showed elevated miR-199a-5p, miR-221-3p and Let-7a-3p levels which could target genes involved in innate immune response and viral replication. In AVH group, miR-125b-5p and miR-3613-3p were up whereas miR-940 was down which might affect cell proliferation via STAT3 pathway. In early fibrosis, miR-34b-3p, miR-1224-3p and miR-1227-3p were up while miR-499a-5p was down which together, possibly mediate chronic inflammation. In advanced fibrosis, miR-1, miR-10b-5p, miR-96-5p, miR-133b and miR-671-5p were up while miR-20b-5p and miR-455-3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver-specific miRNAs exhibited a similar expression pattern in patient sera.
    • Gut bacterial communities across tadpole ecomorphs in two diverse tropical anuran faunas.

      Vences, Miguel; Lyra, Mariana L; Kueneman, Jordan G; Bletz, Molly C; Archer, Holly M; Canitz, Julia; Handreck, Svenja; Randrianiaina, Roger-Daniel; Struck, Ulrich; Bhuju, Sabin; et al. (2016-04)
      Animal-associated microbial communities can play major roles in the physiology, development, ecology, and evolution of their hosts, but the study of their diversity has yet focused on a limited number of host species. In this study, we used high-throughput sequencing of partial sequences of the bacterial 16S rRNA gene to assess the diversity of the gut-inhabiting bacterial communities of 212 specimens of tropical anuran amphibians from Brazil and Madagascar. The core gut-associated bacterial communities among tadpoles from two different continents strongly overlapped, with eight highly represented operational taxonomic units (OTUs) in common. In contrast, the core communities of adults and tadpoles from Brazil were less similar with only one shared OTU. This suggests a community turnover at metamorphosis. Bacterial diversity was higher in tadpoles compared to adults. Distinct differences in composition and diversity occurred among gut bacterial communities of conspecific tadpoles from different water bodies and after experimental fasting for 8 days, demonstrating the influence of both environmental factors and food on the community structure. Communities from syntopic tadpoles clustered by host species both in Madagascar and Brazil, and the Malagasy tadpoles also had species-specific isotope signatures. We recommend future studies to analyze the turnover of anuran gut bacterial communities at metamorphosis, compare the tadpole core communities with those of other aquatic organisms, and assess the possible function of the gut microbiota as a reservoir for protective bacteria on the amphibian skin.