• A human-horse comparative map based on equine BAC end sequences.

      Leeb, Tosso; Vogl, Claus; Zhu, Baoli; de Jong, Pieter J; Binns, Matthew M; Chowdhary, Bhanu P; Scharfe, Maren; Jarek, Michael; Nordsiek, Gabriele; Schrader, Frank; et al. (2006-06)
      In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E
    • HuR Small-Molecule Inhibitor Elicits Differential Effects in Adenomatosis Polyposis and Colorectal Carcinogenesis.

      Lang, Michaela; Berry, David; Passecker, Katharina; Mesteri, Ildiko; Bhuju, Sabin; Ebner, Florian; Sedlyarov, Vitaly; Evstatiev, Rayko; Dammann, Kyle; Loy, Alexander; et al. (American Association for Cancer Research, 2017-05-01)
      HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3' untranslated region of mRNAs encoding oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APCMin mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of Prevotella, Akkermansia, and Lachnospiraceae Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD. Cancer Res; 77(9); 2424-38. ©2017 AACR.
    • Hypoxia Enhances Immunosuppression by Inhibiting CD4+ Effector T Cell Function and Promoting Treg Activity.

      Westendorf, Astrid M; Skibbe, Kathrin; Adamczyk, Alexandra; Buer, Jan; Geffers, Robert; Hansen, Wiebke; Pastille, Eva; Jendrossek, Verena; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment.
    • Identification and quantification of (t)RNA modifications in Pseudomonas aeruginosa by liquid chromatography-tandem mass spectrometry.

      Grobe, Svenja; Doberenz, Sebastian; Ferreira, Kevin; Krueger, Jonas; Brönstrup, Mark; Kaever, Volkhard; Häußler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2019-01-15)
      Transfer RNA (tRNA) modifications impact the structure and function of tRNAs thus affecting the efficiency and fidelity of translation. In the opportunistic pathogen Pseudomonas aeruginosa translational regulation plays an important but less defined role in the adaptation to changing environments. In this study, we explored tRNA modifications in P. aeruginosa using LC-MS/MS based approaches. Neutral Loss Scan (NLS) demonstrated the potential to identify previously unknown modifications, while Multiple Reaction Monitoring (MRM) can detect modifications with high specificity and sensitivity. In this study, the MRM-based external calibration method allowed for quantification of the 4 canonical and 32 modified ribonucleosides, of which 21 tRNA modifications were quantified in the total tRNA pool of P. aeruginosa PA14. We also purified the single tRNA isoacceptors tRNA-ArgUCU, tRNA-LeuCAA and tRNA-TrpCCA and determined, both qualitatively and quantitatively, their specific modification pattern. Deeper insights into the nature and dynamics of tRNA modifications in P. aeruginosa will pave the way for further studies on posttranscriptional gene regulation as a relatively unexplored molecular mechanism of controlling bacterial pathogenicity and life style.
    • Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis

      Eckelt, Elke; Jarek, Michael; Frömke, Cornelia; Meens, Jochen; Goethe, Ralph (2014-12-06)
      Abstract Background Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis. Results In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria. Conclusions Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.
    • Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles.

      Doberenz, Sebastian; Eckweiler, Denitsa; Reichert, Olga; Jensen, Vanessa; Bunk, Boyke; Spröer, Cathrin; Kordes, Adrian; Frangipani, Emanuela; Luong, Khai; Korlach, Jonas; et al. (2017-02-21)
      DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N(6)-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria.
    • Identification of miRNAs associated with dendritic cell dysfunction during Acute and Chronic Hepatitis B virus infection.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; Sarin, Shiv Kumar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-10-27)
      The uptake or expression of hepatitis B virus proteins by Dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease viz., immune active (IA; n=20); low replicative (LR; n=20); HBeAg negative (n=20); acute viral hepatitis (AVH, n=20) and healthy controls (n=20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by qPCR. The miRNA-mRNA regulatory networks identified 19 miRNAs and 12 target gene interactions in MHC and other immune pathways. miR-2278, miR-615-3p and miR-3681-3p were down-regulated in IA group compared to healthy control, miR-152-3p and miR-3613-3p in LR group compared to IA group and miR-152-3p and miR-503-3p in HBe negative compared to LR group. However, miR-7-1-1-3p, miR-192-5p, miR-195-5p and miR-32-5p in LR, miR-342-3p and miR-940 in HBe negative, and miR-34a-5p, miR-130b-3p, miR-221-3p, miR-320a, miR-324-5p and miR-484 in AVH were up-regulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs-mRNAs in DCs seems responsible for impaired immune response during HBV pathogenesis. This article is protected by copyright. All rights reserved.
    • Identification of Ppar-modulated miRNA hubs that target the fibrotic tumor microenvironment.

      Winkler, Ivana; Bitter, Catrin; Winkler, Sebastian; Weichenhan, Dieter; Thavamani, Abhishek; Hengstler, Jan G; Borkham-Kamphorst, Erawan; Kohlbacher, Oliver; Plass, Christoph; Geffers, Robert; et al. (National Academy of Sciences, 2020-01-07)
      Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.
    • Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity.

      Blanka, Andrea; Schulz, Sebastian; Eckweiler, Denitsa; Franke, Raimo; Bielecka, Agata; Nicolai, Tanja; Casilag, Fiordiligie; Düvel, Juliane; Abraham, Wolf-Rainer; Kaever, Volkhard; et al. (2014-01)
      Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative σ factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa.
    • Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic.

      Leschner, Sara; Deyneko, Igor V; Lienenklaus, Stefan; Wolf, Kathrin; Bloecker, Helmut; Bumann, Dirk; Loessner, Holger; Weiss, Siegfried; Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany. sara.leschner@helmholtz-hzi.de (2012-04)
      Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasia.
    • IDH1/2 mutations in acute myeloid leukemia patients and risk of coronary artery disease and cardiac dysfunction—a retrospective propensity score analysis

      Kattih, Badder; Shirvani, Amir; Klement, Piroska; Garrido, Abel Martin; Gabdoulline, Razif; Liebich, Alessandro; Brandes, Maximilian; Chaturvedi, Anuhar; Seeger, Timon; Thol, Felicitas; et al. (Springer Nature, 2020-01-01)
      Clonal hematopoiesis of indeterminate potential (CHIP) is linked to leukemia gene mutations and associates with an increased risk for coronary artery disease and poor prognosis in ischemic cardiomyopathy. Two recurrently mutated genes in CHIP and adult acute myeloid leukemia (AML) encode for isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). Global expression of mutant IDH2 in transgenic mice-induced dilated cardiomyopathy and muscular dystrophy. In this retrospective observational study, we investigated whether mutant IDH1/2 predisposes to cardiovascular disease in AML patients. Among 363 AML patients, IDH1 and IDH2 mutations were detected in 26 (7.2%) and 39 patients (10.7%), respectively. Mutant IDH1 patients exhibited a significantly higher prevalence of coronary artery disease (26.1% vs. 6.4%, p = 0.002). Applying inverse probability-weighting analysis, patients with IDH1/2 mutations had a higher risk for a declining cardiac function during AML treatment compared to IDH1/2 wild type patients [left ventricular ejection fraction pretreatment compared to 10 months after diagnosis: 59.2% to 41.9% (p < 0.001) vs 58.5% to 55.4% (p = 0.27), respectively]. Mechanistically, RNA sequencing and immunostaining in hiPS-derived cardiomyocytes indicated that the oncometabolite R-2HG exacerbated doxorubicin mediated cardiotoxicity. Evaluation of IDH1/2 mutation status may therefore help identifying AML patients at risk for cardiovascular complications during cytotoxic treatment. Similar articles
    • Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG.

      Vordermeier, H Martin; Huygen, Kris; Singh, Mahavir; Hewinson, R Glyn; Xing, Zhou (2006-02-01)
      Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.
    • Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

      Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; et al. (2013-05-07)
      Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.
    • The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

      Felgner, Sebastian; Spöring, Imke; Pawar, Vinay; Kocijancic, Dino; Preusse, Matthias; Falk, Christine; Rohde, Manfred; Häussler, Susanne; Weiss, Siegfried; Erhardt, Marc; et al. (Wiley-Blackwell, 2019-11-21)
      Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune‐stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen‐associated molecular pattern recognized by extra‐ and intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum‐specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest anti‐tumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains.
    • Immunoglobulins drive terminal maturation of splenic dendritic cells.

      Zietara, Natalia; Łyszkiewicz, Marcin; Puchałka, Jacek; Pei, Gang; Gutierrez, Maximiliano Gabriel; Lienenklaus, Stefan; Hobeika, Elias; Reth, Michael; Martins dos Santos, Vitor A P; Krueger, Andreas; et al. (2013-02-05)
      Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig.
    • Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.

      Kehrmann, Jan; Tatura, Roman; Zeschnigk, Michael; Probst-Kepper, Michael; Geffers, Robert; Steinmann, Joerg; Buer, Jan; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany. (2014-07)
      The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+)  CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells.
    • Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia.

      Yun, Haiyang; Damm, Frederik; Yap, Damian; Schwarzer, Adrian; Chaturvedi, Anuhar; Jyotsana, Nidhi; Lübbert, Michael; Bullinger, Lars; Döhner, Konstanze; Geffers, Robert; et al. (2014-06-03)
      Hypomethylating agents are widely used in patients with myelodysplastic syndromes and unfit patients with acute myeloid leukemia. However, it is not well understood why only some patients respond to hypomethylating agents. We found previously that the effect of decitabine on hematopoietic stem cell viability differed between Mll5 wildtype and null cells. We therefore investigated the role of MLL5 expression levels on outcome of acute myeloid leukemia patients who were treated with decitabine. MLL5 above the median expression level predicted longer overall survival independent of DNMT3A mutation status in bivariate analysis (median overall survival for high vs. low MLL5 expression, 292 vs. 167 days, P=.026). In patients who received 3 or more courses decitabine, high MLL5 expression and wildtype DNMT3A independently predicted improved overall survival (median overall survival for high vs. low MLL5 expression, 468 vs. 243 days, P=.012). In transformed murine cells, loss of Mll5 was associated with resistance to low-dose decitabine, less global DNA methylation in promoter regions, and reduced DNA demethylation upon decitabine treatment. Together, these data support our clinical observation of improved outcome in decitabine treated patients who express MLL5 at high levels, and suggest a mechanistic role of MLL5 in the regulation of DNA methylation.
    • Impact of SO(2) on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing.

      Hamisch, Domenica; Randewig, Dörte; Schliesky, Simon; Bräutigam, Andrea; Weber, Andreas P M; Geffers, Robert; Herschbach, Cornelia; Rennenberg, Heinz; Mendel, Ralf R; Hänsch, Robert; et al. (2012-12)
      High concentrations of sulfur dioxide (SO(2) ) as an air pollutant, and its derivative sulfite, cause abiotic stress that can lead to cell death. It is currently unknown to what extent plant fumigation triggers specific transcriptional responses. To address this question, and to test the hypothesis that sulfite oxidase (SO) is acting in SO(2) detoxification, we compared Arabidopsis wildtype (WT) and SO knockout lines (SO-KO) facing the impact of 600 nl l(-1) SO(2) , using RNAseq to quantify absolute transcript abundances. These transcriptome data were correlated to sulfur metabolism-related enzyme activities and metabolites obtained from identical samples in a previous study. SO-KO plants exhibited remarkable and broad regulative responses at the mRNA level, especially in transcripts related to sulfur metabolism enzymes, but also in those related to stress response and senescence. Focusing on SO regulation, no alterations were detectable in the WT, whereas in SO-KO plants we found up-regulation of two splice variants of the SO gene, although this gene is not functional in this line. Our data provide evidence for the highly specific coregulation between SO and sulfur-related enzymes like APS reductase, and suggest two novel candidates for involvement in SO(2) detoxification: an apoplastic peroxidase, and defensins as putative cysteine mass storages.
    • Importance of flagella in acute and chronic Pseudomonas aeruginosa infections.

      Lorenz, Anne; Preuße, Matthias; Bruchmann, Sebastian; Pawar, Vinay; Grahl, Nora; Pils, Marina C; Nolan, Laura M; Filloux, Alain; Weiss, Siegfried; Häussler, Susanne; et al. (Wiley-Blackwell, 2018-11-08)
      Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.
    • In Silico Prediction of Scaffold/Matrix Attachment Regions in Large Genomic Sequences

      Frisch, Matthias; Frech, Kornelie; Klingenhoff, Andreas; Cartharius, Kerstin; Liebich, Ines; Werner, Thomas (Cold Spring Harbor Laboratory Press, 2002-02)