Crenarchaeal Biofilm Formation under Extreme Conditions

Background: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. Methodology: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. Conclusion: The study gives first insights into formation and development of crenarchaeal biofilms in extrem

Positive FP-CIT SPECT (DaTSCAN) in Clinical Alzheimer's Disease – An Unexpected Finding?

Clinically, Alzheimer's disease (AD) is by far the most common cause of dementia. Criteria for the diagnosis of dementia with Lewy bodies (DLB) are highly specific but not at all sensitive, which is reflected by the higher number of DLB cases detected histopathologically at autopsy. Imaging of dopamine transporter with FP-CIT SPECT is one possibility to increase sensitivity. Pathological confirmation was also included in the revised consensus criteria for the diagnosis of DLB. However, in the absence of parkinsonism, one of the core features, a clinical diagnosis of AD is more likely. The role of FP-CIT SPECT in DLB diagnosis remains to be clarified. Based on our 3 case reports and a review of the literature, the utility of this imaging method in the differential diagnosis of AD and DLB is highlighted

Interaction modulation through arrays of clustered methyl-arginine protein modifications

Systematic analysis of human arginine methylation identifies two distinct signaling modes;either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions

Human isotype‐dependent inhibitory antibody responses against Mycobacterium tuberculosis

Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B‐cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB‐exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti‐MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies

Die genomische Deletion von Enhancern enthüllt Prinzipien der kombinatorischen Regulation und zelltyp-spezifischen Genexpression

Transcription factors (TFs) are fundamental to the regulation of genes by binding to genomic enhancer elements and orchestrating the expression of their target genes. However, it is largely unclear which TF binding event(s) contribute to the regulation of a specific gene, how cell type-specific plasticity in gene expression is achieved and what minimal circuitry is required to regulate a gene depending on the activity of a specific TF. Here, I used the glucocorticoid receptor (GR), a hormone-activated TF, as a model system to study the molecular mechanisms that determine the functional role of enhancers. By genomically deleting, either alone or in combination, multiple GR binding sites (GBSs) located within the GILZ enhancer, I systematically investigated the interplay of multiple TF binding sites. This mutational analysis demonstrated that multiple GBSs are bound independently but can act cooperatively on gene expression as a single functional unit, which is only active when all of its GBSs are intact. Furthermore, the deletion of GBSs shared between two different cell types demonstrates how cell type-specific differences in the three-dimensional (3D) genome organization and enhancer blocking can rewire enhancer-promoter contacts. This rewiring enables a GBS bound in two different cell types to direct the expression of distinct transcript variants, thereby contributing to the cell type-specific consequences of glucocorticoid signaling. Finally, I investigated the effect of DNA motif sequence on GR activity, by exchanging the sequence of a single GBS of the GILZ enhancer into different GBS motif variants. Whereas in reporter assays this exchange resulted in quantitative changes in gene expression, no effect was observed upon exchange in the endogenous context. Hence, the genomic context can influence the regulatory potency of individual GBS variants, for example by integrating regulatory information from multiple GBSs. To investigate the role GBS variants in an isolated endogenous context, I integrated a GBS at the promoter region of an endogenously silenced gene, thereby activating its expression in a GR-dependent manner. This demonstrates that a single GBS can be sufficient to induce GR-mediated regulation of an associated gene and further provides a model system to investigate the effect of GBS sequence variants in isolation. Together, genomic editing of GBSs in their endogenous genomic context enables insights into the operating priniciples of combinatorial gene regulation by multiple TF binding sites, but also demonstrates that a single promoter-proximal GBS is sufficient to induce GR-dependent gene expression. Furthermore, a GBS equally bound in two different cell types can contribute to the establishment of cell type-specific gene expression patterns by differences in 3D genome organization and enhancer blocking.Die Bindung von Transkriptionsfaktoren (TF) an genomische Enhancer-Elemente ist elementar für die Regulation von Genen. Bis heute ist es jedoch weitgehend unklar, welche Bindungsereignisse zur Regulation eines spezifischen Zielgenes beitragen, wie unterschiedliche Genexpressionsmuster zelltypspezifisch reguliert und welche minimalen Regulationsanordnungen benötigt werden, um ein Gen TF-abhängig zu regulieren. In meiner Doktorabeit verwende ich als Modelsystem den Glukokortikoidrezeptor (GR), einen hormon-aktivierbaren TF, um die molekularen Mechanismen zu untersuchen, die die funktionelle Rolle von Enhancer-Elementen beeinflussen. Durch die genomische Zerstörung von einzelnen und mehreren GR-Bindestellen (GBS) des GILZ Enhancers, habe ich das regulatorische Zusammenspiel von mehreren TF-Bindestellen systematisch untersucht. Diese Mutationsanalyse zeigte, dass mehrere GBS zwar voneinander unabhängig durch GR gebunden werden, aber dennoch kooperativ als funktionelle Einheit die Genexpression beeinflussen, so lange alle beinhalteten GBS intakt sind. Durch die genomische Zerstörung einer GBS, die in zwei verschiedenen Zelltypen von GR gebunden wird, konnte ich zudem zeigen, dass zelltypspezifische Unterschiede in der dreidimensionalen (3D) Genomorganisation und Enhancer-Blocking Enhancer-Promoter Kontakte neu verbinden kann. Die Verbindung unterschiedlicher Enhancer-Promoter Kontakte ermöglicht die Expression verschiedener Transkriptvarianten eines Genes und kann dadurch zu den zelltypspezifischen Effekten von Glukokortikoiden beitragen. Zudem habe ich den Effekt von DNA Motifsequenzen auf die Aktivität von GR untersucht, indem ich die Sequenz einer GBS des GILZ Enhancers in verschiedene GBS-Motifsequenzen umgewandelt habe. Während in Reporter Assays der Austausch von GBS-Motifvarianten in quantitativen Unterschieden in der Genexpression resultierte, zeigte sich im genomischen Kontext kein Effekt auf die Regulation von GILZ. Demzufolge, kann der genomische Kontext die regulatorische Wirkung von GBS-Motifvarianten beeinflussen, z.B. durch die Integration von regulatorischer Information von mehreren GBS. Um GBS- Motifvarianten in einem isolierten, endogenen Umfeld zu untersuchen, habe ich eine einzelne GBS an der Promoter-Region eines endogen nicht exprimierten Genes platziert. Diese Integration zeigt, dass die Präsenz einer einzelnen GBS ausreichen kann, um ein Gen GR-abhängig zu regulieren und stellt zudem ein Modelsystem dar, um die Rolle von GBS-Motifvarianten in Isolation zu testen. Zusammenfassend kann die genomische Editierung Einblicke in die Funktionsweise der kombinatorischen Regulation durch mehrere TF-Bindestellen ermöglichen und zeigt zudem, dass eine einzige Promoter-proximale GBS ausreichen kann, um ein Gen GR-abhängig zu regulieren. Zusätzlich zeigt diese Arbeit, dass eine GBS, die in zwei verschiedenen Zelltypen gleichermaßen gebunden wird, durch Unterschiede in der 3D Genomorganisation und Enhancer-Blocking zur zelltypspezifischen Genexpression beitragen kann

Nontargeted Profiling of Coenzyme A thioesters in biological samples by tandem mass spectrometry

Coenzyme A (CoA) thioesters are ubiquitously present in metabolic networks and play a pivotal role in enzymatic formation and cleavage of carbon–carbon bonds. We present a method allowing nontargeted profiling of CoA-thioesters in biological samples. The reported UHPLC-MS/MS approach employes ion-pairing chromatography to separate CoA-metabolites carrying different chemical functionalities such as hydroxyl or multiple carboxyl groups and to distinguish between isomers. Selective detection of CoA-thioesters is accomplished by precursor ion scanning on a triple quadrupole mass spectrometer and takes advantage of the abundant fragment with <i>m</i>/<i>z</i> = −408 that originates from the CoA-moiety. We used a mixture of 19 commercially available CoA-derivatives to develop and optimize our method. As a proof of concept we demonstrated detection of the major CoA-intermediates of branched chain amino acid degradation in biological samples. We then applied our method to investigate degradation of lipids in the microorganism <i>Mycobacterium smegmatis</i>. Profiling of CoA-thioesters led to the discovery of a novel intermediate of cholesterol degradation. This demonstrates the power of our method for untargeted profiling of CoA-thioesters and adds a missing link in mycobacterial cholesterol catabolism