10 research outputs found
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