A Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8, Bound to DNA Gyrase

Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets

Interaction of anticancer reduced Schiff base coumarin derivatives with human serum albumin investigated by fluorescence quenching and molecular modeling

The specific binding of five reduced Schiff base derived 7-amino-coumarin compounds with antitumor activity to human serum albumin, the principal binding protein of blood, was studied by fluorescence spectroscopy. Their conditional binding constants were computed and the reversible binding at the Sudlow’s site I was found to be strong (KD ~ 0.03-2.09 M). Based on the data albumin can provide a depot for the compounds and is responsible for their biodistribution and transport processes. The experimental data is complemented by protein– ligand docking calculations for two representatives which support the observations. The proton dissociation constants of the compounds were also determined by UV-Vis spectrophotometric and fluorometric titrations to obtain the actual charges and distribution of the species in the various protonation states at physiological pH

Teilhabe an Gesundheit von Menschen mit Beeinträchtigung während der Corona-Pandemie (TaG-Co-Studie) : Ergebnisbericht

Hintergrund und Zielsetzung: Die derzeitige Corona-Pandemie bringt seit dem Frühjahr 2020 viele Veränderungen für Menschen weltweit mit sich. Menschen mit Beeinträchtigung, die in Einrichtungen der Eingliederungshilfe leben und arbeiten, weisen häufig ein erhöhtes Risiko für einen schwereren Krankheitsverlauf bei der Infektion mit Covid-19 auf und sind in besonderem Maß von den Vorgaben und Einschränkungen während der Corona-Pandemie betroffen. Auch das Fachpersonal in Einrichtungen der Eingliederungshilfe steht seit Beginn der Pandemie vor Herausforderungen. Die mit der Corona-Pandemie einhergehenden Maßnahmen zur Eindämmung der Pandemie haben Auswirkungen auf die Teilhabe an Gesundheit von Menschen mit Beeinträchtigung. Ziel der TaG-Co-Studie ist es, die Teilhabe an Gesundheit von Menschen mit Beeinträchtigung während der Corona-Pandemie zu untersuchen. Dabei stehen neben den Vorgaben zur Expositionsprophylaxe auch der Umgang mit Gesundheitsinformationen, der Zugang und die Inanspruchnahme des Gesundheitswesens, Präventions- und Gesundheitsangebote, das Ernährungsverhalten und der Substanzmittelkonsum sowie die Gestaltung von Kontakten zu Angehörigen und Bezugspersonen während der Pandemie im Zentrum der Studie. Darüber hinaus wird die Rolle der Fachverbände und Einrichtungen für Menschen mit Behinderung währende der Corona-Pandemie bei der Teilhabe an Gesundheit von Menschen mit Beeinträchtigung untersucht. Methodik: Im Rahmen einer qualitativen Primärerhebung wurden bundesweit (außer Berlin, Bremen, Hamburg und Saarland) leitfadengestützte Telefoninterviews mit n=12 Leitungs- und Fachpersonen von Einrichtungen für Menschen mit Behinderung im Bereich Wohnen sowie n=4 Vertreter*innen von Bundes- und Fachverbänden für Menschen mit Beeinträchtigung von November bis Dezember 2020 geführt. Die Interviews wurden mittels der qualitativen Inhaltsanalyse nach Kuckartz ausgewertet. Ergebnisse: Verordnungen zur Expositionsprophylaxe und Kontaktbeschränkung wurden in den Einrichtungen meist gut umgesetzt; eine permanente Einhaltung der Hygienekonzepte stellte für das Fachpersonal aufgrund der schweren Vereinbarkeit mit den Abläufen der Pflege allerdings eine große Herausforderung dar. In vielen Einrichtungen wurden Gesundheitsinformationen in Bezug auf die Corona-Pandemie in „Krisenteams“ besprochen und in Leichter Sprache zur Verfügung gestellt. Die meisten Vorsorgeuntersuchungen und Gesundheitsangebote wurden während der Corona-Pandemie nicht durchgeführt. Die Einrichtungen berichteten von positiven Veränderungen im Ernährungsverhalten bei den Bewohner*innen aufgrund der Besuchs- und Kontaktbeschränkungen. Es konnten keine Veränderungen des Alkohol- oder Tabakkonsums im stationären Bereich bepbachtet werden. Dagegen zeigte sich im ambulant betreuten Wohnbereich eine Zunahme des Alkohol- und Medikamentenkonsums, insbesondere bei Klient*innen mit psychischen Beeinträchtigungen. Zur Gewährleistung der sozialen Teilhabe ermöglichten die Einrichtungen Alternativen, bspw. Gespräche am offenen Fenster oder digitale Formate (z. B. Skype). Einschränkungen wie die Schließungen der Werkstätten, führten bei einigen Bewohner*innen zu Änderungen des Verhaltens, wie sozialer Rückzug oder unruhiges und aggressives Verhalten. Auf Ebene der Fachverbände wurden neue Formate (bspw. Newsletter oder Fachforen) zur Weiterleitung von Informationen während der Corona-Pandemie geschaffen. Als problematisch thematisierten die Fachverbände, dass die technische Ausstattung der Einrichtungen vielfach nicht ausreichend sei und digitale Angebote nicht genutzt werden können. Während der Pandemie steht der Infektionsschutz der Menschen mit Beeinträchtigung neben dem Austausch mit Bundes- und Landesbehörden im Vordergrund der Arbeit der Fachverbände, in dem u. a. Konzepte zur Reduzierung des Ansteckungsrisikos entwickelt werden. Schlussfolgerungen: Auch wenn die Einrichtungen der Eingliederungshilfe die Herausforderungen durch die Corona-Pandemie auf vielfältige Weise bewältigen, besteht großer Bedarf um die Einrichtungen von Seiten der Fachverbände und der Politik zu unterstützen. Einrichtungsleitungen und Fachpersonal in Einrichtungen für Menschen mit Beeinträchtigung benötigen Konzepte, die aus der Praxiserfahrung heraus entwickelt werden, um die Klient*innen vor einer Ansteckung mit dem Coronavirus zu schützen. Darüber hinaus dürfen die sozialen Aspekte der Teilhabe und die individuellen Besonderheiten der Menschen mit Beeinträchtigung nicht vernachlässigt werden. Nur so kann auch während der Corona-Pandemie die Teilhabe an Gesundheit von Menschen mit Beeinträchtigung gewährleistet werden

Использование барий-стронциевого карбонатита при изготовлении сварочных флюсов на основе техногенных отходов металлургического производства

В данной работе рассмотрена возможность использования барий-стронциевого карбонатита при изготовлении сварочных флюсов на основе шлака производства силикомарганца, а так же на основе ковшевых электросталеплавильных шлаков, образованных при производстве рельсовых марок стали. В серии опытов в лабораторных условиях изготавливали и исследовали различные составы сварочных флюсов, были определены химические составы наплавленного металла, проведен металлографический анализ.In this paper the possibility of using barium-strontium carbonatite in the manufacture of welding fluxes on the basis of slag from the production of silicomanganese, and based on ladle steelmaking slags formed in the production of rail steel grades. In a series of experiments in the laboratory have produced and investigated different compositions of welding fluxes, were determined the chemical compositions of the weld metal metallographic analysis

Serum after Autologous Transplantation Stimulates Proliferation and Expansion of Human Hematopoietic Progenitor Cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34+ cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34+, CD133+, CD45−) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool

Frequent loss of endothelin-3 (EDN3) expression due to epigenetic inactivation in human breast cancer

Introduction: Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer. Methods: EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome. Results: Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate (P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA reexpression in vitro. Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression (P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation. Conclusions EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland

The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding

SimR, a TetR-family transcriptional regulator (TFR), controls the export of simocyclinone, a potent DNA gyrase inhibitor made by Streptomyces antibioticus. Simocyclinone is exported by a specific efflux pump, SimX and the transcription of simX is repressed by SimR, which binds to two operators in the simR-simX intergenic region. The DNA-binding domain of SimR has a classical helix-turn-helix motif, but it also carries an arginine-rich N-terminal extension. Previous structural studies showed that the N-terminal extension is disordered in the absence of DNA. Here, we show that the N-terminal extension is sensitive to protease cleavage, but becomes protease resistant upon binding DNA. We demonstrate by deletion analysis that the extension contributes to DNA binding, and describe the crystal structure of SimR bound to its operator sequence, revealing that the N-terminal extension binds in the minor groove. In addition, SimR makes a number of sequence-specific contacts to the major groove via its helix-turn-helix motif. Bioinformatic analysis shows that an N-terminal extension rich in positively charged residues is a feature of the majority of TFRs. Comparison of the SimR–DNA and SimR–simocyclinone complexes reveals that the conformational changes associated with ligand-mediated derepression result primarily from rigid-body rotation of the subunits about the dimer interface

Gene expression profiling reveals different pathways related to Abl and other genes that cooperate with c-Myc in a model of plasma cell neoplasia

<p>Abstract</p> <p>Background</p> <p>To elucidate the genes involved in the neoplastic transformation of B cells, global gene expression profiles were generated using Affymetrix U74Av2 microarrays, containing 12,488 genes, for four different groups of mouse B-cell lymphomas and six subtypes of pristane-induced mouse plasma cell tumors, three of which developed much earlier than the others.</p> <p>Results</p> <p>Unsupervised hierarchical cluster analysis exhibited two main sub-clusters of samples: a B-cell lymphoma cluster and a plasma cell tumor cluster with subclusters reflecting mechanism of induction. This report represents the first step in using global gene expression to investigate molecular signatures related to the role of cooperating oncogenes in a model of Myc-induced carcinogenesis. Within a single subgroup, e.g., ABPCs, plasma cell tumors that contained typical T(12;15) chromosomal translocations did not display gene expression patterns distinct from those with variant T(6;15) translocations, in which the breakpoint was in the <it>Pvt-1 </it>locus, 230 kb 3' of c-<it>Myc</it>, suggesting that c-<it>Myc </it>activation was the initiating factor in both. When integrated with previously published Affymetrix array data from human multiple myelomas, the IL-6-transgenic subset of mouse plasma cell tumors clustered more closely with MM1 subsets of human myelomas, slow-appearing plasma cell tumors clustered together with MM2, while plasma cell tumors accelerated by v-Abl clustered with the more aggressive MM3-MM4 myeloma subsets. Slow-appearing plasma cell tumors expressed <it>Socs1 </it>and <it>Socs2 </it>but v-<it>Abl</it>-accelerated plasma cell tumors expressed 4–5 times as much. Both v-<it>Abl</it>-accelerated and non-v-<it>Ab</it>l-associated tumors exhibited phosphorylated STAT 1 and 3, but only v-Abl-accelerated plasma cell tumors lost viability and STAT 1 and 3 phosphorylation when cultured in the presence of the v-Abl kinase inhibitor, STI-571. These data suggest that the Jak/Stat pathway was critical in the transformation acceleration by v-Abl and that v-Abl activity remained essential throughout the life of the tumors, not just in their acceleration. A different pathway appears to predominate in the more slowly arising plasma cell tumors.</p> <p>Conclusion</p> <p>Gene expression profiling differentiates not only B-cell lymphomas from plasma cell tumors but also distinguishes slow from accelerated plasma cell tumors. These data and those obtained from the sensitivity of v-Abl-accelerated plasma cell tumors and their phosphorylated STAT proteins indicate that these similar tumors utilize different signaling pathways but share a common initiating genetic lesion, a c-<it>Myc</it>-activating chromosome translocation.</p

Favorable prognostic value of SOCS2 and IGF-I in breast cancer

<p>Abstract</p> <p>Background</p> <p>Suppressor of cytokine signaling (SOCS) proteins comprise a protein family, which has initially been described as STAT induced inhibitors of the Jak/Stat pathway. Recent in vivo and in vitro studies suggest that SOCS proteins are also implicated in cancer. The STAT5 induced IGF-I acts as an endocrine and para/autocrine growth and differentiation factor in mammary gland development. Whereas high levels of circulating IGF-I have been associated with increased cancer risk, the role of autocrine acting IGF-I is less clear. The present study is aimed to elucidate the clinicopathological features associated with SOCS1, SOCS2, SOCS3, CIS and IGF-I expression in breast cancer.</p> <p>Methods</p> <p>We determined the mRNA expression levels of SOCS1, SOCS2, SOCS3, CIS and IGF-I in 89 primary breast cancers by reverse transcriptase PCR. SOCS2 protein expression was further evaluated by immuno-blot and immunohistochemistry.</p> <p>Results</p> <p>SOCS2 expression inversely correlated with histopathological grade and ER positive tumors exhibited higher SOCS2 levels. Patients with high SOCS2 expression lived significantly longer (108.7 vs. 77.7 months; P = 0.015) and high SOCS2 expression proved to be an independent predictor for good prognosis (HR = 0.45, 95% CI 0.23 – 0.91, P = 0.026). In analogy to SOCS2, high IGF-I expression was an independent predictor for good prognosis in the entire patient cohort. In the subgroup of patients with lymph-node negative disease, high IGF-I was a strong predictor for favorable outcome in terms of overall survival and relapse free survival (HR = 0.075, 95% CI 0.014 – 0.388, P = 0.002).</p> <p>Conclusion</p> <p>This is the first report on the favorable prognostic value of high SOCS2 expression in primary mammary carcinomas. Furthermore a strong association of high IGF-I expression levels with good prognosis was observed especially in lymph-node negative patients. Our results suggest that high expression of the STAT5 target genes SOCS2 and IGF-I is a feature of differentiated and less malignant tumors.</p