Generation of a vancomycin-intermediate Staphylococcus aureus (VISA) strain by two amino acid exchanges in VraS

Objectives Staphylococcus aureus is a notorious bacterial pathogen and antibiotic-resistant isolates complicate current treatment strategies. We characterized S. aureus VC40, a laboratory mutant that shows full resistance to glycopeptides (vancomycin and teicoplanin MICs ≥32 mg/L) and daptomycin (MIC = 4 mg/L), to gain deeper insights into the underlying resistance mechanisms. Methods Genomics and transcriptomics were performed to characterize changes that might contribute to development of resistance. The mutations in vraS were reconstituted into a closely related parental background. In addition, antimicrobial susceptibility testing, growth analyses, transmission electron microscopy, lysostaphin-induced lysis and autolysis assays were performed to characterize the phenotype of resistant strains. Results Genome sequencing of strain VC40 revealed 79 mutations in 75 gene loci including genes encoding the histidine kinases VraS and WalK that control cell envelope-related processes. Transcriptomics indicated the increased expression of their respective regulons. Although not reaching the measured MIC for VC40, reconstitution of the L114S and D242G exchanges in VraS(VC40) into the susceptible parental background (S. aureus NCTC 8325) resulted in increased resistance to glycopeptides and daptomycin. The expression of VraS(VC40) led to increased transcription of the cell wall stress stimulon, a thickened cell wall, a decreased growth rate, reduced autolytic activity and increased resistance to lysostaphin-induced lysis in the generated mutant. Conclusions We show that a double mutation of a single gene locus, namely vraS, is sufficient to convert the vancomycin-susceptible strain S. aureus NCTC 8325 into a vancomycin-intermediate S. aureu

a clinical study protocol

Introduction The approved analgesic and anti-inflammatory drugs ibuprofen and indometacin block the small GTPase RhoA, a key enzyme that impedes axonal sprouting after axonal damage. Inhibition of the Rho pathway in a central nervous system-effective manner requires higher dosages compared with orthodox cyclooxygenase-blocking effects. Preclinical studies on spinal cord injury (SCI) imply improved motor recovery after ibuprofen/indometacin-mediated Rho inhibition. This has been reassessed by a meta-analysis of the underlying experimental evidence, which indicates an overall effect size of 20.2% regarding motor outcome achieved after ibuprofen/indometacin treatment compared with vehicle controls. In addition, ibuprofen/indometacin may also limit sickness behaviour, non-neurogenic systemic inflammatory response syndrome (SIRS), neuropathic pain and heterotopic ossifications after SCI. Consequently, ‘small molecule’-mediated Rho inhibition after acute SCI warrants clinical investigation. Methods and analysis Protocol of an investigator-initiated clinical open-label pilot trial on high-dose ibuprofen treatment after acute traumatic, motor-complete SCI. A sample of n=12 patients will be enrolled in two cohorts treated with 2400 mg/day ibuprofen for 4 or 12 weeks, respectively. The primary safety end point is an occurrence of serious adverse events, primarily gastroduodenal bleedings. Secondary end points are pharmacokinetics, feasibility and preliminary effects on neurological recovery, neuropathic pain and heterotopic ossifications. The primary safety analysis is based on the incidence of severe gastrointestinal bleedings. Additional analyses will be mainly descriptive and casuistic. Ethics and dissemination The clinical trial protocol was approved by the responsible German state Ethics Board, and the Federal Institute for Drugs and Medical Devices. The study complies with the Declaration of Helsinki, the principles of Good Clinical Practice and all further applicable regulations. This safety and pharmacokinetics trial informs the planning of a subsequent randomised controlled trial. Regardless of the result of the primary and secondary outcome assessments, the clinical trial will be reported as a publication in a peer-reviewed journal. Trial registration number NCT02096913; Pre-results

Genome sequence of the bioplastic-producing ‘‘Knallgas’’ bacterium Ralstonia eutropha H16

The H2-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H2 and CO2 as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism’s ability to produce and store large amounts of poly[R-(–)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility

Large internal dipole moment in InGaN/GaN quantum dots

This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in Appl. Phys. Lett. 97, 063103 (2010) and may be found at https://doi.org/10.1063/1.3477952.Direct observation of large permanent dipole moments of excitonic complexes in InGaN/GaN quantum dots is reported. Characteristic traces of spectral diffusion, observed in cathodoluminescence of InGaN/GaN quantum dots, allow deducing the magnitude of the intrinsic dipole moment. Our experimental results are in good agreement with realistic calculations of quantum dot transition energies for position-dependent external electric fields.DFG, 43659573, SFB 787: Halbleiter - Nanophotonik: Materialien, Modelle, Bauelement

Lateral positioning of InGaAs quantum dots using a buried stressor

This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in Appl. Phys. Lett. 100, 093111 (2012) and may be found at https://doi.org/10.1063/1.3691251.We present a “bottom-up” approach for the lateral alignment of semiconductor quantum dots (QDs) based on strain-driven self-organization. A buried stressor formed by partial oxidation of (Al,Ga)As layers is employed in order to create a locally varying strain field at a GaAs(001) growth surface. During subsequent strained layer growth, local self-organization of (In,Ga)As QDs is controlled by the contour shape of the stressor. Large vertical separation of the QD growth plane from the buried stressor interface of 150 nm is achieved enabling high optical quality of QDs. Optical characterization confirms narrow QD emission lines without spectral diffusion.DFG, 43659573, SFB 787: Halbleiter - Nanophotonik: Materialien, Modelle, Bauelement

Multi-dimensional modeling and simulation of semiconductor nanophotonic devices

Self-consistent modeling and multi-dimensional simulation of semiconductor nanophotonic devices is an important tool in the development of future integrated light sources and quantum devices. Simulations can guide important technological decisions by revealing performance bottlenecks in new device concepts, contribute to their understanding and help to theoretically explore their optimization potential. The efficient implementation of multi-dimensional numerical simulations for computer-aided design tasks requires sophisticated numerical methods and modeling techniques. We review recent advances in device-scale modeling of quantum dot based single-photon sources and laser diodes by self-consistently coupling the optical Maxwell equations with semiclassical carrier transport models using semi-classical and fully quantum mechanical descriptions of the optically active region, respectively. For the simulation of realistic devices with complex, multi-dimensional geometries, we have developed a novel hp-adaptive finite element approach for the optical Maxwell equations, using mixed meshes adapted to the multi-scale properties of the photonic structures. For electrically driven devices, we introduced novel discretization and parameter-embedding techniques to solve the drift-diffusion system for strongly degenerate semiconductors at cryogenic temperature. Our methodical advances are demonstrated on various applications, including vertical-cavity surface-emitting lasers, grating couplers and single-photon sources

Metagenomics of the Deep Mediterranean, a Warm Bathypelagic Habitat

BACKGROUND: Metagenomics is emerging as a powerful method to study the function and physiology of the unexplored microbial biosphere, and is causing us to re-evaluate basic precepts of microbial ecology and evolution. Most marine metagenomic analyses have been nearly exclusively devoted to photic waters. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a metagenomic fosmid library from 3,000 m-deep Mediterranean plankton, which is much warmer (approximately 14 degrees C) than waters of similar depth in open oceans (approximately 2 degrees C). We analyzed the library both by phylogenetic screening based on 16S rRNA gene amplification from clone pools and by sequencing both insert extremities of ca. 5,000 fosmids. Genome recruitment strategies showed that the majority of high scoring pairs corresponded to genomes from Rhizobiales within the Alphaproteobacteria, Cenarchaeum symbiosum, Planctomycetes, Acidobacteria, Chloroflexi and Gammaproteobacteria. We have found a community structure similar to that found in the aphotic zone of the Pacific. However, the similarities were significantly higher to the mesopelagic (500-700 m deep) in the Pacific than to the single 4000 m deep sample studied at this location. Metabolic genes were mostly related to catabolism, transport and degradation of complex organic molecules, in agreement with a prevalent heterotrophic lifestyle for deep-sea microbes. However, we observed a high percentage of genes encoding dehydrogenases and, among them, cox genes, suggesting that aerobic carbon monoxide oxidation may be important in the deep ocean as an additional energy source. CONCLUSIONS/SIGNIFICANCE: The comparison of metagenomic libraries from the deep Mediterranean and the Pacific ALOHA water column showed that bathypelagic Mediterranean communities resemble more mesopelagic communities in the Pacific, and suggests that, in the absence of light, temperature is a major stratifying factor in the oceanic water column, overriding pressure at least over 4000 m deep. Several chemolithotrophic metabolic pathways could supplement organic matter degradation in this most depleted habitat

Transkriptionelle Regulation und Differzierung in Saccharomyces und Aspergillus: jlbA, RPS26, and ARO3/4

Eine steigende Anzahl von Daten aus Genomuntersuchungen belegt, dass die Anzahl von Isogenen innerhalb eines gegebenen Organismus deutlich höher liegt als noch vor 10 Jahren angenommen. In dieser Arbeit haben wir verschiedene Vertreter von Isogenen charakterisiert, welche an der transkriptionellen Regulation und an der Translation als Teil der Ribosomen beteiligt sind. Zwei Isogene wurden charakterisiert, welche an der aromatischen Aminosäurebiosynthese beteiligt sind. Alle untersuchten Gene stammen aus zwei pilzlichen Modellsystemen, aus der Bäckerhefe Saccharomyces cerevisiae und aus dem filamentösen Pilz Aspergillus nidulans. Das jlbA Gen aus dem filamentösen Pilz Aspergillus nidulans codiert für ein neues, potentielles bZIP-Protein der Familie der JUN Isogene. Ein anderer bekannter Vertreter dieser Genfamilie ist das cpcA Gen, welches das Regulatorprotein der ‚Cross-Pathway’ Kontrolle codiert, welches unter Mangelbedingungen für die Aminosäurebiosynthese benötigt wird. Die transkriptionelle Regulierung von jlbA wurde unter induzierten und nicht induzierten Nährstoff- und Umweltbedingungen verglichen. Unter Aminosäuremangel wird die Expression von jlbA stark induziert, was auf eine Regulation durch das CPCA der ‚Cross-Pathway’ Kontrolle schliessen lässt. Zusätzlich wird die jlbA Transkription unter Aminosäuremangelbedingungen aber auch durch eine CPCA unabhängige Regulierung induziert. Die zwei RPS26 Isogene von S. cerevisiae codieren für zwei Proteine der kleinen ribosomalen Untereinheit. Die abgeleitete Aminosäuresequenz der beiden Proteine unterscheidet sich in nur zwei Aminosäureresten. Das Rps26A Protein wurde als ein essentieller Bestandteil der normalen Zellteilung identifiziert. Zusätzlich nimmt Rps26A an der translationellen Regulation von adhäsivem und pseudohyphalem Wachstum teil, welche auf die FLO11 Expression von S. cerevisiae S1278b Wildtyp-Stämmen wirkt. Im Gegensatz dazu scheint das Rps26B Protein bei allen diesen Regulationsmechanismen eine weniger wichtige Rolle zu spielen, es kann aber teilweise die Rps26A Proteinfunktionen übernehmen. Die ARO3 und ARO4 Gene von S. cerevisiae codieren für zwei unterschiedlich regulierte DAHP Synthase-Isoenzyme des ersten Schrittes des Shikimat-Synthese-Weges. Die Aro3 und Aro4 Proteine werden durch Phenylalanin bzw. Tyrosin in ihrer Wirkung gehemmt. Beide Isogene wurden durch ein einzelnes Gen ersetzt, welches eine Tryptophan-regulierte DAHP Synthase codiert. Ein Vergleich des strukturellen und physiologischen Verhaltens aller DAHP Isoenzyme resultierte in einer gezielten Veränderung (‚protein engineering’) des Regulationsverhalten eines Aro4 Pro165Gly Mutanten-Enzymes. Dieses Enzym steht unter einer effizienten, zweifachen Kontrolle. Eine Aro4 Pro165Gly Mutante wird durch Tyrosin und durch Tryptophan in ihrer Aktivität gehemmt. Eine sorgfältige Analyse des Aro4 Wildtyp-Proteins zeigte, dass dieses Enzym ebenfalls moderat durch Tryptophan gehemmt wird