Stress response of a clinical Enterococcus faecalis isolate subjected to a novel antimicrobial surface coating

Emerging antibiotic resistance among pathogenic bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem for effective and long-term decontamination in health-care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the impact of AGXX®, a novel broad-spectrum antimicrobial surface coating consisting of micro-galvanic elements formed by silver and ruthenium, on the transcriptome of Enterococcus faecalis. A clinical E. faecalis isolate was subjected to metal stress by growing it for different periods in presence of the antimicrobial coating or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression in presence of the antimicrobial materials with focus on known stress genes. Exposure to the antimicrobial coating had a large impact on the transcriptome of E. faecalis. After 24min almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to the antimicrobial coating induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES and the Clp proteases, ClpE and ClpB, were among the top up-regulated heat shock genes. Differential expression of thioredoxin, superoxide dismutase and glutathione synthetase genes indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXX®results in a synergistic antimicrobial effect, superior to that of conventional silver coatings.</p

The Involvement of the McsB Arginine Kinase in Clp-Dependent Degradation of the MgsR Regulator in Bacillus subtilis

Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR

Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release

Serotonin is a neurotransmitter in the central nervous system. In the periphery, serotonin functions as a ubiquitous hormone involved in vasoconstriction and platelet function. Serotonin is synthesized independently in peripheral tissues and neurons by two different rate-limiting tryptophan hydroxylase (TPH) isoenzymes. Here, we show that mice selectively deficient in peripheral TPH and serotonin exhibit impaired hemostasis, resulting in a reduced risk of thrombosis and thromboembolism, although the ultrastructure of the platelets is not affected. While the aggregation of serotonin-deficient platelets in vitro is apparently normal, their adhesion in vivo is reduced due to a blunted secretion of adhesive -granular proteins. In elucidating the mechanism further, we demonstrate that serotonin is transamidated to small GTPases by transglutaminases during activation and aggregation of platelets, rendering these GTPases constitutively active. Our data provides evidence for a receptor-independent signaling mechanism, termed herein as "serotonylation," which leads to -granule exocytosis from platelets

Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

<div><p>Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.</p></div

First operation of the KATRIN experiment with tritium

The determination of the neutrino mass is one of the major challenges in astroparticle physics today. Direct neutrino mass experiments, based solely on the kinematics of β β -decay, provide a largely model-independent probe to the neutrino mass scale. The Karlsruhe Tritium Neutrino (KATRIN) experiment is designed to directly measure the effective electron antineutrino mass with a sensitivity of 0.2 eV 0.2 eV (90% 90% CL). In this work we report on the first operation of KATRIN with tritium which took place in 2018. During this commissioning phase of the tritium circulation system, excellent agreement of the theoretical prediction with the recorded spectra was found and stable conditions over a time period of 13 days could be established. These results are an essential prerequisite for the subsequent neutrino mass measurements with KATRIN in 2019

Nf1Prx1 bone tissue shows diminished mechanical strength and increased micro-porosity due to increased osteocyte lacuna size.

<p>(A) Diagram of the analytical setup used for tensile experiment. Measurements were done on laser dissected cortical bone slices from P90 mice. (B) A typical tensile stress-strain pattern in controls showed a linear elastic modulus (E-modulus) phase that is followed by the yield point plateau and the ultimate stress point. Note, increased elasticity and diminished ultimate strength in bone slices of Nf1Prx1 humerus compared to controls. (C) Diminished E-modulus (slope of tensile curve) and (D) ultimate stress point (point of maximal load before tissue failure) in bone tissue slices from 3 months old Nf1Prx1 (n = 14) and control (n = 15) mice. (E) Quantitative microCT analysis of the total (summed) lacunae porosity (Lc.V/BV) within the lacunae range from 100-4000 µm³. Note the significant increase of Lc.V/BV in Nf1Col1 and Nf1Prx1 mice. (F) The summed volume of Ot. lacunae (Lc.V) within the size range 100–4000 µm³ with an increment of 100 µm³ (histogram). Note the shift of summed Lc.V fractions towards higher volume range in Nf1Col1 and Nf1Prx1 mutants. Insert (a) - Nf1Col1 mice demonstrated reduced summed Lc. volume in the intervals 200–300 and 300–400 µm³ and (b) - summed Lc.V in fractions 800–900 and 900–1000 µm³. Insert (c) - Nf1Prx1 mice showed reduced summed Lc. volume in the intervals 100–200 and 200–300 µm³ and (d) - summed Lc.V in fractions 700–800 and 800–900 µm³. (G) The total number of Ot. lacunae (Lc.N) was unaffected in both mouse models. (H) Ot. lacunae morphology (range from 100–4000 µm³) assessed by measuring the x, y and z axis length in a selected volume of ROI E2. Note, lacunae size is increased in all dimensions in Nf1Col1 and Nf1Prx1 mice. The number of analyzed bone samples: Nf1Prx1 n = 5, controls n = 5; Nf1Col1 n = 3, controls n = 3. Statistical analysis was performed with unpaired Student t-test, * p≤0.05 ** p≤0.01.</p

Cortical bone in NF1 tibial dysplasia is characterized by heterogeneous mineralization, diminished collagen thickness and increased micro-porosity.

<p>(A) MicroCT analysis of human cortical bone samples (tibia). Bone mineral density was calibrated according to Houndsfield units (HU) and color coded (see HU scale). HU values measured in ROI1 (cortical bone without blood vessels) and ROI2 (cortical bone near blood vessel) were in control specimen similar. However, in the NF1 bone sample HU values were in proximity of large vessels decreased (ROI2). The analyzed bone sample was obtained pre-fracture from an individual affected by NF1 with tibial bowing. (B) Two further mineralized bone samples were obtained from cortical bone of NF1 individuals with pseudarthrosis. Samples were analyzed by picrosirius red histology and imaged in polarized light (large image) and bright light (inset). Collagen fibers follow osteonal organization in control specimens. Note that in the NF1 cortical bone sample collagen fiber organization appears less orderly with abundant thin (green) collagen fibers. (C) Ot. morphology was visualised with AgNOR staining. Ot. are spindle shaped (inset) and regularly distributed in control cortical bone. In contrast, Ot. are round and irregularly distributed in NF1 cortical bone. (D) Histomorphometry of AgNOR stained bone sections showing: relative Ot. area (Ot.Ar/B.Ar), Ot. number per bone area (Ot.N/B.Ar), individual Ot. area (Ot.Ar) and individual Ot. circumference (Ot.Cir). Presented data are mean values with standard deviations (control n = 3, NF1 n = 2). (E) Volumetric microCT analysis showing increased specific lacunae (Ot.) volume (Lc.Vol) and surface (Lc.Sur) in NF1 tibial dysplasia cortical bone as compared to controls. Statistical analysis was performed with unpaired t-test, ** p≤0.01. Abbreviations: blood vessels (bv) and bone (b). All scale bars represent 50 µm.</p

Diminished organic matrix properties in Nf1Prx1 mice.

<p>(<b>A</b>) Picrosirius red stained bone sections imaged with polarized light. Homogenous red staining in controls indicates highly packed and thick collagen. Heterogeneous red-yellow-green staining of Nf1Prx1 bone sections is indicative of diminished packaging and thickness of bone collagen. Note, there is no picrosirius red staining within non-mineralized bone tissue (osteoid) surrounding blood vessels. Scale bar shows 20 µm. (<b>B</b>) Silver staining (AgNOR) detects the osteocytic network and other accumulations of acidic matrix proteins. Note the large unstained area around blood vessels in Nf1Prx1 humerus. Scale bar shows 50 µm. (<b>C</b>) Sections of humerus cortex stained with Toluidin and Safranin O. Toluidin stained osteocyte (Ot.) appear blue with dark blue nuclei (black arrowhead). Presence of nuclei suggests vitality of cells in the bone cortex. Osteoid (dotted line) near to the blood vessel was stained light blue (Toluidin) or light red (Safranin O). Light blue or red staining indicates that bone lesions are not composed of cartilaginous matrix, which with these methods stains purple (Toluidin) or dark red (Safranin O) (not shown). Ot. in non-mineralized areas are viable (nuclei marked with grey arrow head). Scale bar represents 50 µm. Abbreviations: blood vessels (bv), bone (b), bone marrow (bm), osteoid (o).</p

Diminished bone stiffness, bone mineralization and organic matrix formation in Nf1Prx1 mice.

<p>(<b>A</b>) Scanning acoustic microscopy (SAM) was applied to show elastic properties of the bone matrix. SAM measurements were performed within six ROIs E1a–E3b on anterior (a) and posterior (b) bone cortex. Images illustrate impedance values (Z in MRayl) according to the color scale (left). Nf1Prx1 mutants show diminished impedance values within all analyzed ROIs. (<b>B</b>) Quantitative evaluation of SAM measurements reveals decreased Z values in Nf1Prx1 humerus in all analyzed ROIs (control n = 4, Nf1Prx1 n = 4). Nf1Col1 humeri did not demonstrate diminished Z values (control n = 5, Nf1Col1 n = 4). (<b>C</b>) The cross sections of humerus at the level E2 with color coded BMD values measured by phantom calibrated microCT. The yellow and green coloring indicates low and high BMD values, respectively. Scale bar - 600 µm. (<b>D</b>) Cortical bone mineral density (BMD) was assessed by microCT within region E1–E3 in humeri of Nf1Prx1 (control n = 5, Nf1Prx1 n = 5) mice. BMD was reduced in the Nf1Prx1 model in all ROIs (for definition see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086115#pone.0086115.s001" target="_blank">Fig.S1</a>). Statistical significance - t-test, * p≤0.05 and ** p≤0.01.</p