Comparison of four genotyping assays for epidemiological study of methicillin-resistant Staphylococcus aureus

Twenty-six methicillin-resistant Staphylococcus aureus strains were genetically differentiated by interrepeat PCR and the results compared with those of ribotyping, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA analysis obtained in a previous study for the same strains. The comparison showed that the PCR-mediated assays were as discriminatory as PFGE, whereas ribotyping was the least powerful genotyping method. Due to the ease of performance, PCR fingerprinting may become the method of choice for establishing clonal relationships among Staphylococcus aureus isolates

Mechanistic and structural basis for the actions of the antibacterial gepotidacin against Staphylococcus aureus gyrase

Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 µM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 µM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or with intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound

Three-Dimensional Triple-Resonance NMR of \u3csup\u3e13\u3c/sup\u3eC/\u3csup\u3e15\u3c/sup\u3eN-Enriched Proteins Using Constant-Time Evolution

Recently it has been convincingly demonstrated that 30 triple-resonance NMR provides a practical alternative for obtaining sequential resonance assignments in larger proteins ( 1, 2). This approach requires a set of five or six 30 NMR experiments that correlate the various protein backbone nuclei. Details regarding the mechanisms and technical implementations of these experiments have been described previously ( 3- 5). Two of the experiments used in this approach correlate backbone Hα and Cα resonances with either the intraresidue carbonyl resonance (CO) or the 15N resonance of the succeeding residue and are referred to as HCACO and HCA(CO)N, respectively. The present Communication describes a modification of these experiments which optimizes their sensitivity and removes the F1 antiphase character of correlations

Midwives’ perceptions of the performance- and transition into practice of newly qualified midwives, a focus group study

Problem: Newly qualified midwives in the Netherlands perceive the adaptation to new responsibilities as difficult due to the autonomous nature of- and required accountability for the work they face in practice. Background: All Dutch newly qualified midwives are accountable for their work from the moment of registration while usually working solistically. Aim: This paper explores the perceptions of experienced midwives regarding: (1) the performance- and transition into practice of newly qualified midwives, and (2) their supporting role in this transition. Methods: The design of this study is qualitative with focus groups. Experienced midwives’ perceptions were explored by means of seven semi-structured focus groups (N = 46 participants) with two meetings for each focus group. Findings: Community-based and hospital-based midwives perceived newly qualified midwives as colleagues who did not oversee all their tasks and responsibilities. They perceived newly qualified midwives as less committed to the practice organisation. Support in community-based practices was informally organised with a lack of orientation. In the hospital-based setting, midwives offered an introduction period in a practical setting, which was formally organised with tasks and responsibilities. Experienced midwives recognised the need to support newly qualified midwives; however, in practice, they faced barriers. Discussion: The differences in experienced midwives’ expectations of newly qualified midwives and reality seemed to depend on the newly qualified midwives’ temporary working contracts and -context, rather than the generational differences that experienced midwives mentioned. Dutch midwives prioritised their work with pregnant individuals and the organisation of their practice above supporting newly qualified midwives

The Structure of Mouse Cytomegalovirus m04 Protein Obtained from Sparse NMR Data Reveals a Conserved Fold of the m02-m06 Viral Immune Modulator Family

SummaryImmunoevasins are key proteins used by viruses to subvert host immune responses. Determining their high-resolution structures is key to understanding virus-host interactions toward the design of vaccines and other antiviral therapies. Mouse cytomegalovirus encodes a unique set of immunoevasins, the m02-m06 family, that modulates major histocompatibility complex class I (MHC-I) antigen presentation to CD8+ T cells and natural killer cells. Notwithstanding the large number of genetic and functional studies, the structural biology of immunoevasins remains incompletely understood, largely because of crystallization bottlenecks. Here we implement a technology using sparse nuclear magnetic resonance data and integrative Rosetta modeling to determine the structure of the m04/gp34 immunoevasin extracellular domain. The structure reveals a β fold that is representative of the m02-m06 family of viral proteins, several of which are known to bind MHC-I molecules and interfere with antigen presentation, suggesting its role as a diversified immune regulation module

Cell-matrix biology in vascular tissue engineering

We are developing biocompatible small-calibre vascular substitutes based on polymeric scaffolds that incorporate cell-matrix signals to enhance vascular cell attachment and function. Our graft scaffold comprises an outer electrostatically spun porous polyurethane layer seeded with smooth muscle cells, and a luminal polycaprolactone layer for endothelial cell attachment. Vascular cell adhesion properties of three vascular elastic fibre molecules, tropoelastin, fibrillin-1 and fibulin-5, have been defined, and adhesion fragments optimized. These fragments are being used to coat the scaffolds to enhance luminal endothelial cell attachment, and to regulate smooth muscle cell attachment and function. Tropoelastin-based cell seeding materials are also being developed. In this way, vascular cell-matrix biology is enhancing graft design

Using eye-tracking in applied linguistics and second language research

With eye-tracking technology the eye is thought to give researchers a window into the mind. Importantly, eye-tracking has significant advantages over traditional online processing measures: chiefly that it allows for more ‘natural’ processing as it does not require a secondary task, and that it provides a very rich moment-to-moment data source. In recognition of the technology’s benefits, an ever increasing number of researchers in applied linguistics and second language research are beginning to use it. As eye-tracking gains traction in the field, it is important to ensure that it is established in an empirically sound fashion. To do this it is important for the field to come to an understanding about what eye-tracking is, what eye-tracking measures tell us, what it can be used for, and what different eye-tracking systems can and cannot do. Further, it is important to establish guidelines for designing sound research studies using the technology. The goal of the current review is to begin to address these issues