Determination of Real-Time Efflux Phenotypes in Escherichia coli AcrB Binding Pocket Phenylalanine Mutants Using a 1,2′-Dinaphthylamine Efflux Assay

To evaluate the importance of phenylalanine residues for substrate transport in the Escherichia coli efflux pump protein AcrB, we subjected Phe-to-Ala binding pocket mutants to a real-time efflux assay with the novel near-infrared lipophilic membrane probe 1,2′-dinaphthylamine (1,2′-DNA). All mutations, with the exception of F617A, led to considerable retardation of efflux. F610A was the point mutation with the most pronounced impact, followed by F628A, F615A, F136A, and F178A. This is the first study to demonstrate the importance of single phenylalanine residues within the AcrB binding pocket for real-time substrate transport

Clinically Approved Drugs Inhibit the Staphylococcus aureus Multidrug NorA Efflux Pump and Reduce Biofilm Formation

Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 μM. Moreover, at 0.39 μM, nilotinib, in combination with 8 μg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations

SIN-dependent phosphoinhibition of formin multimerization controls fission yeast cytokinesis

Many eukaryotes accomplish cell division by building and constricting a medial actomyosin-based cytokinetic ring (CR). In Schizosaccharomyces pombe, a Hippo-related signaling pathway termed the septation initiation network (SIN) controls CR formation, maintenance, and constriction. However, how the SIN regulates integral CR components was unknown. Here, we identify the essential cytokinetic formin Cdc12 as a key CR substrate of SIN kinase Sid2. Eliminating Sid2-mediated Cdc12 phosphorylation leads to persistent Cdc12 clustering, which prevents CR assembly in the absence of anillin-like Mid1 and causes CRs to collapse when cytokinesis is delayed. Molecularly, Sid2 phosphorylation of Cdc12 abrogates multimerization of a previously unrecognized Cdc12 domain that confers F-actin bundling activity. Taken together, our findings identify a SIN-triggered oligomeric switch that modulates cytokinetic formin function, revealing a novel mechanism of actin cytoskeleton regulation during cell division. © 2013 Bohnert et al

Determinants of National Guard Mental Health Service Utilization in VA versus Non‐VA Settings

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134155/1/hesr12446.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134155/2/hesr12446-sup-0001-AppendixSA1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134155/3/hesr12446_am.pd

Progression of foot deformity in charcot neuropathic osteoarthropathy

BACKGROUND: Charcot neuropathic osteoarthropathy associated foot deformity can result in joint instability, ulceration, and even amputation. The purpose of the present study was to follow patients with and without active Charcot osteoarthropathy for as long as two years to examine the magnitude and timing of foot alignment changes. METHODS: We studied fifteen subjects with Charcot osteoarthropathy and nineteen subjects with diabetes mellitus and peripheral neuropathy without Charcot osteoarthropathy for one year; eight of the subjects with osteoarthropathy and five of the subjects with diabetes and peripheral neuropathy were followed for two years. Bilateral weight-bearing radiographs of the foot were made at baseline for all subjects, with repeat radiographs being made at six months for the osteoarthropathy group and at one and two years for both groups. Radiographic measurements included the Meary angle, cuboid height, calcaneal pitch, and hindfoot-forefoot angle. RESULTS: The Meary angle, cuboid height, and calcaneal pitch worsened in feet with Charcot osteoarthropathy over one year as compared with the contralateral, uninvolved feet and feet in patients with diabetes and peripheral neuropathy. Cuboid height continued to worsen over the two-year follow-up in the feet with Charcot osteoarthropathy. These feet also had a greater change in the hindfoot-forefoot angle at one year as compared with the feet in patients with diabetes and peripheral neuropathy and at two years as compared with the contralateral, uninvolved feet. CONCLUSIONS: In patients with Charcot neuropathic osteoarthropathy, radiographic alignment measurements demonstrate the presence of foot deformity at the time of the initial clinical presentation and evidence of progressive changes over the first and second years. The six-month data suggest worsening of medial column alignment prior to lateral column worsening. This radiographic evidence of worsening foot alignment over time supports the need for aggressive intervention (conservative bracing or surgical fixation) to attempt to prevent limb-threatening complications. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence

Applications of Two-Body Dirac Equations to the Meson Spectrum with Three versus Two Covariant Interactions, SU(3) Mixing, and Comparison to a Quasipotential Approach

In a previous paper Crater and Van Alstine applied the Two Body Dirac equations of constraint dynamics to the meson quark-antiquark bound states using a relativistic extention of the Adler-Piran potential and compared their spectral results to those from other approaches, ones which also considered meson spectroscopy as a whole and not in parts. In this paper we explore in more detail the differences and similarities in an important subset of those approaches, the quasipotential approach. In the earlier paper, the transformation properties of the quark-antiquark potentials were limited to a scalar and an electromagnetic-like four vector, with the former accounting for the confining aspects of the overall potential, and the latter the short range portion. A part of that work consisted of developing a way in which the static Adler-Piran potential was apportioned between those two different types of potentials in addition to covariantization. Here we make a change in this apportionment that leads to a substantial improvement in the resultant spectroscopy by including a time-like confining vector potential over and above the scalar confining one and the electromagnetic-like vector potential. Our fit includes 19 more mesons than the earlier results and we modify the scalar portion of the potential in such a way that allows this formalism to account for the isoscalar mesons {\eta} and {\eta}' not included in the previous work. Continuing the comparisons made in the previous paper with other approaches to meson spectroscopy we examine in this paper the quasipotential approach of Ebert, Faustov, and Galkin for a comparison with our formalism and spectral results.Comment: Revisions of earlier versio

Neuropathic midfoot deformity: Associations with ankle and subtalar joint motion

BACKGROUND: Neuropathic deformities impair foot and ankle joint mobility, often leading to abnormal stresses and impact forces. The purpose of our study was to determine differences in radiographic measures of hind foot alignment and ankle joint and subtalar joint motion in participants with and without neuropathic midfoot deformities and to determine the relationships between radiographic measures of hind foot alignment to ankle and subtalar joint motion in participants with and without neuropathic midfoot deformities. METHODS: Sixty participants were studied in three groups. Forty participants had diabetes mellitus (DM) and peripheral neuropathy (PN) with 20 participants having neuropathic midfoot deformity due to Charcot neuroarthropathy (CN), while 20 participants did not have deformity. Participants with diabetes and neuropathy with and without deformity were compared to 20 young control participants without DM, PN or deformity. Talar declination and calcaneal inclination angles were assessed on lateral view weight bearing radiograph. Ankle dorsiflexion, plantar flexion and subtalar inversion and eversion were assessed by goniometry. RESULTS: Talar declination angle averaged 34±9, 26±4 and 23±3 degrees in participants with deformity, without deformity and young control participants, respectively (p< 0.010). Calcaneal inclination angle averaged 11±10, 18±9 and 21±4 degrees, respectively (p< 0.010). Ankle plantar flexion motion averaged 23±11, 38±10 and 47±7 degrees (p<0.010). The association between talar declination and calcaneal inclination angles with ankle plantar flexion range of motion is strongest in participants with neuropathic midfoot deformity. Participants with talonavicular and calcaneocuboid dislocations result in the most severe restrictions in ankle joint plantar flexion and subtalar joint inversion motions. CONCLUSIONS: An increasing talar declination angle and decreasing calcaneal inclination angle is associated with decreases in ankle joint plantar flexion motion in individuals with neuropathic midfoot deformity due to CN that may contribute to excessive stresses and ultimately plantar ulceration of the midfoot

Automated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands

A Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei