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Staphylococcus aureus small-colony variants (SCVs) often persist despite antibiotic therapy. Against a 10 8 -CFU/ml methicillinresistant S. aureus (MRSA) (strain COL) population of which 0%, 1%, 10%, 50%, or 100% was an isogenic hemB knockout (Ia48) subpopulation displaying the SCV phenotype, vancomycin achieved maximal reductions of 4.99, 5.39, 4.50, 3.28, and 1.66 log 10 CFU/ml over 48 h. Vancomycin at >16 mg/liter shifted a population from 50% SCV cells at 0 h to 100% SCV cells at 48 h, which was well characterized by a Hill-type model (R 2 > 0.90). Staphylococcus aureus is a virulent pathogen responsible for a myriad of infections ranging from minor community-acquired skin and soft tissue infections to severe nosocomial infections (1). While the current IDSA guidelines recommend vancomycin as the primary agent for treatment of methicillin-resistant S. aureus (MRSA) infections, the utility of the drug has been brought into question due to increasing reports of heterogeneous resistance, treatment failure, and nephrotoxicity (2-4). Despite the global decrease in vancomycin susceptibility, the exact mechanism by which S. aureus develops resistance is not well understood (5). It has been suggested that S. aureus adapts by utilizing an array of genotypic alterations that arise stepwise during the selective pressure of antimicrobial therapy (6, 7).One pathway that S. aureus may exploit during the evolution of antimicrobial resistance is the development of small-colony variants (SCVs) that grow slowly relative to strains of the normal phenotype (NP) (8-10). In vitro testing and macrophage models have confirmed that the SCV phenotype is less susceptible to vancomycin (11, 12). Studies with other antibiotics also suggest that SCV subpopulations may cooperate with NP S. aureus to attenuate antimicrobial activity (13). At present, it is unknown whether SCVs alter vancomycin pharmacodynamics through interactions with NP S. aureus or how the selection of a vancomycin regimen influences the relationship between the two phenotypes. The objective of the current study was, therefore, to utilize reconstructive population biology to determine how the interplay of both phenotypes alters vancomycin pharmacodynamics.The MRSA strain COL (NP) and its isogenic hemB knockout Ia48 (COL hemB::ermB, a stable SCV phenotype) were utilized. The creation of the mutant strain and its features were previously characterized (14). Prior to each experiment, a solution of vancomycin was prepared using analytical-grade powder (Sigma Chemical, St. Louis, MO) in the following concentrations: 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/liter. Brain heart infusion (BHI) broth supplemented with magnesium (12.5 mg/liter) and calcium (25 mg/liter) was used for every experiment. SCV and NP cell suspensions were volumetrically titrated to achieve 5 different starting compositions, with a total bacterial load of 10 8 CFU/ml. Two experiments were conducted exclusively investigating the NP or the SCV phenotype (0% SCV/100% NP cells and 100% SCV/0% NP cells), an...

The ubiquitous α-catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α-catulin has homology to the cytoskeletal linkers α-catenin and vinculin, it appears to be functionally divergent. Here we further investigated α-catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α-catulin has a unique cytoskeletal role. Examination of endogenous α-catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double-immunofluorescence analyses of VEC showed endogenous α-catulin co-localization with vimentin intermediate filaments. Similar to vimentin, α-catulin was found to distribute into detergent-soluble and -insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α-catulin network distribution and altered α-catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α-catulin knock-down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α-catulin depletion was shown to reduce ROCK-induced signaling. These findings indicate that α-catulin has a unique function in co-localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. J. Cell. Physiol. 231: 934-943, 2016. © 2015 Wiley Periodicals, Inc.

Older adults who use NSAIDs may be unaware of potential risks. Counseling older adults may reduce potentially inappropriate use and increase patient risk awareness. Pharmacists can improve their role in recognizing and counseling patients on NSAIDs.