JCTH-4 and CC do not yield apoptotic morphology in HOb and NFF cells.

<p>Nuclear and cellular morphology of (<b>A</b>) HOb and (<b>B</b>) NFF cells after 72 hours of treatment. Cells were treated with JCTH-4, CC, and solvent control (Me₂SO). Post treatment, the cells were stained with Hoechst 33342 dye. Corresponding phase micrographs are shown below the Hoechst micrographs. Apoptotic morphology is evident in cells with bright and condensed nuclei accompanied by apoptotic bodies, as well as cell shrinkage and blebbing. Images were taken at 400× magnification on a fluorescent microscope. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 causes selective cytotoxicity in OS cells in a time and dose-dependent manner.

<p>Effect of JCTH-4 on cellular viability of OS cells was determined by the WST-1 based colorimetric assay. (<b>A</b>) Saos-2 and (<b>B</b>) U-2 OS cells were treated with JCTH-4 and the WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of the control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 versus solvent control (Me₂SO). (<b>C</b>) Effect on cellular viability of HOb and NFF cells treated with JCTH-4 compared to Saos-2 and U-2 OS cells after 72 hours. The WST-1 reagent was used to quantify cellular viability. Absorbance was read at 450 nm and expressed as a percent of the solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.005 versus Saos-2 cells; #<i>p</i><0.005 versus U-2 OS cells.</p

JCTH-4 and CC do not induce autophagy in Hob and NFF cells.

<p>MDC staining was used to detect the presence of autophagic vacuoles in (<b>A</b>) HOb and (<b>B</b>) NFF cells after 72 hours of treatment with JCTH-4, CC, and solvent control (Me₂SO) at the indicated concentrations. Bright blue punctate marks are indicative of autophagic vacuoles. Corresponding phase and PI micrographs are shown below the MDC images. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 dissipates MMP alone and in combination with CC in OS cells.

<p>Effect of JCTH-4 and CC on MMP in (<b>A</b>) Saos-2 cells after 96 hours of treatment and (<b>B</b>) U-2 OS cells after 72 hours of treatment was examined by TMRM staining. Cells were grown on coverslips, treated with the indicated concentrations of JCTH-4, CC, and solvent control (Me₂SO) and stained with TMRM and Hoechst dye. Images were taken at 400× magnification on a fluorescent microscope. Red fluorescent punctuate marks are indicative of mitochondria with intact MMP. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 directly causes mitochondrial ROS production and release of apoptogenic factors independent of caspases.

<p>(<b>A</b>) Saos-2 and (<b>B</b>) U-2 OS isolated mitochondria were treated directly with JCTH-4, CC, PQ, and solvent control (Me₂SO), and incubated with Amplex Red and horseradish peroxidase for 2 hours. Subsequently, fluorescence readings were taken at Ex. 560 nm and Em. 590 nm and expressed as relative fluorescence units (RFU). Statistics were performed using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean ± SD of quadruplicates of 1 independent experiment. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 versus solvent control (Me₂SO); †<i>p</i><0.01 versus 0.25 µM JCTH-4; @<i>p</i><0.01 versus 0.5 µM JCTH-4; #<i>p</i><0.01 versus 5 µM CC. Isolated mitochondria samples treated directly with JCTH-4, CC, and solvent control (Me₂SO) for 2 hours were also centrifuged, producing mitochondrial pellets and post mitochondrial supernatants which were examined for retention and release of apoptogenic factors respectively via western blot analyses; (<b>C</b>) Retention of AIF and release of EndoG by U-2 OS cell mitochondria and (<b>D</b>) release of AIF by Saos-2 cell mitochondria was monitored. Mitochondrial pellets were probed for SDHA to serve as loading controls. Densitometric analyses were performed using ImageJ software and statistics were calculated using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean ± SD of triplicates of one independent experiment. *<i>p</i><0.01, **<i>p</i><0.001 versus solvent control (Me₂SO); †<i>p</i><0.01 versus 0.25 µM JCTH-4; #<i>p</i><0.01 versus 5 µM CC. (<b>E</b>) Saos-2 cells were treated with broad spectrum caspase inhibitor Z-VAD-FMK with and without JCTH-4 for 72 hours. WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.001 versus solvent control (Me₂SO); ns = not significant.</p

JCTH-4 alone and in combination with CC yields apoptotic morphology in OS cells.

<p>Nuclear and cellular morphology of (<b>A</b>) Saos-2 cells after 96 hours of treatment and (<b>B</b>) U-2 OS cells after 72 hours of treatment. Cells were treated with JCTH-4, CC, and solvent control (Me₂SO). Post treatment, the cells were stained with Hoechst 33342 dye. Corresponding phase micrographs are shown below the Hoechst micrographs. Apoptotic morphology is evident in cells with bright and condensed nuclei accompanied by apoptotic bodies, as well as cell shrinkage and blebbing. Images were taken at 400× magnification on a fluorescent microscope. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

CC potentiates the cytotoxicity of JCTH-4 selectively in OS cells.

<p>Effect of JCTH-4 & CC in combination on cellular viability of OS cells was determined by the WST-1 based colorimetric assay. (<b>A</b>) Saos-2 (96 hours), (<b>B</b>) U-2 OS (72 hours), (<b>C</b>) HOb (72 hours), and (<b>D</b>) NFF (72 hours) cells were treated with JCTH-4 and CC and the WST-1 reagent was used to quantify cellular viability. Absorbance was read at 450 nm and expressed as a percent of the solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, versus solvent control (Me₂SO); †<i>p</i><0.001 versus 0.25 µM JCTH-4; ††<i>p</i><0.01 versus 0.5 µM JCTH-4; #<i>p</i><0.001 versus 5 µM CC; @<i>p</i><0.001 versus 0.25 µM JCTH-4+5 µM CC treatment with Saos-2 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028780#pone-0028780-g003" target="_blank">Figure 3A</a>); &<i>p</i><0.01 versus 0.5 µM JCTH-4+5 µM CC treatment with U-2 OS cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028780#pone-0028780-g003" target="_blank">Figure 3B</a>).</p

Comparison of chemical structures.

<p>Structures of (<b>A</b>) Pancratistatin (PST) and (<b>B</b>) JC-TH-acetate-4 (JCTH-4).</p

Clustered multidrug-resistant Bordetella petrii in adult cystic fibrosis patients in Ireland: case report and review of antimicrobial therapies

Introduction: Bordetella petrii is an emerging pathogen. Whilst association with cystic fibrosis (CF) has been described previously, this is the first report to our knowledge of multidrug-resistant B. petrii incidence in an Irish CF patient population. Case presentation: Using a case series of four adult CF patients with varying baselines of health, one of whom was asymptomatic, this report attempts correlation of B. petrii colonization, by one common strain, with incidence of acute exacerbation of symptoms. As definitive guidelines for antimicrobial sensitivity/resistance do not exist for B. petrii, we completed a systematic review of available literature to collate evidence of antimicrobial efficacy against B. petrii. Comparison with the isolates in this study indicated B. petrii sensitivity to piperacillin/ tazobactam and minocycline but resistance to antimicrobials in the macrolide, other b-lactam and fluoroquinolone groups. Conclusion: To our knowledge, this is the first report of multiple CF patients sharing a strain of B. petrii. Furthermore, B. petrii may be under-identified in CF patients and should be considered when evaluating exacerbation of CF symptoms

Fire Promotes Pollinator Visitation: Implications for Ameliorating Declines of Pollination Services

<div><p>Pollinators serve critical roles for the functioning of terrestrial ecosystems, and have an estimated annual value of over $150 billion for global agriculture. Mounting evidence from agricultural systems reveals that pollinators are declining in many regions of the world, and with a lack of information on whether pollinator communities in natural systems are following similar trends, identifying factors which support pollinator visitation and services are important for ameliorating the effects of the current global pollinator crisis. We investigated how fire affects resource structure and how that variation influences floral pollinator communities by comparing burn versus control treatments in a southeastern USA old-field system. We hypothesized and found a positive relationship between fire and plant density of a native forb, <i>Verbesina alternifolia</i>, as well as a significant difference in floral visitation of <i>V. alternifolia</i> between burn and control treatments. <i>V. alternifolia</i> density was 44% greater and floral visitation was 54% greater in burned treatments relative to control sites. When the density of <i>V. alternifolia</i> was experimentally reduced in the burn sites to equivalent densities observed in control sites, floral visitation in burned sites declined to rates found in control sites. Our results indicate that plant density is a proximal mechanism by which an imposed fire regime can indirectly impact floral visitation, suggesting its usefulness as a tool for management of pollination services. Although concerns surround the negative impacts of management, indirect positive effects may provide an important direction to explore for managing future ecological and conservation issues. Studies examining the interaction among resource concentration, plant apparency, and how fire affects the evolutionary consequences of altered patterns of floral visitation are overdue.</p> </div