Characteristics and clinical assessment of antibiotic delivery by chitosan sponge in the high-risk diabetic foot: a case series.

OBJECTIVE: The local delivery of antimicrobials is attractive for a number of reasons. Chitosan, a biodegradable polysaccharide sponge material, has been proposed as medium to deliver antibiotics directly to wounds. In this report we evaluate the safety and practicality of antimicrobial delivery via chitosan sponge. METHOD: We present the clinical course and systemic absorption characteristics of three cases of people with diabetic foot wounds treated with antibiotic soaked chitosan sponge (Sentrex BioSponge, Bionova Medical, Germantown, TN). The antibiotic sponge was made by reconstituting 1.2g tobramycin or 100mg doxycycline in 10-15ml saline and saturating the sponge with the solution. The sponge was then applied to the wounds. Serum levels of each respective antibiotic were evaluated after application. Additional in vitro studies were conducted evaluating elution of antibiotics from the chitosan sponge at established minimum inhibitory concentrations (MIC) for Staphylococcus aureus over 28 days. RESULTS: No patient experienced adverse local or systemic effects due to the sponge treatment. The measured serum levels applied antibiotics remained far less than established minimums after intravenous therapy. Each patient required further treatment, however local infection or contamination resolved during the course of their hospital stay after the chitosan/antibiotic application. CONCLUSION: The use of antibiotic-impregnated chitosan sponges appears a safe and effective mechanism of local delivery of antimicrobials in wounds. Future studies and clinical trials are ongoing to confirm these results and to guide clinical applications.

Decontamination of materials contaminated with Francisella philomiragia or MS2 bacteriophage using PES-Solid, a solid source of peracetic acid.

AIM: The aim of the study was to develop test methods and evaluate survival of Francisella philomiragia cells and MS2 bacteriophage after exposure to PES-Solid (a solid source of peracetic acid) formulations with or without surfactants. METHODS AND RESULTS: Francisella philomiragia cells (≥7·6 log10 CFU) or MS2 bacteriophage (≥6·8 log10 PFU) were deposited on seven different test materials and treated with three different PES-Solid formulations, three different preneutralized samples and filter controls at room temperature for 15 min. There were 0-1·3 log10 CFU (<20 cells) of cell survival, or 0-1·7 log10 (<51 PFU) of bacteriophage survival in all 21 test combinations (organism, formulation and substrate) containing reactive PES-Solid. In addition, the microemulsion (Dahlgren Surfactant System) showed ≤2 log10 (100 cells) of viable F. philomiragia cells, indicating the microemulsion achieved <2 log10 CFU on its own. CONCLUSIONS: Three PES-Solid formulations and one microemulsion system (DSS) inactivated F. philomiragia cells and/or MS2 bacteriophage that were deposited on seven different materials. SIGNIFICANCE AND IMPACT OF THE STUDY: A test method was developed to show that reactive PES-Solid formulations and a microemulsion system (DSS) inactivated >6 log10 CFU/PFU F. philomiragia cells and/or MS2 bacteriophage on different materials.

Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

AIMS: To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. METHODS AND RESULTS: Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. CONCLUSIONS: Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. SIGNIFICANCE AND IMPACT OF THE STUDY: A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials.

Imaging haematopoietic cells recruitment to an acute wound in vivo identifies a role for c-Met signalling.

We have used a direct in vivo imaging strategy to investigate the role of c-Met signalling and kinase activity during the immune response to wounding. Our assay utilizes the optical translucent properties of the zebrafish embryo and demonstrates the versatility of microscopy-based approach to the screening of compounds for inhibition of the wounding response. We have focussed on the c-Met pathway as little is known about the influence of c-Met signalling in immune responses, although it has been suggested that the c-Met tyrosine kinase receptor signalling pathway may be involved in cytokine secretion and directional migration in immune cells. Using both imaging of fixed zebrafish embryos in combination with digital time lapse microscopy of neutrophils recruited to a wound site, we find that pharmacological inhibition of c-Met, using a specific inhibitor, modulates the immune response in zebrafish embryos. We have found that inhibition of c-Met does not prevent the inflammatory response but does appear to limit recruitment and retention of immune cells at the wound site. This work demonstrates the versatility of using direct imaging assays for inhibitor studies and suggests that the HGF/c-Met signalling cascade plays an important role in the migration of haematopoietic cells in vivo.

PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity.

Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of overexpression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harbouring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the glycine-rich loop region of the kinase. Our data suggest that E329K mutation leads to a modest increase in kinase activity, conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition.

Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

AIMS: To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. METHODS AND RESULTS: Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. CONCLUSIONS: Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. SIGNIFICANCE AND IMPACT OF THE STUDY: Hot, humid air is a potential alternative to conventional chemical decontamination.

Sport-specific fitness testing differentiates professional from amateur soccer players where VO2max and VO2 kinetics do not.

AIM: The purpose of this study was to identify if sport-specific and cardiopulmonary exercise testing differentiated professional from amateur soccer players. METHODS: Thirty six men comprising 18 professional (mean±s: age 23.2±2.4 years) and 18 amateur (mean±SD: age 21.1±1.6 years) soccer players participated and performed four tests on separate occasions: 1) a graded exercise test to determine VO2max; 2) four exercise transients from walking to 80%Δ for the determination of VO2 kinetics; 3) the Yo-Yo Intermittent Recovery Test level 2 (Yo-Yo IR2) and 4) a repeated sprint test (RST). RESULTS: The players did not differ in VO2max (professional 56.5±2.9 mL.kg-1.min-1; amateur 55.7±3.5 mL.kg-1.min-1: P=0.484) or VO2 kinetic fundamental measures (τ1 onset, professional 24.5±3.2 s; amateur 24.0±1.8 s: τ1 cessation, professional 28.7±2.8 s; amateur 29.3±3.5 s: P=0.923). However, the amateurs were outperformed in the Yo-Yo IR2 (Professional 966±153 m; Amateur 840±156 m) (P=0.034) and RST (best time, professional 6.46±0.27 s; amateur 6.84±0.24 s, P=0.012). CONCLUSION: Performance indices derived from field-based sport-specific performance tests identified significant differences between professional and amateur players (P<0.05). However, neither tests of VO2 kinetics nor VO2max differentiated between groups, suggesting laboratory tests of cardiorespiratory parameters are probably less consequential to soccer than sport-specific field-based observations.

Decontamination of a hard surface contaminated with Bacillus anthracisΔSterne and B. anthracis Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO2).

AIMS: To evaluate the inactivation of Bacillus anthracisΔSterne and Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO(2)) and compare two sporulation and decontamination methods with regard to cost, safety and technical constraints. METHODS AND RESULTS: Spores were prepared via agar and broth methods and subsequently inoculated and dried onto clean, autoclave-sterilized glass coupons. Bacillus anthracis spore inactivation efficacy was evaluated using the modified three-step method (AOAC 2008.05) and a single-tube extraction method. Spores (7·0 ± 0·5 logs) were inactivated within 1 min at room temperature using freshly prepared eClO(2). Bacillus anthracisΔSterne spores decreased in size after eClO(2) treatment as measured using a Beckman Coulter Multisizer. CONCLUSIONS: eClO(2) saturation of a hard surface was an effective B. anthracis sporicide. Broth sporulation and the single-tube extraction method required less time and fewer steps, yielded a higher percentage of phase-bright spores and showed higher spore recovery efficiency compared with AOAC 2008.05, making it more amenable to biosafety level 3 (BSL3) testing of virulent spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Two test methods demonstrated the sporicidal efficacy of eClO(2). A new single-tube extraction test protocol for decontaminants was introduced.

Rho family GTPases are activated during HGF-stimulated prostate cancer-cell scattering.

An important process in embryogenesis and cancer-cell metastasis is the conversion of epithelial cells to a migratory phenotype, a phenomenon known as epithelial-mesenchymal transition (E-MT). To achieve E-MT, cells dissociate from neighbouring cells and adopt a migratory morphology. This transition requires remodelling of their cell shape and substratum adhesions; activities that require extensive reorganisation of the actin cytoskeleton. Hepatocyte growth factor (HGF)-induced scattering of Madin Darby canine kidney (MDCK) cells is a routinely used model of E-MT, in which actin cytoskeletal rearrangement is known to be dependent on Rho family GTPases. We have developed a novel model of HGF-induced E-MT using the human prostate cancer cell line, DU145. This model overcomes the limitation of using a canine cell line and facilitates the study of E-MT in human cancer. We demonstrate for the first time the scattering response of individual DU145 cells to HGF in real time and have characterised changes in actin cytoskeletal organisation and cell adhesions as these cells respond to HGF. HGF-induced scattering of DU145 cells is dependent on the activity of Rho family GTPases, and using this model, we are able to demonstrate for the first time that endogenous Cdc42 is activated downstream of HGF. Furthermore we have also shown that the response of DU145 cells to HGF is dependent on a phosphatidylinositide 3-kinase pathway.

Identification of carbonic anhydrase activity in bullfrog olfactory receptor neurons: histochemical localization and role in CO2 chemoreception.

The objectives of this study were to determine: (1) the frequency and distribution of carbonic anhydrase (CA) activity in the bullfrog nasal cavities, and (2) whether inhibition of nasal CA affects the olfactory receptor response to CO2 or other odorants. It was found, using Hansson's staining technique, that some olfactory receptor neurons exhibited CA activity and that these CA-positive receptors were distributed throughout the nasal cavity with peak densities in the dorsal and ventral sensory epithelial regions. To test for the role of CA in olfactory transduction, electro-olfacto-grams (EOGs) were recorded from the surface of the ventral sensory epithelium in response to 2-s pulses of 5% CO2 and amyl acetate before and after topical CA inhibition with acetazolamide (10(-3) mol.l-1). In 52 bullfrogs, 1222 sites on the ventral epithelium were tested resulting in 23 locations that exhibited a response to 5% CO2. Inhibition of CA caused an immediate 65% reduction in the EOG response to CO2 while the response to amyl acetate was not affected. These results, along with the histochemical localization of CA in some olfactory receptor neurons, indicate that CA plays a role in the detection of CO2 in frog olfactory neurons and that only a small population of olfactory receptor neurons are CO2 sensitive.

Aggregation pathway of recombinant human keratinocyte growth factor and its stabilization.

Recombinant human keratinocyte growth factor (rhKGF) is prone to aggregation at elevated temperatures. Its aggregation pathway is proposed to proceed initially with a conformational change which perhaps results from repulsion between positively charged residues in clusters forming heparin binding sites. Unfolding of the protein leads to formation of large soluble aggregates. These soluble aggregates then form disulfide cross-linked precipitates. Finally these precipitates are converted to scrambled disulfides and/or non-disulfide cross-linked precipitates. Stabilizers such as heparin, sulfated polysaccharides, anionic polymers and citrate can greatly decrease the rate of aggregation of rhKGF at elevated temperatures. These molecules may all act by reducing charge repulsion on the protein thus stabilizing the native conformation. EDTA, on the other hand, is found to inhibit disulfide formation in aggregates and has only a moderate stabilizing effect on rhKGF.

Measurements of the electron dose distribution near inhomogeneities using a plastic scintillation detector.

PURPOSE: Accurate measurement of the electron dose distribution near an inhomogeneity is difficult with traditional dosimeters which themselves perturb the electron field. We tested the performance of a new high resolution, water-equivalent plastic scintillation detector which has ideal properties for this application. METHODS AND MATERIALS: A plastic scintillation detector with a 1 mm diameter, 3 mm long cylindrical sensitive volume was used to measure the dose distributions behind standard benchmark inhomogeneities in water phantoms. The plastic scintillator material is more water equivalent than polystyrene in terms of its mass collision stopping power and mass scattering power. Measurements were performed for beams of electrons having initial energies of 6 and 18 MeV at depths from 0.2-4.2 cm behind the inhomogeneities. RESULTS: The detector reveals hot and cold spots behind heterogeneities at resolutions equivalent to typical film digitizer spot sizes. Plots of the dose distributions behind air, aluminum, lead, and formulations for cortical and inner bone-equivalent materials are presented. CONCLUSION: The plastic scintillation detector is suited for measuring the electron dose distribution near an inhomogeneity.

The self-efficacy-performance link in maximum strength performance.

To examine whether manipulating self-efficacy affects strength performance on a bench press, and to see if these situation-specific changes would affect levels of physical self-efficacy, 24 undergraduates untrained in weightlifting were randomly assigned to three groups: 'light', who lifted less weight than they believed; 'heavy', who lifted more weight than they believed; and control, for whom there was no manipulation. Self-efficacy measures were taken before and after the manipulation. Physical self-efficacy was measured using the Physical Self-Efficacy Scale (PSE). 'Light' subjects lifted significantly greater increases in weight than the other subjects. 'Heavy' subjects significantly decreased self-efficacy following the manipulation. Initial self-efficacy was found to be a significant predictor of baseline maximum, while manipulated self-efficacy was significant for performance change. The PSE scores did not change pre- to post-study. The results suggest that self-efficacy is a situation-specific construct which can be manipulated, and which relates to both past performance experience and future performance.

Thrombin is a Na(+)-activated enzyme.

The amidase activity of human alpha-thrombin has been studied at steady state as a function of the concentration of several chloride salts, at a constant ionic strength I = 0.2 M. All kinetic steps of the catalytic mechanism of the enzyme have been solved by studies conducted as a function of relative viscosity of the solution. Among all monovalent cations, Na+ is the most effective in activating thrombin catalysis. This effect is observed with different amide substrates and also with gamma-thrombin, a proteolytic derivative of the native enzyme which has little clotting activity but retains amidase activity toward small synthetic substrates. The specific effects observed as a function of Na+ concentration are indicative of a binding interaction of this monovalent cation with the enzyme. The basis of this interaction has been explored by measurements of substrate hydrolysis collected in a three-dimensional matrix of substrate concentration, relative viscosity, and Na+ concentration, keeping the ionic strength constant with an inert cation such as choline or tetraethylammonium. The data have globally been analyzed in terms of a kinetic linkage scheme where Na+ plays the role of an allosteric effector. The properties of the enzyme change drastically upon binding of Na+, with substrate binding and dissociation, as well as deacylation, occurring on a time scale which is 1 order of magnitude faster. The apparent association constants for Na+ binding to the various intermediate forms of the enzyme have all been resolved from analysis of experimental data and are in the range of 50-100 M-1 at 25 degrees C. Studies conducted at different temperatures, in the range 15-35 degrees C, have revealed the enthalpic and entropic components of Na+ binding to the enzyme. The results obtained from steady-state measurements are supported by independent measurements of the intrinsic fluorescence of the enzyme as a function of Na+ concentration at a constant ionic strength I = 0.2 M, over the temperature range 15-35 degrees C. These measurements are indicative of a drastic conformational change of the enzyme upon Na+ binding to a single site. The energetics of Na+ binding derived from analysis of fluorescence measurements agree very well with those derived independently from steady-state determinations. It is proposed that thrombin exists in two conformations, slow and fast, and that the slow-->fast transition is triggered by binding of a monovalent cation. The high specificity in thrombin activation found in the case of Na+ is the result of its higher affinity compared to all other monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Synchrotron-produced ultrasoft X-rays: a tool for testing biophysical models of radiation action.

Ultrasoft X-rays are useful for mechanistic studies of ionizing radiation damage in living cells due to the localized nature of their energy depositions. To date radiobiology experiments in this energy region have relied on characteristic X-rays (mainly Alk and Ck) from X-ray tubes. However, limitations in the photon intensity and the available energies from X-ray tube sources prevent a definitive characterization of the relationship between photon energy and biological damage. Synchrotron radiation has the potential to avoid these limitations, since it produces X-rays with high intensity over a continuous spectrum. We have established a synchrotron-based system for radiation biology studies using the ES-0 exposure station of the Center for X-ray Lithography at the University of Wisconsin Synchrotron Radiation Center storage ring, Aladdin. A characterization of the system including spectral and intensity properties of the photon beam is presented. The first mammalian cell survival curve for synchrotron-produced ultrasoft X-rays was generated and is presented. Cell survival curves of C3H/10T 1/2 cells using synchrotron radiation of 1.48 keV agree with previous data using Alk X-rays (1.49 keV). An RBE of 1.47 +/- 0.30 at the 10% survival level was measured with reference to 250 kVp X-rays.