The impact of potassium peroxymonosulphate and chlorinated cyanurates on biofilms of Stenotrophomonas maltophilia: effects on biofilm control, regrowth, and mechanical properties.

The activity of two chlorinated isocyanurates (NaDCC and TCCA) and peroxymonosulphate (OXONE) was evaluated against biofilms of Stenotrophomonas maltophilia, an emerging pathogen isolated from drinking water (DW), and for the prevention of biofilm regrowth. After disinfection of pre-formed 48 h-old biofilms, the culturability was reduced up to 7 log, with OXONE, TCCA, and NaDCC showing more efficiency than free chlorine against biofilms formed on stainless steel. The regrowth of biofilms previously exposed to OXONE was reduced by 5 and 4 log CFU cm-2 in comparison to the unexposed biofilms and biofilms exposed to free chlorine, respectively. Rheometry analysis showed that biofilms presented properties of viscoelastic solid materials, regardless of the treatment. OXONE reduced the cohesiveness of the biofilm, given the significant decrease in the complex shear modulus (G*). AFM analysis revealed that biofilms had a fractured appearance and smaller bacterial aggregates dispersed throughout the surface after OXONE exposure than the control sample. In general, OXONE has been demonstrated to be a promising disinfectant to control DW biofilms, with a higher activity than chlorine. The results also show the impact of the biofilm mechanical properties on the efficacy of the disinfectants in biofilm control.

Inhibition of cardiac potassium currents by oxidation-activated protein kinase A contributes to early afterdepolarizations in the heart.

Reactive oxygen species (ROS) have been shown to prolong cardiac action potential duration resulting in afterdepolarizations, the cellular basis of triggered arrhythmias. As previously shown, protein kinase A type I (PKA I) is readily activated by oxidation of its regulatory subunits. However, the relevance of this mechanism of activation for cardiac pathophysiology is still elusive. In this study, we investigated the effects of oxidation-activated PKA I on cardiac electrophysiology. Ventricular cardiomyocytes were isolated from redox-dead PKA-RI Cys17Ser knock-in (KI) and wild-type (WT) mice and exposed to H2O2 (200 µmol/L) or vehicle (Veh) solution. In WT myocytes, exposure to H2O2 significantly increased oxidation of the regulatory subunit I (RI) and thus its dimerization (threefold increase in PKA RI dimer). Whole cell current clamp and voltage clamp were used to measure cardiac action potentials (APs), transient outward potassium current (Ito) and inward rectifying potassium current (IK1), respectively. In WT myocytes, H2O2 exposure significantly prolonged AP duration due to significantly decreased Ito and IK1 resulting in frequent early afterdepolarizations (EADs). Preincubation with the PKA-specific inhibitor Rp-8-Br-cAMPS (10 µmol/L) completely abolished the H2O2-dependent decrease in Ito and IK1 in WT myocytes. Intriguingly, H2O2 exposure did not prolong AP duration, nor did it decrease Ito, and only slightly enhanced EAD frequency in KI myocytes. Treatment of WT and KI cardiomyocytes with the late INa inhibitor TTX (1 µmol/L) completely abolished EAD formation. Our results suggest that redox-activated PKA may be important for H2O2-dependent arrhythmias and could be important for the development of specific antiarrhythmic drugs.NEW & NOTEWORTHY Oxidation-activated PKA type I inhibits transient outward potassium current (Ito) and inward rectifying potassium current (IK1) and contributes to ROS-induced APD prolongation as well as generation of early afterdepolarizations in murine ventricular cardiomyocytes.

A direct comparison of experimental methods to measure dimensions of synthetic nanoparticles.

Nanoparticles have properties that depend critically on their dimensions. There are a large number of methods that are commonly used to characterize these dimensions, but there is no clear consensus on which method is most appropriate for different types of nanoparticles. In this work four different characterization methods that are commonly applied to characterize the dimensions of nanoparticles either in solution or dried from solution are critically compared. Namely, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and dynamic light scattering (DLS) are compared with one another. The accuracy and precision of the four methods applied nanoparticles of different sizes composed of three different core materials, namely gold, silica, and polystyrene are determined. The suitability of the techniques to discriminate different populations of these nanoparticles in mixtures are also studied. The results indicate that in general, scanning electron microscopy is suitable for large nanoparticles (above 50 nm in diameter), while AFM and TEM can also give accurate results with smaller nanoparticles. DLS reveals details about the particles' solution dynamics, but is inappropriate for polydisperse samples, or mixtures of differently sized samples. SEM was also found to be more suitable to metallic particles, compared to oxide-based and polymeric nanoparticles. The conclusions drawn from the data in this paper can help nanoparticle researchers choose the most appropriate technique to characterize the dimensions of nanoparticle samples.

Contribution of the cashew gum (Anacardium occidentale L.) for development of layer-by-layer films with potential application in nanobiomedical devices.

The search for bioactive molecules to be employed as recognition elements in biosensors has stimulated researchers to pore over the rich Brazilian biodiversity. In this sense, we introduce the use of natural cashew gum (Anacardium occidentale L.) as an active biomaterial to be used in the form of layer-by-layer films, in conjunction with phthalocyanines, which were tested as electrochemical sensors for dopamine detection. We investigated the effects of chemical composition of cashew gum from two different regions of Brazil (Piauí and Ceará states) on the physico-chemical characteristics of these nanostructures. The morphology of the nanostructures containing cashew gum was studied by atomic force microscopy which indicates that smooth films punctuated by globular features were formed that showed low roughness values. The results indicate that, independent of the origin, cashew gum stands out as an excellent film forming material with potential application in nanobiomedical devices as electrochemical sensors.

Cardiolipin, a key component to mimic the E. coli bacterial membrane in model systems revealed by dynamic light scattering and steady-state fluorescence anisotropy.

The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA-DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for Escherichia coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared with those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.

Musculoskeletal CPD revision: cases from the New Zealand Bone & Soft Tissue Tumour Registry.

This case report has been selected from the New Zealand Bone & Soft Tissue Tumour Registry to highlight some key concepts and findings in musculoskeletal imaging with radiological-pathological correlation. The case is presented in a question and answer format, with clinical information and selected images in one section, followed by the diagnosis, discussion and teaching points.

Peroxynitrite: in vivo cardioprotectant or arrhythmogen?

The factors that determine susceptibility to lethal ventricular arrhythmias during myocardial ischaemia and reperfusion in vivo are complex, but the balance between proarrhythmic and antiarrhythmic endogenous substances is likely to be important. However, in this context, it is not well established what effect endogenously produced peroxynitrite has on arrhythmias that develop during ischaemia/reperfusion in vivo. The study by Kiss et al., published in this issue of the BJP, provides some insights to this problem. We discuss the wider implications of this study as well as issues that still require resolution.

AlphaB crystallin translocation and phosphorylation: signal transduction pathways and preconditioning in the isolated rat heart.

In this program of studies we have characterized in detail the translocation (assessed by Triton-insolubility) and phosphorylation (using serine-45 or -59 phosphospecific antibodies) of alphaB crystallin during myocardial ischemia [both with or without ischemic preconditioning (IPC)]. Pharmacological activators and inhibitors allowed us to characterize the signaling pathways involved in alphaB crystallin phosphorylation during ischemia. Ischemic preconditioning alone caused 30% of the heart's alphaB crystallin pool to translocate, providing a significant translocation 'head-start' in protected tissue. This enhanced translocation is coupled with increased (3-fold) alphaB crystallin phosphorylation at both serine residues. The possible role of alphaB crystallin in the protection afforded by ischemic preconditioning is supported by the signal transduction data; which showed preconditioning-induced alphaB crystallin phosphorylation can be blocked by tyrosine kinase inhibition (using genistein) and by p38 MAP kinase or PKC inhibition (using SB203580 or bisindolylmaleimide, respectively). The activation of both p38 MAP kinase and PKC are recognized requirements for the induction of preconditioning and their inhibition is known to block protection. Western immunoblotting analysis after isoelectric focusing electrophoresis, confirmed the observations made with the phosphospecific antibodies; but also showed that 27+/-4% of total cardiac crystallin was phosphorylated after 30 min of ischemia. AlphaB crystallin exists as large polymeric aggregates in cardiac tissue under basal conditions (approximately 1 MDa as determined by gel filtration chromatography). We induced phosphorylation of alphaB crystallin during aerobic perfusion by the administration of phenylephrine. However this treatment did not alter the molecular aggregate size of alphaB crystallin. It appears that alphaB crystallin molecular aggregate size is not simply regulated by phosphorylation. AlphaB crystallin may have a role to play in the myocardial protection induced by ischemic preconditioning, as both translocation and phosphorylation are both accelerated and enhanced by ischemic preconditioning.

Lipid hydroperoxide modification of proteins during myocardial ischaemia.

OBJECTIVE: Lipid hydroperoxides (LOOH) are lipid peroxidation products formed during oxidative stress. A component of their cytotoxicity is mediated by the direct modification of proteins. OBJECTIVES: (i) To assess whether ischaemia and reperfusion in the isolated rat heart generates LOOH-protein (ii) to characterise the extent, time-course and subcellular localization of any protein adducts formed. METHODS: Using a well-characterised antibody which binds to LOOH-modified proteins and densitometry of Western blots, we quantified the amounts of LOOH-protein in control aerobically perfused rat hearts and those subjected to ischaemia with and without reperfusion. RESULTS: Hearts (n=3/4 group), analysed after various periods (0, 5, 10, 20 and 30 min) of zero-flow global ischaemia, exhibited a time-dependent increase in the LOOH-mediated modification of a number of proteins. In hearts subjected to 30 min of ischaemia and then reperfused for various times (0, 5, 10, 20, 30 or 60 min) no changes in LOOH-protein content achieved during the proceeding ischaemic episode were detected. Reperfusion after short periods of ischaemia (5 or 10 min) also did not result in reperfusion-induced LOOH-protein formation. Administration of mercaptopropionylglycine (1 mM) to hearts for 5 min before the onset of 30 min ischaemia efficiently attenuated the formation of LOOH-protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in situ immunofluorescent studies which showed marked LOOH-protein immunostaining in ischaemic tissue around the sarcolemmal membrane. CONCLUSIONS: We conclude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the pathophysiology of ischaemic injury. In addition, these modifications may be initiators of oxidant-induced signal transduction pathways. These findings are consistent with an oxidant stress occurring during ischaemia which is not exacerbated or reduced during the first 60 min of reperfusion.

LiCB(11)Me(12): a catalyst for pericyclic rearrangements.

[structure: see text] Benzene and 1,2-dichloroethane solutions of the Li(+) salt of the weakly coordinating anion CB(11)Me(12)(-) catalyze the rearrangement of cubane to cuneane, quadricyclane to norbornadiene, basketene to Nenitzescu's hydrocarbon, and diademane to triquinacene. The Claisen rearrangement of phenyl allyl ether is also strongly accelerated.

Differential centrifugation separates cardiac sarcolemmal and endosomal membranes from Langendorff-perfused rat hearts.

The application of subcellular fractionation protocols developed in soft tissues to fibrous organs such as the heart is unsuitable given the substantial differences in subcellular structure these tissues exhibit. The purpose of this study was to develop a simple method for the separation of sarcolemma and endosomes from isolated Langendorff-perfused rat hearts. Hearts were homogenized with either an Ultra-Turrax homogenizer or a hand-held glass tissue grinder. Quantitative immunoblots assessed the enrichment of the sarcolemmal proteins caveolin 3 and the sodium potassium ATPase and the endosomal proteins rab4 and GLUT4 in different membrane fractions. Application of homogenates to sucrose and Percoll density gradients failed to resolve membranes differentially enriched in sarcolemmal or endosomal marker proteins, indicating little difference in density between the sarcolemma and endosomes. However, successive spins of homogenates from a hand-held glass tissue grinder successfully separated the endosomes from the sarcolemma, indicating differences in masses between the two membrane fractions. Approximately 70% of total caveolin 3 and sodium potassium ATPase immunoreactivity was in membrane pellets up to 20,000g and approximately 85% of rab4 and GLUT4 in pellets from 20,000-100,000g. In addition, 86% of ouabain-sensitive ATPase activity (sodium potassium ATPase activity) was in membrane pellets up to 20,000g. Therefore, sarcolemmal membranes were pelleted up to 20,000g, and endosomal membranes between 20,000 and 100,000g. Regional ischemia (40 min) followed by reperfusion (60 min) caused the translocation of GLUT4 (but not rab4) from the endosomal membranes to the sarcolemma in the area of the heart subjected to ischemia.

The need for integrated linkages and long-term monitoring of mercury in Canada.

A nation-wide ecosystem science network for Canada was formed in 1994. At that time, mercury was a re-emerging issue in Canada and the Coordinating Office for the network sought collaboration to assess the issue. The key mechanisms by which the network has added value in addressing this issue are: 1) Information Dissemination, the network has organised, facilitated and co-hosted a number of regional. national and international mercury events (meetings, conferences and workshops) which have served to bring the expertise together, the network also disseminates information on it's web page. and the Coordinating Office hosts an annual National Science Conference: 2) Collaborative Mercury Monitoring, network partners advocated the need for a single hemispheric mercury network which resulted in the development of a compatible Canada-U.S. mercury deposition network, which may also be expanded into Mexico, and 3) Environmental Reporting, the network has collaborated with others to report on current mercury findings through initiatives such as the 1998 Northeast States and Eastern Canadian Mercury Study, a 1999 Mercury Case Study and is presently a partner in the University of Quebec's proposal to form a Collaborative Mercury Ecosystem Research Network in Canada.

Ischemic preconditioning: a potential role for constitutive low molecular weight stress protein translocation and phosphorylation?

We have investigated whether translocation of constitutive low molecular weight stress proteins (alphaB-crystallin and HSP27) to the myofilament/cytoskeletal compartment occurs during ischemic preconditioning and assessed if this is causally associated with cardioprotection. Triton-insoluble preparations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little alphaB-crystallin (96 +/- 43 and 43 +/- 36 units respectively) or HSP27 (177 +/- 32 and 101 +/- 26 units respectively). Three preconditioning cycles of (5 min ischemia + 5 min reperfusion) increased the Triton-insoluble crystallin to 864 +/- 61 units (P<0.05) and HSP27 to 1353 +/- 53 units (P<0.05). Two hours of aerobic perfusion following the preconditioning protocol resulted the return of alphaB-crystallin and HSP27 to near control levels (189 +/- 14 units and 252 +/- 24 units, respectively). Stress protein translocation, comparable to that achieved by the IPC protocol was induced by aerobic perfusion with hypercarbic (pH 6.8) perfusion. Thus, three cycles of 5 min hypercarbia + 5 min normocarbia increased alphaB-crystallin to 628 +/- 30 units (P<0.05) and HSP27 to 1353 +/- 53 units. In parallel functional studies, the recovery of LVDP after 35 min ischemia and 60 min of reperfusion was 43 +/- 7% in the ischemic control group, 61 +/- 3% (P<0.05) in the preconditioned group and 42 +/- 6% in the hypercarbic group. Thus, translocation of alphaB-crystallin and/or is not of-itself sufficient to induce cardioprotection. Using a phospho-specific antibody, we have demonstrated that preconditioning not only translocates alphaB-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to aerobic controls (1616 +/- 402 v 166 +/- 28 units respectively). In contrast, hypercarbia while eliciting a comparable translocation, failed to alter the phosphorylation state of alphaB-crystallin. Preconditioning-induced phosphorylation was significantly attenuated by 50 microM genistein (by 61%), 10 microM SB203580 (by 91%) and 10 microM bisindolylmaleimide (by 68%), but not by 10 microM PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and translocation of constitutive low molecular weight stress proteins, particularly alphaB-crystallin.

Neurocontroller alternatives for "fuzzy" ball-and-beam systems with nonuniform nonlinear friction.

The ball-and-beam problem is a benchmark for testing control algorithms. In the World Congress on Neural Networks, 1994, Prof. L. Zadeh proposed a twist to the problem, which, he suggested, would require a fuzzy logic controller. This experiment uses a beam, partially covered with a sticky substance, increasing the difficulty of predicting the ball's motion. We complicated this problem even more by not using any information concerning the ball's velocity. Although it is common to use the first differences of the ball's consecutive positions as a measure of velocity and explicit input to the controller, we preferred to exploit recurrent neural networks, inputting only consecutive positions instead. We have used truncated backpropagation through time with the node-decoupled extended Kalman filter (NDEKF) algorithm to update the weights in the networks. Our best neurocontroller uses a form of approximate dynamic programming called an adaptive critic design. A hierarchy of such designs exists. Our system uses dual heuristic programming (DHP), an upper-level design. To our best knowledge, our results are the first use of DHP to control a physical system. It is also the first system we know of to respond to Zadeh's challenge. We do not claim this neural network control algorithm is the best approach to this problem, nor do we claim it is better than a fuzzy controller. It is instead a contribution to the scientific dialogue about the boundary between the two overlapping disciplines.

Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart.

4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.

The origins, fate, and ecological significance of free amino compounds released by freshwater pulmonate snails.

The mass-specific accumulation rates (MSAR) of both total (TFAC) and individual free amino compounds (FAC) in conditioned media were measured by HPLC, using the orthophthaldialdehyde (OPA) methods, in the following cases: (a) laboratory-reared freshwater snails (B. glabrata) with chemosterilized shells; (b) Biomphalaria glabrata with non-chemosterilized shells; (c) B. glabrata faeces; (d) isolated shells of B. glabrata; and (e) 10 other species of freshwater gastropods from the Lewes Brooks, East Sussex, U.K. The MSAR values for B. glabrata show that 95% of the TFAC's (predominantly ethanolamine, phosphoserine, and the amino acids leucine, isoleucine, valine, aspartic acid, and glycine/threonine) originated from the snails themselves as the faeces and shells contributed only 5.0 and 0%, respectively. In contrast, epizootic organisms on the shells of all 10 snail species from the Lewes Brooks released significant amounts of FAC with the two smallest species (Planorbis vortex and Planorbis contortus) having the highest MSAR values. The MSAR for isolated B. glabrata mucus was 42.45 micromol x g(-1)h(-1). As 500 mg snails can release 16.67 mg of mucus daily, this could potentially result in the daily loss of 707.5 micromol of FAC. The cost/benefits of mucus secretion and the various anatomical, physiological, biochemical, and ecological mechanisms which allow freshwater snails to recover FAC's lost as a result of a high rate of urine production in their hypotonic environment, are discussed.

4-substituted cubylcarbinylamines: a new class of mechanism-based monoamine oxidase B inactivators.

Cubylcarbinylamine (1a), (4-cyclopropylcubyl)carbinylamine (1b), and (4-phenylcubyl)carbinylamine (1c) were synthesized and shown to be time-dependent, irreversible inactivators of monoamine oxidase B (MAO B). Substrate protects the enzyme from inactivation, but beta-mercaptoethanol does not, suggesting that these compounds are mechanism-based inactivators. All three compounds were also substrates for MAO B with partition ratios ranging from 152 to 536. The 4-substituted analogues were more potent inactivators than the unsubstituted analogue, indicating a benefit to 4-substitution in this class of inactivators.

Rural-urban migration and underemployment among females in the Brazilian Northeast.

PIP: The relationships among rural-urban migration, underemployment, and government policy are explored using data from two surveys on female migration to Fortaleza, Brazil. The results suggest that for this segment of the labor force, the unfavorable responses to government policy predicted by Michael Todaro and others do not occur.^ieng