Tissue Responses to Postoperative Laser Therapy in Diabetic Rats Submitted to Excisional Wounds

In a previous study about low-level laser therapy biomodulation on a full-thickness burn model we showed that single and fractionated dose regimens increased wound healing and leukocyte influx similarly when compared with untreated control. In order to verify if this finding would be similar in an impaired wound model, we investigated the effect of single and multiple irradiations on wound closure rate, type of inflammatory infiltrate, myofibroblasts, collagen deposition, and optical retardation of collagen in diabetic rats. Female Wistar rats in the same estrous cycle had diabetes induced with streptozotocin and an 8-mm excisional wound performed with a punch. The experimental groups were: control group – untreated ulcer; single-dose group – ulcer submitted to single dose of diode laser therapy (λ = 660 ± 2 nm; P = 30 mW; energy density: 4 J/cm²) and fractionated-dose group – ulcer submitted to 1 J/cm² laser therapy on Days 1, 3, 8, and 10. The ulcers were photographed on the experimental days and after euthanasia tissue samples were routinely processed for histological and immunohistochemistry analyses. Independently of the energy density, laser therapy accelerated wound closure by approximately 40% in the first three days in comparison to the control group. Laser therapy increased acute inflammatory infiltrate until Day 3. Both laser groups exhibited more myofibroblasts and better collagen organization than the control group. The findings demonstrate that low-level laser therapy in the immediate postoperative period can enhance the tissue repair process in a diabetes model. Similar effects were achieved with laser therapy applied a single time with an energy density of 4 J/cm² and applied four times with an energy density of 1 J/cm². The application of laser therapy in the inflammatory phase was the most important factor to the enhancement of the tissue repair process.     

Fiber-Optic Microarray for Simultaneous Detection of Multiple Harmful Algal Bloom Species

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 μm) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.     

Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR

The toxic dinoflagellate Alexandrium fundyense is widespread in the northeastern part of North America, including the Gulf of Maine, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. fundyense toxins can accumulate in shellfish and result in paralytic shellfish poisoning (PSP). PSP can be debilitating or lethal to humans and other shellfish consumers and is a public health concern. As a result, accurate measurements of A. fundyense distributions, particularly at low cell density, are critical to continued PSP monitoring and mitigation efforts. Towards this end we have developed a real-time quantitative PCR (qPCR) method to monitor A. fundyense. Laboratory validation indicates that the qPCR assay is sensitive enough to detect 10 cells per sample, and that it does not detect co-occurring dinoflagellates such as Alexandrium ostenfeldii. The qPCR methodology was used to quantify A. fundyense cell densities in samples collected during a spring 2003 transect in the Gulf of Maine, and the data were compared to those obtained in parallel from light microscope and DNA hybridization-based methods. Results show that A. fundyense cell density was low during this period relative to typical cell densities required for PSP contamination of local shellfish, and that qPCR values were comparable to numbers determined by independent methods.     

Parallel Analyses of Alexandrium catenella Cell Concentrations and Shellfish Toxicity in the Puget Sound

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter⁻¹ were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 μg of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.Various species of the dinoflagellate genus Alexandrium, including members of the species complex comprising Alexandrium catenella, Alexandrium fundyense, and Alexandrium tamarense, produce saxitoxins and a number of related derivatives (1). Shellfish that ingest toxic Alexandrium cells accumulate these potent neurotoxins, which can then lead to paralytic shellfish poisoning (PSP) in human consumers of shellfish. As such, paralytic shellfish toxins (PSTs) pose a serious threat to both public health and economically important fisheries (16). Within the Alexandrium genus, A. catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms (HABs) in this region (17). In the Puget Sound, recreational shellfish harvesters collect nearly 2 million pounds of clams and oysters annually, and Washington is also a leading producer of farmed bivalve shellfish in the United States, generating an estimated $77 million in sales a year and supporting thousands of jobs (13).PSTs are not a new problem in the Pacific Northwest; events have been documented as far back as the late 18th century (17). Currently, the Sentinel Monitoring Program of the Washington State Department of Health (WADOH) is in place to provide systematic early warning of harmful levels of PSTs, with caged mussels sampled at as many as 70 sites throughout all basins of Puget Sound at roughly 2-week intervals. Analysis of this long-term shellfish monitoring data indicates that maximum PST levels and PST-related closures have increased over the past 20 years, reaching >10,000 μg of PST per 100 g of shellfish tissue in multiple years and resulting in significant negative impacts on shellfisheries in the region (17).To date, monitoring efforts in the Puget Sound have focused on measuring the level of PSTs present in shellfish tissue. Existing programs do not typically monitor for phytoplankton species composition or abundance. Information on A. catenella distribution and seasonal dynamics is limited for this region, despite its potential value for monitoring and understanding toxic A. catenella blooms and their impacts. Toward this end, we used a previously developed high-throughput quantitative PCR (qPCR) method (5, 6) to detect and enumerate A. catenella cells. We couple this specific and sensitive detection method for A. catenella with PST monitoring efforts to examine changes in A. catenella populations and accompanying shellfish toxicity in the Puget Sound. The data, collected from April through October, span nearly all of the 2006 A. catenella bloom season in the region. These results provide important information on the abundance and dynamics (e.g., possible source populations) of A. catenella cells during a bloom season and on their relationship to PSTs in shellfish. This effort represents a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this region.     

The current state of personal training: managers' perspectives

Fitness managers play an integral role in the day-to-day operation of the facilities in which they work. One of the most critical responsibilities is that of hiring, training, and supervising personal trainers. Research studies have identified a number of qualities and competencies that an effective exercise leader is required to have, but there is little scholarly work addressing managers' attitudes surrounding the performance of personal trainers. Using focus group methodology, managers of personal trainers were recruited to provide viewpoints related to desirable qualities of personal trainers and opinions regarding certification and academic preparation. Responses of the participants were transcribed, coded, and analyzed for themes. Two global themes, Selection Rationale and Negative Characteristics, emerged. Selection Rationale consisted of qualities that influence a manager's decision to hire a particular trainer (e.g., physique, education, social skills). Negative characteristics referred to qualities considered unethical or unprofessional (e.g., sexual comments, misuse of power) and the consequences of those behaviors (e.g., loss of clients, potential for litigation). The results suggest that undergraduate exercise science programs devote additional time toward the development of the affective qualities of future fitness trainers. Managers also recommended licensure for employees.     

Differences in trait compositions between rocky natural and artificial habitats

Question: What are the differences in trait compositions that enable native plants to colonise comparable natural and man‐made habitats? Are these traits independent of phylogenetic relationships between species? Location: Czech Republic. Methods: The relative importance of biological, ecological and distributional traits of native species was studied, using a dataset of 75 species growing in rock and wall habitats in the Czech Republic. Species preferences for individual habitats due to climatic conditions and proportions of different vegetation types in their surroundings were partialled out using partial canonical correspondence analysis. The pattern of plant traits along a gradient from natural rock habitats to secondary wall habitats was analysed using regression trees and generalized linear models with and without phylogenetic correction. Results: The most common native species colonising rock habitats are phanerophytes, mostly woody juveniles, with a CSR life strategy and most are adapted to epizoochory. Summer green leaves, annual life span, CR life strategy, reproduction mostly by seeds and dispersal by ants are all traits positively associated with the ability of species to colonise wall habitats. These species are also characterised by their high demand for nutrients, temperature, base‐rich substrates and light. Biological and ecological traits are more important for colonising new habitats than traits related to species dispersal ability or phylogenetic relationships between species. Biological and ecological traits alone explained 29.3% of variability in the species dataset, while dispersal characteristics and phylogeny alone explained 9.1% and 4.8%, respectively. Conclusions: We outline how the process of environmental filtering determines native species assemblages and identify a set of species traits that enable them to persist in particular habitats. We conclude that although urbanisation generally results in loss of natural habitats, there are new, man‐made habitats potentially suitable for native species.     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

The function of RNase G in Escherichia coli is constrained by its amino and carboxyl termini

RNase G is a homologue of the essential Escherichia coli ribonuclease RNase E. Whereas RNase E plays a key role in the degradation of mRNA and the processing of tRNA and rRNA in E. coli, the biological functions of RNase G appear more limited. We report here that this difference in function is not merely a consequence of the significantly lower cellular concentration of RNase G, but also reflects differences in the intrinsic properties of these ribonucleases, as overproducing wild-type RNase G at a level up to 20 times the usual cellular concentration of RNase E cannot normally compensate for the absence of RNase E in E. coli. Instead, RNase G can sustain significant growth of RNase E-deficient E. coli cells only when it bears an unnatural extension at its amino terminus (e.g. MRKGINM) or carboxyl terminus (e.g. GHHHHHH). These extensions presumably enable RNase G to cleave critically important cellular RNAs whose efficient processing or degradation ordinarily requires RNase E. That extending the amino terminus of RNase G restores growth to E. coli cells lacking RNase E without detectably improving tRNA processing suggests that RNase E is not essential for tRNA production and is required for cell growth because it plays an indispensable role in the maturation or decay of essential E. coli RNAs other than tRNA.     

VE-cadherin-p120 interaction is required for maintenance of endothelial barrier function

Interaction of p120 with juxtamembrane domain (JMD) of VE-cadherin has been implicated in regulation of endothelial cell-cell adhesion. We used a number of approaches to alter the level of p120 available for binding to VE-cadherin as a means to investigate the role of p120-VE-cadherin interaction in regulation of barrier function in confluent endothelial monolayers. Expression of an epitope-tagged fragment corresponding to JMD of VE-cadherin resulted in a decrease in endothelial barrier function as assessed by changes in albumin clearance and electrical resistance. Binding of JMD-Flag to p120 resulted in a decreased level of p120. In addition to decreasing p120 level, expression of JMD also decreased level of VE-cadherin. Expression of JMD also caused an increase in MLC phosphorylation and rearrangement of actin cytoskeleton, which, coupled with decreased cadherin, can contribute to loss of barrier function. Reducing p120 by siRNA resulted in a decrease in VE-cadherin, whereas increasing the level of p120 increased the level of VE-cadherin, demonstrating that p120 regulates the level of VE-cadherin. Overexpression of p120 was, however, associated with decreased barrier function and rearrangement of the actin cytoskeleton. Interestingly, expression of p120 was able to inhibit thrombin-induced increases in MLC phosphorylation, suggesting that p120 inhibits activation of Rho/Rho kinase pathway in endothelial cells. Excess p120 also prevented JMD-induced increases in MLC phosphorylation, correlating this phosphorylation with Rho/Rho kinase pathway. These findings show p120 plays a major role in regulating endothelial barrier function, as either a decrease or increase of p120 resulted in disruption of permeability across cell monolayers.