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A great number of pathogens secrete pore-forming proteins during infection. Such molecules, from either bacterial or viral origin, are considered important virulence factors, which makes them attractive targets in the study of new therapeutic agents. Thus, the inhibitory activity of isatin-Schiff base copper(II) complexes was evaluated against membrane damage activity of Staphylococcus aureus α-hemolysin (α-HL). For this purpose, a standard hemolysis assay with rabbit erythrocytes and micromolar concentrations of the compounds was employed. Additionally, planar artificial lipid membranes with a single α-HL ion channel and molecular docking studies were used to elucidate the molecular mechanism of the complexes. Accordingly, the compounds were observed to possess a significant anti-hemolytic activity, capable of interacting with the constriction region of α-HL channel and blocking it in a potential dependent manner. Based on these results, it is expected that such isatin-Schiff base Copper(II) complexes may be employed as cotherapeutic agents for the treatment of staphylococcal infections., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Recently, it has been suggested an association between red cell distribution width (RDW) and Crohn's disease activity index (CDAI), but its use is not yet performed in daily clinical practice., Objectives: To determine whether RDW can be used as a marker of Crohn's disease (CD) activity., Methods: This was a cross-sectional study including patients with CD, observed consecutively in an outpatient setting between January 1st and September 30th 2013. Blood cell indices, erythrocyte sedimentation rate (ESR), and C-reactive protein were measured. CD activity was determined by CDAI (active disease if CDAI ≥ 150). Associations were analyzed using logistic regression (SPSS version 20)., Results: 119 patients (56% female) were included in the study with a mean age of 47 years (SD 15.2). Twenty patients (17%) had active disease. The median RDW was 14.0 (13-15). There was an association between RDW and disease activity ( p  = 0.044). After adjustment for age and gender, this association remained consistent (OR 1.20, 95% CI 1.03-1.39, p  = 0.016). It was also found that the association between RDW and disease activity was independent of hemoglobin and ESR (OR 1.36, 95% CI 1.08-1.72, p  = 0.01) and of biologic therapy (OR 1.19, 95% CI 1.03-1.37, p  = 0.017). A RDW cutoff of 16% had a specificity and negative predictive value for CDAI ≥ 150 of 88% and 86%, respectively., Conclusion: In this study, RDW proved to be an independent and relatively specific marker of CD activity. These results may contribute to the implementation of this simple parameter, in clinical practice, aiming to help therapeutic decisions.

Construction, in the framework of a nonequilibrium statistical ensemble formalism, of a higher-order generalized hydrodynamics, also referred to as mesoscopic hydrothermodynamics, that is, covering phenomena involving motion of fluids displaying variations short in space and fast in time-unrestricted values of Knudsen numbers, is presented. In that way, an approach is provided enabling the coupling and simultaneous treatment of the kinetics and hydrodynamic levels of descriptions. It is based on a complete thermostatistical approach in terms of the densities of matter and energy and their fluxes of all orders covering systems arbitrarily driven away from equilibrium. The set of coupled nonlinear integrodifferential hydrodynamic equations is derived. They are the evolution equations of the Gradlike moments of all orders, derived from a generalized kinetic equation built in the framework of the nonequilibrium statistical ensemble formalism. For illustration, the case of a system of particles embedded in a fluid acting as a thermal bath is fully described. The resulting enormous set of coupled evolution equations is of unmanageable proportions, thus requiring in practice to introduce an appropriate description using the smallest possible number of variables. We have obtained a hierarchy of Maxwell times, associated to the set of all the higher-order fluxes, which have a particular relevance in the process of providing criteria for establishing the contraction of description.

This cross-sectional study aims to assess through analysis of data suggested by the Social Behavior Schedule (SBS) quantitatively and qualitatively distinguishing between the limitations of social interaction presented by users of substitute services. Analyses were performed of 385 instruments applied to users of Therapeutic Residential Services (SRT) and the Centers for Psychosocial Care (CAPS). Twenty-one items from the Social Behaviour Schedule (SBS, BR). The data indicate that the individuals studied had from zero to 15 social behavior problems, with an average of 2.5 problems per service user with a standard deviation of 2.84. The majority of the sample appeared to have 1-2 problems among those identified by the scale, and the nonspecific behaviors are present in greater proportion in both services, followed by panic attacks among CAPS users and problems with personal appearance and hygiene in SRT users. In this way, the use of tools that make it possible to assess the data on the social aspects of the psychological distress deserves to be encouraged as a way to further research in this area, and rethink ways of effecting social reintegration of these individuals.

Background: Dental caries is a serious public health concern. The high cost of dental treatment can be avoided by effective preventive measures, which are dependent on dentists' adherence. This study aimed to evaluate the factors that drive dentists towards or away from dental caries preventive measures., Methods and Findings: This systematic review was registered in PROSPERO (CRD42012002235). Several databases as well as the reference lists and citations of the included publications were searched according to PRISMA guidelines, yielding 18,276 titles and abstracts, which were assessed to determine study eligibility. Seven qualitative studies and 41 surveys (36,501 participants) remained after data extraction and interpretation. A total of 43 findings were abstracted from the reports and were grouped together into 6 categories that were judged to be topically similar: education and training, personal beliefs, work conditions, remuneration, gender, place of residence and patients. The main findings for adherence based on their calculated frequency effect sizes (ES) were teamwork (21%) and post-graduation (12%), while for non-adherence were biologicism (27%), and remuneration for preventive procedures (25%). Intensity ES were also calculated and demonstrated low prevalence of the findings. Quality assessment of the studies demonstrated that the methodological quality, particularly of surveys, varied widely among studies., Conclusions: Despite the questionable quality of the included reports, the evidence that emerged seems to indicate that further education and training coupled with a fairer pay scheme would be a reasonable approach to change the balance in favor of the provision of dental caries preventive measures by dentists. The results of this review could be of value in the planning and decision making processes aimed at encouraging changes in professional dental practice that could result in the improvement of the oral health care provided to the population in general.

Background: Anterior open bite occurs when there is a lack of vertical overlap of the upper and lower incisors. The aetiology is multifactorial including: oral habits, unfavourable growth patterns, enlarged lymphatic tissue with mouth breathing. Several treatments have been proposed to correct this malocclusion, but interventions are not supported by strong scientific evidence., Objectives: The aim of this systematic review was to evaluate orthodontic and orthopaedic treatments to correct anterior open bite in children., Search Methods: The following databases were searched: the Cochrane Oral Health Group's Trials Register (to 14 February 2014); the Cochrane Central Register of Controlled Trials (CENTRAL)(The Cochrane Library 2014, Issue 1); MEDLINE via OVID (1946 to 14 February 2014); EMBASE via OVID (1980 to 14 February 2014); LILACS via BIREME Virtual Health Library (1982 to 14 February 2014); BBO via BIREME Virtual Health Library (1980 to 14 February 2014); and SciELO (1997 to 14 February 2014). We searched for ongoing trials via ClinicalTrials.gov (to 14 February 2014). Chinese journals were handsearched and the bibliographies of papers were retrieved., Selection Criteria: All randomised or quasi-randomised controlled trials of orthodontic or orthopaedic treatments or both to correct anterior open bite in children., Data Collection and Analysis: Two review authors independently assessed the eligibility of all reports identified. Risk ratios (RRs) and corresponding 95% confidence intervals (CIs) were calculated for dichotomous data. The continuous data were expressed as described by the author., Main Results: Three randomised controlled trials were included comparing: effects of Frankel's function regulator-4 (FR-4) with lip-seal training versus no treatment; repelling-magnet splints versus bite-blocks; and palatal crib associated with high-pull chincup versus no treatment.The study comparing repelling-magnet splints versus bite-blocks could not be analysed because the authors interrupted the treatment earlier than planned due to side effects in four of ten patients.FR-4 associated with lip-seal training (RR = 0.02 (95% CI 0.00 to 0.38)) and removable palatal crib associated with high-pull chincup (RR = 0.23 (95% CI 0.11 to 0.48)) were able to correct anterior open bite.No study described: randomisation process, sample size calculation, there was not blinding in the cephalometric analysis and the two studies evaluated two interventions at the same time. These results should be therefore viewed with caution., Authors' Conclusions: There is weak evidence that the interventions FR-4 with lip-seal training and palatal crib associated with high-pull chincup are able to correct anterior open bite. Given that the trials included have potential bias, these results must be viewed with caution. Recommendations for clinical practice cannot be made based only on the results of these trials. More randomised controlled trials are needed to elucidate the interventions for treating anterior open bite.

Obesity is linked to the development of cancer. Previous studies have suggested that there is a relationship between bariatric surgery and reduced cancer risk. Data sources were from Medline, Embase, and Cochrane Library. From 951 references, 13 studies met the inclusion criteria (54,257 participants). In controlled studies, bariatric surgery was associated with a reduction in the risk of cancer. The cancer incidence density rate was 1.06 cases per 1000 person-years within the surgery groups. In the meta-regression, we found an inverse relationship between the presurgical body mass index and cancer incidence after surgery (beta coefficient -0.2, P < 0.05). Bariatric surgery is associated with reduced cancer risk in morbidly obese people. However, considering the heterogeneity among the studies, conclusions should be drawn with care.

Background: Mortality rates amongst ST segment elevation myocardial infarction (STEMI) patients remain high, especially in developing countries. The aim of this study was to evaluate the factors related with delays in the treatment of STEMI patients to support a strategic plan toward structural and personnel modifications in a primary hospital aligning its process with international guidelines., Methods and Findings: The study was conducted in a primary hospital localized in Foz do Iguaçu, Brazil. We utilized a qualitative and quantitative integrated analysis including on-site observations, interviews, medical records analysis, Qualitative Comparative Analysis (QCA) and System Dynamics Modeling (SD). Main cause of delays were categorized into three themes: a) professional, b) equipment and c) transportation logistics. QCA analysis confirmed four main stages of delay to STEMI patient's care in relation to the 'Door-in-Door-out' time at the primary hospital. These stages and their average delays in minutes were: a) First Medical Contact (From Door-In to the first contact with the nurse and/or physician): 7 minutes; b) Electrocardiogram acquisition and review by a physician: 28 minutes; c) ECG transmission and Percutaneous Coronary Intervention Center team feedback time: 76 minutes; and d) Patient's Transfer Waiting Time: 78 minutes. SD baseline model confirmed the system's behavior with all occurring delays and the need of improvements. Moreover, after model validation and sensitivity analysis, results suggested that an overall improvement of 40% to 50% in each of these identified stages would reduce the delay., Conclusions: This evaluation suggests that investment in health personnel training, diminution of bureaucracy, and management of guidelines might lead to important improvements decreasing the delay of STEMI patients' care. In addition, this work provides evidence that SD modeling may highlight areas where health system managers can implement and evaluate the necessary changes in order to improve the process of care.

Background: The applicability of international risk scores in heart surgery (HS) is not well defined in centers outside of North America and Europe., Objective: To evaluate the capacity of the Parsonnet Bernstein 2000 (BP) and EuroSCORE (ES) in predicting in-hospital mortality (IHM) in patients undergoing HS at a reference hospital in Brazil and to identify risk predictors (RP)., Methods: Retrospective cohort study of 1,065 patients, with 60.3% patients underwent coronary artery bypass grafting (CABG), 32.7%, valve surgery and 7.0%, CABG combined with valve surgery. Additive and logistic scores models, the area under the ROC (Receiver Operating Characteristic) curve (AUC) and the standardized mortality ratio (SMR) were calculated. Multivariate logistic regression was performed to identify the RP., Results: Overall mortality was 7.8%. The baseline characteristics of the patients were significantly different in relation to BP and ES. AUCs of the logistic and additive BP were 0.72 (95% CI, from 0.66 to 0.78 p = 0.74), and of ES they were 0.73 (95% CI; 0.67 to 0.79 p = 0.80). The calculation of the SMR in BP was 1.59 (95% CI; 1.27 to 1.99) and in ES, 1.43 (95% CI; 1.14 to 1.79). Seven RP of IHM were identified: age, serum creatinine > 2.26 mg/dL, active endocarditis, systolic pulmonary arterial pressure > 60 mmHg, one or more previous HS, CABG combined with valve surgery and diabetes mellitus., Conclusion: Local scores, based on the real situation of local populations, must be developed for better assessment of risk in cardiac surgery.

Background: Trends in the prevalence of acute myocardial infarction in sub-Saharan Africa have not been well described, despite growing recognition of the increasing burden of cardiovascular disease in low- and middle-income countries. The aim of this systematic review was to describe the prevalence of acute myocardial infarction in sub-Saharan Africa., Methods: We searched PubMed, EMBASE, Global Health Archive, CINAHL, and Web of Science, and conducted reference and citation analyses. Inclusion criteria were: observational studies, studies that reported incidence or prevalence of acute myocardial infarction, studies conducted in sub-Saharan Africa, and studies that defined acute myocardial infarction by EKG changes or elevation of cardiac biomarkers. Studies conducted prior to 1992 were excluded. Two independent reviewers analyzed titles and abstracts, full-texts, and references and citations. These reviewers also performed quality assessment and data extraction. Quality assessment was conducted with a validated scale for observational studies., Findings: Of 2292 records retrieved, seven studies met all inclusion criteria. These studies included a total of 92,378 participants from highly heterogeneous study populations in five different countries. Methodological quality assessment demonstrated scores ranging from 3 to 7 points (on an 8-point scale). Prevalence of acute myocardial infarction ranged from 0.1 to 10.4% among the included studies., Interpretation: There is insufficient population-based data describing the prevalence of acute myocardial infarction in sub-Saharan Africa. Well-designed registries and surveillance studies that capture the broad and diverse population with acute myocardial infarction in sub-Saharan Africa using common diagnostic criteria are critical in order to guide prevention and treatment strategies., Registration: Registered in International Prospective Register of Systematic Reviews (PROSPERO) Database #CRD42012003161.

Background: Road traffic injuries (RTI) are a major public health epidemic killing thousands of people daily. Low and middle-income countries, such as Brazil, have the highest annual rates of road traffic fatalities. In order to improve road safety, this study mapped road traffic fatalities on a Brazilian highway to determine the main environmental factors affecting road traffic fatalities., Methods and Findings: Four techniques were utilized to identify and analyze RTI hotspots. We used spatial analysis by points by applying kernel density estimator, and wavelet analysis to identify the main hot regions. Additionally, built environment analysis, and principal component analysis were conducted to verify patterns contributing to crash occurrence in the hotspots. Between 2007 and 2009, 379 crashes were notified, with 466 fatalities on BR277. Higher incidence of crashes occurred on sections of highway with double lanes (ratio 2∶1). The hotspot analysis demonstrated that both the eastern and western regions had higher incidences of crashes when compared to the central region. Through the built environment analysis, we have identified five different patterns, demonstrating that specific environmental characteristics are associated with different types of fatal crashes. Patterns 2 and 4 are constituted mainly by predominantly urban characteristics and have frequent fatal pedestrian crashes. Patterns 1, 3 and 5 display mainly rural characteristics and have higher prevalence of vehicular collisions. In the built environment analysis, the variables length of road in urban area, limited lighting, double lanes roadways, and less auxiliary lanes were associated with a higher incidence of fatal crashes., Conclusions: By combining different techniques of analyses, we have identified numerous hotspots and environmental characteristics, which governmental or regulatory agencies could make use to plan strategies to reduce RTI and support life-saving policies.

It is of current interest the identification of appropriate matrices for growing mesenchymal stem cells (MSC). These cells are able not only to regenerate themselves but also to differentiate into other type of functional cells, and so they have been extensively used in tissue engineering. In this work, we have evaluated the use of electric impedance spectroscopy (EIS) to follow the adhesion of MSC from Wharton's jelly of the human umbilical cord (hWJMSC) on sugarcane biopolymers (SCB). Impedance spectra of the systems were obtained in the frequency range of 10(2)-10(5) Hz. An EIS investigation showed that when deposited on a metallic electrode SCB films prevent the passage of electrons between the solution and the metallic interface. The impedance spectra of hWJMSCs adhered on SCB revealed that there is a significant increase in the magnitude of the impedance when compared to that of pure SCB. The corresponding resistance (real part of the impedance) was even higher for the SCB-hWJMSC system than for SCB without cells on their surface, in an indication of an increased blockage to the electron transfers. The resistance charge transfer is extracted by curve-fitting the impedance spectra to an equivalent circuit model. Also, a shift of the phase angle to higher frequencies was obtained for SCB-hWJMSC system as a result from hWJMSC adhesion. Our study demonstrates that EIS is an appropriate method to evaluate the adhesion of MSC. SCB can be considered as a promising biomaterial for tissue engineering.

Unlabelled: Gene therapy can induce angiogenesis in ischemic tissues. The aim of this study was to assess safety, feasibility, and results, both clinical and on myocardial perfusion, of gene therapy in refractory angina. This was a phase I/II, prospective, temporal-controlled series, clinical trial. Thirteen patients were maintained for minimum 6 months under optimized clinical management, and then received intramyocardial injections of 2000 μg plasmid vascular endothelial growth factor 165 and were followed by single-photon emission computed tomography (SPECT), treadmill tests, Minnesota quality of life questionnaire (QOL), and New York Heart Association (NYHA) functional plus Canadian Cardiovascular Society (CCS) angina classifications. There were no deaths, early or late. During the optimized clinical treatment, we observed worsening of rest ischemia scores on SPECT (p<0.05). After treatment, there was a transitory increase in myocardial perfusion at the third-month SPECT under stress (pre-operative [pre-op] 18.38 ± 7.51 vs. 3 months 15.31 ± 7.30; p<0.01) and at the sixth month under rest (pre-op 13.23 ± 7.98 vs. 6 months: 16.92 ± 7.27; p<0.01). One year after, there were improvements in treadmill test steps (pre-op 2.46 ± 2.07 vs.12 months 4.15 ± 2.23; p<0.01) and oxygen consumption (pre-op 7.66 ± 4.47 vs.12 months 10.89 ± 4.65; p<0.05), QOL (pre-op 48.23 ± 18.35 vs.12 months 28.31 ± 18.14; p<0.01) scores, and CCS (pre-op 3 [3-3.5] vs.12 months 2 [1-2.5]; p<0.01) and NYHA (pre-op 3 [3-3] vs. 2 [2-2] vs. 12 months 2 [1-2]; p<0.01) classes. Gene therapy demonstrated to be feasible and safe in this advanced ischemic cardiomyopathy patient sample. There were improvements in clinical evaluation parameters, and a transitory increase in myocardial perfusion detectable by SPECT scintigraphy., Clinical Trial Registration: NCT00744315 http://clinicaltrials.gov/

Background: Vascular endothelial growth factor (VEGF) induces mobilization of endothelial progenitor cells (EPCs) with the capacity for proliferation and differentiation into mature endothelial cells, thus contributing to the angiogenic process., Objective: We sought to assess the behavior of EPCs in patients with ischemic heart disease and refractory angina who received an intramyocardial injections of 2000 µg of VEGF 165 as the sole therapy., Methods: The study was a subanalysis of a clinical trial. Patients with advanced ischemic heart disease and refractory angina were assessed for eligibility. Inclusion criteria were as follows: signs and symptoms of angina and/or heart failure despite maximum medical treatment and a myocardial ischemic area of at least 5% as assessed by single-photon emission computed tomography (SPECT). Exclusion criteria were as follows: age > 65 years, left ventricular ejection fraction < 25%, and a diagnosis of cancer. Patients whose EPC levels were assessed were included. The intervention was 2000 µg of VEGF 165 plasmid injected into the ischemic myocardium. The frequency of CD34+/KDR+ cells was analyzed by flow cytometry before and 3, 9, and 27 days after the intervention., Results: A total of 9 patients were included, 8 males, mean age 59.4 years, mean left ventricular ejection fraction of 59.3% and predominant class III angina. The number of EPCs on day 3 was significantly higher than that at baseline (p = 0.03); however, that on days 9 and 27 was comparable to that at baseline., Conclusion: We identified a transient mobilization of EPCs, which peaked on the 3th day after VEGF 165 gene therapy in patients with refractory angina and returned to near baseline levels on days 9 and 27.

Background: Research coaching program focuses on the development of abilities and scientific reasoning. For health professionals, it may be useful to increase both the number and quality of projects and manuscripts., Objective: To evaluate the initial results and implementation methodology of the Research and Innovation Coaching Program of the Research on Research group of Duke University in the Brazilian Society of Cardiology., Methods: The program works on two bases: training and coaching. Training is done online and addresses contents on research ideas, literature search, scientific writing and statistics. After training, coaching favors the establishment of a collaboration between researchers and centers by means of a network of contacts. The present study describes the implementation and initial results in reference to the years 2011-2012., Results: In 2011, 24 centers received training, which consisted of online meetings, study and practice of the contents addressed. In January 2012, a new format was implemented with the objective of reaching more researchers. In six months, 52 researchers were allocated. In all, 20 manuscripts were published and 49 more were written and await submission and/or publication. Additionally, five research funding proposals have been elaborated., Conclusion: The number of manuscripts and funding proposals achieved the objectives initially proposed. However, the main results of this type of initiative should be measured in the long term, because the consolidation of the national production of high-quality research is a virtuous cycle that feeds itself back and expands over time.

This paper investigates the extent of socioeconomic inequalities in antenatal care use and related medical procedures in Brazil and India, which represent transition economies with contrasting geographical and sociocultural composition and health care provision. Concentration indices and regression analyses applied on recent Demographic Health Survey data reveal high and proportionate distribution of antenatal coverage in Brazil, whereas the Indian case present problems of both scale and equity. Inequalities in access to four or more antenatal visits are significantly pronounced in India, and in Brazil the differences are significant only for those who had six or more visits. Brazil's universal healthcare model which proved effective in promoting equitable distribution of antenatal care could be implemented in India. Future interventions should emphasis quality of care in monitoring essential antenatal services especially targeting the poor and deprived communities., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Severe ischemic heart disease with refractory angina, occurs in increasing incidence. Alternative forms of treatment, in an attempt to reduce myocardial ischemia and relief of symptoms has been studied. In this context, gene therapy is an option, for the possibility of inducing angiogenesis, establish collateral circulation and reperfuse ischemic myocardium. Several clinical trials have been conducted and, except for specific cases of adverse effects, there is indication of safety, feasibility and potential effectiveness of therapy. The clinical benefit, however, is not yet well established. In this article we review the clinical trials of gene therapy for patients with ischemic heart disease. The approach includes: (1) myocardial ischemia and angiogenesis on the pathophysiological aspects involved, (2) growth factors, dealing with specific aspects and justifying the use in cardiac patients with no option for conventional therapy, (3) controlled clinical trials, where a summary of the main studies involving gene therapy for severe ischemic heart disease is presented, (4) our experience, especially on preliminary results of the first gene therapy clinical trial in Brazil and (5) future prospects.

Purpose: The study aims to clinically validate the defining characteristics (DCs) of the nursing diagnosis (ND) of Activity Intolerance for patients with ischemic heart disease and refractory angina., Methods: Cross-sectional study was used, involving 22 patients with ND of Activity Intolerance. The Fehring method was used to validate the ND., Findings: Most DCs presented reliability indexes between 0.5 and 0.79. Three DCs presented reliability indexes ≥ 0.8., Conclusion: All DCs were validated, and electrocardiographic changes indicating ischemia, verbal report of fatigue, and abnormal rate response to activity were considered as DC major., Implications for Nursing Practice: This study is relevant in daily nursing practice for guidance in establishing the care plan and describing the assistance for this group of patients., (© 2011, The Authors. International Journal of Nursing Terminologies and Classifications © 2011, NANDA International.)

Despite extensive research in the nanopore-sensing field, there is a paucity of experimental studies that investigate specific ion effects in confined spaces, such as in nanopores. Here, the effect of halogen anions on a simple bimolecular complexation reaction between monodisperse poly(ethylene glycol) (PEG) and α-hemolysin nanoscale pores have been investigated at the single-molecule level. The anions track the Hofmeister ranking according to their influence upon the on-rate constant. An inverse relationship was demonstrated for the off-rate and the solubility of PEG. The difference among anions spans several hundredfold. Halogen anions play a very significant role in the interaction of PEG with nanopores although, unlike K(+), they do not bind to PEG. The specific effect appears dominated by a hydration-dehydration process where ions and PEG compete for water. Our findings provide what we believe to be novel insights into physicochemical mechanisms involved in single-molecule interactions with nanopores and are clearly relevant to more complicated chemical and biological processes involving a transient association of two or more molecules (e.g., reception, signal transduction, enzyme catalysis). It is anticipated that these findings will advance the development of devices with nanopore-based sensors for chemical and biological applications., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Objectives: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells., Methods: Involvement of ion channels in cell volume regulation was studied using video-microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used., Results: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V(max) and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G(0) /G(1) phase boundary. Electrophysiological study confirmed presence of volume-activated Cl(-) channels and K(+) channels in plasmatic membranes of the cells., Conclusion: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells., (© 2010 Blackwell Publishing Ltd.)

Objective: Safety, feasibility and early myocardial angiogenic effects evaluation of transthoracic intramyocardial phVEGF165 administration for refractory angina in no option patients., Methods: Cohort study, in which 13 patients with refractory angina under optimized clinical treatment where included, after cineangiograms had been evaluated and found unfeasible by surgeon and interventional cardiologist. Intramyocardial injections of 5 mL solution containing plasmidial VEGF165 where done over the ischemic area of myocardium identified by previous SPECT/Sestamibi scan. Evaluations included a SPECT scan, stress test, Minnesota QOL questionnaire and NYHA functional class and CCS angina class determinations., Results: There were no deaths or new interventions during the study period. There were no significant variations in SPECT scans, QOL scores and stress tests results during medical treatment in the included patients. After the 3rd post operative month, there was improvement in SPECT segmental scores, SSS (18.38 ± 7.51 vs. 15.31 ± 7.29, P = 0.003) and SRS (11.92 ± 7.49 vs. 8.53 ± 6.68, P = 0.002). The ischemic area extension, however, had non-significant variation (23.38 ± 13.12% vs. 20.08 ± 13.88%, P = 0.1). Stress tests METs varied from 7.66 ± 4.47 pre to 10.29 ± 4.36 METs post-op (P = 0.08). QOL score improved from 48.23 ± 18.35 pre to 30.15 ± 20.13 post-op points (P = 0.02). NYHA class was 3.15 ± 0.38 pre vs. 1.77 ± 0.83 post-op (P = 0.001) and angina CCS class, 3.08 ± 0.64 vs. 1.77 ± 0.83 (P = 0.001)., Conclusions: Intramyocardial VEGF165 therapy for refractory angina, in this small trial of no option patients, resulted feasible and safe. Early clinical and scintilographic data showed improvements in symptoms and myocardial perfusion, with regression of ischemia severity in treated areas.

The aim of this paper is to analyze how the social position of families affects self-reported health status, based on data from the 1998 and 2003 National Sample Household Survey of the Brazilian Institute of Geography and Statistics (PNAD-IBGE). The method was based on descriptive statistics and logistic regression to capture the conditional relationship between health status, social position, and other control variables, such as age, sex, race/color, income, education, and place of residence. The results show that the same hierarchy of the occupational classes is reproduced in self-reported health status. There was an increase in inequalities in the period, with a reduction in the prevalence of healthy workers in the lower social strata and an increase in the upper strata. Although there were more healthy individuals in non-agricultural classes, the probability of good self-reported health was higher among agricultural workers, after the other control variables were added to the regression models. The results emphasize the need to analyze health inequalities from the perspective of social stratification.

The mechanisms of KCl-induced enhancement in identification of individual molecules of poly(ethylene glycol) using solitary alpha-hemolysin nanoscale pores are described. The interaction of single molecules with the nanopore causes changes in the ionic current flowing through the pore. We show that the on-rate constant of the process is several hundred times larger and that the off-rate is several hundred times smaller in 4 M KCl than in 1 M KCl. These shifts dramatically improve detection and make single molecule identification feasible. KCl also changes the solubility of poly(ethylene glycol) by the same order of magnitude as it changes the rate constants. In addition, the polymer-nanopore interaction is determined to be a strong non-monotonic function of voltage, indicating that the flexible, nonionic poly(ethylene glycol) acts as a charged molecule. Therefore, salting-out and Coulombic interactions are responsible for the KCl-induced enhancement. These results will advance the development of devices with sensor elements based on single nanopores.

We introduce a two-dimensional method for mass spectrometry in solution that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) molecules that enter a single alpha-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of molecules in solution.

The capture and release of single poly(ethylene glycol) molecules by the alpha-Hemolysin pore are observed as time-resolved reversible steps in ion conductance. The capture on rate, inferred from the step frequency, decreases monotonically with polymer size. However, the polymer residence time shows a crossover behavior, first increasing and then decreasing with molecular weight. Our interpretation is that, in the case of polymers which are too large to be accommodated within the pore, the out-of-the-pore part of the molecule pulls on the trapped part, thus acting as an entropic spring.

Closing linear poly(ethylene glycol) (PEG) into a circular "crown" dramatically changes its dynamics in the alpha-hemolysin channel. In the electrically neutral crown ether (C2H4O)6, six ethylene oxide monomers are linked into a circle that gives the molecule ion-complexing capacity and increases its rigidity. As with linear PEG, addition of the crown to the membrane-bathing solution decreases the ionic conductance of the channel and generates additional conductance noise. However, in contrast to linear PEG, both the conductance reduction (reporting on crown partitioning into the channel pore) and the noise (reporting on crown dynamics in the pore) now depend on voltage strongly and nonmonotonically. Within the whole frequency range accessible in channel reconstitution experiments, the noise power spectrum is "white", showing that crown exchange between the channel and the bulk solution is fast. Analyzing these data in the framework of a Markovian two-state model, we are able to characterize the process quantitatively. We show that the lifetime of the crown in the channel reaches its maximum (a few microseconds) at about the same voltage (approximately 100 mV, negative from the side of protein addition) where the crown's reduction of the channel conductance is most pronounced. Our interpretation is that, because of its rigidity, the crown feels an effective steric barrier in the narrowest part of the channel pore. This barrier together with crown-ion complexing and resultant interaction with the applied field leads to behavior usually associated with voltage-dependent binding in the channel pore.

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.

Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha-toxin in planar lipid membranes is a heptamer.

Asymmetrical (one-sided) application of penetrating water-soluble polymers, polyethylene glycols (PEGs), to a well-defined channel formed by Staphylococcus aureus alpha-toxin is shown to probe channel pore geometry in more detail than their symmetrical (two-sided) application. Polymers added to the cis side of the planar lipid membrane (the side of protein addition) affect channel conductance differently than polymers added to the trans side. Because a satisfactory theory quantitatively describing PEG partitioning into a channel pore does not exist, we apply the simple empirical rules proposed previously (, J. Membr. Biol. 161:83-92) to gauge the size of pore openings as well as the size and position of constrictions along the pore axis. We estimate the radii of the two openings of the channel to be practically identical and equal to 1. 2-1.3 nm. Two apparent constrictions with radii of approximately 0. 9 nm and approximately 0.6-0.7 nm are inferred to be present in the channel lumen, the larger one being closer to the cis side. These structural findings agree well with crystallographic data on the channel structure (, Science. 274:1859-1866) and verify the practicality of polymer probing. The general features of PEG partitioning are examined using available theoretical considerations, assuming there is no attraction between PEG and the channel lumen. It is shown that the sharp dependence of the partition coefficient on polymer molecular weight found under both symmetrical and asymmetrical polymer application can be rationalized within a "hard sphere nonideal solution model." This finding is rather surprising because PEG forms highly flexible coils in water with a Kuhn length of only several Angstroms.

The effects of heparin on ion channels formed by Staphylococcus aureus alpha-toxin (ST channel) in lipid bilayers were studied under voltage clamp conditions. Heparin concentrations as small as 100 pM induced a sharp dose-dependent increase in channel voltage sensitivity. This was only observed when heparin was added to the negative-potential side of lipid bilayers in the presence of divalent cations. Divalent cations differ in their efficiency: Zn2+>Ca2+>Mg2+. The apparent positive gating charge increased 2-3-fold with heparin addition as well as with acidification of the bathing solution. 'Free' carboxyl groups and carboxyl groups in ion pairs of the protein moiety are hypothesized to interact with sulfated groups of heparin through divalent cation bridges. The cis mouth of the channel (that protrudes beyond the membrane plane on the side of ST addition and to which voltage was applied) is less sensitive to heparin than the trans-mouth. It is suggested that charged residues which interact with heparin at the cis mouth of ST channels and which contribute to the effective gating charge at negative voltage may be physically different from those at the trans mouth and at positive voltage.

The effective size of colicin Ia channel was tested by a recently described method (FEMS, Microbiology and Immunology (1992). 105: 93-100) in which the nonelectrolyte molecules with different hydrodynamic diameters (0.52 to 5.0 nm) were used as molecular tools. We have shown that despite low conductance (55-105 pS at 1.5 M KCl, pH 7.0) the ion channels formed by colicin Ia have a fairly large water pore diameter equal to 1.66-1.88 nm. The results are discussed in terms of an energetic barrier for ions passing into the channel lumen.

The selectivity of the planar lipid bilayers modified by two channel-forming proteins (alpha-toxin S. aureus and colicin Ia) was examined. It was established that in all cases the value of zero current potential depended on the amount of open ion channels and increased with the number of channels (from one to about 5-7). These facts point out both the interactions among ion channels and their non stochastic distribution on the membrane surface.

Antigen F1 is a protein of 17 kDa produced by Yersinia pestis when it is cultured at 37 degrees C. When incorporated into planar lipid bilayer membranes this protein induces fluctuations on membrane conductance typical of the formation of ionic channels. These fluctuations reveal two distinct unitary conductance sizes, one in the range of 800 to 1400 pS and the other in the range of 140 to 600 pS. Zero current potential measurements in the presence of a salt gradient show that the channel is not significantly ion selective. The reversal potential measured in the presence of 0.5 M KCl on the cis side and 0.1 M KCl on the trans side was 3.58 +/- 3.98 mV (N = 7). The non-selectivity of the channel, in addition to its large conductance, suggests that it forms large aqueous pores. The present results, taken together with other data showing that antigen F1 inhibits the activity of phagocytic cells, suggest that antigen F1 acts by forming aqueous pores in the membrane of these target cells.

Genes for the blue copper proteins Populus nigra var. italica plastocyanin and Pseudomonas aeruginosa azurin have been constructed by a stepwise procedure. The leader sequence for azurin has been placed before the genes directing plastocyanin and azurin transport to the periplasmic space when the genes are expressed in Escherichia coli. Site-saturation mutagenesis has been used to alter two copper-binding residues of azurin (Met-121 and His-46) and Met-92 of plastocyanin. While the plastocyanin mutants do not appear to bind copper, the azurin variants all bind copper and show characteristic type I blue copper centers. In particular, the electronic spectra reflect the dominance of the charge transfer interaction between copper and the thiolate of Cys-112, being relatively insensitive to changes in Met-121 or His-46. In contrast, removal of Met-121 appreciably alters the EPR spectra of the mutants, although, to a first order, the spectra of all mutants are themselves similar, suggesting a more distorted geometry around copper in the mutants than in the wild type.