Evaluation of curcumin and cisplatin-induced DNA damage in PC12 cells by the alkaline comet assay

A very appropriate method for antigenotoxicity evaluation of antioxidants is the comet assay, since this analytical method detects initial DNA lesions that are still subject to repair; in other words, lesions that are very associated to damages resulting from the generation and subsequent action of reactive species. However, a solid evaluation should be developed in order to avoid inexact interpretations. In our study, besides the association of curcumin with cisplatin, curcumin and cisplatin agents were also tested separately. Classical genotoxic compounds, when tested by the comet assay, present an increase in the nucleoid tail; however, the cisplatin treatment has resulted in a decrease of DNA migration. This was an expected effect, as the cross-links between cisplatin and DNA decrease the DNA electrophoretic mobility. A similar effect was observed with the curcumin treatment, which decreased the nucleoid tail. Such effect was not expected and reinforced the necessity of including in the study, separate treatment groups with potentially antigenotoxic substances. The comet assay results have been analyzed using specific software for image analysis, as well as the classical visual analysis, and we have observed that the effect of decrease in DNA electrophoretic mobility was more easily observed when the data were analyzed by the software.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. All rights reserved.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Preparation, characterization and in vitro cytotoxicity of BSA-based nanospheres containing nanosized magnetic particles and/or photosensitizer

This study reports on the preparation, characterization and in vitro toxicity test of a new nano-drug delivery system (NDDS) based on bovine serum albumin (BSA) nanospheres which incorporates surface-functionalized magnetic nanoparticles (MNP) and/or the silicon(IV) phthalocyanine (NzPc). The new NDDS was engineered for use in photodynamic therapy (PDT) combined with hyperthermia (HPT) to address cancer treatment. The BSA-based nanospheres, hosting NzPc, MNP or both (NzPc and MNP), present spherical shape with hydrodynamic average diameter values ranging from 170 to 450 nm and zeta potential of around -23 mV. No difference on the fluorescence spectrum of the encapsulated NzPc was found regardless of the presence of MNP. Time-dependent fluorescence measurements of the encapsulated NzPc revealed a bi-exponential decay for samples incorporating only NzPc and NzPc plus MNP, in the time window ranging from 1.70 to 5.20 ns. The in vitro assay, using human fibroblasts, revealed no cytotoxic effect in all samples investigated, demonstrating the potential of the tested system as a synergistic NDDS. (C) 2009 Elsevier B.V. All rights reserved.

Identification of trail pheromone compounds from the labial glands of the stingless bee Geotrigona mombuca

Foragers of several species of stingless bees deposit pheromone spots in the vegetation to guide recruited nestmates to a rich food source. Recent studies have shown that Trigona and Scaptotrigona workers secrete these pheromones from their labial glands. An earlier report stated that species within the genus Geotrigona use citral from their mandibular glands for scent marking. Since convincing experimental proof for this conjecture is lacking, we studied the glandular origin of the trail pheromone of Geotrigona mombuca. In field bioassays, newly recruited bees were diverted by artificial scent trails that branched off from the natural scent trail deposited by their nestmates only when they were baited with extracts from the foragers` labial glands. Compounds extracted from the mandibular glands, however, did not release trail following behavior. This demonstrates that the trail pheromone of G. mombuca is produced in the labial glands, as in Trigona and Scaptotrigona. Furthermore, in chemical analyses citral was identified exclusively in the foragers` mandibular glands, which disproves its supposed role as a trail pheromone. The labial glands contained a series of terpene- and wax type esters, with farnesyl butanoate as major constituent. We, therefore, postulate that the trail pheromone of G. mombuca is composed of a blend of esters.

Improvement in Insulin Sensitivity and B-Cell Function Following Ileal Interposition with Sleeve Gastrectomy in Type 2 Diabetic Patients: Potential Mechanisms

Bariatric surgery in morbidly obese type 2 diabetic (T2DM) patients is associated with high rates of diabetes remission. We investigated the mechanisms of the anti-diabetic effect of the laparoscopic ileal interposition with sleeve gastrectomy (LII-SG) in normal weight (NW), overweight (OW) and obese (OB) T2DM patients. Ninety-four patients (aged 54 +/- 8 years) with long-standing (median 10 years), treated diabetes (median HbA(1c) = 8.6%), who were NW (15), OW (64) or OB (15) based on BMI, underwent LII-SG. Insulin sensitivity and parameters of -cell function were measured from an Oral Glycaemic Tolerance Test pre- and post-operatively. At a median of 13.4 months post-operatively, weight loss averaged 9.4 +/- 1.3, 16.8 +/- 0.8 and 23.2 +/- 1.7 kg in NW, OW and OB subjects, respectively (p < 0.0001). Insulin sensitivity was fully restored (395 [108] vs 208 [99] ml min(-1) m(-2)), fasting insulin secretion rate decreased (68 [52] vs 146 [120] pmol min(-1) m(-2)) and total insulin output increased (52 [26] vs 39 [28] nmol m(-2), all p a parts per thousand currency signaEuro parts per thousand 0.001). -cell glucose sensitivity doubled (37 [33] vs 18 [24] mol min(-1) m(-2) mM(-1), p < 0.0001). The only parameter predicting remission of diabetes was a lower baseline insulin sensitivity (p = 0.005). LII-SG induced changes on T2DM by mechanisms in part distinct from weight loss, principally involving restoration of insulin sensitivity and improvement of -cell function.

Identification of novel L2HGDH gene mutations and update of the pathological spectrum

L-2-hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a neurometabolic disorder caused by the toxic accumulation of high concentration of L-2-hydroxyglutaric acid in plasma and cerebrospinal fluid. Distinct mutations on the L2HGDH gene have been associated with the clinical and biochemical phenotype. Here we present three novel mutations (Gln197X, Gly211Val and c.540+1 G>A), which increase the present deleterious collection of L2HGDH gene up to 35 mutations that we have compiled in this study. In addition, we used the haplotypic information based on polymorphic markers to demonstrate the common origin of Gly57Arg harboring chromosomes. Journal of Human Genetics (2010) 55, 55-58; doi: 10.1038/jhg.2009.110; published online 13 November 2009

Leishmania (Viannia) braziliensis identification by PCR in the state of Para, Brazil

The incidence of cutaneous leishmaniasis (CL) is increasing and there is limited surveillance of Leishmania species throughout the world. We identified the species associated with CL in a region of Amazonia, an area recognized for its Leishmania species variability. Clinical findings were analyzed and correlated with the species identified in 93 patients. PCR assays were based on small subunit ribosomal DNA (SSU-rDNA) and G6PD, and were performed in a laboratory located 3,500 km away. Leishmania (V.) braziliensis was identified in 53 patients (57%). The other 40 patients (43%) carried a different species (including six cases of L (L) amazonensis). Molecular methods can be employed, using special media, to allow transport to distant laboratories. L (V.) braziliensis is the most common species in the area of Para. The location of ulcers can suggest CL species (C) 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Computed tomographic assessment of lung weights in trauma patients with early posttraumatic lung dysfunction

Introduction: Quantitative computed tomography (qCT)-based assessment of total lung weight (M(lung)) has the potential to differentiate atelectasis from consolidation and could thus provide valuable information for managing trauma patients fulfilling commonly used criteria for acute lung injury (ALI). We hypothesized that qCT would identify atelectasis as a frequent mimic of early posttraumatic ALI. Methods: In this prospective observational study, M(lung) was calculated by qCT in 78 mechanically ventilated trauma patients fulfilling the ALI criteria at admission. A reference interval for M(lung) was derived from 74 trauma patients with morphologically and functionally normal lungs (reference). Results are given as medians with interquartile ranges. Results: The ratio of arterial partial pressure of oxygen to the fraction of inspired oxygen was 560 (506 to 616) mmHg in reference patients and 169 (95 to 240) mmHg in ALI patients. The median reference M(lung) value was 885 (771 to 973) g, and the reference interval for M(lung) was 584 to 1164 g, which matched that of previous reports. Despite the significantly greater median M(lung) value (1088 (862 to 1,342) g) in the ALI group, 46 (59%) ALI patients had M(lung) values within the reference interval and thus most likely had atelectasis. In only 17 patients (22%), Mlung was increased to the range previously reported for ALI patients and compatible with lung consolidation. Statistically significant differences between atelectasis and consolidation patients were found for age, Lung Injury Score, Glasgow Coma Scale score, total lung volume, mass of the nonaerated lung compartment, ventilator-free days and intensive care unit-free days. Conclusions: Atelectasis is a frequent cause of early posttraumatic lung dysfunction. Differentiation between atelectasis and consolidation from other causes of lung damage by using qCT may help to identify patients who could benefit from management strategies such as damage control surgery and lung-protective mechanical ventilation that focus on the prevention of pulmonary complications.

Extrapolation from ten sections can make CT-based quantification of lung aeration more practicable

Clinical applications of quantitative computed tomography (qCT) in patients with pulmonary opacifications are hindered by the radiation exposure and by the arduous manual image processing. We hypothesized that extrapolation from only ten thoracic CT sections will provide reliable information on the aeration of the entire lung. CTs of 72 patients with normal and 85 patients with opacified lungs were studied retrospectively. Volumes and masses of the lung and its differently aerated compartments were obtained from all CT sections. Then only the most cranial and caudal sections and a further eight evenly spaced sections between them were selected. The results from these ten sections were extrapolated to the entire lung. The agreement between both methods was assessed with Bland-Altman plots. Median (range) total lung volume and mass were 3,738 (1,311-6,768) ml and 957 (545-3,019) g, the corresponding bias (limits of agreement) were 26 (-42 to 95) ml and 8 (-21 to 38) g, respectively. The median volumes (range) of differently aerated compartments (percentage of total lung volume) were 1 (0-54)% for the nonaerated, 5 (1-44)% for the poorly aerated, 85 (28-98)% for the normally aerated, and 4 (0-48)% for the hyperaerated subvolume. The agreement between the extrapolated results and those from all CT sections was excellent. All bias values were below 1% of the total lung volume or mass, the limits of agreement never exceeded +/- 2%. The extrapolation method can reduce radiation exposure and shorten the time required for qCT analysis of lung aeration.

Urodynamic evaluation in diabetic patients with prostate enlargement and lower urinary tract symptoms

Introduction: Lower urinary tract symptoms ( LUTS) are common in men over 50 years of age due to prostate enlargement. Diabetes mellitus is also more prevalent in this group. LUTS may result from bladder outlet obstruction ( BOO) secondary to prostate enlargement or bladder dysfunction secondary to diabetes or even from a combination of both. Objectives: The objective of this study was to determine the prevalence of BOO and other urodynamic abnormalities in diabetic patients with LUTS and enlarged prostate. A secondary objective was to assess the predictive value of non-invasive tests for BOO diagnosis in this group of patients. Patients and Methods: 50 consecutive diabetic patients with enlarged prostate and LUTS were evaluated by the International Prostate Symptom Score ( IPSS), ultra sonography and urodynamics. BOO diagnosis was based on pressure/ flow measurements according to the International Continence Society`s standards. Results: Of the 50 patients in the study, 23 ( 46%) had BOO. There was no correlation between the IPSS, uroflowmetry, post- voiding residual urine or prostate volume and the presence of BOO ( p > 0.05). Conclusions: There is a relatively low prevalence of BOO in diabetic patients with prostate enlargement and LUTS. Non- invasive tests did not allow the identification of these subjects. Only urodynamic evaluation is able to determine symptom etiology. Copyright (c) 2008 S. Karger AG, Basel.

CD4+T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

P>Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Strategies for genetic model specification in the screening of genome-wide meta-analysis signals for further replication

Background Meta-analysis is increasingly being employed as a screening procedure in large-scale association studies to select promising variants for follow-up studies. However, standard methods for meta-analysis require the assumption of an underlying genetic model, which is typically unknown a priori. This drawback can introduce model misspecifications, causing power to be suboptimal, or the evaluation of multiple genetic models, which augments the number of false-positive associations, ultimately leading to waste of resources with fruitless replication studies. We used simulated meta-analyses of large genetic association studies to investigate naive strategies of genetic model specification to optimize screenings of genome-wide meta-analysis signals for further replication. Methods Different methods, meta-analytical models and strategies were compared in terms of power and type-I error. Simulations were carried out for a binary trait in a wide range of true genetic models, genome-wide thresholds, minor allele frequencies (MAFs), odds ratios and between-study heterogeneity (tau(2)). Results Among the investigated strategies, a simple Bonferroni-corrected approach that fits both multiplicative and recessive models was found to be optimal in most examined scenarios, reducing the likelihood of false discoveries and enhancing power in scenarios with small MAFs either in the presence or in absence of heterogeneity. Nonetheless, this strategy is sensitive to tau(2) whenever the susceptibility allele is common (MAF epsilon 30%), resulting in an increased number of false-positive associations compared with an analysis that considers only the multiplicative model. Conclusion Invoking a simple Bonferroni adjustment and testing for both multiplicative and recessive models is fast and an optimal strategy in large meta-analysis-based screenings. However, care must be taken when examined variants are common, where specification of a multiplicative model alone may be preferable.

Most of the patients presenting myocardial infarction would not be eligible for intensive lipid-lowering based on clinical algorithms or plasma C-reactive protein

Objective: The study we assessed how often patients who are manifesting a myocardial infarction (MI) would not be considered candidates for intensive lipid-lowering therapy based on the current guidelines. Methods: In 355 consecutive patients manifesting ST elevation MI (STEMI), admission plasma C-reactive protein (CRP) was measured and Framingham risk score (FRS), PROCAM risk score, Reynolds risk score, ASSIGN risk score, QRISK, and SCORE algorithms were applied. Cardiac computed tomography and carotid ultrasound were performed to assess the coronary artery calcium score (CAC), carotid intima-media thickness (cIMT) and the presence of carotid plaques. Results: Less than 50% of STEMI patients would be identified as having high risk before the event by any of these algorithms. With the exception of FRS (9%), all other algorithms would assign low risk to about half of the enrolled patients. Plasma CRP was <1.0 mg/L in 70% and >2 mg/L in 14% of the patients. The average cIMT was 0.8 +/- 0.2 mm and only in 24% of patients was >= 1.0 mm. Carotid plaques were found in 74% of patients. CAC > 100 was found in 66% of patients. Adding CAC >100 plus the presence of carotid plaque, a high-risk condition would be identified in 100% of the patients using any of the above mentioned algorithms. Conclusion: More than half of patients manifesting STEMI would not be considered as candidates for intensive preventive therapy by the current clinical algorithms. The addition of anatomical parameters such as CAC and the presence of carotid plaques can substantially reduce the CVD risk underestimation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Analyzing the connectivity between regions of interest: An approach based on cluster Granger causality for NRI data analysis

The identification, modeling, and analysis of interactions between nodes of neural systems in the human brain have become the aim of interest of many studies in neuroscience. The complex neural network structure and its correlations with brain functions have played a role in all areas of neuroscience, including the comprehension of cognitive and emotional processing. Indeed, understanding how information is stored, retrieved, processed, and transmitted is one of the ultimate challenges in brain research. In this context, in functional neuroimaging, connectivity analysis is a major tool for the exploration and characterization of the information flow between specialized brain regions. In most functional magnetic resonance imaging (fMRI) studies, connectivity analysis is carried out by first selecting regions of interest (ROI) and then calculating an average BOLD time series (across the voxels in each cluster). Some studies have shown that the average may not be a good choice and have suggested, as an alternative, the use of principal component analysis (PCA) to extract the principal eigen-time series from the ROI(s). In this paper, we introduce a novel approach called cluster Granger analysis (CGA) to study connectivity between ROIs. The main aim of this method was to employ multiple eigen-time series in each ROI to avoid temporal information loss during identification of Granger causality. Such information loss is inherent in averaging (e.g., to yield a single ""representative"" time series per ROI). This, in turn, may lead to a lack of power in detecting connections. The proposed approach is based on multivariate statistical analysis and integrates PCA and partial canonical correlation in a framework of Granger causality for clusters (sets) of time series. We also describe an algorithm for statistical significance testing based on bootstrapping. By using Monte Carlo simulations, we show that the proposed approach outperforms conventional Granger causality analysis (i.e., using representative time series extracted by signal averaging or first principal components estimation from ROIs). The usefulness of the CGA approach in real fMRI data is illustrated in an experiment using human faces expressing emotions. With this data set, the proposed approach suggested the presence of significantly more connections between the ROIs than were detected using a single representative time series in each ROI. (c) 2010 Elsevier Inc. All rights reserved.