Quality control of Toxoplasma gondii in meat packages: Standardization of an ELISA test and its use for detection in rabbit meat cuts

Toxoplasma gondii causes severe disease both to man and livestock and its detection in meat after slaughtering requires PCR or biological tests. Meat packages contain retained exudate that could be used for serology due to its blood content. Similar studies reported false negative assays in those tests. We standardized an anti-T. gondii IgG ELISA in muscle juices from experimentally infected rabbits, with blood content determination by cyanhemoglobin spectrophotometry. IgG titers and immunoblotting profiles were similar in blood, serum or meat juice, after blood content correction. These assays were adequate regardless of the storage time up to 120 days or freeze-thaw cycles, without false negative results. We also found 1.35% (1/74) positive sample in commercial Brazilian rabbit meat cuts, by this assay. The blood content determination shows ELISA of meat juice may be useful for quality control for toxoplasmosis monitoring. (C) 2011 Elsevier Ltd. All rights reserved.

Clinical usefulness of B-type natriuretic peptide in the diagnosis of pleural effusions due to heart failure

Background and objective: Light`s criteria are frequently used to evaluate the exudative or transudative nature of pleural effusions. However, misclassification resulting from the use of Light`s criteria has been reported, especially in the setting of diuretic use in patients with heart failure (HF). The objective of this study was to evaluate the utility of B-type natriuretic peptide (BNP) measurements as a diagnostic tool for determining the cardiac aetiology of pleural effusions. Methods: Patients with pleural effusions attributable to HF (n = 34), hepatic hydrothorax (n = 10), pleural effusions due to cancer (n = 21) and pleural effusions due to tuberculosis (n = 12) were studied. Diagnostic thoracentesis was performed for all 77 patients. Receiver operating characteristic (ROC) curves were constructed to determine the diagnostic accuracy of plasma BNP and pleural fluid BNP for the prediction of HF. Results: The areas under the ROC curves were 0.987 (95% CI 0.93-0.998) for plasma BNP and 0.949 (95% CI 0.874-0.986) for pleural fluid BNP, for distinguishing between patients with pleural effusions caused by HF (n = 34) and those with pleural effusions attributable to other causes (n = 43). The cut-off concentrations with the highest diagnostic accuracy for the diagnosis of HF as the cause of pleural effusion were 132 pg/mL for plasma BNP (sensitivity 97.1%, specificity 97.4%) and 127 pg/mL for pleural fluid BNP (sensitivity 97.1%, specificity 87.8%). Conclusions: In patients with pleural effusions of suspected cardiac origin, measurements of BNP in plasma and pleural fluid may be useful for the diagnosis of HF as the underlying cause.

Global Analysis of Protein Palmitoylation in African Trypanosomes

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.

Intracranial vascular lesions associated with small epidural hematomas

OBJECTIVE: We studied the angiographic findings in patients with small epidural hematomas and cranial fractures crossing over the trajectory of the middle meningeal artery and its branches. Additionally, the Occurrence of traumatic vascular lesions and their clinical relevance and treatment are discussed. METHODS: A consecutive analysis was performed for 24 patients who harbored small epidural hematomas in middle meningeal artery topography associated with cranial fractures. Computed tomographic scans and plain x-ray studies were used to diagnose linear cranial fractures. Patients with large epidural hematomas or associated traumatic lesions were excluded from the study. Selective ipsilateral external carotid angiograms were obtained, and an endovascular procedure was performed if any vascular injury was evidenced. RESULTS: In all patients with cranial fractures crossing over the middle meningeal artery and its branches, some kind of vascular lesion was seen. Two types of findings were noted: active extravasation of the contrast medium (71%) and pseudoaneurysms (29%). Early filling of diploic vessels was found in 8.3% of fractures concomitantly with active extravasation. Embolization was performed in all patients. No additional enlargement of the epidural hematoma was observed, and the postoperative period was uneventful. CONCLUSION: This study shows that pseudoaneurysms and active extravasation of contrast are common findings in this subset of patients. Although the natural history of these lesions is still poorly understood, additional investigation with ipsilateral external carotid angiography may be recommended, considering the potentially catastrophic consequences of late rupture.

Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation

Glioblastoma multiforme (GBM) is a highly invasive and radioresistant brain tumor. Aiming to study how glioma cells respond to gamma-rays in terms of biological processes involved in cellular responses, we performed experiments at cellular context and gene expression analysis in U343-MG-a GBM cells irradiated with 1 Gy and collected at 6 h post-irradiation. The survival rate was approximately 61% for 1 Gy and was completely reduced at 16 Gy. By performing the microarray technique, 859 cDNA clones were analyzed. The Significance Analysis of Microarray algorithm indicated 196 significant expressed genes (false discovery rate (FDR) = 0.42%): 67 down-regulated and 97 up-regulated genes, which belong to several classes: metabolism, adhesion/cytoskeleton, signal transduction, cell cycle/apoptosis, membrane transport, DNA repair/DNA damage signaling, transcription factor, intracellular signaling, and RNA processing. Differential expression patterns of five selected genes (HSPA9B, INPP5A, PIP5K1A, FANCG, and TPP2) observed by the microarray analysis were further confirmed by the quantitative real time RT-PCR method, which demonstrated an up-regulation status of those genes. These results indicate a broad spectrum of biological processes (which may reflect the radio-resistance of U343 cells) that were altered in irradiated glioma cells, so as to guarantee cell survival.

Radon-domain detection of the nipple and the pectoral muscle in mammograms

In this paper, methods are presented for automatic detection of the nipple and the pectoral muscle edge in mammograms via image processing in the Radon domain. Radon-domain information was used for the detection of straight-line candidates with high gradient. The longest straight-line candidate was used to identify the pectoral muscle edge. The nipple was detected as the convergence point of breast tissue components, indicated by the largest response in the Radon domain. Percentages of false-positive (FP) and false-negative (FN) areas were determined by comparing the areas of the pectoral muscle regions delimited manually by a radiologist and by the proposed method applied to 540 mediolateral-oblique (MLO) mammographic images. The average FP and FN were 8.99% and 9.13%, respectively. In the detection of the nipple, an average error of 7.4 mm was obtained with reference to the nipple as identified by a radiologist on 1,080 mammographic images (540 MLO and 540 craniocaudal views).

Thomasius on the toleration of heresy

Over two thousand years the Christian Church identified a wider range and a greater number of heresies than most other religions and, when secular authorities did not protected the heretics, took drastic measures to persuade the heretic to recant and to extirpate the false doctrine. Heresy, of course, is a word like a box [End Page 201] that at different times may hold many different ideas and so some articles are dealing with definitions and identifications that are not the same. The editors suggest that the articles show a profound change in culture in the eighteenth century which means that present day scholars can barely imagine the mind-set that produced medieval attitudes to heresy. This is the task some of the authors have set themselves while others seek to explain how the change came about as part of the historical search for truth.

Quantitative three-dimensional power Doppler angiography: a flow-free phantom experiment to evaluate the relationship between color gain, depth and signal artifact

Objectives To evaluate the presence of false flow three-dimensional (3D) power Doppler signals in `flow-free` models. Methods 3D power Doppler datasets were acquired from three different flow-free phantoms (muscle, air and water) with two different transducers and Virtual Organ Computer-aided AnaLysis was used to generate a sphere that was serially applied through the 3D dataset. The vascularization flow index was used to compare artifactual signals at different depths (from 0 to 6 cm) within the different phantoms and at different gain and pulse repetition frequency (PR F) settings. Results Artifactual Doppler signals were seen in all phantoms despite these being flow-free. The pattern was very similar and the degree of artifact appeared to be dependent on the gain and distance from the transducer. False signals were more evident in the far field and increased as the gain was increased, with false signals first appearing with a gain of 1 dB in the air and muscle phantoms. False signals were seen at a lower gain with the water phantom (-15 dB) and these were associated with vertical lines of Doppler artifact that were related to PRF, and disappeared when reflections were attenuated. Conclusions Artifactual Doppler signals are seen in flow-free phantoms and are related to the gain settings and the distance from the transducer. In the in-vivo situation, the lowest gain settings that allow the detection of blood flow and adequate definition of vessel architecture should be used, which invariably means using a setting near or below the middle of the range available. Additionally, observers should be aware of vertical lines when evaluating cystic or liquid-containing structures. Copyright (C) 2010 ISUOC. Published by John Wiley & Sons, Ltd.

Two-step tuberculin skin test and booster phenomenon prevalence among Brazilian medical students

SETTING: Five medical schools in three cities in Rio de Janeiro State, Brazil, with different tuberculosis (TB) incidence rates. OBJECTIVE: To evaluate the prevalence of the booster phenomenon and its associated factors in a voting universally BCG-vaccinated TB-exposed population. DESIGN: A two-step tuberculin skin test (TST) was performed among undergraduate medical students. Boosting was defined as an induration >= 10 mm in the second TST (TST2), with an increase of at least 6 mm over the first TST (TST1). The association of boosting with independent variables was evaluated using multivariate analysis. RESULTS: Of the 764 participants (mean age 21.9 +/- 2.7 years), 672 (87.9%) had a BCG scar. The overall booster SUMMARY phenomenon prevalence was 8.4% (95%CI 6.5-10.6). Boosting was associated with TST1 reactions of 1-9 mm (aOR 2.5, 95%CI 1.04-5.9) and with BCG vaccination, mostly after infancy, i.e., after age two years (aOR 9.1, 95%,CI 1.2-70.7). CONCLUSION: The prevalence of the booster phenomenon was high. A two-step TST in young BCG-vaccinated populations, especially in those with TST1 reactions of 1-9 mm, can avoid misdiagnosis as a false conversion and potentially reduce unnecessary treatment for latent TB infection.

A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay

At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme Alwl. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RI-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RI-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. (C) 2010 Elsevier B.V. All rights reserved.

Diazepam decreases leukocyte-endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass)

Ethnopharmacological relevance: The essential oil (EO) from Cymbopogon citratus (DC) Stapf is reported to have a wide range of biological activities and is widely used in traditional medicine as an infusion or decoction. However, despite this widely use, there are few controlled studies confirming its biological activity in central nervous system. Materials and methods: The anxiolytic-like activity of the EO was investigated in light/dark box (LDB) and marble-burying test (MBT) and the antidepressant activity was investigated in forced-swimming test (FST) in mice. Flumazenil, a competitive antagonist of benzodiazepine binding and the selective 5-HT(1A) receptor antagonist WAY100635 was used in experimental procedures to determine the action mechanism of EO. To exclude any false positive results in experimental procedures, mice were submitted to the rota-rod test. We also quantified some neurotransmitters at specific brain regions after EO oral acute treatment. Results: The present work found anxiolytic-like activity of the EO at the dose of 10 mg/kg in a LDB. Flumazenil, but not WAY100635, was able to reverse the effect of the EO in the LOB, indicating that the EO activity occurs via the GABA(A) receptor-benzodiazepine complex. Only at higher doses did the EO potentiate diethyl-ether-induced sleeping time in mice. In the FST and MBT, EO showed no effect. Finally, the increase in time spent in the light chamber, demonstrated by concomitant treatment with ineffective doses of diazepam (DZP) and the EO, revealed a synergistic effect of the two compounds. The lack of activity after long-term treatment in the LDB test might be related to tolerance induction, even in the DZP-treated group. Furthermore, there were no significant differences between groups after either acute or repeated treatments with the EO in the rota-rod test. Neurochemical evaluation showed no amendments in neurotransmitter levels evaluated in cortex, striatum, pons, and hypothalamus. Conclusions: The results corroborate the use of Cymbopogon citratus in folk medicine and suggest that the anxiolytic-like effect of its EO is mediated by the GABA(A) receptor-benzodiazepine complex. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

LC-MS/MS quantitation of plasma progesterone in cattle

Quantitation of progesterone (P(4)) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P(4) in plasma of cattle with no sample derivatization. The quantitation of plasma P(4) released from three nonbiodegradable, commercial, intravaginal P(4)-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P(4) kinetics (P > 0.05) whereas results of P(4) quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P(4) limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P(4) quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies. (C) 2011 Elsevier Inc. All rights reserved.

A New Periapical Index Based on Cone Beam Computed Tomography

The purpose of this study was to evaluate a new periapical index based on cone beam computed tomography (CBCT) for identification of apical periodontitis (AP). The periapical index proposed in this study (CBCTPAI) was developed on the basis of criteria established from measurements corresponding to periapical radiolucency interpreted on CBCT scans. Radiolucent images suggestive of periapical lesions were measured by using the working tools of Planimp software on CBCT scans in 3 dimensions: buccopalatal, mesiodistal, and diagonal. The CBCTPAI was determined by the largest lesion extension. A 6-point (0-5) scoring system was used with 2 additional variables, expansion of cortical bone and destruction of cortical bone. A total of 1014 images (periapical radiographs and CBCT scans) originally taken from 596 patients were evaluated by 3 observers by using the CBCTPAI criteria. AP was identified in 39.5% and 60.9% of cases by radiography and CBCT, respectively (P<.01). The CBCTPAI offers an accurate diagnostic method for use with high-resolution images, which can reduce the incidence of false-negative diagnosis, minimize observer interference, and increase the reliability of epidemiologic studies, especially those referring to AP prevalence and severity. (J Endod 2008;34:1325-1331)

Mindreading and moral awareness in popular and rejected preschoolers

This study sought to examine links among young children's peer relations, their moral understanding in terms of the ability to distinguish lies from mistakes, and their theory-of-mind development. Based on sociometric measures, 109 children with a mean age of 4.8 years were divided into groups of popular and rejected preschoolers. Rejected children who had a stable mutual friend scored higher on measures of moral understanding and theory of mind than did rejected children without such friendships. Similarly, popular children who had a stable mutual friendship outperformed other popular children on mindreading, although their moral understanding was no better than that of the popular group who lacked mutual friends. Hierarchical multiple regression analyses revealed that peer popularity was a significant independent predictor of children's moral understanding after any effects of verbal maturity, age and theory-of-mind were statistically controlled. Moreover, having a reciprocal stable friendship made a significant independent contribution to the explanation of individual differences in mindreading, over and above age and verbal maturity, which also contributed significantly. These results are discussed in terms of conversational, cognitive, and emotional processes in the development of social cognition.