Usefulness of a quick decalcification of bone sections embedded in methyl metacrylate: an improved method for immunohistochemistry

Immunohistochemistry of undecalcified bone sections embedded in methyl methacrylate (MMA) is not commonly employed because of potential destruction of tissue antigenicity by highly exothermic polymerization. The aim of the present study was to describe a new technique in which a quick decalcification of bone sections embedded in MMA improves the results for immunohistochemistry. The quality of interleukin 1 alpha (IL-1 alpha) immunostaining according to the present method was better than the conventional one. Immunostaining for osteoprotegerin (OPG) and the receptor activator of NF-kappa B ligand (RANKL) in bone sections of chronic kidney disease patients with mineral bone disorders (CKD-MBD) was stronger than in controls (postmortem healthy subjects). The present study suggested that this method is easy, fast, and effective to perform both histomorphometry and immunohistochemistry in the same bone fragment, yielding new insights into pathophysiological aspects and therapeutic approaches in bone disease.

A method for drug infusion into the lateral median eminence and arcuate nucleus of sheep

The role of catecholamines in the control of the GnRH pulse generator is unclear as studies have relied on the use of peripheral or intracerebroventricular injections, which lack specificity in relation to the anatomical site of action. Direct brain site infusions have been used, however, these are limited by the ability to accurately target small brain regions. One such area of interest in the control of GnRH is the median eminence and arcuate nucleus within the medial basal hypothalamus. Here we describe a method of stereotaxically targeting this area in a large animal (sheep) and an infusion system to deliver drugs into unrestrained conscious animals. To test our technique we infused the dopamine agonist, quinpirole or vehicle into the medial basal hypothalamus of ovariectomised ewes. Quinpirole significantly suppressed LH pulsatility only in animals with injectors located close to the lateral median eminence. This in vivo result supports the hypothesis that dopamine inhibits GnRH secretion by presynaptic inhibition in the lateral median eminence. Also infusion of quinpirole into the medial basal hypothalamus suppressed prolactin secretion providing in vivo evidence that is consistent with the hypothesis that there are stimulatory autoreceptors on tubero-infundibular dopamine neurons. (C) 1997 Elsevier Science B.V.

A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum)

Six Burkholderia solanacearum (formerly Pseudomonas solanacearum) genomic DNA fragments were isolated, using RAPD techniques and cloning, from the three genetically diverse strains: ACH092 (Biovar 4), ACH0158 (Biovar 2) and ACH0171 (Biovar 3) (1). One of these cloned fragments was selected because it was present constantly in all bacterial strains analysed. The remaining five clones were selected because Southern hybridisation revealed that each showed partial or complete specificity towards the strain of origin. A seventh genomic fragment showing a strain-specific distribution in Southern hybridisations was obtained by differential restriction, hybridisation and cloning of genomic DNA. Each of these clones was sequenced and primers to amplify the insert were designed. When DNA from the strain of origin was used as template, PCR amplification for each of these fragments yielded a single band on gel analysis. One pair of primers amplified the species-constant fragment of 281 bp from DNA of all B. solanacearum strains investigated, from DNA of the closely related bacterium which causes ''blood disease'' of banana (BDB) and in P. syzigii. The sensitivity of detection of B. solanacearum using these ubiquitous primers was between 1.3 and 20 bacterial cells. The feasibility and reliability of a PCR approach to detection and identification of B. solanacearum was tested in diverse strains of the bacterium in several countries and laboratories.

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r = 0.8486, p < 0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25.207.3 +/- 6548.6. H2O, 52,487.4 +/- 16,284.9, p < 0.05; X-Gal: control 41.31 +/- 7.0%, H2O2 92.97 +/- 2.8%, p < 0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture. (C) 2007 Elsevier Inc. All rights reserved.

Structure optimization combining soft-core interaction functions, the diffusion equation method, and molecular dynamics

Smoothing the potential energy surface for structure optimization is a general and commonly applied strategy. We propose a combination of soft-core potential energy functions and a variation of the diffusion equation method to smooth potential energy surfaces, which is applicable to complex systems such as protein structures; The performance of the method was demonstrated by comparison with simulated annealing using the refinement of the undecapeptide Cyclosporin A as a test case. Simulations were repeated many times using different initial conditions and structures since the methods are heuristic and results are only meaningful in a statistical sense.

Hyperplane navigation: A method to set individual scores in fMRI group datasets

Recent studies have demonstrated that spatial patterns of fMRI BOLD activity distribution over the brain may be used to classify different groups or mental states. These studies are based on the application of advanced pattern recognition approaches and multivariate statistical classifiers. Most published articles in this field are focused on improving the accuracy rates and many approaches have been proposed to accomplish this task. Nevertheless, a point inherent to most machine learning methods (and still relatively unexplored in neuroimaging) is how the discriminative information can be used to characterize groups and their differences. In this work, we introduce the Maximum Uncertainty Linear Discrimination Analysis (MLDA) and show how it can be applied to infer groups` patterns by discriminant hyperplane navigation. In addition, we show that it naturally defines a behavioral score, i.e., an index quantifying the distance between the states of a subject from predefined groups. We validate and illustrate this approach using a motor block design fMRI experiment data with 35 subjects. (C) 2008 Elsevier Inc. All rights reserved.

Relative dispersion of intravascular transit times during isolated human limb perfusions for recurrent melanoma

Aims We have characterized the relative dispersion of vascular and extravascular markers in the limbs of three patients undergoing isolated limb perfusions with the cytotoxic melphalan for recurrent malignant melanoma both before and after melphalan dosing. Methods A bolus of injectate containing [Cr-51] labelled red blood cells, [C-14]-sucrose and [H-3]-water was injected into an iliac or femoral artery and outflow samples collected at 1 s intervals by a fraction collector. The radioactivity due to each isotype was analysed by either gamma [Cr-51] or beta [C-14 and H-3] counting. The moments of the outflow fraction-time profiles were estimated by a nonparametric (numerical integration) method and a parametric model (sum of two inverse Gaussian functions). Results The availability, mean transit time and normalised variance (CV2) obtained for labelled red blood cells, sucrose and water were similar before and after melphalan dosing and with the two methods of calculation but varied between the patients. Conclusions The vascular space is not well-stirred but characterized by a CV2 similar that reported previously for in situ rat hind limb and rat liver perfusions. A flow-limited blood-tissue exchange was observed for the permeating indicators. Administration of melphalan did not influence the distribution characteristics of the indicators.

Validation of a Spectrophotometric Method for Quantification of Carboxyhemoglobin

Universidade de São Paulo - LIM[40/HC-FM-USP]

Scanning electron microscopy as an auxiliary method in the study of exhumed bones

Scanning electronmicroscopy (SEM) has been used in forensic science in many ways. The reports of cases in which SEM has been used as an auxiliary method in the investigation of exhumed bones are rare. In this article, we report an exhumation that was made to determine if a seized weapon could have been used in a homicide. We used SEM to analyze a fracture in the interior of the skull of the victim. The findings described in this article showed us that it is possible to develop new researches in this field. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

A method to immunolabel rodent spinal cord neurons and glia for molecular study in specific laser microdissected cells involved in neurodegenerative disorders

Neuron-glia interaction is involved in physiological function of neurons, however, recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Laser microdissection offers a unique opportunity to study molecular regulation in specific immunolabeled cell types. However, an adequate protocol to allow morphological and molecular analysis of rodent spinal cord astrocyte, microglia and motoneurons remains a big challenge. In this paper we present a quick method to immunolabel those cells in flash frozen sections to be used in molecular biology analyses after laser microdissection and pressure catapulting.

Collagen V and vascular injury promote lung architectural changes in systemic sclerosis

Background: Systemic sclerosis (SSc) is a multisystem disorder characterized by inflammation, fibrosis and vascular damage. The aim of this study was to evaluate the interactions between basement membrane disruption, endothelial injury and collagen V deposition on the vascular wall, as well as their association with pulmonary function tests in patients with SSc. Method: The endothelial apoptosis was assessed by TUNEL and electron microscopy, and quantified through the point-counting technique. To evaluate basement membrane integrity, laminin immunostaining and electron microscopy were used. Immunofluorescence and morphometric analysis were used to determine the amount of collagen V in the vascular walls in 23 open lung biopsies of patients with SSc without pulmonary hypertension. Normal lung tissue was obtained from five individuals who had died of traumatic injuries. Results: The apoptosis index in SSc was higher in the endothelial cells (13.83 +/- 6.83) when compared with the control (2.51 +/- 2.06) group (P < 0.001) and confirmed by electron microscopy. We observed an important disruption of the basement membrane on the vascular wall shown by discontinuous laminin immunostaining and electron microscopy. An increase in collagen V on the vascular wall of the SSc group was observed (45.28 +/- 13.21), when compared with control group (22.90 +/- 4.13, P < 0.001), and this difference was statistically significant. An inverse correlation was found between vital capacity, forced vital capacity, forced expiratory volume in 1 s, vascular collagen V and endothelial apoptosis (P < 0.05). Conclusions: We conclude that the endothelial apoptosis and vascular collagen V interaction reinforce the vascular pathway in the SSc pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential. Please cite this paper as: Parra ER, Aguiar AC Jr, Teodoro WR, de Souza R, Yoshinari NH and Capelozzi VL. Collagen V and vascular injury promote lung architectural changes in systemic sclerosis. The Clinical Respiratory Journal 2009; 3: 135-142.

A method for better positioning bipolar electrodes for lower limb EMG recordings during dynamic contractions

To obtain a high quality EMG acquisition, the signal must be recorded as far away as possible from muscle innervations and tendon zones, which are known to shift during dynamic contractions. This study describes a methodology, using commercial bipolar electrodes, to identify better electrode positions for superficial EMG of lower limb muscles during dynamic contractions. Eight female volunteers participated in this study. Myoelectric signals of the vastus lateralis, gastrocnemius medialis, peroneus longus and tibialis anterior muscles were acquired during maximum isometric contractions using bipolar electrodes. The electrode positions of each muscle were selected assessing SENIAM and then, other positions were located along the length of muscle up and down the SENIAM site. The raw signal (density), the linear envelopes, the RMS value, the motor point site, the position of the IZ and its shift during dynamic contractions were taken into account to select and compare electrode positions. For vastus lateralis and peroneus longus, the best sites were 66% and 25% of muscle length, respectively (similar to SENIAM location). The position of the tibialis anterior electrodes presented the best signal at 47.5% of its length (different from SENIAM location). The position of the gastrocnemius medialis electrodes was at 38% of its length and SENIAM does not specify a precise location for signal acquisition. The proposed method should be considered as another methodological step in every EMG study to guarantee the quality of the signal and subsequent human movement interpretations. (C) 2009 Elsevier B.V. All rights reserved.

Inhibin A Production after Gonadotropin Stimulus: A New Method to Detect Ovarian Tissue in Ovotesticular Disorder of Sex Development

Background/Aims: While laboratory methods for the detection of testicular tissue are well standardized, currently there is no available test to demonstrate the presence of ovarian tissue. We evaluated the effectiveness of gonadal stimulation with luteinizing hormone (LH)/follicle-stimulating hormone (FSH) for the detection of ovarian tissue in patients with disorders of sex development (DSD). Methods: Ten patients with congenital adrenal hyperplasia (CAH) as ovarian-positive controls, 10 with cryptorchidism (ovarian-negative controls), 13 patients with DSD of no defined etiology and 7 patients with ovotesticular DSD (true hermaphroditism, TH) were included in the study. They underwent a daily injection of both LH and FSH on 3 consecutive days. LH, FSH, estradiol, testosterone and inhibin A were measured before treatment, 24 h after the 1st dose and 24 h after the 3rd dose. Results: Estradiol increased in all CAH and TH patients, with a median value of 155.1 and 92.6 pg/ml, respectively, after the 3rd injection. Inhibin A also increased in all CAH and TH patients, with a median value of 70.4 and 32.2 pg/ml, respectively, after the 3rd injection. There was no change in these hormones in the other groups. Conclusion: The LH/FSH stimulation test might be a useful method to detect the presence of ovarian tissue. Copyright (C) 2009 S. Karger AG, Basel

Prognostic Impact of MYCN, DDX1, TrkA, and TrkC Gene Transcripts Expression in Neuroblastoma

Background. The aims of this study were to define the mRNA expression profiles of MYCN, DDX1, TrkA, and TrkC in biopsy tumor samples from 64 Brazilian patients with neuroblastomas of different risk stages and to correlate altered expression with prognostic values. Procedure. Patients were retrospectively classified into low- (n = 11), intermediate- (n = 18), and high-risk (n = 35) groups using standard criteria. The mRNA levels of the above genes were measured by quantitative real-time polymerase chain reaction. Univariate analyses were performed and survival curves were plotted by the Kaplan-Meier method. Results. Of the 64 patients, 53% were female and 62.5% were older than 18 months. The 5-year overall survival (OS) for the entire cohort was 40.3%, with inferior median OS in patients identified in the intermediate- and high-risk groups. A significant difference in OS with respect to TrkA mRNA expression was found for the high-risk group vs. either the low- or intermediate-risk groups (P < 0.01, log rank test). Within the intermediate-risk group, neuroblastoma patients with positive TrkA mRNA expression had better clinical outcomes than patients with no TrkA transcript expression (P = 0.004). Another difference in OS was only found between the intermediate- and high-risk groups (P < 0.027, log rank test). No significant correlation of mRNA expression and survival outcome could be detected for the MYCN, DDX1. Conclusions. Positive expression of TrkA mRNA may be a clinically useful addition to the current risk classification system, allowing the identification of NB tumors with favorable prognosis. Pediatr Blood Cancer 2011; 56: 749-756. (c) 2010 Wiley-Liss, Inc.