ADAPTAÇÃO METODOLÓGICA PARA ESTUDOS DE ORGANISMOS ZOOPLANCTÔNICOS"IN SITU", APLICADOS À PISCICULTURA

Os organismos zooplanctônicos desempenham um importante papel na alimentação de larvas e pós-larvas de peixes, tanto na natureza, como em cativeiro em tanques de piscicultura. No aspecto do cultivo de zooplâncton cm massa, somente conhecendo-se os valores de alguns parâmetros importantes do ciclo de vida de um único indivíduo, em situações diversas, é que se pode avaliar se este estará respondendo de maneira adequada, ou não, a determinado tratamento aplicado. O trabalho mostra uma adaptação de mamadeiras plásticas comerciais para a utilização no estudo individualizado de microcrustáceos zooplanctônicos (cladóceros), em laboratório (aquários), viveiros de piscicultura, ou ambientes naturais (lagos e igarapés).

RAPD em Pau-rosa (Aniba rosaeodora Ducke): adaptação do método para coleta de amostras in situ, ajuste das condições de PCR e apresentação de um processo para selecionar bandas reprodutíveis

O Pau-rosa (Aniba rosaeodora Ducke) é uma espécie importante economicamente para a Região Amazônica, porque sua madeira é fonte de linalol, insumo utilizado pelas perfumarias. Esta espécie foi explorada durante décadas e, ainda assim, o conhecimento acerca da diversidade genética intra-específica é muito restrito. Foram objetivos deste trabalho: 1) validar um protocolo para coleta de folhas de pau-rosa que permitisse preservar a integridade do DNA até a estocagem em "freezer"; 2) selecionar um protocolo para extração de DNA em quantidade e qualidade adequadas para geração de bandas RAPD e 3) desenvolver um critério para avaliar o grau de reprodutibilidade que pudesse auxiliar a seleção de bandas RAPD úteis para análises de diversidade genética. Imediatamente após a coleta, as folhas foram acondicionadas em tubos de polietileno com sílica gel e aí permaneceram por até 10 dias. Foram testados três protocolos para a extração de ácidos nucléicos destas folhas, condições ideais para as PCR e a reprodutibilidade dos padrões RAPD. Critérios para a eliminação das bandas que mais contribuíram para o afastamento dos resultados do ideal da reprodutibilidade total foram desenvolvidos e a significância estatística das diferenças geradas pela aplicação dos critérios ao conjunto de dados foi testada. DNA com qualidade e em quantidade suficiente para a geração de padrões RAPD, nas condições ideais definidas para as PCRs, foi obtido. A eliminação de bandas com reprodutibilidade menor que 70% não diferiu do controle. A eliminação de bandas com reprodutibilidade menor que 90% diferiu dos demais tratamentos em todos os arranjos testados (P < 5%).

Time-variations of equivalent water heights'from Grace Mission and in-situ river stages in the Amazon basin

Gravity Recovery and Climate Experiment (GRACE) mission is dedicated to measuring temporal variations of the Earth's gravity field. In this study, the Stokes coefficients made available by Groupe de Recherche en Géodésie Spatiale (GRGS) at a 10-day interval were converted into equivalent water height (EWH) for a ~4-year period in the Amazon basin (from July-2002 to May-2006). The seasonal amplitudes of EWH signal are the largest on the surface of Earth and reach ~ 1250mm at that basin's center. Error budget represents ~130 mm of EWH, including formal errors on Stokes coefficient, leakage errors (12 ~ 21 mm) and spectrum truncation (10 ~ 15 mm). Comparison between in situ river level time series measured at 233 ground-based hydrometric stations (HS) in the Amazon basin and vertically-integrated EWH derived from GRACE is carried out in this paper. Although EWH and HS measure different water bodies, in most of the cases a high correlation (up to ~80%) is detected between the HS series and EWH series at the same site. This correlation allows adjusting linear relationships between in situ and GRACE-based series for the major tributaries of the Amazon river. The regression coefficients decrease from up to down stream along the rivers reaching the theoretical value 1 at the Amazon's mouth in the Atlantic Ocean. The variation of the regression coefficients versus the distance from estuary is analysed for the largest rivers in the basin. In a second step, a classification of the proportionality between in situ and GRACE time-series is proposed.

Myocardial Scintigraphy in the Evaluation of Cardiac Events in Patients without Typical Symptoms

AbstractBackground:Cardiovascular disease is a leading cause of death in the world and in Brazil. Myocardial scintigraphy is an important noninvasive method for detecting ischemia in symptomatic patients, but its use in asymptomatic ones or those with atypical symptoms is yet to be defined.Objective:To verify the presence of major cardiac events in asymptomatic patients or those with atypical symptoms (atypical chest pain or dyspnea) that underwent myocardial scintigraphy (MS), over a period of 8 years. Secondary objectives were to identify cardiac risk factors associated with myocardial scintigraphy abnormalities and possible predictors for major cardiac events in this group.Methods:This was a retrospective, observational study using the medical records of 892 patients that underwent myocardial scintigraphy between 2005 and 2011 and who were followed until 2013 for assessment of major cardiac events and risk factors associated with myocardial scintigraphy abnormalities. Statistical analysis was performed by Fisher’s exact test, logistic regression and Kaplan-Meyer survival curves, with statistical significance being set at p ≤ 0.05.Results:Of the total sample, 52.1% were men, 86.9% were hypertensive, 72.4% had hyperlipidemia, 33.6% were diabetic, and 12.2% were smokers; 44.5% had known coronary artery disease; and 70% had high Framingham score, 21.8% had moderate and 8% had low risk. Of the myocardial scintigraphies, 58.6% were normal, 26.1% suggestive of fibrosis and 15.3% suggestive of ischemia. At evolution, 13 patients (1.5%) had non-fatal myocardial infarction and six individuals (0.7%) died. The group with normal myocardial scintigraphy showed longer period of time free of major cardiac events, non-fatal myocardial infarction (p = 0.036) and death. Fibrosis in the myocardial scintigraphy determined a 2.4-fold increased risk of non-fatal myocardial infarction and five-fold higher risk of death (odds ratio: 2.4 and 5.7, respectively; p = 0.043).Conclusion:The occurrence of major cardiac events in 8 years was small. Patients with fibrosis at MS had more major events, whereas patients with normal MS result had fewer major cardiac events, with higher survival.

Medium-term protocols for in vivo evaluation of chemical modifiers of carcinogenesis

Cancer development is a long-term multistep process which allows interventional measure before the clincial disease emerges. the detection of natural substances which can block the process of carcinogenesis is a important as the identification of anti-tumoral drugs since they might be used in chemoprevention of cancer in high-risk groups. In vivo rodent models of chemical caecinogenesis have been used to study plant-derived inhibitors of carcinofenesis such as indols, coumarins, isothiocyanates, flavones, phenols and allyl-sulfides. Since the standard in vivo rodent bioassay is prolonged and expensive, shorter reliable protocols are needed. Two in vivo medium-term protocols for evaluation of modifiers of carcinogenesis are presented, one related to liver and the other to bladder cancer. Both protocols use rats, last 8 and 36 weeks and are based on the two-step concept of carcinogenesis: initiation and promotion. The protocols use respectively the development of altered foci of hepatocytes expressing immunochistochemically the placental form of gluthation S-transferase and the appearence of pre-neoplastic urothelium and papillomas as the "end-points". the use of these protocols for detection of plantpderived inhibitors of carcinogenesis appear warranted.

Human papillomavirus detection in genital lesions by in situ hybridization and ultrastructural observations

Detection of papillomavirus DNA in sity hybridization technique was perfomed in 29 symptomatic patients (6 males and 23 females) during the period of 1989-1991 at the Clinic for Sexually Transmitted Diseases, Universidade Federal Fluminense, State of rio de Janeiro. All the male patients had condyloma acuminata. Only HPV 6/11 were found in these lesions. Clinical features inthe female patients included vulvar condyloma acuminata, bowenoid populosis, flat cervical condyloma, cervical condyloma acuminatum and cervical intraepithelialneoplasia grade II (CIN II). We also found cases of condyloma acuminata associated to vulvar intraepithelial neoplasia grade III (VIN III), as well as to vaginal invasive carcinoma. HPV 6/11 and 16/18 were found in vulvar condyloma acuminata. Mixed infection by 6/11-16/18 HPV were also seen in these lesions as well as in the patient who had cervical condyloma acuminatum. HPV 16/18 were found in the condyloma acuminatum plus VIN III and in the CIN II lesions. We have found HPV31/33/51 in the specimen of condyloma acuminatum plus invasive carcinoma. In order to investigate the ultrastructural aspects of HPV infection in genital tissue, the biopsies of three female patients were observed under electron microscope.Mature virus particles were found in the cells of a condyloma acuminatum as wellas in the condyloma acuminatum plus invasive carcinoma case. In another sample, chromosome breakages were found in the nuclei of the infected cells although no viral particles were observed.

In vitro evaluation of the activity of aromatic nitrocompounds against Trypanosoma cruzi

Fourteen compounds were evaluated for their activity against Trypanosoma cruzi blood stream forms at the concentration of 500 µg/ml. Six compounds were active and re-tested at lower concentrations.

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

Erythrovirus B19 infection in acquired immunodeficiency syndrome: screening by histopathology, immunohistochemistry, and in situ hybridization

Erythrovirus B19 infects erythrocytic progenitors, transiently interrupting erythropoiesis. In AIDS patients it causes chronic anemia amenable to treatment. We looked for evidences of B19 infection in stored bone marrow material from patients with acquired immunodeficiency syndrome. Histological sections were made from stored paraffin blocks from 33 autopsies (39 blocks) and 35 biopsies (45 blocks, 30 patients) performed from 1988 to 2002. They were examined after hematoxylin-eosin (HE) staining, immunohistochemical (IHC), and in situ hybridization. HE revealed intra-nuclear inclusion bodies ("lantern cells") suggesting B19 infection in 19 sections corresponding to 19 of 63 patients examined with this test. Seven of 78 sections subjected to immunohistochemistry were positive, corresponding to 7 of 58 patients examined with this test. Fourteen sections corresponding to 13 of the 20 HE and/or IHC positive patients were subjected to in situ hybridization, with six positives results. Among the 13 patients subjected to the three techniques, only one gave unequivocal positive results in all and was considered a true positive. The frequency of B19 infection (1/63 patients) in the material examined can be deemed low.

Clinical versus ultrasound examination in the evaluation of hepatosplenic schistosomiasis mansoni in endemic areas

The best way to appraise the size of abdominal organs remains undefined. Herein we compare the size of liver and spleen in hepatosplenic schistosomiasis using clinical and ultrasound (US) examination, and the size of the organs measured by US with their visualization below the costal margin ("palpable by US"). For this study, 411 individuals from an endemic area for schistosomiasis mansoni in Brazil have been selected. We found that palpable spleens and left liver lobes are larger than non palpable ones. Also, 23% of normal spleens measured by US were palpable on clinical examination, and 22% of spleens increased in size on US were non palpable. A total of 21% of normal spleens were "palpable by US". We also found 54% of normal sized right liver lobes palpable on clinical examination, whilst 54% of the increased livers, measured by US, were non palpable. About 76% of normal right liver lobes were "palpable by US". We conclude that the association of clinical, ultrasound and magnetic resonance imaging (MRI) examinations, in the near future, should give the investigators the necessary tools to perform a more accurate clinical diagnosis of hepatosplenic schistosomiasis mansoni.

Imaging techniques and histology in the evaluation of liver fibrosis in hepatosplenic schistosomiasis mansoni in Brazil: a comparative study

Few publications have compared ultrasound (US) to histology in diagnosing schistosomiasis-induced liver fibrosis (LF); none has used magnetic resonance (MR). The aim of this study was to evaluate schistosomal LF using these three methods. Fourteen patients with hepatosplenic schistosomiasis admitted to hospital for surgical treatment of variceal bleeding were investigated. They were submitted to upper digestive endoscopy, US, MR and wedge liver biopsy. The World Health Organization protocol for US in schistosomiasis was used. Hepatic fibrosis was classified as absent, slight, moderate or intense. Histology and MR confirmed Symmers' fibrosis in all cases. US failed to detect it in one patient. Moderate agreement was found comparing US to MR; poor agreement was found when US or MR were compared to histology. Re-classifying LF as only slight or intense created moderate agreement between imaging techniques and histology. Histomorphometry did not separate slight from intense LF. Two patients with advanced hepatosplenic schistosomiasis presented slight LF. Our data suggest that the presence of the characteristic periportal fibrosis, diagnosed by US, MR or histology, associated with a sign of portal hypertension, defines the severity of the disease. We conclude that imaging techniques are reliable to define the presence of LF but fail in grading its intensity.

Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

Candida parapsilosis, currently divided into three distinct species, proliferates in glucose-rich solutions and has been associated with infections resulting from the use of medical devices made of plastic, an environment common in dialysis centres. The aims of this study were (i) to screen for Candida orthopsilosis and Candida metapsilosis (100 environmental isolates previously identified as C. parapsilosis), (ii) to test the ability of these isolates to form biofilm and (iii) to investigate the in vitro susceptibility of Candida spp biofilms to the antifungal agents, fluconazole (FLC) and amphotericin B (AMB). Isolates were obtained from a hydraulic circuit collected from a haemodialysis unit. Based on molecular criteria, 47 strains were re-identified as C. orthopsilosis and 53 as C. parapsilosis. Analyses using a formazan salt reduction assay and total viable count, together with microscopy studies, revealed that 72 strains were able to form biofilm that was structurally similar, but with minor differences in morphology. A microtitre-based colorimetric assay used to test the susceptibility of fungal biofilms to AMB and FLC demonstrated that the C. parapsilosis complex displayed an increased resistance to these antifungal agents. The results from these analyses may provide a basis for implementing quality controls and monitoring to ensure the microbiological purity of dialysis water, including the presence of yeast.

Ultrasound versus biological markers in the evaluation of periportal fibrosis in human Schistosoma mansoni

In this paper, the authors review the literature and share their experience of the principal biological markers of fibrosis for the evaluation of periportal fibrosis (PPF) caused by mansoni schistosomiasis. These biological markers are compared to diagnostic ultrasound (US) scans as means of grading PPF. We also review procollagen type I and III, collagen type IV, laminin, hyaluronic acid (HA), immunoglobulin G, platelets, aspartate aminotransferase to platelet ratio index (APRI) and gamma-glutamyl transpeptidase as markers of the disease. Although there are several good markers for evaluating PPF and portal hypertension, such as HA, platelets or APRI, none can yet replace US. These markers may, however, be used to identify patients at greater risk of developing advanced disease in endemic areas and determine who will need further care and US studies.