A novel colorimetric assay of beta-D-glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter plate

Basidiomycete strains synthesize several types of beta-D-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these beta-D-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these beta-D-glucans present some disadvantages, a novel colorimetric assay method for beta-D-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (similar to 14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of beta-D-glucans in the range of 0-0.8 mu g, with a slope of 44.15 x 10(-2) and a limit of detection of 0.017 mu g/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for beta-1,3-D-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red beta-D-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify beta-D-glucans from other biological sources. (C) 2015 American Institute of Chemical Engineers

Assessment and control of nanoparticles exposure in welding operations by use of a control banding tool

This paper describes the use of a Control Banding Tool to assess and further control of exposure of nanoparticles emitted during welding operations. The tool was applied to Metal Active Gas (MAG) arc welding of mild and stainless steel, providing semi-quantitative data on the process, so that protection measures could be derived, e.g. exhaust gas ventilation by hoods, local ventilation devices and containment measures. This tool is quite useful to compare and evaluate the characteristics of arc welding procedures so that more eco-friendly processes could be preferred over the more potentially noxious ones.

Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminaata by in situ hybridization with biotinylated DNA probes

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with bioti-nylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.

Possibility of in-hospital infection by Cryptococcus neoformans in patients with AIDS

The objective of the present work was to carry out a survey of soil samples taken from different areas of a hospital of infectious disease located in the city of Cordoba, where three AIDS patients were hospitalized during different periods in the same ward. The three of them returned with meningeal cryptococcosis between three or five months after having been discharged. Cryptococcus neoformans was isolated in 8/10 samples collected outside the hospital, near the pigeon house. The samples collected from the AIDS patients ward and its surroundings were negative. These findings suggest that the patients may have been infected by the fungus during their first stay in hospital.

Schistosoma mansoni: protective immunity in mice cured by chemotherapy at the chronic phase of the disease

Aiming at demonstrating a decrease of acquired immunity after chemotherapeutic cure, a group of mice was infected with 25 Schistosoma mansoni cercariae (LE strain). A part of these animals was treated with 400 mg/kg oxamniquine, at 120 days after infection. Challenge infections were carried out at 45, 90 and 170-day-intervals after treatment (185, 210 and 290 days after primoinfection, respectively). Recovery of worms at 20 days after reinfections showed that a residual immunity remains up to 90 days after treatment, and disappears at 170 days after cure. Using the ELISA method, it was possible to detect a decrease of antibody levels (total IgG) in the treated group, when antigens from different evolutive stages of S. mansoni were used. The epidemiological implications of the present results, and the possible mechanisms involved in the decrease of acquired immunity after treatment are discussed.

Liver dysfunction in patients bitten by Crotalus durissus terrificus (Laurenti, 1768) snakes in Botucatu (State of São Paulo, Brazil)

Thirty-two patients bitten by venomous snakes sixteen by Bothrops spp. and sixteen by Crotalus durissus terrificus were studied. The group comprised thirty males and two females, aged eight to sixty-three years (mean 33±15). Bromsulphalein tests were increased in the majority of patients bitten by Crotalus durissus terrificus. The correlation coefficient of Spearman was positive between bromsulphalein tests and alanine aminotransferase levels, and between alanine aminotransferase and aspartate aminotransferase levels only in the Crotalus group. The only patient who died was bitten by Crotalus durissus terrificus and showed hydropic degeneration and mitochondrial injury in the liver. It was concluded that the hepatic damage might have been caused by at least two possible mechanisms: venom effect on liver mitochondria and cytokine effects on hepatocyte, specially interleukin-6.

Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients.

Bacterial antigen detection in cerebrospinal fluid by the latex agglutination test

Eighty purulent cerebrospinal fluid (CSF) samples from patients with clinical evidence of meningitis were studied using the Directigen latex agglutination (LA) kit to determine the presence of bacterial antigen in CSF. The results showed a better diagnostic performance of the LA test than bacterioscopy by Gram stain, culture and counterimmunoelectrophoresis (CIE), as far as Neisseria meningitidis groups B and C, and Haemophilus influenzae type b are concerned, and a better performance than bacterioscopy and culture considering Streptococcus pneumoniae. Comparison of the results with those of culture showed that the LA test had the highest sensitivity for the Neisseria meningitidis group C. Comparing the results with those of CIE, the highest levels of sensitivity were detected for N. meningitidis groups B and C. Regarding specificity, fair values were obtained for all organisms tested. The degree of K agreement when the LA test was compared with CIE exhibited better K indices of agreement for N. meningitidis groups B and C.

A comparative study of severe scorpion envenomation in children caused by Tityus bahiensis and Tityus serrulatus

From January 1984 to May 1994, 17 of 239 children under 15 years old stung by Tityus serrulatus (15.1%) or Tityus bahiensis (84.9%) presented severe envenoming. Of these 17 patients (1-11 years old; median=2 yr) 14 were stung by T.serrulatus and three by T.bahiensis. All of them received scorpion antivenom i.v. at times ranging from 45 min. to 5 h after the accident (median=2h). On admission, the main clinical manifestations and laboratory and electrocardiographic changes were: vomiting (17), diaphoresis (15), tachycardia (14), prostration (10), tachypnea (8), arterial hypertension (7), arterial hypotension (5), tremors (5), hypothermia (4), hyperglycemia (17), leukocytosis (16/16), hypokalemia (13/17), increased CK-MB enzyme activity (>6% of the total CK, 11/12), hyperamylasemia (11/14), sinusal tachycardia (16/17) and a myocardial infarction-like pattern (11/17). Six patients stung by T.serrulatus had depressed left ventricular systolic function assessed by means of echocardiography. Of these, five presented pulmonary edema and four had shock. A child aged two-years old presented severe respiratory failure and died 65 h after being stung by T.serrulatus. Severe envenomations caused by T.serrulatus were 26.2 times more frequent than those caused by T.bahiensis (p<0.001).

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia) braziliensis

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Environmental impact assessment in higher education and training in Portugal

Journal of Cleaner Production, nº 16, p. 639-645

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Congenital cytomegalovirus infection in a neonatal intensive care unit in Brazil evaluated by PCR and association with perinatal aspects

Cytomegalovirus (CMV) infection is the most common congenital infection, affecting 0.4% to 2.3% newborns. Most of them are asymptomatic at birth, but later 10% develop handicaps, mainly neurological disturbances. Our aim was to determine the prevalence of CMV shed in urine of newborns from a neonatal intensive care unit using the polymerase chain reaction (PCR) and correlate positive cases to some perinatal aspects. Urine samples obtained at first week of life were processed according to a PCR protocol. Perinatal data were collected retrospectively from medical records. Twenty of the 292 cases (6.8%) were CMV-DNA positive. There was no statistical difference between newborns with and without CMV congenital infection concerning birth weight (p=0.11), gestational age (p=0.11), Apgar scores in the first and fifth minutes of life (p=0.99 and 0.16), mother's age (p=0.67) and gestational history. Moreover, CMV congenital infection was neither related to gender (p=0.55) nor to low weight (<2,500g) at birth (p=0.13). This high prevalence of CMV congenital infection (6.8%) could be due to the high sensitivity of PCR technique, the low socioeconomic level of studied population or the severe clinical status of these newborns.

Transverse Vibrations in Beams Supported by a Piece-Wise Homogeneous Visco- Elastic Foundation

Transversal vibrations induced by a load moving uniformly along an infinite beam resting on a piece-wise homogeneous visco-elastic foundation are studied. Special attention is paid to the additional vibrations, conventionally referred to as transition radiations, which arise as the point load traverses the place of foundation discontinuity. The governing equations of the problem are solved by the normalmode analysis. The solution is expressed in a form of infinite sum of orthogonal natural modes multiplied by the generalized coordinate of displacement. The natural frequencies are obtained numerically exploiting the concept of the global dynamic stiffness matrix. This ensures that the frequencies obtained are exact. The methodology has restrictions neither on velocity nor on damping. The approach looks simple, though, the numerical expression of the results is not straightforward. A general procedure for numerical implementation is presented and verified. To illustrate the utility of the methodology parametric optimization is presented and influence of the load mass is studied. The results obtained have direct application in analysis of railway track vibrations induced by high-speed trains when passing regions with significantly different foundation stiffness.