Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE

The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism`s pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.

The competing roles of extensional viscosity and normal stress differences in complex flows of elastic liquids

In various attempts to relate the behaviour of highly-elastic liquids in complex flows to their rheometrical behaviour, obvious candidates for study have been the variation of shear viscosity with shear rate, the two normal stress differences N(1) and N(2) especially N(1), and the extensional viscosity eta(E). In this paper, we shall be mainly interested in `constant-viscosity` Boger fluids, and, accordingly, we shall limit attention to N(1) and eta(E). We shall concentrate on two important flows - axisymmetric contraction flow and ""splashing"" (particularly that which arises when a liquid drop falls onto the free Surface of the same liquid). Modem numerical techniques are employed to provide the theoretical predictions. It is shown that the two obvious manifestations of viscoelastic rheometrical behaviour can sometimes be opposing influences in determining flow characteristics. Specifically, in an axisymmetric contraction flow, high eta(E) , can retard the flow, whereas high N(1) can have the opposite effect. In the splashing experiment, high eta(E) can certainly reduce the height of the so-called Worthington jet, thus confirming some early suggestions, but, again, other rheometrical influences can also have a role to play and the overall picture may not be as clear as it was once envisaged.

Increased Viability of Odontoblast-Like Cells Subjected to Low-Level Laser Irradiation

Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm(2) were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO(2) at 37 degrees C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm(2) + 5% FBS; G2: 1.5 J/cm(2) + 10% FBS; G3: 5 J/cm(2) + 5% FBS; G4: 5 J/cm(2) + 10% FBS; G5: 19 J/cm(2) + 5% FBS; G6: 19 J/cm(2) + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm(2). These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.

Oxidative stress protection of Trypanosomes requires selenophosphate synthase

Selenoproteins are characterized by the incorporation of at least one amino acid selenocysteine (Sec-U) encoded by in-frame UGA stop codons. These proteins, as well as the components of the Sec synthesis pathway, are present in members of the bacteria, archaea and eukaryote domains. Although not a ubiquitous pathway in all organisms, it was also identified in several protozoa, including the Kinetoplastida. Genetic evidence has indicated that the pathway is non-essential to the survival of Trypanosoma growing in non-stressed conditions. By analyzing the effects of RNA interference of the Trypanosoma brucei selenophosphate synthetase SPS2, we found a requirement under sub-optimal growth conditions. The present work shows that SPS2 is involved in oxidative stress protection of the parasite and its absence severely hampers the parasite survival in the presence of an oxidizing environment that results in an apoptotic-like phenotype and cell death. (C) 2011 Elsevier B.V. All rights reserved.

In silico screening of HIV-1 non-nucleoside reverse transcriptase and protease inhibitors

Two targets, reverse transcriptase (RT) and protease from HIV-1, were used during the past two decades to the discovery of non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI) that belong to the arsenal of the antiretroviral therapy. Herein these enzymes were chosen as templates for conducting a computer-aided ligand design. Ligand and structure-based drug designs were the starting points to select compounds from a database bearing more than five million compounds by means of cheminformatic tools. New promising lead structures are retrieved from the database, which are open to acquisition and test. Classes of molecules already described as NNRTI or PI in the literature also came out and were useful to prove the reliability of the workflow, and thus validating the work carried out so far. (c) 2007 Elsevier Masson SAS. All rights reserved.

Effect of low-level laser irradiation on odontoblast-like cells

Low-level laser therapy (LLLT), also referred to as therapeutic laser, has been recommended for a wide array of clinical procedures, among which the treatment of dentinal hypersensitivity. However, the mechanism that guides this process remains unknown. Therefore, the objective of this study was to evaluate in vitro the effects of LLL irradiation on cell metabolism (MTT assay), alkaline phosphatase (ALP) expression and total protein synthesis. The expression of genes that encode for collagen type-1 (Col-1) and fibronectin (FN) was analyzed by RT-PCR. For such purposes, oclontoblast-like cell line (MDPC-23) was previously cultured in Petri dishes (15000 cells/cm(2)) and submitted to stress conditions during 12 h. Thereafter, 6 applications with a monochromatic near infrared radiation (GaAlAs) set at predetermined parameters were performed at 12-h intervals. Non-irradiated cells served as a control group. Neither the MTT values nor the total protein levels of the irradiated group differed significantly from those of the control group (Mann-Whitney test; p > 0.05). On the other hand, the irradiated cells showed a decrease in ALP activity (Mann-Whitney test; p < 0.05). RT-PCR results demonstrated a trend to a specific reduction in gene expression after cell irradiation, though not significant statistically (Mann-Whitney test; p > 0.05). It may be concluded that, under the tested conditions, the LLLT parameters used in the present study did not influence cell metabolism, but reduced slightly the expression of some specific proteins.

False-positive results of a rapid K39-based strip test and Chagas disease

Background: The definitive diagnosis of visceral. leishmaniasis (VL) requires invasive procedures with demonstration of amastigotes in tissue or promastigotes in culture. Unfortunately, these approaches require laboratory materials not available in poor countries where the disease is endemic. The correct diagnosis of VL is important, and made more difficult by the fact that several common tropical diseases such as malaria, disseminated tuberculosis, and enteric fever share the same clinical presentation. Serological tests have been developed to replace parasitological diagnosis in the field. A commercially available K39-based strip test for VL has been developed for this purpose. The endemic area of leishmaniasis in Brazil overlaps the endemic area of Chagas disease, a disease that can cause false-positive serological test results. The aim of this study was to evaluate the incidence of false-positive exams using a rapid test for VL in patients with Chagas disease. Methods: A rapid test based on the recombinant K39 antigen of Leishmania was used in: (1) 30 patients with confirmed Chagas disease, (2) 30 patients with a serological diagnosis of Chagas disease by ELISA, indirect immunofluorescence, indirect hemagglutination, and chemiluminescence, (3) 30 healthy patients from a non-endemic area as the control group, (4) 30 patients with confirmed VL, and (5) 20 patients with proved cutaneous leishmaniasis. Results: The sensitivity and specificity of the rapid strip test were 100% when compared with healthy volunteers and those with confirmed Chagas disease. One false-positive result occurred in the group with Chagas disease diagnosed by serological tests (specificity of 96%). Conclusion: The rapid test based on recombinant K39 is a useful diagnostic assay, and a false-positive result rarely occurs in patients with a serological diagnosis of Chagas disease. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A fast and robust statistical test based on likelihood ratio with Bartlett correction to identify Granger causality between gene sets

We propose a likelihood ratio test ( LRT) with Bartlett correction in order to identify Granger causality between sets of time series gene expression data. The performance of the proposed test is compared to a previously published bootstrapbased approach. LRT is shown to be significantly faster and statistically powerful even within non- Normal distributions. An R package named gGranger containing an implementation for both Granger causality identification tests is also provided.

Occupational airborne contamination in South Brazil: 2. Oxidative stress detected in the blood of workers of incineration of hospital residues

One of the most useful methods for elimination of solid residues of health services (SRHS) is incineration. However, it also provokes the emission of several hazardous air pollutants such as heavy metals, furans and dioxins, which produce reactive oxygen species and oxidative stress. The present study, which is parallel to an accompanied paper (Avila Jr. et al., this issue), investigated several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of vitamin E, lipoperoxidation = TBARS, reduced glutathione = GSH, oxidized glutathione = GSSG, and activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in three different groups (n = 20 each) exposed to airborne contamination associated with incineration of SRHS: workers directly (ca. 100 m from the incinerator) and indirectly exposed (residents living ca. 5 km the incineration site), and controls (non-exposed subjects). TBARS and GSSG levels were increased whilst GSH, TG and alpha-tocopherol contents were decreased in workers and residents compared to controls. Increased GST and CAT activities and decreased GPx activities were detected in exposed subjects compared to controls, while GR did not show any difference among the groups. In conclusion, subjects directly or indirectly exposed to SRHS are facing an oxidative insult and health risk regarding fly ashes contamination from SRHS incineration.

Antioxidant therapy attenuates oxidative stress in the blood of subjects exposed to occupational airborne contamination from coal mining extraction and incineration of hospital residues

Coal mining and incineration of solid residues of health services (SRHS) generate several contaminants that are delivered into the environment, such as heavy metals and dioxins. These xenobiotics can lead to oxidative stress overgeneration in organisms and cause different kinds of pathologies, including cancer. In the present study the concentrations of heavy metals such as lead, copper, iron, manganese and zinc in the urine, as well as several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of lipoperoxidation = TBARS, protein carbonyls = PC, protein thiols = PT, alpha-tocopherol = AT, reduced glutathione = GSH, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of six different groups (n = 20 each) of subjects exposed to airborne contamination related to coal mining as well as incineration of solid residues of health services (SRHS) after vitamin E (800 mg/day) and vitamin C (500 mg/day) supplementation during 6 months, which were compared to the situation before the antioxidant intervention (Avila et al., Ecotoxicology 18:1150-1157, 2009; Possamai et al., Ecotoxicology 18:1158-1164, 2009). Except for the decreased manganese contents, heavy metal concentrations were elevated in all groups exposed to both sources of airborne contamination when compared to controls. TBARS and PC concentrations, which were elevated before the antioxidant intervention decreased after the antioxidant supplementation. Similarly, the contents of PC, AT and GSH, which were decreased before the antioxidant intervention, reached values near those found in controls, GPx activity was reestablished in underground miners, and SOD, CAT and GST activities were reestablished in all groups. The results showed that the oxidative stress condition detected previously to the antioxidant supplementation in both directly and indirectly subjects exposed to the airborne contamination from coal dusts and SRHS incineration, was attenuated after the antioxidant intervention.

Occupational airborne contamination in south Brazil: 1. Oxidative stress detected in the blood of coal miners

Reactive oxygen species and nitrogen species have been implicated in the pathogenesis of coal dust-induced toxicity. The present study investigated several oxidative stress biomarkers (Contents of lipoperoxidation = TBARS, reduced = GSH, oxidized = GSSG and total glutathione = TG, alpha-tocopherol, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of three different groups (n = 20 each) exposed to airborne contamination associated with coal mining activities: underground workers directly exposed, surface workers indirectly exposed, residents indirectly exposed (subjects living near the mines), and controls (non-exposed subjects). Plasma TBARS were increased and whole blood TG and GSH levels were decreased in all groups compared to controls. Plasma alpha-tocopherol contents showed approximately half the values in underground workers compared to controls. GST activity was induced in workers and also in residents at the vicinity of the mining plant, whilst CAT activity was induced only in mine workers. SOD activity was decreased in all groups examined, while GPx activity showed decreased values only in underground miners, and GR did not show any differences among the groups. The results showed that subjects directly and indirectly exposed to coal dusts face an oxidative stress condition. They also indicate that people living in the vicinity of the mine plant are in health risk regarding coal mining-related diseases.

A home environment test battery for status assessment in patients with motor fluctuations

Background and aims Evaluating status in patients with motor fluctuations is complex and occasional observations/measurements do not give an adequate picture as to the time spent in different states. We developed a test battery to assess advanced Parkinson patients' status consisting of diary assessments and motor tests. This battery was constructed and implemented on a handheld computer with built-in mobile communication. In fluctuating patients, it should typically be used several times daily in the home environment, over periods of about one week. The aim of this battery is to provide status information in order to evaluate treatment effects in clinical practice and research, follow up treatments and disease progression and predict outcome to optimize treatment strategy. Methods Selection of diary questions was based on a previous study with Duodopa® (DIREQT). Tapping tests (with and without visual cueing) and a spiral drawing test were added. Rapid prototyping was used in development of the user interface. An evaluation with two pilot patients was performed before and after receiving new treatments for advanced disease (one received Duodopa® and one received DBS). Speed and proportion missed taps were calculated for the tapping tests and entropy of the radial drawing velocity was calculated for the spiral tests. Test variables were evaluated using non-parametric statistics. Results Post-treatment improvement was detected in both patients in many of the test variables. Conclusions Although validation work remains, preliminary results are promising and the test battery is currently being evaluated in a long-term health economics study with Duodopa® (DAPHNE).

Simplified follow-up after medical abortion using a low-sensitivity urinary pregnancy test and a pictorial instruction sheet in Rajasthan, India : study protocol and intervention adaptation of a randomised control trial

The World Health Organisation suggests that simplification of the medical abortion regime will contribute to an increased acceptability of medical abortion, among women as well as providers. It is expected that a home-based follow-up after a medical abortion will increase the willingness to opt for medical abortion as well as decrease the workload and service costs in the clinic. Trial design The study is a randomised, controlled, non-superiority trial . Methods Women screened to participate in the study are those with unwanted pregnancies and gestational ages equal to or less than nine weeks. Eligible women randomised to the home-based assessment group will use a low-sensitivity pregnancy test and a pictorial instruction sheet at home, while the women in the clinic follow-up group will return to the clinic for routine follow-up carried out by a doctor. The primary objective of the study is to evaluate the effectiveness of home-based assessment using a low-sensitivity pregnancy test and a pictorial instruction sheet 10-14 days after an early medical abortion. Providers or research assistants will not be blinded during outcome assessment. To ensure feasibility of the self-assessment intervention an adaption phase took place at the selected study sites before study initiation. This was to optimise and tailor-make the intervention and the study procedures and resulted in the development of the pictorial instruction sheet for how to use the low-sensitivity pregnancy test and the danger signs after a medical abortion. Discussion In this paper, we will describe the study protocol for a randomised control trial investigating the efficacy of simplified follow-up in terms of home-based assessment, 10-14 days after a medical abortion. Moreover, a description of the adaptation phase is included for a better understanding of the implementation of the intervention in a setting where literacy is low and the road-connections are poor. Trial registration: Clinicaltrials.gov NCT01827995. Registered 04 May 2013

An empirical test of the gravity p-median model

A customer is presumed to gravitate to a facility by the distance to it and the attractiveness of it. However regarding the location of the facility, the presumption is that the customer opts for the shortest route to the nearest facility.This paradox was recently solved by the introduction of the gravity p-median model. The model is yet to be implemented and tested empirically. We implemented the model in an empirical problem of locating locksmiths, vehicle inspections, and retail stores ofv ehicle spare-parts, and we compared the solutions with those of the p-median model. We found the gravity p-median model to be of limited use for the problem of locating facilities as it either gives solutions similar to the p-median model, or it gives unstable solutions due to a non-concave objective function.

Acceptability of home-assessment post medical abortion and medical abortion in a low-resource setting in Rajasthan, India : Secondary outcome analysis of a non-inferiority randomized controlled trial

Background: Studies evaluating acceptability of simplified follow-up after medical abortion have focused on high-resource or urban settings where telephones, road connections, and modes of transport are available and where women have formal education. Objective: To investigate women's acceptability of home-assessment of abortion and whether acceptability of medical abortion differs by in-clinic or home-assessment of abortion outcome in a low-resource setting in India. Design: Secondary outcome of a randomised, controlled, non-inferiority trial. Setting Outpatient primary health care clinics in rural and urban Rajasthan, India. Population: Women were eligible if they sought abortion with a gestation up to 9 weeks, lived within defined study area and agreed to follow-up. Women were ineligible if they had known contraindications to medical abortion, haemoglobin < 85mg/l and were below 18 years. Methods: Abortion outcome assessment through routine clinic follow-up by a doctor was compared with home-assessment using a low-sensitivity pregnancy test and a pictorial instruction sheet. A computerized random number generator generated the randomisation sequence (1: 1) in blocks of six. Research assistants randomly allocated eligible women who opted for medical abortion (mifepristone and misoprostol), using opaque sealed envelopes. Blinding during outcome assessment was not possible. Main outcome measures: Women's acceptability of home-assessment was measured as future preference of follow-up. Overall satisfaction, expectations, and comparison with previous abortion experiences were compared between study groups. Results: 731 women were randomized to the clinic follow-up group (n = 353) or home-assessment group (n = 378). 623 (85%) women were successfully followed up, of those 597 (96%) were satisfied and 592 (95%) found the abortion better or as expected, with no difference between study groups. The majority, 355 (57%) women, preferred home-assessment in the event of a future abortion. Significantly more women, 284 (82%), in the home-assessment group preferred home-assessment in the future, as compared with 188 (70%) of women in the clinic follow-up group, who preferred clinic follow-up in the future (p < 0.001). Conclusion: Home-assessment is highly acceptable among women in low-resource, and rural, settings. The choice to follow-up an early medical abortion according to women's preference should be offered to foster women's reproductive autonomy.