Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability

Involvement of the p62/NRF2 signal transduction pathway on erythrophagocytosis

This deposit is composed by the main article plus the supplementary materials of the publication.Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.Fundação para a Ciência e a Tecnologia grants: (HMSP-ICT/0024/2010, UID/Multi/04462/2013, SFRH/BD/62197/2009, SFRH/BD/90258/2012, SFRH /BD/51877/2012, SFRH/BD/52293/2013, PTDC/SAU-TOX/116627/2010, HMSP-ICT/0022/2010 ); European Union FEDER support: (COMPETE, QREN, PT2020 Partnership Agreement), ERC grant: (ERC-2011-AdG 294709-DAMAGECONTROL).info:eu-repo/semantics/publishedVersio

Resistance of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis to nitric oxide correlates with disease severity in Tegumentary Leishmaniasis

BACKGROUND: Nitric oxide (NO(•)) plays a pivotal role as a leishmanicidal agent in mouse macrophages. NO(• )resistant Escherichia coli and Mycobacterium tuberculosis have been associated with a severe outcome of these diseases. METHODS: In this study we evaluated the in vitro toxicity of nitric oxide for the promastigote stages of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis parasites, and the infectivity of the amastigote stage for human macrophages. Parasites were isolated from patients with cutaneous, mucosal or disseminated leishmaniasis, and NO(• )resistance was correlated with clinical presentation. RESULTS: Seventeen isolates of L. (L.) amazonensis or L. (V.) braziliensis promastigotes were killed by up to 8 mM of more of NaNO(2 )(pH 5.0) and therefore were defined as nitric oxide-susceptible. In contrast, eleven isolates that survived exposure to 16 mM NaNO(2 )were defined as nitric oxide-resistant. Patients infected with nitric oxide-resistant Leishmania had significantly larger lesions than patients infected with nitric oxide-susceptible isolates. Furthermore, nitric oxide-resistant L. (L.) amazonensis and L. (V.) braziliensis multiplied significantly better in human macrophages than nitric oxide-susceptible isolates. CONCLUSION: These data suggest that nitric oxide-resistance of Leishmania isolates confers a survival benefit for the parasites inside the macrophage, and possibly exacerbates the clinical course of human leishmaniasis

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Viagra (R) and Cialis (R) blister packaging fingerprinting using Fourier transform infrared spectroscopy (FTIR) allied with chemometric methods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The production of counterfeit drugs is a criminal problem that carries serious risks to public health worldwide. Herein, the chemical fingerprinting of blister packaging using Fourier transform infrared spectroscopy (FTIR) of authentic and counterfeit samples of Viagra (R) and Cialis (R) is demonstrated. Fifteen commercial samples (Viagra (R) and Cialis (R)) and thirty two counterfeit samples (Viagra and Cialis) were analyzed, and the FTIR data was subjected to chemometric treatment via unsupervised pattern recognition methods (principal component analysis, and hierarchical cluster analysis) and a supervised pattern recognition method (partial least squares discriminant analysis). ATR-FTIR spectra of the blister packaging of authentic Cialis (R) and counterfeit Cialis samples showed bands at 2976, 2904, 1431, 1326, 1243, 973, 691 and 608 cm(-1), suggesting the presence of polyvinyl chloride (PVC) in its chemical composition. For authentic Viagra (R) and counterfeit Viagra samples, several distinct chemical profiles were observed in the ATR-FTIR spectra. Using unsupervised methods, samples were separated into three large groups: (i) counterfeit Viagra (seven samples made of PVC); (ii) authentic Viagra (R) (three samples made of poly(ethylene terephthalate)); (iii) Cialis (authentic and counterfeit) and some samples of Viagra (thirty seven made of PVC with additives of stearic acid derivatives, butyl hydroxy toluene or bisphenol A). Therefore, this suggests that three different types of forming films are used in the market for blister packaging used to contain inhibitors of PDE-5. Using supervised methods, all samples were correctly classified into their respective classes.6827222728Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FINEPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Monitoring the polyamide 11 degradation by thermal properties and X-ray fluorescence spectrometry allied to chemometric methods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The influence of temperature (110 and 120 degrees C) on the ageing of piping made from polyamide 11 (PA-11) containing 1012% of plasticizer was studied using deionized water (pH approximate to 7.0). A clean analytical methodology has been employed for quality control of polymeric materials: energy-dispersive X-ray fluorescence spectrometry (ED-XRF). It provides a fast and suitable technique to characterize chemical elements because of its multielemental capability, good sensitivity, high precision, short analytical time, and nondestructive nature. Herein, the content of additive in PA-11 was monitored from ED-XRF measurements where the abundance of the S line is directly related to the ageing time, agreeing with the thermogravimetric analysis. The XRF data were allied to chemometric treatment to classify PA-11 samples according to the amount of additive and weight average molar mass change, predicting the ageing time, and viscosity values of PA-11. Therefore, the XRF can be used as a clean analytical methodology to monitor the PA-11 degradation, thus eliminating the use of toxic organic solvents (necessary to viscosity measurements) and reducing the working time. Also, the effect of hydrolysis on the structure over time and the material morphology were monitored through measurements of dynamic mechanical analysis and differential scanning calorimetry. Copyright (c) 2012 John Wiley & Sons, Ltd.4227986PETROBRAS/CENPESConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FINEPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Humanized mouse models for HIV-1 infection of the CNS

Since the onset of the HIV epidemic, there has been a shift from a deadly diagnosis to the management of a chronic disease. This shift is the result of the development of highly effective drugs that are able to suppress viral replication for years. The availability of these regimens has also shifted the neurocognitive pathology associated with infection from potentially devastating to a much milder phenotype. As the disease outcome has changed significantly with the availability of antiretroviral therapy, there is an opportunity to re-evaluate the currently available models to address the neurocognitive pathology seen in suppressed patients. In the following, we seek to summarize the current literature on humanized mouse models and their utility in understanding how HIV infection leads to changes in the central nervous systems (CNS). Also, we identify some of the unanswered questions regarding HIV infection of the CNS as well as the opportunities and limitations of currently existing models to address those questions. Finally, our conclusions indicate that the earlier humanized models used to study HIV infection in the CNS provided an excellent foundation for the type of work currently being performed using novel humanized mouse models. We also indicate the potential of some humanized mouse models that have not been used as of this time for the analysis of HIV infection in the brain