White Light-Photolysis for the Removal of Polycyclic Aromatic Hydrocarbons from Proximity Firefighting Protective Clothing.

The presence of polycyclic aromatic hydrocarbons (PAHs) on firefighters' personal protective equipment is a concern. One form of preventing from these compounds is to decontaminate proximity firefighting protective clothing (PFPC). Traditional decontamination methods do not promote total removal of pollutants and alter the properties of PFPC. The objective of this work was to evaluate the effectiveness of white light-photolysis (WLP), an advanced oxidation process (AOP), for removing PAHs from PFPC, while maintaining the integrity of the fabric fibers. Experiments were carried out, varying reaction time and concentration of H2O2. With WLP (without H2O2), it was possible to remove more than 73% of the PAHs tested from the outer layer of PFPC in 3 days. The WLP provided the greatest removal of PAHs, compared with the most common mechanical decontamination techniques (laundering and wet-soap brushing). The fibers' integrity after exposure to the white light was evaluated with infrared spectroscopy and scanning electron microscopy/energy dispersive X-ray spectrometry. In addition, a tearing strength test was performed. No remarkable fabric degradation was observed, indicating a possible, routine-compatible, simple, and inexpensive method of decontamination of PFPC, based on photolysis, which is effective in the degradation of PAHs and maintains the integrity of fabric fibers.

Restarting Elective Bariatric and Metabolic Surgery Under a Security Protocol During the COVID-19 Pandemic-a Prospective Observational Cohort Study.

BACKGROUND: During the SARS-CoV-2 pandemic, in order to protect the patient and to save hospital beds, cancelation of elective surgeries has become a great challenge. Considering that obesity is a chronic disease and the possible effect imposed by quarantine on weight gain with worsening rates of obesity and metabolic comorbidities, the creation of a protocol for a safe return to bariatric surgery became essential. OBJECTIVE: The aim of this study was to identify the incidence of new-onset severe acute respiratory syndrome coronavirus (SARS-CoV-2) symptoms in patients who underwent bariatric procedures during the declining curve period. SETTING: Private practice METHODS: A prospective observational cohort study was conducted and included patients with indications for bariatric surgery during the decreasing curve period of the SARS-CoV-2 pandemic who underwent surgery under a hospital security protocol. Patients were asked to answer a questionnaire and had a swab PCR test for SARS-CoV-2 detection. The primary outcome measure was the presence of 14-day and 30-day postoperative symptoms associated with COVID-19. Mortality was also analyzed. RESULTS: Three hundred patients with negative RT-PCR were operated on from May to June 2020. Seventeen patients had their surgery postponed because of a positive RT-PCR test or close contact. None of the patients developed new-onset SARS-CoV-2 symptomatic infection after 30 days of observation. No deaths were reported. Eleven had complications not related to SARS-CoV-2. CONCLUSIONS: Even though this population may have a poorer outcome when infected with SARS-CoV-2, this security protocol has shown that the procedure can be safely performed during the outbreak.

Development and validation of analytical method for identification of new psychoactive substances using linear retention indexes and gas chromatography-mass spectrometry.

New Psychoactive Substances (NPS) are quickly developing to evade legislation, posing unprecedented challenges to public health and law enforcement authorities around the world. The aim of this work was to develop and validate a simple and reliable non-target gas chromatography/mass spectrometry (GC/MS) analytical method based on linear retention indexes for the expeditious identification of NPS without the need of analytical standards. The method was optimized and validated for 22 different drugs covering ten categories: phenethylamines (amphetamine, MDMA, methamphetamine, 25CNBOMe, 2-FA, 5-MAPB), "classic" drugs (cocaine, ephedrine, THC, heroine), synthetic cannabinoids (JWH-081, AM-2201, JWH-210, MAM-2201), piperazines (o-CPP, p-CPP), tryptamines (5-MeO-MiPT), synthetic cathinones (N-ethylpentylone), synthetic opioids (U-47700), aminoindanes (5-IAI), plant-based substances (Salvinorin-A) and "other" (methiopropamine). Three figures of merit (Selectivity, Precision and Robustness) were evaluated with retention index confidence intervals ranging from 0.5 to 20.6 i.u. and relative standard deviations in the range of 0.003% to 0.027% (repeatability) and 0.02% to 0.29% (intermediate precision). A general equation for estimating linear retention index variation as a function of retention time tolerance has been derived. This result in combination with a 2III6-3 fractional factorial design allowed to conclude column polarity to be only statistically relevant factor as compared to gas flow, split ratio, injection temperature, temperature program offset and column brand.

Trend analysis of leprosy indicators in a hyperendemic Brazilian state, 2001-2015.

OBJECTIVE: To analyze the temporal trend of leprosy indicators in a hyperendemic state of Brazil, from 2001-2015. METHODS: This is a time-series study of leprosy indicators in the state of Maranhão, Northeastern region of Brazil. The study used data from the Brazilian National System of Reportable Diseases, for the period between 2001 and 2015. The following indicators were evaluated: (i) detection coefficient in the general population; (ii) detection coefficient in people under 15 years old; (iii) rate of cases with grade 2 physical disability in the diagnosis; (iv) rate of examined contacts, and (v) proportion of healing . The Prais-Winsten regression model was used for trend analysis. Analyses were performed for the state and by each health region. RESULTS: 77,697 leprosy cases were analyzed in the general population and 7,599 in individuals under 15 years old. The detection coefficient in the general population ranged from 80.7/100 thousand inhabitants in 2001 to 51.2/100 thousand inhabitants in 2015. The coefficient in the general population presented a downward trend (annual percentage variation [APV] = -2.98; 95%CI -4.15- -1.79). For the population under 15 years old, the rate was 24.9/100 thousand inhabitants in 2001, and 19.9/100 thousand inhabitants in 2015, with downward trend (APV = -3.07; 95%CI -4.95- -1.15). It was observed upward trend in rate of contacts examined (APV = 2.35; 95%CI 0.58-4.15) and rate of cases with grade 2 disability (APV = 2.19; 95%CI 0.23-4.19). Stationary trend was observed in the proportion of healing (APV = -0.10; 95%CI -0.50-0.30). Regional differences were found in the performance of the indicators. CONCLUSIONS: A downward trend for the detection coefficients in the general population and in individuals under 15 years old was found in Maranhão. Despite this result, the rates are still very high, demanding efforts from all spheres of public administration and health professionals to reduce the burden of the disease in the state.

Redox titration on foldable paper-based analytical devices for the visual determination of alcohol content in whiskey samples.

This report describes the development of foldable paper-based analytical devices (PADs) to perform redox titrations. Paper devices were designed to contain three spot tests, which were wax printed and folded to create a three-layer structured platform and to promote the sample transport based on vertical flow. The proposed devices were explored for the visual determination of the alcoholic content in whiskey samples. For this purpose, a classical permanganometry reaction was employed to allow the indirect determination of ethanol based on the required amount of oxalic acid to react with the excess of permanganate in acidic medium. The endpoint of the redox titrations performed in different alcoholic concentrations was measured and revealed a good linear behavior for the ethanol concentration range between 0% and 50% (R2 = 0.992), achieving a limit of detection equal to 2.1%. The alcoholic content was determined in a total of 44 whiskey samples seized by the Brazilian Federal Police. When compared to genuine samples and using an established cut-off limit, 73% of the seized samples were correctly classified as whiskeys containing adulterated alcoholic content. The proposed method was compared to a reference protocol and no difference was observed at the confidence level of 95%. The instrumental simplicity, the low cost, the sample volume requirement, the short analysis time and mainly the inherent portability make these devices quite attractive for on-site forensic applications.

Trend analysis of leprosy indicators in a hyperendemic Brazilian state, 2001-2015 / Análise da tendência dos indicadores da hanseníase em estado brasileiro hiperendêmico, 2001-2015

ABSTRACT OBJECTIVE To analyze the temporal trend of leprosy indicators in a hyperendemic state of Brazil, from 2001-2015. METHODS This is a time-series study of leprosy indicators in the state of Maranhão, Northeastern region of Brazil. The study used data from the Brazilian National System of Reportable Diseases, for the period between 2001 and 2015. The following indicators were evaluated: (i) detection coefficient in the general population; (ii) detection coefficient in people under 15 years old; (iii) rate of cases with grade 2 physical disability in the diagnosis; (iv) rate of examined contacts, and (v) proportion of healing . The Prais-Winsten regression model was used for trend analysis. Analyses were performed for the state and by each health region. RESULTS 77,697 leprosy cases were analyzed in the general population and 7,599 in individuals under 15 years old. The detection coefficient in the general population ranged from 80.7/100 thousand inhabitants in 2001 to 51.2/100 thousand inhabitants in 2015. The coefficient in the general population presented a downward trend (annual percentage variation [APV] = -2.98; 95%CI -4.15- -1.79). For the population under 15 years old, the rate was 24.9/100 thousand inhabitants in 2001, and 19.9/100 thousand inhabitants in 2015, with downward trend (APV = -3.07; 95%CI -4.95- -1.15). It was observed upward trend in rate of contacts examined (APV = 2.35; 95%CI 0.58-4.15) and rate of cases with grade 2 disability (APV = 2.19; 95%CI 0.23-4.19). Stationary trend was observed in the proportion of healing (APV = -0.10; 95%CI -0.50-0.30). Regional differences were found in the performance of the indicators. CONCLUSIONS A downward trend for the detection coefficients in the general population and in individuals under 15 years old was found in Maranhão. Despite this result, the rates are still very high, demanding efforts from all spheres of public administration and health professionals to reduce the burden of the disease in the state.

RESUMO OBJETIVO Analisar a tendência temporal dos indicadores da hanseníase em estado brasileiro hiperendêmico, no período de 2011 a 2015. MÉTODOS Trata-se de um estudo de série temporal dos indicadores de hanseníase no estado do Maranhão, região Nordeste, que utilizou dados do Sistema Nacional de Agravos de Notificação, no período de 2001 a 2015. Foram avaliados os seguintes indicadores: (i) coeficiente de detecção na população em geral; (ii) coeficiente de detecção em menores de 15 anos; (iii) proporção de casos com grau 2 de incapacidade física no diagnóstico; (iv) proporção de contatos examinados e (v) proporção de cura. Para análise da tendência foi utilizado o modelo de regressão de Prais Winsten . As análises foram realizadas para o Estado e por região de saúde. RESULTADOS Foram analisados 77.697 casos de hanseníase na população geral e 7.599 em menores de 15 anos. O coeficiente de detecção na população geral variou de 80,7/100 mil habitantes em 2001 para 51,2/100 mil habitantes em 2015. Verificou-se tendência decrescente do coeficiente na população geral (variação percentual anual [VPA] = -2,98; IC95% -4,15- -1,79). Na população menor de 15 anos, o coeficiente foi de 24,9/100 mil habitantes em 2001 para 19,9/100 mil habitantes em 2015, com tendência decrescente (VPA = -3,07; IC95% -4,95- -1,15). Observou-se tendência crescente na proporção de contatos examinados (VPA = 2,35; IC95%: 0,58-4,15) e na proporção de casos com grau 2 de incapacidade (VPA = 2,19; IC95% 0,23-4,19). Verificou-se tendência estacionária na proporção de cura de hanseníase (VPA = -0,10; IC95% -0,50-0,30). Observaram-se diferenças regionais no desempenho dos indicadores. CONCLUSÕES A tendência dos coeficientes de detecção na população geral e em menores de 15 anos mostrou-se decrescente no Maranhão. Apesar disso, as taxas ainda são muito elevadas, o que exige esforços de todas as esferas de gestão e profissionais da saúde para redução da carga da doença no estado.

Discrimination of whisky brands and counterfeit identification by UV-Vis spectroscopy and multivariate data analysis.

The discrimination of whisky brands and counterfeit identification were performed by UV-Vis spectroscopy combined with partial least squares for discriminant analysis (PLS-DA). In the proposed method all spectra were obtained with no sample preparation. The discrimination models were built with the employment of seven whisky brands: Red Label, Black Label, White Horse, Chivas Regal (12years), Ballantine's Finest, Old Parr and Natu Nobilis. The method was validated with an independent test set of authentic samples belonging to the seven selected brands and another eleven brands not included in the training samples. Furthermore, seventy-three counterfeit samples were also used to validate the method. Results showed correct classification rates for genuine and false samples over 98.6% and 93.1%, respectively, indicating that the method can be helpful for the forensic analysis of whisky samples.

Brazilian Federal Police drug chemical profiling - the PeQui project.

Over the past six years the Brazilian Federal Police has undertaken major efforts in order to implement and to develop its own drug chemical profiling program. This paper aims to provide a broad perspective regarding the managerial strategies and some examples of subsequent technical issues involved in the implementation of such a project. Close collaboration with local drug enforcement and investigation teams, establishment of proper worldwide partnerships with well recognized institutions in the field of drug analysis and the attainment of suitable funding and human resources are shown to be key success factors. Some preliminary results concerning the chemical profile of cocaine seizures in Brazil during this process are presented.

Correlation of cocaine hydrochloride samples seized in Brazil based on determination of residual solvents: an innovative chemometric method for determination of linkage thresholds.

Cocaine sample correlation provides important information in the identification of traffic networks. However, available methods for estimating if samples are linked or not require the use of previous police investigation and forensic expert knowledge regarding the number of classes and provide thresholds that are both static and data set specific. In this paper, a novel unsupervised linkage threshold method (ULT) based on chemometric analysis is described and applied to the analysis of headspace gas chromatography mass spectrometry (HS-GC/MS) data of more than 250 real cocaine hydrochloride samples seized by Brazilian Federal Police. The method is capable of establishing linkage thresholds that do not require any prior information about the number of classes or distribution of the samples and can be dynamically updated as the data set changes. It is envisaged that the ULT method may also be applied to other forensic expertise areas where limited population knowledge is available and data sets are continually modified with the inflow of new information.

Demystifying "oxi" cocaine: Chemical profiling analysis of a "new Brazilian drug" from Acre State.

Recent information from various sources suggests that a new illicit drug, called "oxi", is being spread across Brazil. It would be used in the smoked form and it would look like to crack cocaine: usually small yellowish or light brown stones. As fully released in the media, "oxi" would differ from crack cocaine in the sense that crack would contain carbonate or bicarbonate salts whereas "oxi" would include the addition of calcium oxide and kerosene (or gasoline). In this context, this work presents a chemical profiling comparative study between "oxi" street samples seized by the Civil Police of the State of Acre (CP/AC) and samples associated with both international and interstate drug trafficking seized by the Brazilian Federal Police in Acre (FP/AC). The outcome of this work assisted Brazilian authorities to stop inaccurate and alarmist releases on this issue. It may be of good use by the forensic community in order to better understand matters in their efforts to guide local law enforcement agencies in case such claims reach the international illicit market.

Rinofima: opções cirúrgicas utilizadas no serviço de cirurgia plástica do Hospital Agamenon Magalhães: PE / Rhinophyma: surgical options used at the service of plastic surgery of Hospital Agamenon Magalhães: PE

Introdução: A rinofima é uma inflamação crônica dos tecidos do nariz com acometimento decor, textura e vascularização, apresentando crescimento exofítico irregular e telangiectasias.Objetivo: Levantar dados epidemiológicos de pacientes portadores de rinofima e avaliar osresultados dos diversos tratamentos para esta patologia realizados no Serviço de CirurgiaPlástica do Hospital Agamenon Magalhães (HAM), Recife, PE, Brasil, comparando-os comos tratamentos descritos na literatura. Método: Estudo retrospectivo, baseado na análise deprontuários de 28 pacientes submetidos a tratamento do rinofima, entre março de 2002 emarço de 2010. Resultados: A média de idade dos pacientes foi 50,1 anos, sendo 72,72%do sexo masculino e 17,28% do sexo feminino. O tratamento mais utilizado foi associaçãode decorticação/ dermoabrasão e eletrocoagulação, em 96% dos casos. Foram observadas asseguintes complicações: 1 caso de hipopigmentação, 2 de hiperpigmentação e 1 de cicatrizinestética. Em 3 casos, houve necessidade de mais de uma cirurgia para complementação dotratamento. Nenhum caso de câncer foi verificado nas peças cirúrgicas. O grau de satisfaçãodos pacientes foi de 100%. Conclusões: A rinofima é uma doença que possui tratamentosimples e eficaz, com excelentes resultados cosméticos.

Introduction: Rhinophyma is a chronic inflammation of the nose with involvement of color,texture and vascularization, with irregular exophytic growth and telangiectasia. Objective:To analyze epidemiological data of patients with rhinophyma and evaluate the results ofvarious treatments for this disease at the Service of Plastic Surgery of Hospital Agamenon Magalhães, Recife, PE, Brasil, comparing them with the treatments described in the literature.Methods: A retrospective study using analysis of medical records of 28 patients undergoing treatment of rhinophyma, between March 2002 and March 2010. Results: The average patient age was 50.1 years, with 72.72% male and 17.28% female. The most common treatment was an association of decortication/ dermabrasion and electrocoagulation,in 96% of cases. Complications observed: one case of hypopigmentation, two cases of hyperpigmentation and one case of unsightly scar. In three cases, there was need for more surgery to completion of treatment. No cancer was found on surgical specimens. The degree of patient satisfaction was 100%. Conclusion: Rhinophyma is a disease that has simple and effective treatment with excellent cosmetic results.

Acylation-dependent export of Trypanosoma cruzi phosphoinositide-specific phospholipase C to the outer surface of amastigotes.

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.

Survival and stimulation of neurite outgrowth in a serum-free culture of spiral ganglion neurons from adult mice.

We have developed a reliable protocol for the serum-free dissociation and culture of spiral ganglion neurons from adult mice, an important animal model for patients with post-lingual hearing loss. Pilot experiments indicated that the viability of spiral ganglion cells in vitro depended critically on the use of Hibernate medium with B27 supplement. With an optimized protocol, we obtained 2 x 10(3) neurons immediately after dissociation, or about one-fifth of those present in the intact spiral ganglion. After four days in culture, 4% of the seeded neurons survived without any exogenous growth factors other than insulin. This yield was highly reproducible in five independent experiments and enabled us to measure systematically the numbers and lengths of the regenerating neurites. Furthermore, the survival rate compared well to the few published protocols for culturing adult spiral ganglion neurons from other species. Enhanced survival and neurite outgrowth upon the addition of brain-derived neurotrophic factor and leukemia inhibitory factor demonstrated that both are potent stimulants for damaged spiral ganglion neurons in adults. This responsiveness to exogenous growth factors suggested that our culture protocol will facilitate the screening of molecular compounds as potential treatments for sensorineural hearing loss.

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte.

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.

Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi.

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope-tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.

The H(+)-pyrophosphatase of Rhodospirillum rubrum is predominantly located in polyphosphate-rich acidocalcisomes.

Acidocalcisomes are acidic, calcium storage compartments with a H(+) pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H(+)-pyrophosphatase (H(+)-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H(+)-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H(+)-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes.

A pyrophosphatase regulating polyphosphate metabolism in acidocalcisomes is essential for Trypanosoma brucei virulence in mice.

We report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg(2+) or Zn(2+), both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PP(i)) (k(cat) = 385 s(-1)) or tripolyP (polyP(3)) and polyphosphate (polyP) of 28 residues (polyP(28)) with k(cat) values of 52 and 3.5 s(-1), respectively. An unusual N-terminal domain of 160 amino acids, containing a putative calcium EF-hand-binding domain, is involved in protein oligomerization. Using double-stranded RNA interference methodology, we produced an inducible bloodstream form (BF) deficient in the TbVSP1 protein (BFiVSP1). The long-chain polyP levels of these mutants were reduced by 60%. Their phenotypes revealed a deficient polyP metabolism, as indicated by their defective response to phosphate starvation and hyposmotic stress. BFiVSP1 did not cause acute virulent infection in mice, demonstrating that TbVSP1 is essential for growth of bloodstream forms in the mammalian host.

Oxidative phosphorylation and rotenone-insensitive malate- and NADH-quinone oxidoreductases in Plasmodium yoelii yoelii mitochondria in situ.

Respiration, membrane potential, and oxidative phosphorylation of mitochondria of Plasmodium yoelii yoelii trophozoites were assayed in situ after permeabilization with digitonin. ADP induced an oligomycin-sensitive transition from resting to phosphorylating respiration in the presence of oxidizable substrates. A functional respiratory chain was demonstrated. In addition, the ability of the parasite to oxidize exogenous NADH, as well as the insensitivity of respiration to rotenone and its sensitivity to flavone, suggested the presence of an alternative NADH-quinone (NADH-Q) oxidoreductase. Rotenone-insensitive respiration and membrane potential generation in the presence of malate suggested the presence of a malate-quinone oxidoreductase. These results are in agreement with the presence of genes in P. yoelii encoding for proteins with homology to NADH-Q oxidoreductases of bacteria, plant, fungi, and protozoa and malate-quinone oxidoreductases of bacteria. The complete inhibition of respiration by antimycin A and cyanide excluded the presence of an alternative oxidase as described in other parasites. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin and GTP but was unaffected by carboxyatractyloside. These results provide the first biochemical evidence of the presence of an alternative NADH-Q oxidoreductase and a malate-quinone oxidoreductase and confirm the operation of oxidative phosphorylation in malaria parasites.

Formation and remodeling of inositolphosphoceramide during differentiation of Trypanosoma cruzi from trypomastigote to amastigote.

Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [(3)H]palmitic acid or [(3)H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using (3)H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A(1), phospholipase A(2), inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes.

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.