RESUMEN

Low-density lipoprotein cholesterol (LDL-C), which makes up about 70% of the cholesterol in the blood, is critical in the formation of arteriosclerotic plaques, increasing the risk of heart disease. LDL-C levels are estimated using Friedewald, Martin and Sampson equations, though they have limitations with high triglycerides. Our aim is to compare the effectiveness of these equations versus the ultracentrifugation technique in individuals with and without dyslipidemia and identify precision. There were 113 participants, 59 healthy controls and 54 dyslipidemic patients. Samples were collected after fasting. LDL-C was estimated using the Friedewald, Martin and Sampson equations. The purified LDL-C, ultracentrifugated and dialysized control group without dyslipidemia vs. patients with coronary artery disease (CAD) showed differences in age, HDL-C, triglycerides and glucose non-HDL-C (p = 0.001 in all). There were correlations in CGWD between ultracentrifugation and Sampson R-squared (R2) = 0.791. In the dyslipidemia control group, ultracentrifugation and Friedewald R2 = 0.911. In patients with CAD, correlation between ultracentrifugation and Sampson R2 = 0.892; Bland-Altman confirmed agreement in controls without dyslipidemia. The Martin and Sampson equations are interchangeable with ultracentrifugation. Conclusion: The role of LDL analysis using precise techniques is necessary to obtain better control of disease outcomes after the use of precise therapies and suggests verifying its importance through clinical trials.

RESUMEN

Gram-negative bacteria are major pathogens that develop antibiotic resistance and cause distinct types of infections. According to the WHO (World Health Organization), bacterial resistance is considered a public health problem. Multiresistance Klebsiella pneumoniae and E. coli are prevalent in many hospitals. These bacteria produce outer membrane vesicles (OMVs) these vesicles can contribute to bacterial survival by eliminating toxic compounds, removing misfolded periplasmic proteins, establishing a colonization niche, biofilm formation, drug delivery, and modulation of host defense and response. This work aimed to evaluate the production and extraction of OMVs produced by E. coli and K. pneumoniae, comparing two different gel filtration and ultracentrifugation methods. Furthermore, we evaluated the interference of OMVs on the growth of enteropathogenic E. coli, uropathogenic E. coli, and hematophatogenic K. pneumoniae strains verifying the viability test. The gel filtration extraction system obtained an elevated concentration when compared to ultracentrifugation method. We observed the presence of OmpA by eletrophoresis and Dot blotting, responsible for the structure of OMVs. The results showed interferences of OMV EC086-UC, EC086-GF and OMV-KP70 on E2348/69 growth were directly proportional to the concentration of OMVs but inversely proportional to the concentration of OMVs with OMV-KP70 on EC086 and KP70 growth. We concluded that vesicle interference on EC086, E2348/69, and KP70 growth should not be based only on optical density parameters but complemented with the viability test of each bacterium strain. The use of biotechnology tools like genetic modifications, OMVs can perform multiple functions carrying molecules with many capabilities to be applied in many fields such as immunology, diagnostics, clinical medicine.

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As bactérias Gram-negativas são patógenos que apresentam resistência a diversos antibióticos. De acordo com a Organização Mundial da Saúde (OMS), a resistência antimicrobiana é um problema de saúde pública. A Escherichia coli e a Klebsiella pneumoniae são multirresistentes e prevalentes nos ambientes hospitalares. Estas bactérias produzem vesículas de membrana externa (OMVs) e possuem diversas funções como: contribuição para a sobrevivência das bactérias através da neutralização de componentes tóxicos, eliminação de proteínas desnaturadas, estabelecimento do nicho de colonização, formação de biofilme, entrega de drogas, modulação do sistema de defesa e resposta imune. Este trabalho avaliou a produção e extração de OMVs produzidas por cepas de E. coli e K. pneumoniae comparando os métodos de extração das mesmas por da gel filtração e ultracentrifugação. Além disso, através do teste de viabilidade bacteriana avaliamos a interferência das OMVs no crescimento de E. coli enteropatogência, E. coli uropatogênica e K. pneumoniae hematopatogênica. A purificação das OMVs por gel filtração apresentou uma concentração elevada quando comparada com o método de ultracentrifugação. Identificamos, para ambos os métodos, através da eletroforese e imunoensaio a presença da proteína de membrana externa a OmpA, responsável pela estrutura das OMVs. Demonstramos pelos resultados que as EC086-UC, EC086-GF e OMV-KP70 interferiram no crescimento da cepa E2348/69 com padrão diretamente proporcional quando comparado com a concentração das vesículas. O contrário ocorreu na interferência da OMV KP70-UC, atuando sobre o crescimento de EC086 e KP70 de forma inversamente proporcional quando comparado com a concentração das vesículas. Concluímos que a interferência das vesículas sobre o crescimento deve levar em conta os parâmetros de densidade óptica e a viabilidade bacteriana. Assim, com as diversas ferramentas da biotecnologia como a engenharia genética podemos desenvolver OMVs capazes de carrear diversas moléculas e ser aplicada em vários campos da imunologia, diagnósticos e na medicina clínica.

RESUMEN

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca2+ and the enzyme kinetics showed a hyperbolic dependence on ATP (Km = 12.16 ± 2.25 µM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (Km). The presence of Ca2+ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.

RESUMEN

Wastewater-based epidemiology has been described as a valuable tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a community. However, there is no consensus on the best concentration method to allow reliable detection of SARS-CoV-2 in this matrix, considering different laboratory facilities. This study compares two viral concentration methods, ultracentrifugation (ULT) and skimmed-milk flocculation (SMF), for detecting SARS-CoV-2 in wastewater samples. The analytical sensitivity (limits of detection and quantification [LoD/LoQ]) of both methods was evaluated using a bovine respiratory syncytial virus (BRSV) as a surrogate. Three different approaches were conducted to establish LoD of each method based on the assays on the standard curve (ALoDsc), on the dilution of internal control (ALoDiC), and the processing steps (PLoD). For PLoD, ULT method had the lowest value (1.86 × 103 genome copy/microliter [GC/µL]) when compared to the SMF method (1.26 × 107 GC/µL). The LoQ determination showed a mean value of 1.55 × 105 GC/µL and 3.56 × 108 GC/µL to ULT and SMF, respectively. The detection of SARSCoV-2 in naturally contaminated wastewater revealed 100% (12/12) and 25% (3/12) of detection using ULT and SMF with quantification ranging from 5.2 to 7.2 log10 genome copy/liter (GC/L) and 5.06 to 5.46 log10 GC/L, respectively. The detection success rate of BRSV used as an internal control process was 100% (12/12) for ULT and 67% (8/12) for SMF, with an efficiency recovery rate ranging from 12 to 38% and 0.1 to 5%, respectively. Our data consolidates the importance of assessing the methods used; however, further analysis should be carried out to improve low-cost concentration methodologies, essential for use in low-income and developing countries.

Asunto(s)

COVID-19 , Virus , Animales , Bovinos , SARS-CoV-2/genética , COVID-19/diagnóstico , Aguas Residuales , Límite de Detección , ARN Viral

RESUMEN

Dengue is an infectious disease caused by Dengue Virus, mainly transmitted by Aedes aegypti mosquitoes. Severe dengue is a potentially fatal syndrome in consequence of overwhelmed inflammation, in which thrombocytopenia and increased vascular permeability are frequently observed. Several experimental evidences point to the participation of both microvesicles (MVs) and circulating lipoproteins in inflammatory amplification in dengue pathogenesis. On this regard, many protocols for isolating plasma MVs have shown lipoproteins as the main contaminant. This is a limitation to studies aiming at the functional characterization of MVs, since both MVs and lipoproteins can modulate inflammatory responses. Here, we describe a biphasic density-based gradient ultracentrifugation as a tool for concomitant isolation of MVs and lipoproteins without cross-contamination. Flow cytometry for MVs quantification and western blot for detection of apoB100 may be used to confirm the isolation and purity of the MVs.

Asunto(s)

Micropartículas Derivadas de Células , Animales , Dengue , Citometría de Flujo , Lipoproteínas

RESUMEN

Wastewater-based epidemiology (WBE) may be successfully used to comprehensively monitor and determine the scale and dynamics of some infections in the community. We monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in raw wastewater samples from Porto Alegre, Southern Brazil. The samples were collected and analyzed every week between May 2020 to May 2021. Meanwhile, different social restrictions were applied according to the number of hospitalized patients in the region. Weekly samples were obtained from two wastewater treatment plants (WWTP), named Navegantes and Serraria. To determine the SARS-CoV-2 RNA titers in wastewater, we performed RT-qPCR analysis targeting the N gene (N1). The highest titer of SARS-CoV-2 RNA was observed between epidemiological weeks (EWs) 33-37 (August), 42-43 (October), 45-46 (November), 49-51 (December) in 2020, and 1-3 (January), 7-13 (February to March) in 2021, with viral loads ranging from 1 × 106-3 × 106 genomic copies/Liter. An increase in positive confirmed cases followed such high viral loads. Depending on the sampling method used, positive cases increased in 6-7 days and 15 days after the rise of viral RNA titers in wastewater, with composite sampling methods showing a lower time lag and a higher resolution on the analyses. The results showed a direct relation between strict social restrictions and the loads of detected RNA reduction in wastewater, corroborating the number of confirmed cases. Differences in viral loads between different sampling points and methods were observed, as composite samples showed more stable results during the analyzed period. Besides, viral loads obtained from samples collected at Serraria WWTP were consistently higher than the ones obtained at Navegantes WWTP, indicating differences in local dynamics of SARS-CoV-2 spread in different regions of Porto Alegre. In conclusion, wastewater sampling to monitor SARS-CoV-2 is a robust tool to evaluate the viral loads contributing to hospitalized patients' data and confirmed cases. In addition, SARS-CoV-2 detection in sewage may inform and alert the government when there are asymptomatic or non-tested patients.

RESUMEN

Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

Asunto(s)

Cistatinas/química , Inhibidores de Cisteína Proteinasa/química , Humulus/química , Proteínas de Plantas/química , Clonación Molecular , Cristalografía por Rayos X , Cistatinas/genética , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Concentración de Iones de Hidrógeno , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica

RESUMEN

One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.

Asunto(s)

Ingeniería Genética , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Lentivirus/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Citometría de Flujo , Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Terapia Genética , Vectores Genéticos/aislamiento & purificación , Humanos , Inmunoterapia Adoptiva , Transducción Genética , Transfección , Transgenes , Ultracentrifugación/métodos

RESUMEN

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Asunto(s)

Catepsina D/aislamiento & purificación , Schistosoma mansoni/enzimología , Animales , Proteasas de Ácido Aspártico/biosíntesis , Proteasas de Ácido Aspártico/química , Proteasas de Ácido Aspártico/aislamiento & purificación , Proteasas de Ácido Aspártico/metabolismo , Catepsina D/biosíntesis , Catepsina D/química , Catepsina D/metabolismo , Catepsinas/biosíntesis , Catepsinas/química , Catepsinas/aislamiento & purificación , Catepsinas/metabolismo , Cromatografía en Gel , Dimerización , Células HEK293 , Humanos , Cinética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ultracentrifugación/métodos

RESUMEN

Extracellular vesicles (EVs) are heterogeneous membrane-surrounded structures that participate in cellular communications, which comprise exosomes and microvesicles. These vesicles have different biogenesis, and their physiological and pathological roles in chronic and infectious diseases are under constant investigation. In Chagas disease, Trypanosoma cruzi EVs have been described using different approaches. The isolation of T. cruzi-derived EVs has been done mainly using the differential centrifugation technique, and different strategies have been employed for characterization of them. Here, we describe the method to isolate EVs by differential centrifugation and a detection protocol for EVs in T. cruzi-host cell interaction to allow further investigations about this parasite.

Asunto(s)

Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/parasitología , Vesículas Extracelulares/metabolismo , Interacciones Huésped-Parásitos , Trypanosoma cruzi/fisiología , Animales , Línea Celular , Vesículas Extracelulares/química , Humanos , Proteínas/análisis , Trypanosoma cruzi/química , Trypanosoma cruzi/metabolismo , Ultracentrifugación/métodos

RESUMEN

INTRODUCTION: High-density lipoprotein cholesterol comprises a group of heterogeneous subfractions that might have differential effects on atherosclerosis. Moreover, prior investigations suggest that the presence of diabetes (T2D) modifies the impact of some subfractions on atherosclerosis. In this study, we aimed to evaluate the association between high-density lipoprotein cholesterol subfractions and carotid intima-media thickness in the baseline assessment of the Brazilian Longitudinal Study of Adult Health participants from the São Paulo investigation centre. METHODS: We evaluated 3930 individuals between 35 and 74 years without previous cardiovascular disease not using lipid-lowering drugs. High-density lipoprotein cholesterol subfractions (HDL2-C and HDL3-C) were measured by vertical ultracentrifugation (vertical auto profile). The relationship between each high-density lipoprotein cholesterol subfraction and carotid intima-media thickness was analysed by multiple linear regression models. RESULTS: Total high-density lipoprotein cholesterol, as well as HDL2-C and HDL3-C, was negatively associated with carotid intima-media thickness after adjustment for demographic data (all p < 0.001) and traditional risk factors (all p < 0.05). When stratified by T2D status, the HDL2-C/HDL3-C ratio showed a negative association with carotid intima-media thickness in participants with T2D ( p = 0.032), even after fully controlling for confounding variables, including total high-density lipoprotein cholesterol. CONCLUSION: HDL2-C, HDL3-C and HDL2/HDL3-C ratio are inversely associated with carotid intima-media thickness after adjustment for traditional risk factors. Association of the HDL2-C/HDL3-C ratio is modified by the presence of diabetes, being more pronounced in diabetic individuals.

Asunto(s)

Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Grosor Intima-Media Carotídeo , HDL-Colesterol/sangre , Diabetes Mellitus/sangre , Dislipidemias/sangre , Adulto , Anciano , Biomarcadores/sangre , Brasil/epidemiología , Enfermedades de las Arterias Carótidas/epidemiología , Estudios Transversales , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiología , Dislipidemias/diagnóstico , Dislipidemias/epidemiología , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Factores de Riesgo

RESUMEN

In a recently published study, Anna Krichevsky and colleagues raise the important question of whether results of in vitro extracellular RNA (exRNA) studies, including extracellular vesicle (EV) investigations, are confounded by the presence of RNA in cell culture medium components such as foetal bovine serum (FBS). The answer, according to their data, is a resounding "yes". Even after lengthy ultracentrifugation to remove bovine EVs from FBS, the majority of exRNA in FBS remained. Although technical factors may affect the degree of depletion, residual EVs and exRNA in FBS could influence the conclusions of in vitro studies: certainly, for secreted RNA, and possibly also for cell-associated RNA. In this commentary, we critically examine some of the literature in this field, including a recent study from some of the authors of this piece, in light of the Wei et al. study and explore how cell culture-derived RNAs may affect what we think we know about EV RNAs. These findings hold particular consequence as the field moves towards a deeper understanding of EV-RNA associations and potential functions.

RESUMEN

Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.

Asunto(s)

Microbiología de Alimentos , Virus/aislamiento & purificación , Microbiología del Agua , Adenoviridae/genética , Adenoviridae/fisiología , Enterovirus/genética , Enterovirus/aislamiento & purificación , Concentración de Iones de Hidrógeno , Lactuca/virología , Levivirus/genética , Levivirus/aislamiento & purificación , Norovirus/genética , Norovirus/aislamiento & purificación , Fagos Pseudomonas/genética , Fagos Pseudomonas/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Aguas del Alcantarillado/virología , Ultracentrifugación , Ultrafiltración , Virus/genética

RESUMEN

This chapter is derived from our experience in the study of stress-Inducible Protein 1 (STI1) in extracellular vesicles. We used different techniques to isolate, explore, and characterize the extracellular vesicles that contained this protein. Ultracentrifugation and gel chromatography were used to isolate extracellular vesicles of different sizes, nanotracking particle analysis (NTA) determined number and size of vesicles, while flow cytometry and ELISA were used to determine the specific protein content of vesicles.

Asunto(s)

Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico/metabolismo , Fraccionamiento Celular , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Transporte de Proteínas , Ultracentrifugación

RESUMEN

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

Asunto(s)

Animales , Ratas , Glucólisis , Trypanosoma/enzimología , Tripanosomiasis/parasitología , Modelos Animales de Enfermedad , Filogenia , Ratas Sprague-Dawley , Especificidad de la Especie , Trypanosoma/clasificación , Trypanosoma/genética , Ultracentrifugación

RESUMEN

The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/ß)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of ß-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.

Asunto(s)

NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Trypanosoma cruzi/enzimología , Cristalografía por Rayos X , Modelos Moleculares , NADPH Deshidrogenasa/genética , Conformación Proteica , Especificidad por Sustrato , Termodinámica

RESUMEN

ABSTRACT The main of the present study was analyze the use of a french press device to obtain genetic material from staphylococci and to detect genes involved with chromosomal and plasmid resistance to the following antimicrobial drugs: oxacillin, gentamicin, kanamycin and vancomycin. The agar disc diffusion method was conducted initially for 50 strains and the bacterial susceptibility was confirmed by means of PCR reactions. The results from the antibiogram method revealed high sensibility to gentamicin and kanamycin (4%) and oxacillin (8%). All strains were susceptible to vancomycin. The DNA from the bacteria was obtained by means of physical lyses using a french press device. The genes mecA and aph3 IIIa were detected on the staphylococci chromosome and the gene aac(6)Ie + aph(2) was observed either in the chromosome and in the plasmid content of the staphylococci analyzed. Based on the obtained results one can conclude that the methodology used to extract the genomic genetic material using the french press device was efficient and allowed a simple method to detect by PCR and to locate by ultracentrifugation, the staphylococci antibiotic resistance genes.

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RESUMO Este trabalho teve por objetivo analisar o uso da prensa francesa para se adquirir material genético de estafilococos e detectar possíveis genes de resistência cromossomais e plasmidiais aos antimicrobianos oxacilina, gentamicina, canamicina e vancomicina. O método da difusão de discos em ágar foi realizado, inicialmente, para 50 linhagens de estafilococos e a susceptibilidade antimicrobiana foi confirmada por meio de Reação em Cadeia da Polimerase (PCR). Os resultados obtidos pelo antibiograma constataram alta susceptibilidade para gentamicina e canamicina (4%) e oxacilina (8%). Todas as linhagens foram susceptíveis à vancomicina. O DNA bacteriano foi obtido por lise física a partir da prensa francesa. Os genes mecA e aph3IIIa foram detectados no cromossomo dos estafilococos e o gene aac(6) Ie + aph (2") foi observado tanto no cromossomo como no plasmidio destas bactérias. Pelos resultados pode-se concluir que a metodologia utilizada para a extração de DNA genômico, por meio da prensa francesa, foi barata e eficiente, pois possibilitou a detecção por PCR e a localização, por ultracentrifugação, de genes de resistência em estafilococos.