Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p>Chagas disease is a neglected illness, with limited treatments, caused by the parasite <it>Trypanosoma cruzi</it>. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by <it>T. cruzi</it>. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action.</p> <p>Results</p> <p>The differential genes expression of a sensitive <it>Trypanosoma cruzi </it>strain and its induced <it>in vitro </it>benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC₅₀ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ.</p> <p>Conclusion</p> <p>Here, we propose NTR as the major marker of the appearance of resistance to BZ.</p

High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate

Evaluación del impacto social de la Licenciatura en Educación Especial en dos subregiones de Antioquia, Colombia

This research paper presents the results of the evaluation of the social impact of the Special Education undergraduate program, of the University of Antioquia’s Education Faculty, in the East and Southwest subregions of the Antioquia department (Colombia). The main objective was to evaluate the impact of the Program among the graduates and other audiences. The methodology was carried out under a qualitative design, in the evaluative research modality, Social Programs impact type of evaluation. The results of the research were structured under the following categories: Assessment of the quality of the Program, the role of the Special Educator, significant experiences and their impact, and location and working conditions of the graduates. Finally, it poses recommendations to various academic units of the University.Keywords: special education, evaluation, social impact, graduates, occupational performance, assessment.En el presente artículo se dan a conocer los resultados de la evaluación del impacto social del programa de Licenciatura en Educación Especial, Facultad de Educación de la Universidad de Antioquia, en las subregiones de Oriente y Suroeste del departamento de Antioquia (Colombia), cuyo objetivo principal fue evaluar el impacto del Programa desde los egresados y otras audiencias. La metodología se llevó a cabo bajo un diseño cualitativo, modalidad de investigación evaluativa, tipo de evaluación de impacto de programas sociales. Los resultados se estructuraron bajo las categorías: valoración de la calidad del Programa, rol del educador especial, experiencias significativas y su impacto, y ubicación y condiciones laborales de los egresados y egresadas. Finalmente, se plantean recomendaciones a diferentes unidades académicas de la Universidad

Molecular surveillance and phylogenetic traits of Babesia bigemina and Babesia bovis in cattle (Bos taurus) and water buffaloes (Bubalus bubalis) from Colombia

Abstract Background Babesia bigemina and B. bovis are two economically important hemoparasites affecting both cattle and buffaloes involved in dairy and beef production. In Colombia, although some parasitological and serological studies suggest an endemicity of these pathogens in areas under 1000 m, little is known about its molecular prevalence in different host. The objective of this study was to estimate the prevalence and molecular traits of these parasites in cattle and buffaloes from two Colombian regions. Methods Between 2014 and 2016, a three-point longitudinal survey was designed in farms from Caribbean and Orinoquia regions to evaluate the molecular prevalence of B. bigemina and B. bovis using a nested PCR (n-PCR) targeting hypothetical protein (hyp) and rhoptry-associated protein (RAP-1) genes, respectively. A total of 1432 cattle, 152 buffalo and 1439 Rhipicephalus microplus samples were analyzed. Moreover, phylogenetic relationship of isolates was analyzed using the 18S rRNA gene. Results A molecular prevalence of 31.6% (24.2% for B. bigemina and 14.4% for B. bovis), 23.6% (6.5% for B. bigemina and 17.7% for B. bovis) and 4.3% (3.5% for B. bigemina and 1.0% for B. bovis) was observed in cattle, buffaloes and Rhipicephalus microplus, respectively. Higher values of infection were observed during the wet season and late wet season; nevertheless, other variables such as age, production type, sex, breed and babesiosis control were also significantly associated with infection. Prevalence analysis showed that B. bovis infection was higher in cattle that coexist with buffaloes, when compared to those which did not. For each species, phylogenetic analyses revealed a high genetic diversity of isolates without clusters related to the isolation source. Conclusions To our knowledge, this is the first longitudinal survey that evaluates through molecular methods, the infection of B. bigemina and B. bovis in two important livestock regions from Colombia. This study reveals that the prevalence of infection by Babesia spp., in cattle and buffaloes are modulated by seasonal variations, host factors and vector traits. Our results provide new insights on the epidemiological aspects of infection of Babesia spp., in cattle and buffaloes, which must be taken into consideration when babesiosis control programs are implemented in the study area

Efecto del inhibidor de fosfodiesterasa tipo 4 -rolipram, sobre la maduración in vitro de oocitos bovinos

Gonadotropic follicle stimulating hormone (FSH) and lutenizing hormone (LH) induce intracellular cyclic AMP (cAMP) production during the in vitro maturation (IVM) of bovine oocytes. Cyclic AMP exerts a dual effect, where high intraoocyte cAMP levels are responsible for oocyte meiotic blockage, while high cAMP levels into the granulose cells induce oocyte maturation. Intracellular cAMP levels are regulated by phosphodiesterases (PDE)-mediated hydrolysis, enzymes having a specific follicle expression pattern. Oocyte expresses typo 3 PDE (PDE 3), while granulose cells expresses type 4 PDE (PDE 4). With the aim to test the effect of the specific PDE 4 inhibitor rolliprom on percentage in vitro nuclear maturation (IVNM) of bovine oocytes, 629 cumulus-oocyte complexes (COC) were cultured at 38.5 °C/CO2 5%/24 h on TCM-199 medium with pFSH and hrLH with or without rolipram. Experimental groups were: gonadotrophins alone, gonadotropins + rolipram (25, 50, or 75 µM), rolipram 50 µM + gonadotrophins, and control (media without stimulus). In order to determinate the nuclear maturation percentage by the first polar body expulsion, oocytes were dyed with DAPI and evaluated by fluorescence. Rolipram 50 µM stimulated bovine oocyte nuclear maturation in a similar way to gonadotrophins stimulus (76.83 vs. 79.46%, p>0.05) did, but in a higher way than rolipram 25 µM (31.25%) or 75 µM (28.61%). The COC cultured with rolipram 50 µM+gonadotrophins maturated in a lower proportion (63.74%) than did with gonadotropins (p0.05), porém em maior medida que a observada com rolipram 25 y 75 µM (31.25, y 28.61%, respectivamente). Os CCOs cultivados na presença de rolipram 50 µM+Gonadotrofinas maturaram em menor proporção (63.74%) quando comparado com gonadotrofinas (p0.05), pero en mayor medida que la observada con rolipram 25 y 75 µM (31.25, y 28.61%, respectivamente). Los CCOs cultivados en presencia de rolipram 50 µM+Gonadotropinas maduraron en menor proporción (63.74%) comparada con gonadotropinas (p<0.01) o rolipram 50 µM (p<0.05). Los resultados permiten concluir que el porcentaje de maduración nuclear in vitro de oocitos bovinos depende de la dosis de rolipram utilizada, donde la concentración de rolipram 50 µM presentó un comportamiento similar a las gonadotropinas en la maduración del oocito. Además, la presencia del estímulo gonadotrópico y del inhibidor de PDE puede inducir una respuesta de desensibilización de la vía del AMPc, reflejada en una disminución del porcentaje de maduración

Efecto del inhibidor de fosfodiesterasa tipo 4 -rolipram, sobre la maduración in vitro de oocitos bovinos Efeito do inibidor de fosfodiesterasa tipo 4 -rolipram, sobre a maturação in vitro de oocitos bovinos Effect of phosphodiesterase type 4-inhibitor rolipram on in vitro maturatrion of bovine oocytes

Durante el proceso de maduración in vitro de oocitos, las gonadotropinas FSH y LH inducen la producción de AMPc. El AMPc tiene efecto dual, donde los altos niveles de AMPc intraoocitario mantienen su bloqueo meiótico, mientras que en las células de la granulosa inducen la maduración del oocito. Los niveles de AMPc son regulados por hidrólisis mediada por fosfodiesterasas (PDE), las cuales presentan expresión específica en el folículo, el oocito expresa la PDE 3, mientras que las células de la granulosa PDE 4. Con el objetivo de evaluar el efecto del rolipram, un inhibidor específico de PDE 4, sobre el porcentaje de maduración nuclear in vitro (MNIV) de oocitos bovinos, 629 complejos cúmulo oocito (CCO) fueron cultivados a 38.5 °C/5% CO2/24 h, en medio TCM-199 con la adición de pFSH y hrLH, o rolipram. Los grupos experimentales fueron: adición de gonadotropinas, rolipram (25, 50 ó 75 µM), rolipram 50 µM + gonadotropinas, o control sin estímulo. Los oocitos fueron teñidos con DAPI y evaluados bajo fluorescencia para determinar el porcentaje de maduración nuclear por la expulsión del primer cuerpo polar. El rolipram 50µM estimuló la maduración nuclear de oocitos bovinos de una manera similar a la obtenida con las gonadotropinas (76.83 vs 79.46%, p>0.05), pero en mayor medida que la observada con rolipram 25 y 75 µM (31.25, y 28.61%, respectivamente). Los CCOs cultivados en presencia de rolipram 50 µM+Gonadotropinas maduraron en menor proporción (63.74%) comparada con gonadotropinas (p<0.01) o rolipram 50 µM (p<0.05). Los resultados permiten concluir que el porcentaje de maduración nuclear in vitro de oocitos bovinos depende de la dosis de rolipram utilizada, donde la concentración de rolipram 50 µM presentó un comportamiento similar a las gonadotropinas en la maduración del oocito. Además, la presencia del estímulo gonadotrópico y del inhibidor de PDE puede inducir una respuesta de desensibilización de la vía del AMPc, reflejada en una disminución del porcentaje de maduración.<br>Durante o processo de maturação in vitro de oocitos, as gonadotrofinas FSH e LH induzem a produção de AMPc. O AMPc tem duplo efeito, pois os altos níveis de AMPc intraoocitario mantém o bloqueio meiótico, enquanto que nas células da granulosa induzem a maturação do oocito. Os níveis de AMPc são regulados pela hidrólise mediada das fosfodiesterasas (PDE), as quais apresentam expressão específica no folículo, o oocito expressa a PDE 3, enquanto que as células da granulosa PDE 4. Com o objetivo de avaliar o efeito do rolipram, um inibidor específico de PDE 4, sobre a percentagem de maturação nuclear in vitro (MNIV) de oocitos bovinos, 629 complexos cúmulo oocito (CCO) foram cultivados a 38.5 °C/5% CO2/24 h, em meio TCM-199 com a adição de pFSH e hrLH, o rolipram. Os grupos experimentais foram: adição de gonadotrofinas, rolipram (25, 50 ó 75 µM), rolipram 50 µM + gonadotrofinas, ou controle sem estímulo. Os oocitos foram tingidos com DAPI e avaliados sob fluorescência para determinar a percentagem de maturação nuclear pela expulsão do primeiro corpo polar. O rolipram 50µM estimulou a maturação nuclear de oocitos bovinos de maneira similar a obtida com as gonadotrofinas (76.83 vs 79.46%, p>0.05), porém em maior medida que a observada com rolipram 25 y 75 µM (31.25, y 28.61%, respectivamente). Os CCOs cultivados na presença de rolipram 50 µM+Gonadotrofinas maturaram em menor proporção (63.74%) quando comparado com gonadotrofinas (p<0.01) ou rolipram 50 µM (p<0.05). Os resultados permitem concluir que a percentagem de maturação nuclear in vitro de oocitos bovinos depende da doses de rolipram utilizada, sendo que a concentração de rolipram 50 µM apresentou um comportamento similar às gonadotrofinas na maturação do oocito. Ale disto, a presença do estímulo gonadotrópico e do inibidor de PDE podem induzir uma resposta de diminuição na sensibilização da via do AMPc, refletida numa diminuição da percentagem de maturação.<br>Gonadotropic follicle stimulating hormone (FSH) and lutenizing hormone (LH) induce intracellular cyclic AMP (cAMP) production during the in vitro maturation (IVM) of bovine oocytes. Cyclic AMP exerts a dual effect, where high intraoocyte cAMP levels are responsible for oocyte meiotic blockage, while high cAMP levels into the granulose cells induce oocyte maturation. Intracellular cAMP levels are regulated by phosphodiesterases (PDE)-mediated hydrolysis, enzymes having a specific follicle expression pattern. Oocyte expresses typo 3 PDE (PDE 3), while granulose cells expresses type 4 PDE (PDE 4). With the aim to test the effect of the specific PDE 4 inhibitor rolliprom on percentage in vitro nuclear maturation (IVNM) of bovine oocytes, 629 cumulus-oocyte complexes (COC) were cultured at 38.5 °C/CO2 5%/24 h on TCM-199 medium with pFSH and hrLH with or without rolipram. Experimental groups were: gonadotrophins alone, gonadotropins + rolipram (25, 50, or 75 µM), rolipram 50 µM + gonadotrophins, and control (media without stimulus). In order to determinate the nuclear maturation percentage by the first polar body expulsion, oocytes were dyed with DAPI and evaluated by fluorescence. Rolipram 50 µM stimulated bovine oocyte nuclear maturation in a similar way to gonadotrophins stimulus (76.83 vs. 79.46%, p>0.05) did, but in a higher way than rolipram 25 µM (31.25%) or 75 µM (28.61%). The COC cultured with rolipram 50 µM+gonadotrophins maturated in a lower proportion (63.74%) than did with gonadotropins (p<0.01) or rolipram 50 µM (p<0.05). A dose-dependent response of percentage of IVNM of bovine oocytes was detected. Thus rolipram 50 µM, exerts a similar effect of gonadotropins on oocyte maturation. In addition, the presence of gonadotropic stimulus and a PDE inhibitor may induce a desensitization response of the cAMP way, as suggested by the reduction of the percentage of maturation

Aldo-keto reductase and alcohol dehydrogenase contribute to benznidazole natural resistance in Trypanosoma cruzi

The improvement of Chagas disease treatment is focused not only on the development of new drugs but also in understanding mechanisms of action and resistance to drugs conventionally used. Thus, some strategies aim to detect specific changes in proteins between sensitive and resistant parasites and to evaluate the role played in these processes by functional genomics. In this work, we used a natural Trypanosoma cruzi population resistant to benznidazole, which has clones with different susceptibilities to this drug without alterations in the NTR I gene. Using 2DE-gel electrophoresis, the aldo-keto reductase and the alcohol dehydrogenase proteins were found up regulated in the natural resistant clone and therefore their possible role in the resistance to benznidazole and glyoxal was investigated. Both genes were overexpressed in a drug sensitive T. cruzi clone and the biological changes in response to these compounds were evaluated. The results showed that the overexpression of these proteins enhances resistance to benznidazole and glyoxal in T. cruzi. Moreover, a decrease in mitochondrial and cell membrane damage was observed, accompanied by a drop in the intracellular concentration of reactive oxygen species after treatment. Our results suggest that these proteins are involved in the mechanism of action of benznidazole.Fil: González, Laura. Universidad de Antioquia; ColombiaFil: García Huertas, Paola. Universidad de Antioquia; ColombiaFil: Triana Chávez, Omar. Universidad de Antioquia; ColombiaFil: Garcia, Gabriela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Murta, Silvane Maria Fonseca. Centro de Pesquisas Rene Rachou; BrasilFil: Mejía Jaramillo, Ana M.. Universidad de Antioquia; Colombi

Amplification results from field samples (Table S1) showing one or two peaks in the melting curve.

<p>Tm1: melting temperature of peak 1, Tm2: melting temperature of peak 2, SD: standard deviation, HRM %C: confidence percentage of HRM analysis, ND: not determined or not recognized, -: No secondary peak.</p

Source of the sample of the standard species used in the HRM analysis.

<p>-: sample not used.</p

Blastn results of ten insects collected from the field.

<p>For each sample the identity with the standard species used for HRM identification is shown.</p