Tuning surface interactions on MgFe2O4 nanoparticles to induce interfacial hyperactivation in Candida rugosa lipase immobilization.

Lipase adsorption on solid supports can be mediated by a precise balance of electrostatic and hydrophobic interactions. A suitable fine-tuning could allow the immobilized enzyme to display high catalytic activity. The objective of this work was to investigate how pH and ionic strength fluctuations affected protein-support interactions during immobilization via physical adsorption of a Candida rugosa lipase (CRL) on MgFe2O5. The highest amount of immobilized protein (IP) was measured at pH 4, and an ionic strength of 90 mM. However, these immobilization conditions did not register the highest hydrolytic activity (HA) in the biocatalyst (CRLa@MgFe2O4), finding the best values also at acidic pH but with a slight shift towards higher values of ionic strength around 110 mM. These findings were confirmed when the adsorption isotherms were examined under different immobilization conditions so that the maximum measurements of IP did not coincide with that of HA. Furthermore, when the recovered activity was examined, a strong interfacial hyperactivation of the lipase was detected towards acidic pH and highly charged surrounding environments. Spectroscopic studies, as well as in silico molecular docking analyses, revealed a considerable involvement of surface hydrophobic protein-carrier interactions, with aromatic aminoacids, especially phenylalanine residues, playing an important role. In light of these findings, this study significantly contributes to the body of knowledge and a better understanding of the factors that influence the lipase immobilization process on magnetic inorganic oxide nanoparticle surfaces.

Interfacial Hyperactivation of Candida rugosa Lipase onto Ca2Fe2O5 Nanoparticles: pH and Ionic Strength Fine-Tuning to Modulate Protein-Support Interactions.

The current study provides a comprehensive look of the adsorption process of Candida rugosa lipase (CRL) on Ca2Fe2O5 iron oxide nanoparticles (NPs). Protein-support interactions were identified across a broad range of pH and ionic strengths (mM) through a response surface methodology, surface charge determination, and spectroscopic and in silico analyses. The maximum quantity of immobilized protein was achieved at an ionic strength of 50 mM and pH 4. However, this condition did not allow for the greatest hydrolytic activity to be obtained. Indeed, it was recorded at acidic pH, but at 150 mM, where evaluation of the recovered activity revealed hyperactivation of the enzyme. These findings were supported by adsorption isotherms performed under different conditions. Based on zeta potential measurements, electrostatic interactions contributed differently to protein-support binding under the conditions tested, showing a strong correlation with experimentally determined immobilization parameters. Raman spectra revealed an increase in hydrophobicity around tryptophan residues, whereas the enzyme immobilization significantly reduced the phenylalanine signal in CRL. This suggests that this residue was involved in the interaction with Ca2Fe2O2 and molecular docking analysis confirmed these findings. Fluorescence spectroscopy showed distinct behaviors in the CRL emission patterns with the addition of Ca2Fe2O5 at pH 4 and 7. The calculated thermodynamic parameters indicated that the contact would be mediated by hydrophobic interactions at both pHs, as well as by ionic ones at pH 4. In this approach, this work adds to our understanding of the design of biocatalysts immobilized in iron oxide NPs.

Nuclear Medicine Departments in the Era of COVID-19.

From the outset of the COVID-19 pandemic we, the nuclear medicine (NM) community, expediently mobilized to enable continuity of essential services to the best of our abilities. For example, we effectuated adapted guidelines for NM standard operating procedures (SOPs) and enacted heightened infection protection measures for staff, patients, and the public, alike. Challenges in radionuclide supply chains were identified and often met. NM procedural volumes declined globally and underwent restoration of varying degrees, contingent upon local contexts. Serial surveys have gauged and chronicled such geographical variance of the impact of COVID-19 on NM service delivery and, though it may be too early to fully understand the long-term consequences of reduced NM services, overall, we can certainly expect that this era adversely affected the management of many patients afflicted with non-communicable diseases. Today we are unquestionably better prepared to face unforeseen outbreaks, but a degree of uncertainty lingers. Which lessons learned will endure in the form of permanent NM pandemic preparedness procedures and protocols? In this spirit, the present manuscript presents a revision of prior recommendations issued mid-pandemic to NM centers, some of which may become mainstays in NM service delivery and implementation. Discussed herein are (1) comparative worldwide survey results of the measurable impact of COVID-19 on the practice of nuclear medicine (2) the definitions of a pandemic and its phases (3) relevant, recently developed or updated guidelines specific to nuclear medicine (4) incidental findings of COVID-19 on hybrid nuclear medicine studies performed primarily for oncologic indications and (5) how pertinent pedagogical methods for medical education, research, and development have been re-invented in a suddenly more virtual world. NM professionals shall indefinitely adopt many of the measures implemented during this pandemic, to enable continuity of essential services while preventing the spread of the virus. Which ones? Practices must remain ready for possible new peaks or variants of the roiling COVID-19 contagion and for the emergence of potential new pathogens that may incite future outbreaks or pandemics. Communications technologies are here to stay and will continue to be used in a broad spectrum of applications, from telemedicine to education, but how best? NM departments must align synergistically with these trends, considering what adaptations to a more virtual professional environment should not only last but be further innovated. The paper aims to provide recent history, analysis, and a springboard for continued constructive dialogue. To best navigate the future, NM must continue to learn from this crisis and must continue to bring new questions, evidence, ideas, and warranted systematic updates to the figurative table.

An Exploratory Study for Assessment of Multimodal Semantic Memory in Colombian Children.

Semantic memory (SM) is a type of long-term memory associated with the storage of general information about the world. Here we assessed the characteristics of the SM battery, developed by Catricalà et al. (2013), in a sample of Colombian children. This battery was originally conceived to evaluate adults, and features six subtests that assess SM in different modalities, using a common set of 48 stimuli in both living and nonliving categories. The design of the current study is of a cross-sectional and exploratory type. The sample was composed of 111 children, 57 boys (51%) and 54 girls (49%), who were 6 (n = 68) and 7 (n = 43) years old and had no intellectual disability. Robust linear regression models and correlation networks were used. We found an effect of age on general intelligence after correcting for gender, and no differences on the six subtest scores after corrections for gender and age were performed. Furthermore, age was found to be positively associated with the naming of colored photographs (ß = .75, p = .039), naming in response to an oral description (ß = 1.81, p = .039), picture sorting at four levels (ß = 7.22, p = .029), and sentence verification (ß = 26.66, p = .01). In addition, there were differences between the results obtained in adults in the original study and in the children of our study. This exploratory study supports the feasibility of the Spanish translation of the Catricalà et al. (2013) battery to assess SM in children with a nonclinical condition. Future studies are needed to evaluate the psychometric properties of this SM battery, and to corroborate and expand our findings in a larger sample of control children, and in children with some degree of intellectual disability or suffering of some neurodegenerative or psychiatric conditions.

La memoria semántica (SM) es un tipo de memoria a largo plazo asociada con el almacenamiento de información general sobre el mundo. Aquí evaluamos las características de la batería SM, desarrollada por Catricalà et al. (2013), en una muestra de niños colombianos. Esta batería fue concebida originalmente para evaluar adultos, y presenta seis subpruebas que evalúan SM en diferentes modalidades, utilizando un conjunto común de 48 estímulos en las categorías de vida y no vida. El diseño del presente estudio es de tipo transversal y exploratorio. La muestra estaba compuesta por 111 niños, 57 niños (51%) y 54 niñas (49%), que tenían 6 (n = 68) y 7 (n = 43 ) años y no tenían discapacidad intelectual. Se utilizaron modelos de regresión lineal robustos y redes de correlación. Encontramos un efecto de la edad en la inteligencia general después de corregir por género, y no hubo diferencias en las seis puntuaciones de la subprueba después de realizar correcciones por género y edad. Además, se encontró que la edad se asociaba positivamente con el nombramiento de fotografías en color (ß = .75, p = .039), nombrando en respuesta a una descripción oral (ß = 1.81, p = .039), clasificación de imágenes en cuatro niveles (ß = 7.22, p = .029) y verificación de oraciones (ß = 26.66, p = .01). Además, hubo diferencias entre los resultados obtenidos en adultos en el estudio original y en los niños de nuestro estudio. Este estudio exploratorio respalda la viabilidad de la traducción al español de Catricalà et al. (2013) batería para evaluar SM en niños con una condición no clínica. Se necesitan estudios futuros para evaluar las propiedades psicométricas de esta batería SM, y para corroborar y expandir nuestros hallazgos en una muestra más grande de niños control, y en niños con algún grado de discapacidad intelectual o que padecen algunas condiciones neurodegenerativas o psiquiátricas.

Growth arrest and morphological changes triggered by emodin on Trypanosoma cruzi epimastigotes cultivated in axenic medium.

Emodin is an anthraquinone obtained from Rheum palmatum rootstocks. Here we tested the cytotoxic effects of emodin on Trypanosoma cruzi epimastigotes, as well as the morphological changes that were induced by this compound in the parasite. Emodin was permeable and blocked in vitro cell division of T. cruzi epimastigotes in axenic medium, causing growth arrest in a dose-dependent but reversible manner. Emodin-exposed epimastigotes underwent duplication of organelles, such as the nucleus, kinetoplast and flagellum, but were incapable of completing cytokinesis. Neither elongation of the parasite body nor appearance of the regular longitudinal cleavage furrow was displayed, suggesting that emodin is most likely affecting components of the parasite cytoskeleton. Moreover, drug-treated parasites acquired alterations such as protuberances, folds and indentations on their membrane surface. Since emodin has been shown to be a potent protein kinase CK2 inhibitor, and we have previously described an association between tubulin and CK2 in T. cruzi epimastigotes (De Lima et al. Parasitology132, 511-523, 2006), we also measured the indirect effect of the drug on tubulin. Incubation of epimastigotes with axenic medium containing emodin hindered the endogenous phosphorylation of tubulin in whole-cell parasite extracts. All our results suggested that the parasite CK2 may be important for the maintenance of the morphology and for the regulation of mitosis-cytokinesis transition in T. cruzi epimastigotes.

Cambios en la mucosa nasal de los médicos por exposición al humo por electrocoagulación / Changes in the nasal mucosa of physicians due to exposure to smoke from electrocoagulation / Mudanças na mucosa nasal por exposição à fumaça por electrocoagulação

Objetivo: demostrar que la exposición al humo, producto de la electrocoagulación, origina cambios en la mucosa nasal en médicos en formación de un hospital público en México. Metodología: se realizó un estudio de cohorte fija prospectivo, cuyo universo de trabajo estuvo conformado por un total de 43 médicos, 20 corresponden a médicos de especialidades no quirúrgicas (no expuestos a la inhalación de humo del cauterio) y 23 médicos de especialidades quirúrgicas (expuestos a la inhalación de humo del cauterio), a quienes se les realizó una biopsia nasal al inicio y otra al finalizar los 4 años de su formación como especialistas. Las biopsias fueron revisadas por el Jefe de Patología del hospital, se calculó incidencia de cambios en la mucosa nasal, en los grupos expuesto y no expuesto, índice de exposición y riesgo relativo. Resultados: el total de los médicos especialistas en formación incluidos en el estudio, presentaron biopsia sin daños en la mucosa nasal al inicio del estudio; mismos que al término de sus 4 años de especialidad presentaron lo siguiente: el 70% de los médicos residentes expuestos tuvieron algún cambio histopatológico en la mucosa nasal (hiperplasia o metaplasia escamosa), mientras que solo el 5% (1/20) de los no expuestos lo presentó; el factor de riesgo de presentar daño a la mucosa nasal por la exposición en estudio se calculó en 13,8. Las lesiones más frecuentes por la exposición al humo producido por la electrocoagulación fueron la hiperplasia y la metaplasia escamosa. Conclusiones: nuestros resultados demuestras que los residentes expuestos al humo producido por la electrocoagulación presentan cambios en la mucosa nasal.

Objective: to prove that exposure to smoke resulting from electrocoagulation causes changes in the nasal mucosa of physicians in training at a public hospital in Mexico. Methodology: a prospective fixed cohort study was conducted with a working universe consisting of 43 physicians distributed as follows: a group of 20 professionals with non-surgical specialties (thus unexposed to electrocautery smoke inhalation), and another group of 23 with surgical specialties (thus they were exposed to electrocautery smoke inhalation). They underwent two nasal biopsies: one at the beginning of the study and another after training as specialists for four years. The biopsies were reviewed by the hospital´s chief of Pathology and the incidence of changes in the nasal mucosa in both groups was calculated together with exposure index and the relative risk. Results: the biopsies performed at baseline showed that none of the specialists in training included in this study had damages in the nasal mucosa. The final biopsies, performed after the four-year medical training, had the following results: 70% of the medical residents, who were exposed, showed some histopathological changes in the nasal mucosa (hyperplasia or squamous metaplasia), whereas only 5% (1/20) of the unexposed individuals had them; the risk factor for nasal mucosa damage by exposure was estimated at 13.8. The most common lesions resulting from exposure to smoke from electrocoagulation were hyperplasia and squamous metaplasia. Conclusions: our results demonstrate that residents exposed to smoke produced by electrocoagulation have changes in the nasal mucosa.

Objetivo: Demonstrar que a exposição à fumaça produzida pela electrocoagulação gera mudanças na mucosa nasal de médicos estudantes de um hospital público do México. Metodologia: Realizou-se um estudo prospectivo de coorte fixa, com um universo de trabalho de 43 médicos. 20 médicos de especialidades não cirúrgicas (não expostos à fumaça do cautério), e 23 médicos de especialidades cirúrgicas (expostos à fumaça do cautério). Realizou-se uma biopsia nasal no início e outra no final, após 4 anos da sua formação como especialistas. As biopsias foram realizadas pelo chefe de Patologia do hospital e os resultados foram processados com o programa Social Sciences (SPSS Statistics), versão 18. Foi possível avaliar a incidência das mudanças na mucosa nasal dos grupos expostos e dos grupos não expostos, também se calculou o índice de exposição e o risco relativo. Resultados : Nenhum dos médicos especialistas em formação incluídos no estudo tinha danos na sua mucosa nasal, segundo a biopsia feita no início do estudo. Depois de 4 anos na especialização: 70% dos médicos residentes expostos teve alguma mudança histopatológica na mucosa nasal (hiperplasia o metaplasia escamosa), enquanto que somente 5% (1/20) dos não expostos apresentou mudança. O fator de risco do dano na mucosa nasal pela exposição estudada se calculou em 13,8. Os prejuízos mais frequentes pela exposição à fumaça produzida pela electrocoagulação foram a hiperplasia e a metaplasia escamosa. Conclusão: Nossos resultados demonstram que os residentes expostos à fumaça produzida pela electrocoagulação apresentam mudanças na sua mucosa nasal.

Relación entre cambios en la expresión de proteasas y metaciclogénesis espontánea asociadas a las condiciones de mantenimiento de Trypanosoma cruzi en el laboratorio / Relationship between changes in the expression of proteases and spontaneous metacyclogenesis associated to Trypanosoma cruzi maintenance conditions in the laboratory

La metaciclogénesis de Trypanosoma cruzi es la transformación del estadio epimastigota en tripomastigota metacíciclico que ocurre en la ampolla rectal del insecto vector. Durante este proceso se ha implicado una activación del metabolismo proteico, con probable participación de proteasas en la degradación de proteínas del parásito. En el presente trabajo se determinó el efecto de las condiciones de mantenimiento de T. cruzi en el laboratorio sobre la actividad de proteasas y su relación sobre la metaciclogénesis espontánea en cultivos axénicos. Se trabajó con el clon Dm30L de Trypanosoma cruzi mantenido en el laboratorio mediante pases trimestrales chipo-ratón (condición triatomino: CT) o repiques semanales durante 15 años en medio LITB (condición cultivo: CC). En ambas condiciones, se hizo curva de crecimiento durante 30 días en medio LITB suplementado con 10, 24 y 48 mM glucosa, calculándose el porcentaje de metacíclicos espontáneos y determinación de la actividad de proteasas. En 10 mM glucosa, parásitos de la CT presentaron mayor expresión de proteasas y porcentaje de metacíclicos respecto a la CC. En la CT, el crecimiento en 48 mM glucosa redujo las variables antes mencionadas, mientras que no se evidenció actividad proteolítica ni metaciclogénesis espontánea en parásitos de la CC en altas concentraciones de glucosa. En todos los casos se evidenció mayor actividad de cisteín proteasas durante la fase estacionaria del cultivo. No se reportó actividades metaloproteasas en ninguna de las condiciones estudiadas, pero sí en el clon Dm28c utilizado como control de la expresión de este tipo de enzima. Estos resultados sugieren que la expresión de proteasas está influenciada por la constitución genética del parásito y que la CC provoca disminución de la actividad proteolítica que persisten aún en condiciones de agotamiento de carbohidratos del medio, con una concomitante reducción significativa en el porcentaje de metacíclicos espontáneos.

Trypanosoma cruzi metacyclogenesis is the transformation of epimastigote stage to trypomastigote metacyclic that happens in the rectal ampulla of the insect vector. During this process it has been implicated protein metabolism activation, with proteases likely involvement in parasite protein degradation. In this study we determined the effect of T. cruzi keeping conditions in the laboratory on the activity of proteases and their relationship on spontaneous metacyclogenesis in axenic culture. We worked with Trypanosoma cruzi Dm30L clone maintained in the laboratory by quarterly passes triatomine-mouse (triatomine condition: TC) or serial culture media during 15 years in LITB medium (culture condition: CC). In both conditions, growth curve was made for 30 days in LITB medium supplemented with 10, 24 and 48 mM glucose, calculating the percentage of spontaneous metacyclics and determining proteases activity. In 10 mM glucose, TC parasites showed higher expression of proteases and metacyclic percentage relative to the CC. In TC, the growth in 48 mM glucose reduced the above variables, while no evidenced proteolytic activity nor spontaneous metacyclogenesis on CC parasites in high glucose concentrations. In all cases there was greater cysteine proteases activity during the stationary phase of the culture. Metalloprotease activity was not reported in any of the conditions studied, but it was detected in clone Dm28c used as control of expression of this type of enzyme. These results suggest that the expression of proteases is influenced by the genetic makeup of the parasite and the CC causes decrease of proteolytic activity which persists even in carbohydrate depleted conditions of the medium, with a concomitant significant reduction in the percentage of spontaneous metacyclics.

Psychosis induced by the interaction between disulfiram and methylphenidate may be dose dependent.

There are few studies describing psychiatric symptoms occurring when methylphenidate and disulfiram are used together. The authors report a case of disulfiram and methylphenidate interaction in which psychotic symptoms could be dose dependent. The patient, diagnosed of alcohol and cocaine dependence and attention deficit hyperactivity disorder (ADHD), started treatment with methylphenidate increasing doses and disulfiram 250 mg/day over 4 weeks. During the first 2 weeks at doses of 36 mg/day of methylphenidate and maintaining disulfiram, side effects were not observed. However, by increasing to 54 mg/day, psychotic symptoms were detected. The authors reported that the effects are dose dependent. This is the first report about dose-dependent side effects in substance use disorder with ADHD.

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes.

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.

Parasitosis intestinales y su relación con factores socioeconómicos y condiciones de habitat en niños de Neuquén, Patagonia, Argentina / Intestinal parasitosis in relation to socioeconomic factors and habitat conditions in children of Neuquen, Patagonia, Argentina

Se investigó la prevalencia y distribución de parásitos intestinales (PI) en niños de 2 poblaciones de diferente nivel socioeconómico del área periurbana de la ciudad de Neuquén (Sectores I y II) a fin de evaluar su relación con las condiciones de hábitat y factores socio-económicos. Se procesaron muestras seriadas de materia fecal y de escobillado anal de 126 niños entre 2 y 14 años de edad. Se registraron datos acerca de condiciones de hábitat y factores socioeconómicos mediante visitas domiciliarias y encuestas observaciones estructuradas. Se detectó presencia de PI en el 50,7 por ciento de los niños del Sector I (barrio suburbano con adecuadas condiciones sanitarias y nivel socioeconómico medio o medio-bajo) y en el 92,9 por ciento de los niños del Sector II (asentamiento marginal con deficientes condiciones sanitarias y bajo nivel socioeconómico). Se identificaron 7 especies de protozoos intestinales y 4 especies de helmintos. Blastocystis hominis fue la especie más frecuente encontrada en ambas poblaciones. No se encontraron helmintos diferentes de Enterobius vermicularis en el Sector I y la prevalencia de tales especies fue muy baja en el Sector II. Las condiciones de hábitat deficientes y los bajos parámetros socioeconómicos se relacionaron con una mayor prevalencia de PI de transmisión directa como protozoos y E. vermicularis en las poblaciones estudiadas. Sin embargo, aún en ese contexto favorable a la transmisión, las especies parasitarias que requieren estadíos intermedios de maduración en el suelo no encuentran un hábitat adecuado para su diseminación en esta región patagónica.

The prevalence and distribution of intestinal parasites (IP) were investigated in children from two populations of different socioeconomic level, located in the same area of the city of Neuquén, in order to evaluate their relationship with habitat conditions and socioeconomic factors. Serial samples of faeces and anal scraping of 126 children between 2 and 14 years from two sectors of the suburban area of Neuquen (Sector I and Sector II) were analyzed. Data concerning habitat conditions and socioeconomic parameters were obtained by home visits and an observational structured survey. Presence of IP was detected in 50.7% of children from Sector I (suburban neighborhood with adequate sanitary conditions and middle or middle low socioeconomic level) and in 92.9% from children of Sector II (marginal settlement with poor sanitary conditions and low socioeconomic status). Seven intestinal protozoan and 4 helminth species were identified. Blastocystis hominis was the most frequent species found in both populations. No helminths different from Enterobius vermicularis were found in Sector I and the prevalence of such species was very low in Sector II. Deficient habitat conditions and low socioeconomic parameters showed relation with a higher prevalence of IP of direct transmission as protozoan and E.vermicularis in the studied populations. Nevertheless, even in this context favourable to transmission, the parasitic species which require intermediate stages of development in soil, don't find an adequate habitat for dissemination in this region

Producción y purificación de anticuerpos (IgY) a partir de huevos de gallinas inmunizadas con epimastigotas de trypanosoma cruzi / Production and purification of IgY antibodies from eggs laid by hens inmunized with trypanosoma cruzi epimastigotes

No obstante las ventajas de producir anticuerpos en gallinas, no se ha reportado inducción y purificación de anticuerpos contra estadios de Trypanosoma cruzi en el modelo aviario. La inducción de anticuerpos anti-estadios de T. cruzi en gallina puede garantizar la producción de reactivos útiles en diagnóstico y terapéutica de la enfermedad de Chagas. Aquí reportamos la obtención y rápido aislamiento de IGY procedente de yema de huevos de gallinas inmunizadas contra epimastigotas de T. cruzi a los 7, 22 y 61 días post-inmunización. Se compararon tres estrategias de purificación de IgY: el método de la dilución acuosa, el método del polietilén glicol (PEG) y el método del cloroformo-PEG, respecto a un estuche comercial. El método del cloroformo-PEG dio un rendimiento de 6,4 mg/mL de proteínas de yema de huevo de 61 días con una sensibilidad tal que 7 ng de IgY detectaron 1 µg de antígeno (ELISA). Por Western blot, 5 µg/mL de IgY revelaron 11 antígenos diferentes en 8 µg de proteína total de epimastigotas. El análisis por SDS-PAGE demostró que las IgY extraídas contienen dos bandas proteícas dominantes de 70/57 y 37/35 kDa y dos tenues de 81 y 41 kDa, las cuales pueden ser eliminadas por re-precipitación con PEG. Para la extracción de IgY el método del cloroformo-polietilén glicol dio la mejor combinación por facilidad de ejecución y bajo costo. Si bien rindió 50 por ciento menos que el estuche comercial bajo las mismas condiciones, la sensibilidad de los anticuerpos fue 4 veces mayor. Estos resultados evidencian la factibilidad del modelo aviario para producir anticuerpos contra estadios de T. cruzi y muestran la experticia para purificarlos usando una tecnología local de bajo costo

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi.

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37ºC. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37ºC for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90 percent of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes.

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37 degrees C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37 degrees C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi.

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Separación de polipéptidos antigénicos de trypanosoma cruzi usando soluciones de pH diferentes / Separation of antigenic polypeptides of tripanosoma cruzi using defferential solubility in different pHs

Fracciones antigénicas útiles para el diagnóstico de la enfermedad de Chagas son obtenidas utilizando metodologías costosas, por eso es necesario desarrollar tecnologías que reduzcan los costos de producción. Previamente mostramos que los polipéptidos de trypanosoma cruzi se solubilizan diferencialmente en soluciones con valores de pH diferentes. El Propósito en este estudio fue obtener fracciones enriquecidas en polipéptidos de T. cruzi antigénicamente funcionales en base a su solubilidad diferencial. Los perfiles polipeptídicos de los epimastigotas fueron identicados por electroforesis en geles de poliacrilamida con dodecil sulfato de sodio (SDS-PAGE), coloreados por el método de la plata metálica. La antigenicidad de los polipéptidos se estudió mediante Western Bot usando un suero específico anti-epimastigotas. Se encontró que: a) la solubilidad de los polipéptidos no fue afectada por cambios en la fuerza iónica entre 0 y 250 mM NaCl; b) todos los polipéptidos se precipitaron a pH 4,0 mientras que el incremento secuencial del pH de la solución de resuspensión produjo un enriquecimiento diferencial de polipéptidos de mayor peso molecular; c) las fracciones enriquecidas, procedentes de soluciones con valores pH menores que 7,0, mostraron una fuerte actividad proteolítica la cual fue inhibida a pH alcalino y; d) los polipéptidos conservaron su antigenicidad. Estos resultados muestran una metodología sencilla y relativamente económica para producir fracciones antigénicas. Están en progreso experimentos para reducir la actividad proteolítica y obtener polipéptidos útiles para el diagnóstico de la enfermedad de Chagas