Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi.

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment. Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques. We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

Update on the Biological Relevance of Lysine Acetylation as a Novel Drug Target in Trypanosomatids.

The number of acetylated proteins identified from bacteria to mammals has grown exponentially in the last ten years, and it is now accepted that acetylation is a key component in most eukaryotic signaling pathways and is as important as phosphorylation. The enzymes involved in this process are well described in mammals; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids, several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this review article, we present an update of the most important known acetylated proteins in trypanosomatids, analyzing the acetylomes available. Also, we summarize the information available regarding acetyltransferases and deacetylases in trypanosomes and their potential use as chemotherapeutic targets.

Separation and enrichment of sodium-motile bacteria using cost-effective microfluidics.

Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use of synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied example is the use of the sodium-powered PomA-PotB chimeric stator unit in the naturally proton-powered Escherichia coli. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eightfold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement the selection of highly motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems.

Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression.

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

Evolution of the Stator Elements of Rotary Prokaryote Motors.

The bacterial flagellar motor is driven by an ion flux that is converted to torque by motor-attendant complexes known as stators. The dynamics of stator assembly around the motor in response to external stimuli have been the subject of much recent research, but less is known about the evolutionary origins of stator complexes and how they select for specific ions. Here, we review the latest structural and biochemical data for the stator complexes and compare these with other ion transporters and microbial motors to examine possible evolutionary origins of the stator complex.

Fibrobroncoscopía en pacientes con hemoptisis no masiva y radiografía de tórax más tomografía de tórax no localizadoras / Fiberoptic Bronchoscopy in Patients with non Massive Haemoptysis and non Explainable Images in Chest Radiography and Tomography

Introducción: La necesidad de la fibrobroncoscopía (FBC) en pacientes con hemoptisis y radiografía de tórax (RxTx) no localizadora (NL) es todavía controversial, aunque en general continúa siendo el procedimiento recomendado como rutina de evaluación. Objetivo: Determinar la rentabilidad diagnóstica de la FBC en pacientes con hemoptisis y RxTxNL. Material y métodos: Se analizaron en forma retrospectiva los registros estandarizados de 340 FBC realizadas en forma programada entre enero de 2008 y diciembre 2012 en un único centro. La hemoptisis fue clasificada como trivial (esputo hemoptoico), leve (menos de 100 ml en 24 horas) y moderada (100-500 mL en 24 horas). El procedimiento se realizo en los primeros 7 días de hemoptisis, y se consideraba positiva si se obtenía el diagnóstico final. Resultados: De 396 pacientes, 102 (25,7%) tenían indicación de fibrobroncoscopia por hemoptisis, de los cuales, 52 presentaba Rx de Tórax no localizadora. En el 42.3% de los casos, la tomografía proveyó información diagnóstica adicional. 30 pacientes (29,4% de todos los casos de hemoptisis) presentaron radiografía y tomografía no localizadora. En un paciente el hallazgo broncoscópico fue un carcinoma bronquial no sospechado. 7 pacientes tenían hallazgos endobronquiales benignos. Todos los pacientes que presentaron hallazgos positivos en la fibrobroncoscopía eran tabaquistas, mayores de 40 años, tenían alguna sospecha clínica previa o presentaban hemoptisis por más de 1 semana. Todos los pacientes asintomáticos, menores de 40 años y con hemoptisis menor a los 7 días, la fibrobroncoscopía fue normal. Conclusión: En los pacientes con hemoptisis, mayores de 40 años, con antecedentes de tabaquismo y síntomas constitucionales se obtiene una alta rentabilidad diagnóstica cuando se asocian la Tomografía computada de tórax y la FBC, pero pueden evitarse en ausencia de estas condiciones o si existe la sospecha clínica de otras causas identificables.

Introduction: The exact role of fiberoptic bronchoscopy (FOB) in the diagnosis of patients presenting with haemoptysis and with normal or non-localizing chest radiography and CT scan has not been clearly defined. Objective: The aim of this study was to examine the yield of FOB in patients with haemoptysis and normal findings on chest roentgenogram and CT scan. Methods: We reviewed the medical records of all consecutive patients undergoing elective flexible bronchoscopy between January 2008 and December 2012. All patients underwent FOB within 7 days of active bleeding. Amount of bleeding was classified as trivial (drops of blood, bloody sputum), mild < 100 mL in 24 h and moderate 100-500 mL in 24 h. Bronchoscopy was considered positive if it led to the final diagnosis. We also assessed whether the bronchoscopic evaluation located the bleeding site. Results: Amongst 396 patients examined with FOB, in 102 patients (25.7%) FOB was prescribed for evaluation of haemoptysis; in 52 patients with haemoptysis, the chest radiography was normal. In 42.3% of these patients, CT scan provided additional diagnostic information. In 30 patients (29.4% of all the haemoptysis cases), CXR and CT did not show images which could explain the haemoptysis . Bronchoscopy findings showed an unsuspected bronchial carcinoma in one patient. In seven patients FOB disclosed some benign abnormality. All patients with a positive finding in the FOB were smokers, older than 40 years, had some clinical suspicion and had suffered haemoptysis for more than one week. FOB was normal in asymptomatic patients, who were younger than 40 years and with hemoptysis lasting less than one week. Conclusion: In patients older than 40 years, smokers, with constitutional symptoms or with hemoptysis lasting for more than one week the combined use of bronchoscopy and chest CT gives the best diagnostic yield. In younger, asymptomatic, non-smoking patients with haemoptysis and normal CT scan, further investigations may not be justified, but these results need to be confirmed in a prospective multicenter study.

Fibrobroncoscopía en pacientes con hemoptisis no masiva y radiografía de tórax más tomografía de tórax no localizadoras

Introducción: La necesidad de la fibrobroncoscopía (FBC) en pacientes con hemoptisis y radiografía de tórax (RxTx) no localizadora (NL) es todavía controversial, aunque en general continúa siendo el procedimiento recomendado como rutina de evaluación. Objetivo: Determinar la rentabilidad diagnóstica de la FBC en pacientes con hemoptisis y RxTxNL. Material y métodos: Se analizaron en forma retrospectiva los registros estandarizados de 340 FBC realizadas en forma programada entre enero de 2008 y diciembre 2012 en un único centro. La hemoptisis fue clasificada como trivial (esputo hemoptoico), leve (menos de 100 ml en 24 horas) y moderada (100-500 mL en 24 horas). El procedimiento se realizo en los primeros 7 días de hemoptisis, y se consideraba positiva si se obtenía el diagnóstico final. Resultados: De 396 pacientes, 102 (25,7

) tenían indicación de fibrobroncoscopia por hemoptisis, de los cuales, 52 presentaba Rx de Tórax no localizadora. En el 42.3

de los casos, la tomografía proveyó información diagnóstica adicional. 30 pacientes (29,4

Introduction: The exact role of fiberoptic bronchoscopy (FOB) in the diagnosis of patients presenting with haemoptysis and with normal or non-localizing chest radiography and CT scan has not been clearly defined. Objective: The aim of this study was to examine the yield of FOB in patients with haemoptysis and normal findings on chest roentgenogram and CT scan. Methods: We reviewed the medical records of all consecutive patients undergoing elective flexible bronchoscopy between January 2008 and December 2012. All patients underwent FOB within 7 days of active bleeding. Amount of bleeding was classified as trivial (drops of blood, bloody sputum), mild < 100 mL in 24 h and moderate 100-500 mL in 24 h. Bronchoscopy was considered positive if it led to the final diagnosis. We also assessed whether the bronchoscopic evaluation located the bleeding site. Results: Amongst 396 patients examined with FOB, in 102 patients (25.7

) FOB was prescribed for evaluation of haemoptysis; in 52 patients with haemoptysis, the chest radiography was normal. In 42.3

of these patients, CT scan provided additional diagnostic information. In 30 patients (29.4

de todos los casos de hemoptisis) presentaron radiografía y tomografía no localizadora. En un paciente el hallazgo broncoscópico fue un carcinoma bronquial no sospechado. 7 pacientes tenían hallazgos endobronquiales benignos. Todos los pacientes que presentaron hallazgos positivos en la fibrobroncoscopía eran tabaquistas, mayores de 40 años, tenían alguna sospecha clínica previa o presentaban hemoptisis por más de 1 semana. Todos los pacientes asintomáticos, menores de 40 años y con hemoptisis menor a los 7 días, la fibrobroncoscopía fue normal. Conclusión: En los pacientes con hemoptisis, mayores de 40 años, con antecedentes de tabaquismo y síntomas constitucionales se obtiene una alta rentabilidad diagnóstica cuando se asocian la Tomografía computada de tórax y la FBC, pero pueden evitarse en ausencia de estas condiciones o si existe la sospecha clínica de otras causas identificables.(AU)

of all the haemoptysis cases), CXR and CT did not show images which could explain the haemoptysis . Bronchoscopy findings showed an unsuspected bronchial carcinoma in one patient. In seven patients FOB disclosed some benign abnormality. All patients with a positive finding in the FOB were smokers, older than 40 years, had some clinical suspicion and had suffered haemoptysis for more than one week. FOB was normal in asymptomatic patients, who were younger than 40 years and with hemoptysis lasting less than one week. Conclusion: In patients older than 40 years, smokers, with constitutional symptoms or with hemoptysis lasting for more than one week the combined use of bronchoscopy and chest CT gives the best diagnostic yield. In younger, asymptomatic, non-smoking patients with haemoptysis and normal CT scan, further investigations may not be justified, but these results need to be confirmed in a prospective multicenter study.(AU)

An operative dengue risk stratification system in Argentina based on geospatial technology.

Based on an agreement between the Ministry of Health and the National Space Activities Commission in Argentina, an integrated informatics platform for dengue risk using geospatial technology for the surveillance and prediction of risk areas for dengue fever has been designed. The task was focused on developing stratification based on environmental (historical and current), viral, social and entomological situation for >3,000 cities as part of a system. The platform, developed with open-source software with pattern design, following the European Space Agency standards for space informatics, delivers two products: a national risk map consisting of point vectors for each city/town/locality and an approximate 50 m resolution urban risk map modelling the risk inside selected high-risk cities. The operative system, architecture and tools used in the development are described, including a detailed list of end users' requirements. Additionally, an algorithm based on bibliography and landscape epidemiology concepts is presented and discussed. The system, in operation since September 2011, is capable of continuously improving the algorithms producing improved risk stratifications without a complete set of inputs. The platform was specifically developed for surveillance of dengue fever as this disease has reemerged in Argentina but the aim is to widen the scope to include also other relevant vector-borne diseases such as chagas, malaria and leishmaniasis as well as other countries belonging to south region of Latin America.

[Interstitial lymphoid pneumonia associated with common variable immunodeficiency]. / Neumonía intersticial linfoidea asociada a inmunodeficiencia común variable.

The interstitial lymphoid pneumonia (LIP) is an uncommon disorder, described as non-neoplastic lung lymphoid tissue hyperplasia and classified as an interstitial lung disease. It has been described in association with HIV infection, autoimmune disorders, policlonal hypergammaglobulinemia and less frequently, with hypogammaglobulinemia. We report the case of a 66 year old female patient with a history of diabetes, Sjogren syndrome and hypertension. She was referred to our hospital due to a dry cough and dyspnea (FC II-III). The physical examination showed bilateral dry crackles and splenomegaly. Laboratory studies showed thrombocytopenia (50 000/ mm3) and hypogammaglobulinemia. A computed tomography thoracic scan showed interstitial bilateral basal lung infiltrates with small peribroncovascular nodules. A lung biopsy was made by thoracoscopy and reported LIP. Initial treatment consisted of oral methilprednisone, 40 mg daily, and once a month intravenous gammaglobulin 500 mg/kg, with good clinical and radiological response. A splenectomy was done due to persistent thrombocytopenia associated with hypersplenism, with good response of the platelets counts. No lymphoid clonal or other associated disease was detected.

Neumonía intersticial linfoidea asociada a inmunodeficiencia común variable / Interstitial lymphoid pneumonia associated with common variable immunodeficiency

La neumonía intersticial linfoidea (NIL) es una rara entidad, descripta como hiperplasia no neoplásica del tejido linfoide pulmonar y clasificada dentro de las enfermedades intersticiales idiopáticas. Se la ha descripto asociada a infección por HIV, fenómenos autoinmunes, hipergamaglobulinemia policlonal o menos frecuentemente a hipogammaglobulinemia. Presentamos una paciente de 66 años de edad con diabetes mellitus, síndrome de Sjögren e hipertensión arterial, derivada a nuestro centro por tos seca y disnea clase funcional II-III. En el examen físico presentaba rales tipo “velcro” bibasales y esplenomegalia. Los estudios de laboratorio evidenciaron plaquetopenia (50 000/mm³) e hipogammaglobulinemia; la tomografía axial computarizada (TAC) de tórax mostró compromiso intersticial bibasal, con nódulos peribroncovasculares menores a un centímetro. Se realizó una videotoracoscopia con biopsia pulmonar, la anatomía patológica mostró hallazgos compatibles con neumonía intersticial linfoidea. Se inició tratamiento con metilprednisona 40 mg/día vía oral e infusión endovenosa de gammaglobulina 500 mg/kg, mensual, evolucionando con mejoría clínico-radiológica. Por persistencia de plaquetopenia, asociada a hiperesplenismo, se realizó esplenectomía con buena respuesta. En la anatomía patológica no se observo clonalidad linfoide.

The interstitial lymphoid pneumonia (LIP) is an uncommon disorder, described as non-neoplastic lung lymphoid tissue hyperplasia and classified as an interstitial lung disease. It has been described in association with HIV infection, autoimmune disorders, policlonal hypergammaglobulinemia and less frequently, with hypogammaglobulinemia. We report the case of a 66 year old female patient with a history of diabetes, Sjögren syndrome and hypertension. She was referred to our hospital due to a dry cough and dyspnea (FC II-III). The physical examination showed bilateral dry crackles and splenomegaly. Laboratory studies showed thrombocytopenia (50 000/ mm³) and hypogammaglobulinemia. A computed tomography thoracic scan showed interstitial bilateral basal lung infiltrates with small peribroncovascular nodules. A lung biopsy was made by thoracoscopy and reported LIP. Initial treatment consisted of oral methilprednisone, 40 mg daily, and once a month intravenous gammaglobulin 500 mg/kg, with good clinical and radiological response. A splenectomy was done due to persistent thrombocytopenia associated with hypersplenism, with good response of the platelets counts. No lymphoid clonal or other associated disease was detected.