Mejora e innovación en los procedimientos de detección y cuantificación de los métodos de análisis de toxinas PSP

La presente Tesis Doctoral se centra en el grupo de toxinas paralizantes (PSP). Se evaluó la eficacia del método de cromatografía líquida de alta eficacia con detección por fluorescencia (HPLC-FLD) y oxidación post-columna (PCOX) cuando se aplica a diferentes especies marinas. Además se realizó una comparación entre los dos métodos HPLC-FLD validados por la AOAC (pre-columna y post-columna) en muestras reales. Por otra parte se presentan dos nuevos métodos basados en el uso de una columna de carbono grafitizado poroso; uno HPLC-FLD con oxidación post-columna y otro de cromatografía líquida con detección por espectrometría de masas (LC-MS/MS), junto con un nuevo proceso de limpieza de muestras

Determination of Gonyautoxin-4 in Echinoderms and Gastropod Matrices by Conversion to Neosaxitoxin Using 2-Mercaptoethanol and Post-Column Oxidation Liquid Chromatography with Fluorescence Detection

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction stepThis research was partially funded by the Portuguese Fundation of Science and Technology (FCT) project UID/Multi/04423/2013 and by the projects MARBIOTECH (reference NORTE-07-0124-FEDER-000047) within the Scientific Resaerch and Technological Development (SR&TD) Integrated Program. MARVALOR—Building research and innovation capacity for improved management and valorizationof marine resources, supported by the Programa Operacional Regional do Norte (ON.2-O Novo Norte) and NOVOMAR (reference 0687-NOVOMAR-1-P), supported by the European Regional Development Fund. Marisa Silva also acknowledges FCT for the grant SFRH/BD/73269/2010. The spanish research leading to these results has received funding from the following European Fund for Economic and Regional Development (FEDER) cofunded-grants. From Centro para el Desarrollo Tecnológico Industrial (CDTI) and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under India&Spain Innovating Program (ISIP) Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEAS

Influence of Different Shellfish Matrices on the Separation of PSP Toxins Using a Postcolumn Oxidation Liquid Chromatography Method

The separation of PSP toxins using liquid chromatography with a post-column oxidation fluorescence detection method was performed with different matrices. The separation of PSP toxins depends on several factors, and it is crucial to take into account the presence of interfering matrix peaks to produce a good separation. The matrix peaks are not always the same, which is a significant issue when it comes to producing good, reliable results regarding resolution and toxicity information. Different real shellfish matrices (mussel, scallop, clam and oyster) were studied, and it was seen that the interference is not the same for each individual matrix. It also depends on the species, sampling location and the date of collection. It was proposed that separation should be accomplished taking into account the type of matrix, as well as the concentration of heptane sulfonate in both solvents, since the mobile phase varies regarding the matrix. Scallop and oyster matrices needed a decrease in the concentration of heptane sulfonate to separate GTX4 from matrix peaks, as well as dcGTX3 for oysters, with a concentration of 6.5 mM for solvent A and 6.25 mM for solvent B. For mussel and clam matrices, interfering peaks are not as large as they are in the other group, and the heptane sulfonate concentration was 8.25 mM for both solvents. Also, for scallops and oysters, matrix interferences depend not only on the sampling site but also on the date of collection as well as the species; for mussels and clams, differences are noted only when the sampling site variesThe research leading to these results received funding from the following FEDER cofunded-grants: CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX; from CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD; and from the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEAS

Liquid Chromatography with a Fluorimetric Detection Method for Analysis of Paralytic Shellfish Toxins and Tetrodotoxin Based on a Porous Graphitic Carbon Column

The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, Xunta de Galicia Axencia Galega de Innovación, ITC-20133020 SINTOX, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEAS

Anti-Müllerian hormone and inhibin B levels as markers of premature ovarian aging and transition to menopause in type 1 diabetes mellitus

BACKGROUNDSerum anti-Müllerian hormone (AMH) levels decrease early during the transition to menopause and women with type 1 diabetes mellitus (DM1) experience menopause at a younger age. We hypothesized that older women with DM1 will have lower AMH levels than controls.METHODSWe studied ovarian function in women with DM1 (n = 66) and healthy controls (n = 58), all 33 years (4.1 ± 4.2 versus 9.5 ± 7.9 pmol/l, mean ± SD, P = 0.006). A higher proportion of women with DM1 showed AMH levels in the menopausal range compared with controls (16.7 versus 3.4, respectively, P = 0.02). For all patients, those with DM1 exhibited lower inhibin B levels than controls (89.3 ± 51.7 versus 113.2 ± 76.0 ng/ml, P < 0.05). FSH and estradiol were similar in both groups. Regression analysis showed an earlier decline in AMH levels in women with DM1 than controls. Even after age adjustment, DM1 was a significant factor for the determination of inhibin B and AMH levels.CONCLUSIONSLower AMH levels in women with DM1 during the fourth decade of life suggest the presence of an earlier decline in the ovarian follicle pool in these women. Further studies are needed to evaluate the mechanism of this complication.Fil: Soto, Néstor. San Borja Arriarán Clinical Hospital; ChileFil: Iñiguez, Germán. Universidad de Chile; ChileFil: López, Patricia. Universidad de Chile; ChileFil: Larenas, Gladys. Universidad de La Frontera; ChileFil: Mujica, Verónica. Hospital Regional de Talca; Chile. Universidad de Talca; ChileFil: Rey, Rodolfo Alberto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Codner, Ethel. Universidad de Chile; Chil

Análisis de la sección 35 Transición a la NIIF para PYMES en los estados financieros de la entidad Materiales de Construcción El Obrero, S.A al periodo 2018 para el año 2019

Esta investigación consiste en la aplicación de la sección No. 35 “Transición a las NIIF para PYMES” en la Entidad Materiales de Construcción “El Obrero, S.A” del departamento de Managua para el periodo finalizado al 31 de diciembre de 2018, En este trabajo se describe la importancia que tiene el estar actualizados a NIIF para PYMES contablemente y que beneficios este puede traer al negocio y como puede mejorar el mismo. El método utilizado en el trabajo fue el método investigativo con un enfoque de estudio en las normas y el giro del negocio con el fin de que se obtengan los mejores resultados, para esto se solicitó a la empresa se nos brindara la información necesaria para poder aplicar y realizar dicha transición logrando los resultados esperados en el trabajo y de esta manera ayudar a la empresa en tener un mejor manejo de su contabilidad. En Nicaragua la mayor parte de las empresas estaban elaborando y presentando sus Estados Financieros de conformidad con los PCGA los cuales no estaban presentando la realidad de las operaciones en cuanto a medición, reconocimiento, presentación e información a revelar. Las NIIF para PYMES establecen los requerimientos que ayudan a que la información financiera de una empresa muestre la realidad de las operaciones. De acuerdo a la investigación se determina que al estar informados sobre NIIF para PYMES contablemente se obtienen mayores beneficios al reconocer de forma adecuada sus activos y pasivos, presentando sus cifras a un valor razonable y actualizado que trae al negocio tanto para su mejoramiento como su expansión. Con la adopción de las NIIF para PYMES se obtiene información financiera eficaz y eficiente. Este trabajo da pautas a las empresas que no han realizado un cambio o transición para que consideren los puntos a tomar en cuenta al momento de realizarla. Palabras claves: NIIF para PYMES, transición, sección 35, estados financieros, alcance, reconocimient

First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum

During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 103 cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumersThe research leading to USC results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. In addition from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 315285 CIGUATOOLS and 312184 PHARMASEA. Inés Rodriguez is supported by a fellowship from Subprograma de Formación de Personal Investigador (AGL2012-40185-CO2-01), Spain. In depth investigation of the toxic episodes leading to the results and publication of the present work was undertaken by the Greek National Reference Laboratory of Marine Biotoxins (NRLMB) to fulfill the requirements of EU Regulation 178/2002/EC (Articles 6 and 7) regarding risk analysis and communication and scientific information needed for risk assessment and EU Regulation 882/2004/EC (article 7) with regard to transparency and information to the public. Collaboration of all the staff of the NRLMB is greatly appreciated. Thanks are also expressed to all the Greek regional veterinary services for their contribution to the shellfish samplings and for provision of the seawater analyses results for the presence of potentially toxic microalgae. The use of cell counts’ data within the period 2006–2009 and 2012 regarding P. minimum presence in seawater, derived from the Greek “National Programme for Monitoring of Bivalve Molluscs’ Production Areas for the presence of Marine Biotoxins” and conducted by the Laboratory Unit of Toxic Marine Microalgae (LUTMM), Department of Biology, Aristotle University of Thessaloniki (scientific coordinator: G. Nikolaidis (until February 2010) and M. Arsenakis (March 2010–to date)), as well as the restrictions of LUTMM regarding the use of data produced within 2013–2015 due to contract terms and ISO 17025 requirements are acknowledgedS

Efecto protector del azul de metileno en la retinopatía del prematuro experimental

Introducción: La hipoxia-isquemia por asfixia perinatal (AP) es causa de lesiones en la retina, pudiendo llevar a la ceguera por retinopatía proliferativa isquémica (RPI). En estudios previos hemos demostrado neurodegeneración, gliosis y neovascularización compatibles con la ROP, e identificado que el sistema nitrérgico está involucrado en su fisiopatología. Objetivos: Analizar la aplicación de azul de metileno como potencial estrategia terapéutica, dado que inhibe a la enzima óxido nítrico sintasa. Métodos: Se estudió la actividad enzimática y expresión de nNOS mediante Western blot, histoquímica de NADPHd e inmunohistoquímica contra nNOS, en la retina de animales de 30 días en un modelo de AP. Resultados: Se observó que a los 30 días postnatal, la nNOS aumenta significativamente su actividad y expresión en aquellos animales sometidos a AP al ser comparados con el grupo control (CTL). En el grupo AP tratado con azul de metileno (AZM) no se encontraron diferencias significativas en la actividad de NOS constitutiva con respecto al grupo CTL, mientras que su expresión evaluada por western blot fue menor. Los estudios realizados con histoquímica para NADPHd e inmunohistoquímica contra nNOS refuerzan estos resultados. Conclusiones: El NO es un radical libre que participa como neurotóxico en el desarrollo de la ROP por AP. La aplicación de azul de metileno como estrategia terapéutica, tiene un potente efecto bloqueante en la actividad de la enzima NOS constitutiva, y en la expresión de la misma. Este hallazgo alienta estudios futuros más exhaustivos para evaluar el uso del azul de metileno con el fin de atenuar o evitar el daño retiniano.Introduction: Hipoxia-ischemia by perinatal asphyxia (PA) is cause of retinal lesions, and can produce blindness by ischemic proliferative retinopathy (IPR). Previously, we have demonstrated retinal neurodegeneration, gliosis and neovascularization. Furthermore, we have identified the involvement of the nitregic system in it physiopatology. Objectives: Studying the participation of nitric oxide (NO) through the neuronal isoform of the enzyme nitric oxide sinthase (nNO) as trigger of the structural and molecular alterations previously observed by our group of research, and analyzing the application of methylene blue (MB) a therapeutic strategy. Methods: The studies were performed in retina by using enzymatic activity, NADPHd histochemical method and immunohistochemistry against nNOS. The studies were applied in three diferent groups (control, PA and MB) of 30 postnatal day animals. Results: At 30 postnatal day, we observed a significant increase of nNOS ativity and expression in retinas of those animals subjected to PA, when compared with the control group. In the group treated with methylene blue we did not found significant diferences in constitutive NOS activity, meanwhile it expression evaluated by western blot was lower than controls. The histochemical and immunohistochemical studies support these results. Conclusions: NO, is a free radical that act as a neurotoxic agent, seems to have a crucial role in the development of ROP after PA. The application of methylene blue as a therapeutic strategy showed a strong protective efect inhibiting the constitutive NOS activity and it expression. This founds stimulates future studies with the aim of using MB in order to avoid or decrease retinal damage.Fil: Rey Funes, Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Fernández, J. C.. Universidad de Buenos Aires. Facultad de Medicina. Cátedra de Farmacología; ArgentinaFil: Ibarra, Mariano Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Peña, Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Contartese, Daniela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Inserra, Pablo Ignacio Felipe. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: López, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Dorfman, Verónica Berta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Loidl, Cesar Fabian. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Avaliação da degradação da penicilina no leite através da utilização da enzima beta-lactamase

Antibiotics are used in dairy farms for the treatment and prevention of diseases in dairy cattle, such as mastitis, lameness, respiratory infections and diarrhea. Antibiotic residues in milk can generate problems in the population in the form of allergies and antimicrobial resistance, as well as in industry and the environment. There are national and international regulations that prohibit the processing of milk with antibiotics, so it is important to study alternatives for their disposal. The aim of this work was to study the effect of treatment with β-lactamase (BL) on the degradation of penicillin G (PG) in milk. The trials were performed on skimmed milk powder contaminated with three antibiotic concentrations: 6, 12 and 30 ppb. Three concentrations of BL (0.1, 0.5 and 1.0 mU/mL) were tested. Samples were taken at 3, 6, and 9 hours for detection and quantification of antibiotics. The results obtained indicate that in three hours the PG was degraded using 0.5 mU/mL of β-lactamase, and in six hours with a lower concentration of enzime (0.1 mU/mL) in all the antibiotic concentrations analyzed. The β-lactamase studied have a high potential to degrade penicillin in milk in dairy farms before final disposal.&nbsp; Key words: antibiotic degradation, microbial enzymes, milkLos antibióticos son utilizados en los tambos para el tratamiento y prevención de enfermedades en ganado bovino lechero, como la mastitis, cojeras, infecciones respiratorias y diarrea. Los residuos de antibióticos en leche pueden generar problemas en la población en forma de alergias y resistencia antimicrobiana, así como en la industria y el medio ambiente. Existen reglamentaciones nacionales e internacionales que prohíben el procesamiento de leche con antibióticos, por lo que es de importancia el estudio de alternativas para su disposición. El objetivo de este trabajo fue estudiar el efecto del tratamiento con β-lactamasa (BL) en la degradación de penicilina G (PG) en leche. Los ensayos se realizaron en leche en polvo descremada contaminada con tres concentraciones de antibiótico: 6, 12 y 30 ppb. Se probaron tres concentraciones de BL (0.1, 0.5 y 1.0 mU/mL). Se extrajeron muestras a las 3, 6, y 9 horas la para detección y cuantificación de antibióticos. Los resultados obtenidos indican que en tres horas se degradó la PG utilizando 0,5 mU/mL de β-lactamasa y 6 horas con una concentración menor (0,1 mU/mL) en todas las concentraciones estudiadas. Las enzimas estudiadas presentan un alto potencial para degradar antibióticos en leche en tambos antes de su disposición final. Palabras clave: degradación de antibióticos, enzimas microbianas, lech

Treatment of nonmetastatic unilateral retinoblastoma in children

IMPORTANCE: Multi-institutional collaborative studies that include large patient populations for the management of retinoblastoma with histopathological risk factors could provide important information for patient management. OBJECTIVE: To evaluate the implementation of a strategy for the management of nonmetastatic unilateral retinoblastoma in children based on standardized diagnostic and treatment criteria. DESIGN, SETTING, AND PARTICIPANTS: This single-arm prospective study applied a strategy based on a single-center experience. The setting was a multicenter study in Latin America (Grupo de America Latina de Oncologia Pediatrica [GALOP]). Participants were children with nonmetastatic unilateral retinoblastoma (staged with the International Retinoblastoma Staging System). The study opened on July 1, 2008, and closed on December 31, 2014. Follow-up was updated until June 30, 2017. INTERVENTIONS: Stage 0 patients (without enucleation) were given conservative therapy without a protocol. Stage I patients (with enucleation and no residual tumor) were divided into a high-risk group (retrolaminar invasion and/or scleral invasion) and a low-risk group (all remaining patients). High-risk children received adjuvant chemotherapy with 4 alternating cycles of regimen 1 (cyclophosphamide [65mg/kg/d] [plus sodium-2-mercaptoethane sulfonate], idarubicin hydrochloride [10mg/m2/d], and vincristine sulfate [0.05mg/kg/d]) and 4 cycles of regimen 2 (carboplatin [500mg/m2/d, days 1 and 2] and etoposide [100mg/m2/d, days 1-3]). Low-risk children did not receive adjuvant therapy. Children with buphthalmia received neoadjuvant and adjuvant chemotherapy for a total of 8 cycles. MAIN OUTCOMES AND MEASURES: Probability of event-free survival (extraocular relapse and death from any cause were considered events). RESULTS: Among 187 children registered in the study, 175 were evaluable (92 [52.5%] female; median age, 22 months; age range, 3-100 months). Forty-two were stage 0 children, 84 were stage I low-risk children, and 42 were stage I high-risk children; there were 7 children in the buphthalmia group. With a median follow-up of 46 months, the 3-year probability of event-free survival was 0.97 (95%CI, 0.94-0.99), and the probability of overall survival was 0.98 (95%CI, 0.94-1.00). Stage 0 patients had no events, stage I low-risk patients had 1 event (orbital relapse treated with second-line therapy), stage I high-risk patients had 2 events (1 central nervous system relapse and 1 death from sepsis), and the buphthalmia group had 1 event (orbital relapse, followed by central nervous relapse and death). CONCLUSIONS AND RELEVANCE: Adjuvant therapymay be effective for high-risk unilateral retinoblastoma but is toxic, and neoadjuvant chemotherapy for buphthalmus appears feasible.Fil: Pérez, Verónica. Hospital San Juan de Dios; ChileFil: Sampor, Claudia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Rey, Guadalupe. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Parareda Salles, Andreu. Hospital Sant Joan de Déu; EspañaFil: Kopp, Katherine. Hospital Dr. Luis Calvo Mackenna Hospital; ChileFil: Dabezies, Agustín P.. Hospital Pereyra Rossell; UruguayFil: Dufort, Gustavo. Hospital Pereyra Rossell; UruguayFil: Zelter, Marta. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: López, Juan P.. Hospital Calvo Mackenna; ChileFil: Urbieta, Marcelo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Alcalde Ruiz, Elisa. Hospital Dr. Luis Calvo Mackenna Hospital; ChileFil: Catala Mora, Jaume. Hospital Sant Joan de Déu; EspañaFil: Suñol, Mariona. Hospital Sant Joan de Déu; EspañaFil: Ossandon, Diego. Hospital San Juan de Dios; ChileFil: Fandiño, Adriana Cristina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Croxatto, Juan Oscar. Fundación Oftalmología Argentina "J. Malbrán"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: De Dávila, María T. G.. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Reaman, Gregory. Center for Drug Evaluation and Research; Estados UnidosFil: Ravindranath, Yaddanapudi. Children’s Hospital of Michigan; Estados UnidosFil: Chantada, Guillermo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentin