Caracterización Estructural y Funcional de una Metaloproteasa del Veneno de Bothrops roedingeri por Aproximación Proteómica

Las especies pertenecientes al género Bothrops son muy importantes en el campo de la investigación por poseer enzimas proteolíticas, la cual tiene como características principales tener una variada acción biológica y contribuir con desórdenes en coagulación, necrosis y formación de hemorragias por ende se necesita una plataforma moderna en el estudio de estas enzimas para así poder sugerir un modelo metodológico actualizado en el estudio de todas las proteínas expresadas por un sistema biológico que nos permita conocer el estado actual de la especie, así como utilizar esta información en la construcción biotecnológica de informaciones con posibles fines terapéuticos futuros. El primer objetivo trazado es purificar y caracterizar una de las enzimas proteolíticas presentes en el veneno de la serpiente peruana Bothrops roedingeri como es la metaloproteasa para lo cual se trabajó inicialmente mediante cromatografía de Interacción Hidrofóbica con columnas del sistema HITrap en la cual nos dio como resultado la presencia de 6 picos, siendo el pico 2 aquel que mostró actividad hemorrágica siendo denominada HFBr, con esta fracción obtenida se realizó una electroforesis SDS PAGE en condición reductora con uso de DTT que evidenció una masa molecular de 23 kDa y en condición no reductora una masa de ~27 kDa. , para la determinación de la masa intacta del veneno recurrimos al método de Espectrometría de Masas obteniendo nuestros resultados crudos e intactos por Electrospray (ESI) dando un resultado de 27342.6641 Da. Se realizó una caracterización de la estructura primaria de “novo” por hidrolisis tríptica (MALDI/TOF) obteniendo 6 secuencias de aminoácidos para posteriormente realizar un análisis Bioinformático a través de la Base de datos SWISS-PROT para luego realizar un análisis de homología secuencial con uso del Software DNAStar especificando que la proteína estudiada es una Metaloproteasa de la familia de las Hemorraginas. Finalmente se hizo pruebas para su caracterización biológica para determinar la actividad hemorrágica con un método de inoculación intradérmica en ratones obteniendo los valores de hemorragia inducida de Bothrops roedingeri correspondiendo a 9.5 mm ± 0.15 mm para el veneno total y de 8 ± 0.12 mm para la fracción HFBr, respectivamente. El procedimiento realizado como es la “Proteómica” nos sirvió para la obtención y caracterización de metaloproteasas procedentes de la especie Bothrops para un futuro fin científico, tecnológico y terapéutico. Palabras clave: HITrap, Cromatografía, SDS PAGE, Espectrometría de Masas, ESI, MALDI/TOF, Hidrolisis, Actividad hemorrágica, Proteómica.Tesi

Caracterización estructural y funcional de una metaloproteasa del veneno de Bothrops roedingeri por aproximación proteómica

The species belonging to the genus Bothrops are very important in the field of research for possessing proteolytic enzymes, which has as main characteristics to have a varied biological action and contribute to disorders in coagulation, necrosis and hemorrhage formation therefore a modern platform is needed in the study of these enzymes to be able to suggest an updated methodological model in the study of all the proteins expressed by a biological system that allows us to know the current status of the species, as well as to use this information in the biotechnological construction of information with possible future therapeutic purposes. The first objective is to purify and characterize one of the proteolytic enzymes present in the venom, such as metalloprotease, for which we initially worked with Hydrophobic Interaction chromatography with columns from the HITrap system, which resulted in the presence of 6 peaks, being peak 2 that showed hemorrhagic activity being called HFBr, with this obtained fraction SDS PAGE electrophoresis was performed in reductive condition using DTT that showed a molecular mass of 23 kDa and in a non-reducing condition a mass of ~ 27 kDa. , for the determination of the intact mass of the poison we resorted to the method of Mass Spectrometry obtaining our raw and intact results by Electrospray (ESI) giving a result of 27342.6641 Da. A characterization of the primary structure of "novo" was carried out by tryptic hydrolysis (MALDI / TOF), obtaining 6 amino acid sequences to subsequently carry out a Bioinformatic analysis through the SWISS-PROT database to then carry out a sequential homology analysis with Use of the DNAStar Software specifying that the protein studied is a Metalloprotease of the Bleeding family. Finally, tests were done for their biological characterization to determine the hemorrhagic activity with a method of intradermal inoculation in mice obtaining the values of induced hemorrhage of Bothrops roedingeri corresponding to 9.5 mm ± 0.15 mm for the total venom and 8 ± 0.12 mm for the fraction HFBr, respectively.Las especies pertenecientes al género Bothrops son muy importantes en el campo de la investigación por poseer enzimas proteolíticas, la cual tiene como características principales tener una variada acción biológica y contribuir con desórdenes en coagulación, necrosis y formación de hemorragias por ende se necesita una plataforma moderna en el estudio de estas enzimas para así poder sugerir un modelo metodológico actualizado en el estudio de todas las proteínas expresadas por un sistema biológico que nos permita conocer el estado actual de la especie, así como utilizar esta información en la construcción biotecnológica de informaciones con posibles fines terapéuticos futuros. El primer objetivo trazado es purificar y caracterizar una de las enzimas proteolíticas presentes en el veneno como es la metaloproteasa para lo cual se trabajó inicialmente mediante cromatografía de Interacción Hidrofóbica con columnas del sistema HITrap en la cual nos dio como resultado la presencia de 6 picos, siendo el pico 2 aquel que mostró actividad hemorrágica siendo denominada HFBr, con esta fracción obtenida se realizó una electroforesis SDS PAGE en condición reductora con uso de DTT que evidenció una masa molecular de 23 kDa y en condición no reductora una masa de ~27 kDa. , para la determinación de la masa intacta del veneno recurrimos al método de Espectrometría de Masas obteniendo nuestros resultados crudos e intactos por Electrospray (ESI) dando un resultado de 27342.6641 Da. Se realizó una caracterización de la estructura primaria de “novo” por hidrolisis tríptica (MALDI/TOF) obteniendo 6 secuencias de aminoácidos para posteriormente realizar un análisis Bioinformático a través de la Base de datos SWISS-PROT para luego realizar un análisis de homología secuencial con uso del Software DNAStar especificando que la proteína estudiada es una Metaloproteasa de la familia de las Hemorraginas. Finalmente se hizo pruebas para su caracterización biológica para determinar la actividad hemorrágica con un método de inoculación intradérmica en ratones obteniendo los valores de hemorragia inducida de Bothrops roedingeri correspondiendo a 9.5 mm ± 0.15 mm para el veneno total y de 8 ± 0.12 mm para la fracción HFBr, respectivamente

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Prevalencia y causas de ceguera en Perú: encuesta nacional

OBJETIVO: Estimar la prevalencia de ceguera y deficiencia visual en adultos de Perú y precisar sus causas, evaluar la cobertura y la calidad de los servicios de cirugía de catarata y determinar las barreras que impiden acceder a esos servicios. MÉTODOS: Estudio poblacional transversal con muestreo aleatorio por conglomerado en dos pasos de personas de 50 años o más, representativo de todo el país, mediante la metodología estándar de la Evaluación Rápida de Ceguera Evitable. Se midió la agudeza visual y se examinó el cristalino y el polo posterior por oftalmoscopía directa. Se calculó la cobertura de cirugía de catarata y se evaluó su calidad, además de las causas de tener una agudeza visual < 20/60 y las barreras para acceder a ese tratamiento. RESULTADOS:Se examinaron 4 849 personas. La prevalencia de ceguera fue 2,0% (intervalo de confianza de 95%: 1,5-2,5%). La catarata fue la causa principal de ceguera (58,0%), seguida por el glaucoma (13,7%) y la degeneración macular relacionada con la edad (11,5%). Los errores de refracción no corregidos fueron la principal causa de deficiencia visual moderada (67,2%). La cobertura de cirugía de catarata fue de 66,9%, y 60,5% de los ojos operados de catarata logró una AV ≥ 20/60 con la corrección disponible. Las principales barreras para someterse a la cirugía de catarata fueron el alto costo (25,9%) y no saber que el tratamiento es posible (23,8%). CONCLUSIONES: La prevalencia de ceguera y deficiencia visual en Perú es similar a la de otros países latinoamericanos. La baja cobertura de cirugía de catarata y el envejecimiento poblacional indican que para aumentar el acceso a estos servicios se debe mejorar la educación de la población en salud ocular y la capacidad resolutiva de los servicios oftalmológicos y de cirugía de catarata, y reducir su costo