Structure-based identification of salicylic acid derivatives as malarial threonyl tRNA-synthetase inhibitors.

Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Threonyl t-RNA synthetase (ThrRS) is one of the enzymes involved in this pathway, and it has been validated as an anti-malarial drug target. Here, we present 9 structurally diverse low micromolar Plasmodium falciparum ThrRS inhibitors that were identified using high-throughput virtual screening (HTVS) and were verified in a FRET enzymatic assay. Salicylic acid-based compound (LE = 0.34) was selected as a most perspective hit and was subjected to hit-to-lead optimisation. A total of 146 hit analogues were synthesised or obtained from commercial vendors and were tested. Structure-activity relationship study was supported by the crystal structure of the complex of a salicylic acid analogue with a close homologue of the plasmodium target, E. coli ThrRS (EcThrRS). Despite the availability of structural information, the hit identified via virtual screening remained one of the most potent PfThrRS inhibitors within this series. However, the compounds presented herein provide novel scaffolds for ThrRS inhibitors, which could serve as starting points for further medicinal chemistry projects targeting ThrRSs or structurally similar enzymes.

Tailoring FPOX enzymes for enhanced stability and expanded substrate recognition.

Fructosyl peptide oxidases (FPOX) are deglycating enzymes that find application as key enzymatic components in diabetes monitoring devices. Indeed, their use with blood samples can provide a measurement of the concentration of glycated hemoglobin and glycated albumin, two well-known diabetes markers. However, the FPOX currently employed in enzymatic assays cannot directly detect whole glycated proteins, making it necessary to perform a preliminary proteolytic treatment of the target protein to generate small glycated peptides that can act as viable substrates for the enzyme. This is a costly and time consuming step. In this work, we used an in silico protein engineering approach to enhance the overall thermal stability of the enzyme and to improve its catalytic activity toward large substrates. The final design shows a marked improvement in thermal stability relative to the wild type enzyme, a distinct widening of its access tunnel and significant enzymatic activity towards a range of glycated substrates.

Synthesis and evaluation of an agrocin 84 toxic moiety (TM84) analogue as a malarial threonyl tRNA synthetase inhibitor.

An analogue of a toxic moiety (TM84) of natural product agrocin 84 containing threonine amide instead of 2,3-dihydroxy-4-methylpentanamide was prepared and evaluated as a putative Plasmodium falciparum threonyl t-RNA synthetase (PfThrRS) inhibitor. This TM84 analogue features submicromolar inhibitory potency (IC50 = 440 nM) comparable to that of borrelidin (IC50 = 43 nM) and therefore complements chemotypes known to inhibit malarial PfThrRS, which are currently limited to borrelidin and its analogues. The crystal structure of the inhibitor in complex with the E. coli homologue enzyme (EcThrRS) was obtained, revealing crucial ligand-protein interactions that will pave the way for the design of novel ThrRS inhibitors.

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Síndrome de Sjögren y evaluación de la calidad de vida / Sjögren's syndrome and quality of life assessment

RESUMEN Introducción: El síndrome de Sjögren (SS) es una enfermedad autoinmune, con compromiso multisistémico, especialmente de la función de las glándulas exocrinas. Los pacientes cursan con una alta carga de la enfermedad que compromete su calidad de vida, asociado sobre todo a fatiga crónica, ansiedad, depresión y disfunción sexual, entre otras dificultades. Objetivo: Describir las generalidades de las principales escalas (específicas y no específicas) para la evaluación de la calidad de vida en pacientes con SS. Métodos: Se realizó una revisión narrativa y ampliada de la literatura. Se hizo una búsqueda en las diferentes bases de datos (PubMed, Embase, Cochrane, BVS) y de literatura gris, seleccionando los artículos relacionados con el tema y realizando una extracción de datos de estos. Resultados: Se encontraron 3 escalas realizadas específicamente para pacientes con SS: Sjögren's Syndrome Patient Reported Index, Psychometric Testing of the Primary Sjögren's Syndrome Quality of Life Questionnaire y Profile of Fatigue and Discomfort, y 14 escalas realizadas en el contexto de otras enfermedades (no específicas), utilizadas en pacientes con SS. Conclusiones: Como en otras afecciones, en pacientes con SS es importante la evaluación de la calidad de vida. A tal efecto se cuenta con múltiples escalas, a parte de las 3 citadas, elaboradas específicamente para este tipo de pacientes, sin dejar de lado las escalas no específicas, como el Beck Depression Inventory y el Ocular Surface Disease Index, que tienen su mayor utilidad en la evaluación específica de síntomas asociados a esta enfermedad. Resultados: Se encontraron 3 escalas realizadas específicamente para pacientes con SS: Sjögren's Syndrome Patient Reported Index, Psychometric Testing of the Primary Sjögren's Syndrome Quality of Life Questionnaire y Profile of Fatigue and Discomfort, y 14 escalas realizadas en el contexto de otras enfermedades (no específicas), utilizadas en pacientes con SS.

ABSTRACT Introduction: Sjögren's syndrome (SS) is an autoimmune disease, with multisystemic invol- vement, particularly compromising the function of exocrine glands. The patients have a high burden of the disease associated with chronic fatigue, anxiety, depression, sexual dysfunction, among others. Objective: To describe the generalities of the main scales (specific and non-specific) for the evaluation of quality of life in patients with SS. Methods: A narrative review and expanded review was performed, including a search conducted in the different databases (PubMed, Embase, Cochrane, VHL) and grey literature. Articles related to the topic were selected and data extracted from them. Results: Three scales were developed specifically for patients with SS, and included Sjögren's Syndrome Patient Reported Index, Psychometric Testing of the Primary Sjögren's Syndrome Quality of Life Questionnaire, and Profile of Fatigue and Discomfort, and 14 scales performed in context of other diseases (non-specific) used in patients with SS. Conclusions: As is the case in other diseases, it is important to evaluate the quality of life in patients with SS. There are multiple scales for this evaluation, including the 3 scales mentioned before, specifically developed for these types of patients. The non-specific scales worth noting are, the Beck Depression Inventory and the Ocular Surface Disease Index, having their greatest use in the specific evaluation of symptoms associated with this disease.

Frecuencia del aislamiento bacteriano y patrones de sensibilidad en yeguas criollas colombianas diagnosticadas con endometritis / Bacterial Isolation Frequency and Sensitivity Patterns in Colombian Creole Mares Diagnosed with Endometritis

Resumen La endometritis bacteriana en las yeguas es uno de los principales problemas en la reproducción equina. A causa de diagnósticos inadecuados, los tratamientos antibióticos generalmente fracasan, lo que aumenta los casos de infertilidad en las hembras. En esta investigación se analizaron 90 yeguas que tenían signos de subfertilidad como abortos, retención placentaria, repetición de celos, secreción vulvar, entre otros. Fueron diagnosticadas por examen clínico reproductivo, ultrasonografía y cultivo microbiológico, mediante la técnica de hisopado uterino, y se les realizó antibiograma, en el cual se evaluaron los sensidiscos de penicilina G procaínica, gentamicina, ceftiofur, cefquinoma, amikacina y ampicilina. Según los resultados obtenidos, en el laboratorio microbiológico se encontró que E. coli fue la bacteria que creció con mayor frecuencia, con 21,11 % (19/90), seguida de Staphylococcus spp., con 11,11 % (10/90). En el aislamiento de E. coli se obtuvo que la bacteria presentó una mayor sensibilidad hacia amikacina, seguido de ceftiofur y cefquinoma. A su vez, Staphylococcus spp. fue más sensible frente a antibióticos como ceftiofur y cefquinoma. Los agentes bacterianos de espectro gramnegativo presentaron altos porcentajes de sensibilidad con respecto a antibióticos como amikacina, ceftiofur, cefquinoma y gentamicina.

Abstract Bacterial endometritis in mares is one of the main problems in equine reproduction. Due to inadequate diagnoses, antibiotic treatments generally fail, increasing the cases of infertility in females. The present study was carried out with 90 mares which presented signs of subfertility such as abortions, placental retention, repetition of jealousy, vulvar secretion, among others; They were diagnosed by clinical reproductive examination, ultrasound, microbiological culture using the uterine swab technique, and an antibiogram was performed in which the sensidisks of procaine Penicillin G, Gentamicin, Ceftiofur, Cefquinoma, Amikacin and Ampicillin were evaluated. According to the results obtained in the microbiological laboratory, it was found that E. coli was the bacterium that grew most frequently with 21.11 % (19/90), followed by Staphylococcus spp. 11.11 % (10/90). In the isolation of E. coli it was found that the bacteria showed a greater sensitivity to amikacin, followed by ceftiofur and cefquinoma; In turn, Staphylococcus spp was more sensitive against antibiotics such as ceftiofur and cefquinoma. The large negative spectrum bacterial agents presented high percentages of sensitivity with respect to antibiotics such as amikacin, ceftiofur, cefquinoma and gentamicin.

Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Šresolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.

Analysis of a program for the prevention and control of wild rabies and its role in the number of cattle outbreaks during years 2001 to 2011 / Análisis del programa de prevención y control de rabia de origen silvestre y su papel en el número de focos bovinos en el periodo 2001-2011 / Analise do programa de prevenção e controle da raiva de origem silvestre e sua importância no número de casos bovinos no período 2001-2011

Bovine rabies is an endemic disease in Colombia. A program has been established in this country for preventing rabies through vaccination and to control the disease by vector control, monitoring of outbreaks and characterization of risk zones. This paper examines how these strategies have influenced the number of outbreaks during years 2001 to 2011. This retrospective observational study analyzed databases built with information reported by Instituto Colombiano Agropecuario (ICA), World Animal Health Information System (WAHIS), Secretaria de Agricultura de Antioquia, and Federación Colombiana de Ganaderos (FEDEGAN). Positive foci, confirmed by laboratory analysis, were referenced and arranged both chronologically and by province. Resolutions and other documents that frame the program for the prevention and control of wild rabies were also consulted and analyzed. 968 outbreaks reported and confirmed as positive evidence of bovine rabies reveal an increasing trend of the disease, which is present in 62.5% of Colombian provinces, especially Antioquia, Arauca, Atlantic, Casanare, Cesar, Chocó and Córdoba. Vaccination covers 9.95% of the population. Economic impact of the disease has not been specified. There is no continuous availability of trained personnel. The program is supported by a well-described methodology and internationally recognized diagnostic methodologies; however, the trend of the disease reveals implementation difficulties and low coverage of vaccination.

En Colombia la rabia bovina es una enfermedad endémica para la cual se estableció un programa que busca su control y prevención a través de vacunación, control de vectores, seguimiento de focos y caracterización de zonas de riesgo. Este trabajo evalúa cómo la implementación de estas estrategias influye en el número de focos en el periodo 2001-2011. Se realizó un estudio observacional retrospectivo, para el cual se construyeron y analizaron bases de datos a partir de información reportada por páginas oficiales del Instituto Colombiano Agropecuario ICA, la interfase-WAHID de la organización mundial de la salud animal-OIE, información suministrada por la Secretaria de Agricultura de Antioquia y el portal FEDEGAN. Se referenciaron focos confirmados como positivos por laboratorio, clasificados cronológicamente y por departamento. Adicionalmente se consultan y analizan resoluciones y documentos anexos que enmarcan el programa de prevención y control de la rabia de origen silvestre. Durante el estudio se evidencia una tendencia al aumento con 968 focos de rabia bovina notificados y confirmados como positivos, hay presencia de la enfermedad en el 62,5% de los departamentos, especialmente en Antioquia, Arauca, Atlántico, Casanare, Cesar, Chocó y Córdoba. La vacunación cubre el 9,95% de la población, el impacto económico no se ha precisado y el personal capacitado no es constante. El programa se respalda en una metodología bien descrita y un diagnóstico internacionalmente reconocido, sin embargo la tendencia de la enfermedad revela dificultades de ejecución y la baja cobertura de vacunación como método de prevención en la ganadería es un factor determinante.

Na Colômbia a raiva bovina é uma doença endémica para a qual se estabeleceu um programa que pretende seu controle e prevenção a traves da vacinação, controle de vetores, seguimento de focos e caracterização de zonas de risco. Esta pesquisa avalio quanto da implementação de estas estratégias influenciam o número de focos no período 2001-2011. Realizou-se um estudo observacional retrospectivo, para o qual construíram-se e analisaram-se bases de dados a partir de informação reportada por páginas oficiais do Instituto Colombiano Agropecuário, ICA, a interfase-WAHID da organização mundial de saúde animal-OIE, informação subministrada pela Secretaria de Agricultura de Antioquia e pela portal web da Federação Nacional de Produtores de Gado, FEDEGAN. Referenciaram-se focos confirmados como positivos por laboratório, classificados cronologicamente e por departamento. Adicionalmente se consultam e analisam resoluções e arquivos anexos que demarcam o programa de prevenção e controle da raiva de origem silvestre. Durante o estudo evidenciou-se uma tendência ao aumento com 968 focos de raiva bovina notificados e confirmados como positivos, tendo presença da doença em 62,5% dos departamentos, especialmente Antioquia, Arauca, Atlántico, Casanare, Cesar, Chocó e Córdoba. A vacinação abrange o 9,95% da população, o impacto econômico da doença não tem-se precisado e o pessoal capacitado não é constante. O programa tem como suporte uma metodologia bem descrita e um diagnóstico internacionalmente reconhecido, embora a tendência da doença deixa ver dificuldades na execução e a baixa cobertura da vacinação como método de prevenção nas fazendas de gado, é um fator determinante.

Levels of nitrates in a urea fertilized Kikuyu (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone) pasture on the high plains of Antioquia, Colombia / Niveles de nitrato en pasto Kikuyo (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone) fertilizado con urea en el antiplano de Antioquia, Colombia / Niveles de nitrato en pasto Kikuyo (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone) fertilizado con urea en el antiplano de Antioquia, Colombia

Abstract Fertilization is one of the factors that can contribute to build-up of nitrates in kikuyu (Cenchrus clandestinus) pastures of the tropics. Field studies to evaluate the influence of nitrogen fertilization on yield and nitrate concentrations were conducted between December 2012 and June 2013 at the Agricultural Station of the National University at Santa Elena (Antioquia). The experimental design was a 4 x 4 block design with four replications in a monoculture paddock of kikuyu grass. Treatments were four levels of nitrogen applied as topdressing urea granules (0, 50, 100 and 200 kg/ha per plot). Whole grass samples (blades and stems) were collected at 42 days post-fertilization to mimic cattle grazing exposure. Biomass was estimated as dry matter yield per hectare and nitrates were measured by ion chromatography and expressed as parts per million (ppm) on dry matter content. No significant difference in biomass was observed between urea application rates; however, all treated plots produced more biomass than non-treated plots (mean 2.14 vs 1.12 Tons/ha, p<0,05). Mean ± SEM concentrations of NO3 for all time intervals combined increased from 369 ± 216 ppm with no fertilization, to 878 ± 881 ppm at the highest application rate. In spite of the large variation within each group, the highest NO3 level attained was 5600 ppm NO3, which are considered safe for cattle consumption. It can be concluded that nitrate levels in kikuyu pastures fertilized with 4 times the usual rate of nitrogen did not accumulate to pose a health risk in cattle; however, other factors that could also influence nitrate accumulation were not determined in this study.

Resumen La fertilización es uno de los factores que contribuye a la acumulación de nitratos en el pasto kikuyo (Cenchrus clandestinus) en el trópico. Este estudio, realizado entre diciembre de 2012 y junio de 2013 en la Hacienda Paysandú de la Universidad Nacional, ubicada en Santa Elena-Medellín (Antioquia), evaluó la influencia de la fertilización con nitrógeno sobre la producción y las concentraciones de nitratos. El diseño experimental fue en bloques de 4 x 4 con cuatro repeticiones de parcelas monocultivo de pasto kikuyo. Los tratamientos fueron cuatro niveles de nitrógeno aplicado como gránulos de urea sobre la superficie (0, 50, 100 y 200 kg/ha por corte). Las muestras de pasto completo (hojas y tallos) se colectaron a los 42 días posfertilización, con el fin de simular la exposición al pastoreo con ganado. La biomasa se midió como la producción de materia seca por hectárea; los nitratos se midieron por cromatografía iónica, expresada en partes por millón (ppm) sobre el contenido de materia seca. No hubo diferencia significativa en la producción de biomasa entre las diferentes tasas de aplicación de urea; sin embargo todas las parcelas tratadas produjeron más biomasa comparadas con las parcelas no tratadas (promedio 2.14 vs 1.12 ton/ha, p<0,05). Las concentraciones promedio ± SEM de NO3 para todos los tiempos de muestreo combinados, se incrementaron de 369 ± 216 ppm sin fertilización a 878 ± 881 ppm con la más alta tasa de fertilización aplicada. A pesar de la gran variación dentro de cada grupo, los niveles más altos de NO3 obtenidos fueron de 5600 ppm NO3, los cuales se consideran seguros para el consumo en ganado. Se puede concluir que los niveles de nitrato en el pasto kikuyu fertilizado con cuatro veces la tasa normal de nitrógeno, no se acumulan como para plantear un riesgo para la salud en el ganado. Sin embargo, es importante señalar que otros factores que pueden influir en la acumulación de nitratos no se evaluaron en este estudio.

Resumo A adubação é um dos fatores que contribuem na acumulação de nitratos da pastagem kikuyo (Cenchrus clandestinus) do trópico. Este estudo foi realizado entre dezembro de 2012 e junho de 2013 na fazenda Paysandu da Universidade Nacional da Colômbia, localizada no corregimento Santa Elena da cidade de Medellín, Antioquia. Avaliou-se a influência da adubação com nitrogênio sobre a produção e as concentrações de nitratos. O desenho experimental foi de blocos de 4 x 4 com quatro repetições nas parcelas da monocultura de pastagem kikuyo. Os tratamentos foram quatro níveis de nitrogênio aplicado como grânulos de uréia sobre a superfície (0, 50, 100 e 200 kg/ha por safra). As amostras da pastagem completa (folhas e talhos) coletaram-se aos 42 dias após adubação, com o intuito de simular a exposição ao pastoreio com gado. A biomassa mediou-se com a produção de matéria seca por hectare; os nitratos mediram-se por cromatografia iônica, expressada em partes por milhão (ppm) sobre o conteúdo de matéria seca. Não houve diferença significativa na produção de biomassa entre as diferentes taxas de aplicação de uréia; embora, todas as parcelas experimentais produziram maior biomassa comparada com as parcelas não tratadas (media 2.14 vs 1.12 ton/ha, p<0,05). As concentrações medias ± SEM de NO3 para todos os tempos de amostragem combinadas, se incrementaram de 369 ± 216 ppm sem adubação até 878 ± 881 ppm com a maior adubação aplicada. Ainda que houve grande variação dentro de cada grupo, os níveis maiores de NO3 obtidos foram de 5600 ppm NO3, os quais se consideram seguros para o consumo do gado. Pode-se concluir que os níveis de nitrato na pastagem kikuyo adubado com quatro vezes a taxa normal de nitrogênio, não apresentam acumulação como para ter um risco de saúde quando é consumido pelo gado. Embora, outros fatores são importantes para ter em conta, pois eles podem influir na acumulação de nitratos que não foram avaliados neste estudo.