Generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cellbased molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirusactivatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus geneticallyencoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging.Vicerrectoría de Investigación, Universidad de Costa Rica, proyecto 803-B9-505 International Centre for Genetic Engineering and Biotechnology Grant CRP/CRI18-02.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Uma aproximação teórica e operacional ao construto de inteligência espiritual

La teoría de las inteligencias múltiples ha ampliado el concepto tradicional de inteligencia abriendo la posibilidad de considerar los aspectos emocionales y espirituales de la misma. El concepto de inteligencia espiritual ha sido recientemente propuesto por algunos autores, pero de una manera ecléctica e imprecisa. Este trabajo tiene por objetivo integrar y esclarecer dicho concepto, sentando las bases teóricas para el desarrollo de una serie de reactivos preliminares direccionados a la evaluación de la inteligencia espiritual.The theory of multiple intelligences has broadened the traditional concept of intelligence opening the possibility of considering the emotional and spiritual aspects of it. The concept of spiritual intelligence has recently been proposed by some authors, but in an eclectic and imprecise manner. This work aims to integrate and clarify the concept, laying the theoretical foundations for the development of a series of preliminary reactive directed to the evaluation of spiritual intelligence.A teoria das inteligências múltiplas ampliou o conceito tradicional de inteligência abrindo a possibilidade de considerar os aspectos emocionais e espirituais desta. O conceito de inteligência espiritual foi proposto recentemente por alguns autores, porém de forma eclética e imprecisa. Este trabalho tem como objetivo integrar e esclarecer o conceito, lançando as bases teóricas para o desenvolvimento de uma série de reagentes preliminares direcionados para a avaliação da inteligência espiritual.Fil: Arias, Rodrigo. Universidad Adventista del Plata; ArgentinaFil: Lemos, Viviana Noemí. Universidad Adventista del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Saavedra 15. Centro Interdisciplinario de Investigaciones En Psicología Matemática y Experimental; Argentin

Determinación del riesgo potencial de estrés térmico del ganado bovino en cuatro localidades del centro y sur de Chile

Datos climáticos del periodo diciembre a febrero de los años 1999 al 2007 provenientes de estaciones meteorológicas situadas en Curicó, Chillán, Temuco y Osorno fueron utilizados para calcular un índice temperatura-humedad ajustado por el efecto del viento y de la radiación (THIadj-max). Las variables climáticas utilizadas para calcular este índice incluyeron: velocidad del viento, temperatura máxima del aire, humedad relativa y un valor estimado de la radiación solar. Cuando los valores de THIadj-max fueron superiores a 75, estos días fueron considerados como indicadores de “alerta” y riesgo potencial de estrés térmico en el ganado. Adicionalmente, el impacto en la producción animal fue estimado sobre la base de pérdidas potenciales de producción de leche para cada localidad, utilizando dos ecuaciones para su estimación. Las localidades de la región central, Curicó y Chillán, presentaron valores promedios de THIadj-max similares (79,4 y 79,1, respectivamente), pero ambos superiores a las de la región sur (Temuco y Osorno 77,1 y 68,1, respectivamente; P 75 fue mayor en la región central. Asimismo, la producción de leche mostró un mayor potencial de pérdidas en las regiones centrales de Chile (1,5 a 4,3 y 1,2 a 3,6 L*vaca-1*d-1 para Curicó y Chillán, respectivamente). Por otra parte, las regiones del sur, Temuco y Osorno, presentaron menores pérdidas potenciales (0,9 a 2,6 y 0,1 a 0,6 L*vaca-1*d-1, respectivamente). En conclusión, el riesgo potencial de estrés térmico para el ganado bovino es mayor en Curicó y Chillán, es moderado en Temuco, y es mínimo en Osorno. Finalmente, la cuantificación del impacto directo que tiene el clima sobre la productividad y el bienestar de los bovinos en Chile necesita ser estudiada.Meteorological data from December to January of years 1999 to 2007 from weather stations located in four localities in Chile (Curicó, Chillán, Temuco and Osorno) were used to calculate the adjusted temperature-humidity index (THIadj-max). Climate variables used to calculate the index were: wind speed, air temperature, relative humidity and an estimated value of solar radiation. When the values of THIadj-max were greater than 75, those days were considered as alert and as potential risk of heat stress in cattle. Additionally, the impact of climate on animal production was assessed by means of potential losses in milk production for each locality estimated by two equations. The localities of Curicó and Chillán presented similar values of THIadj-max (79.4 and 79.1, respectively), and both were higher than those for Temuco and Osorno (77.1 and 68.1, respectively, P 75 was greater in the Central region. In addition, the estimated mean milk production loss was greater in the Central regions (1.5 to 4.3 and 1.2 to 3.1 L*cow-1*d-1 for Curicó and Chillán, respectively). Conversely, the Southern regions, Temuco and Osorno, presented lower potential losses with 0.9 to 2.6 and 0.1 to 0.6 L*cow-1*d-1, respectively. In conclusion, the potential risk of heat stress of cattle seems greater in Curicó and Chillán, moderate in Temuco and minimum in Osorno. Additional research is needed in the order to quantify the impacts of environmental conditions in productivity and welfare of cattle in Chile

Evaluation of Four Thermal Comfort Indices and Their Relationship with Physiological Variables in Feedlot Cattle

Climatic data from different years and experiments conducted in Nebraska were used to estimate four comfort thermal indices and to predict the risk of heat stress and its relationship with pen surface temperature (PST). These included the temperature–humidity index (THI), the adjusted THI (THIadj), the heat load index (HLI), and THIPST using pen surface temperature instead of air temperature. Respiration rates (RR), tympanic temperatures (TT), and panting scores (PS) were also collected in each year and from each location. During 2007, mean values of soil temperature, PST, outgoing shortwave radiation, and TT were greater than in 2008 (p \u3c 0.011). However, HLI, relative humidity, and incoming and outgoing long-wave radiation were greater during 2008 (p \u3c 0.012). The TT was positively correlated with THIPST and THIadj (0.75 and 0.70, respectively), whereas RR had a moderate correlation with THI, THIadj, and HLI (0.32, 0.27, and 0.34, respectively; p \u3c 0.001). Thermal comfort indices showed a positive correlation with TT, especially the THIPST. These relationships vary with location. However, all of the thermal indices showed weak relationships with the observed RR. This would confirm the different roles that TT and RR have as indicators of heat stress. The THIPST was the best index for predicting TT across years

Pen Surface Temperature as a Predictor of DailyWater Intake and Tympanic Temperature in Steers Finished in Feedlots

Adequate estimation of water demand in cattle production feed yards is of high importance, especially due to reduced water availability because of changes in rain precipitation patterns and amounts. The pen surface in feed yards receives and reflects solar radiation, affecting along with other factors the microclimate to which cattle are exposed. This study aimed to describe the relationship between the pen surface temperature with the daily water intake and the tympanic temperature of finishing steers. Climate variables, including solar radiation, air temperature, relative humidity, and wind speed plus pen surface temperature and soil temperature at 10.2 cm depth were recorded. Data were collected from a weather station located in the feedlot in Concord NE, whereas daily water intake was estimated from a set of experimental pens sharing a waterer in two adjacent pens. Simple and polynomial linear regressions were assessed from data collected in different experiments conducted from 2003 to 2006. Two models to predict daily water intake were developed for finishing steers using the pen surface temperature as the predictor variable. The first one included data for the period May-October (overall model) and the second one for the summer period (June-August). The best fit for the overall model was a quadratic fit (r2 = 0.86), whereas the best-fit model for the summer model was the cubic (r2 = 0.72). Subsequently, both models were validated with data from an independent experiment conducted in the summer of 2007 in the same facilities. Both models tended to slightly overestimate daily water intake when they were validated (14.6% and 12.6%, respectively). For tympanic temperature, the best-fit model was linear, explaining 76% of the observed variability. When the dataset was split into night-time and daytime the best-fit model for the night period was a quadratic one and a linear one for the daytime, both improving the explanation of the variability observed. In conclusion, pen surface temperature can be used to predict both daily water intake and tympanic temperature in feedlot steers without access to shade

Modeling Daily Water Intake in Cattle Finished in Feedlots

Simple regression and multiple regression analyses were conducted to estimate factors affecting daily water intake(DWI) of finishing cattle. Seasonal simple linear regression equations were very poor predicting DWI (r2 \u3c 0.15). Best results were obtained with the overall simple regression. The multiple regression analysis showed that daily minimum temperature (or THI), solar radiation, and dry matter intake were the most important factors affecting DWI in cattle finished in feedyards. The following prediction equation was developed: daily water intake, gal*d-1 = -0.52677+ (0.1229 *DMI, lb*d-1) + (0.01137*solar radiation, kcal*d-1) + (0.06529*daily minimum temperature, °F)

Environmental Factors Affecting Water Intake in Steers Finishing in Feedlots

Simple and multiple regression analyses were executed using records of six experiments conducted from 1999 to 2006 at the University of Nebraska Northeast Research and Extension Center. The objective of the study was to obtain the best equation to predict water intake of feedlot steers under summer and winter weather conditions. The analysis permitted regression equations to be obtained for summer, winter and both seasons (overall model). From simple regression analysis, the best predictor of water intake was minimum temperature with r2= 0.61 in the overall model. Whereas, from multiple regression analysis the overall model with the best fit had R2 = 0.70. This model included 4 factors; daily mean minimum temperature, solar radiation, dry matter intake and wind speed