Análisis de expresión de genes de Mycobacterium tuberculosis durante la infección a macrófagos

Los factores bacterianos responsables de la patogenicidad de M. tuberculosis no han sido plenamente identificados. En este trabajo, se analizó el efecto citotóxico producido por esta bacteria sobre macrófagos humanos en cultivo, así como la expresión de algunos genes bacterianos que pudieran estar involucrados en el proceso de infección. Monocapas en cultivo de la línea celular de monocitos humanos (THP-1) diferenciada a macrófagos por inducción con forbol-miristrato-acetato (PMA) se infectaron con M. tuberculosis cepas CDC1551, H37Rv y DR689, con una multiplicidad de infección (MDI) de 10:1, 1:1 y 0.1:1 (bacterias:macrófagos). El efecto citotóxico se observó como aglomeración celular y áreas de lisis. El daño dependió de la concentración del inóculo y del tiempo de incubación. En infecciones realizadas con una MDI de 10:1, se observó destrucción total de los cultivos a 24 h de incubación. Utilizando MDI menores se observó un daño menor en el cultivo celular y aglomeración de células en varias zonas, los cuales no se presentaron en los cultivos testigo. Las monocapas celulares infectadas con una MDI de 1:1 y 0.1:1 conservaron parcialmente su integridad a las 72 h de incubación. Para cuantificar el efecto citotóxico producido por M. tuberculosis, se desarrolló un método colorimétrico basado en la característica particular de M. tuberculosis de no absorber el cristal violeta. De esta manera la perdida de células en el cultivo debido al efecto citotóxico se refleja cuantitativamente en las lecturas de absorbancia. En base a los resultados de citotoxicidad se seleccionó la MDI de 1:1 para realizar los estudios de expresión durante la infección. La extracción del RNA bacteriano se realizó de bacterias obtenidas a los 7 días de crecimiento en medio de cultivo Middlebrook 7H9, de bacterias cultivadas en medio RPMI completo o de bacterias infectando macrófagos a las 2, 6 y 24 h de incubación. La selección de genes para analizar su expresión se hizo tomando como base genes de M. tuberculosis reportados como probables factores de virulencia o genes sin función definida en M. tuberculosis pero con homología con factores de virulencia en otros patógenos. Los genes seleccionados para buscar su expresión se nombraron de acuerdo a la nomenclatura dada en http://www.sanger.ac.uk/Projects/M_tuberculosis/: MT0003, MT0024, MT0040, MT0259, MT0655, MT0876, MT1524, MT3917 (erp), MT2783 (sigB), MT2415 (plcB) y MT2414 (plcC). El testigo de expresión constitutiva fue el gene MT3989 (esat6), el cual se expresó en todas las condiciones analizadas. Como testigo de expresión inducida durante la infección se seleccionó el gene MT2416 (plcA), el cual se expresó de manera semejante a lo reportado previamente por otros autores. Todos los genes a excepción del MT0259 se expresaron en todas las condiciones, pero se observó variabilidad en la intensidad de las bandas lo que representa una posible expresión diferencial que debe establecerse mediante métodos cuantitativos. El gene MT0259 con función aparente de oxidoreductasa se expresó hasta las 24 h de incubación tanto en medio de cultivo como en células infectadas pero no mostró expresión determinada por RT-PCR a los 7 días de crecimiento en medio Middlebrook. Con estos hallazgos, se concluyó que M. tuberculosis tiene un efecto citotóxico sobre la monocapa de macrófagos cuantificable por la tinción con cristal violeta y que la bacteria expresa selectivamente ciertos genes dependiendo de las condiciones del medio de cultivo. Abstract M. tuberculosis virulence factors have not been completely identified. In this work, the cytotoxic effect produced by M. tuberculosis on human macrophages and the expression of specific bacterial genes during macrophage infection that may be involved in mycobacterial virulence was analysed. THP-1 cells monolayers differentiated to macrophages by phorbol myristate acetate (PMA) were infected with M. tuberculosis strains CDC1551, H37Rv and DR689 with a variable multiplicity of infection (MOI): 10:1, 1:1 and 0.1:1 (bacteria:macrophage) and incubated at various time points. Cytotoxicity was observed as areas of clearing and cell agglomeration and the effect was dependent on bacilli inoculum and incubation time. MOI of 10:1 produced a complete destruction of the cell monolayer after 24 h post-infection. Uninfected cultures kept their integrity throughout the experiment. Cell monolayers infected with MOI’s of 1:1 and 0.1:1 were partially disrupted after 72 h post-infection. In order to measure the cytotoxic effect, we developed an in vitro colorimetric assay based on M. tuberculosis inability to absorbe crystal violet dye, such that any lost of cells caused by M. tuberculosis in infected cultures would produce a change in absorbance values. Based on the cytotoxicity results, the MOI of 1:1 was selected to recover bacteria, isolate their RNA and perform the expression analysis. RNA was isolated from bacteria grown in Middlebrook 7H9 medium after 7 days or in RPMI medium at 2, 6 and 24 h, as well as RNA from bacteria infecting macrophages at the same time points. Selection of analysed genes was based on genes reported as probable virulence factors of M. tuberculosis or genes with undefined function in M. tuberculosis but having a nucleotide sequence homologous to virulence genes from others pathogens. Selected genes for this study were named according to the nomenclature given in http://www.sanger.ac.uk/Projects/M_tuberculosis/: MT0003, MT0024, MT0040, MT0259, MT0655, MT0876, MT1524, MT3917 (erp), MT2783 (sigB), MT2415 (plcB) and MT2414 (plcC). Gene MT3989 (esat6) was used as control of constitutive expression and gene MT2416 (plcA) as control of induced expression during infection. Gene MT0259 with a putative oxidoreductase activity did not show expression after 7 days of growth in 7H9 medium as determined by RT-PCR. But, it did express when cultivated in RPMI or during the macrophage infection at least till 24 h. All the rest of the genes analysed were expressed in all the studied conditions. Although, variability was observed on the intensity of bands, indicating a probable upregulation or dowregulation of expression, it is necessary to make quantitative analysis to get conclusive results. These findings indicate that M. tuberculosis has a cytotoxic effect on macrophage monolayers, which can be measured by crystal violet dye and that M. tuberculosis selectively express genes depending on culture conditions

Bluetongue in white-tailed deer (Odocoileus virginianus) in Northeastern Mexico

Abstract Bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) are two distinct viral hemorrhagic diseases of domestic and wild ruminants caused by members of the family Reoviridae and transmitted by Culicoides midges. These conditions have been recognized in Canada and the United States for many years, but not in Mexico. Although in Mexico there is serologic evidence of EHD virus (EHDV) and bluetongue virus (BTV) in domestic and wild ruminants, to our knowledge, there have never been reports of clinical illness or fatalities attributed to either of these viruses. Two free ranging white-tailed deer (Odocoileus virginianus) in two licensed hunting ranches in Northern Mexico near the Texas border died unexpectedly. Postmortem and microscopic examinations revealed hemorrhagic lesions compatible with viral hemorrhagic disease (Reoviridae: Orbivirus). Tissues from one animal tested positive by RT-PCR for BTV but negative for EHDV. To our knowledge, this is the first time in Mexico where deer dying with hemorrhagic lesions consistent with Bluetongue tested positive for BTV by PCR. Key words: hemorrhagic disease, white-tailed deer, Orbivirus, Mexico

Effective Reduction of Radiation Exposure during Cardiac Catheterization

Exposure to ionizing radiation during cardiac catheterization can have harmful consequences for patients and for the medical staff involved in the procedures. Minimizing radiation doses during the procedures is essential. We investigated whether fine-tuning the radiation protocol reduces radiation doses in the cardiac catheterization laboratory. In January 2016, we implemented a new protocol with reduced radiation doses in the Hospital de Jerez catheterization laboratory. We analyzed 170 consecutive coronary interventional procedures (85 of which were performed after the new protocol was implemented) and the personal dosimeters of the interventional cardiologists who performed the procedures. Overall, the low-radiation protocol reduced air kerma (dose of radiation) by 44.9% (95% CI, 18.4%–70.8%; P=0.001). The dose-area product decreased by 61% (95% CI, 30.2%– 90.1%; P <0.001) during percutaneous coronary interventions. We also found that the annual deep (79%, P=0.026) and shallow (62.2%, P=0.035) radiation doses to which primary operators were exposed decreased significantly under the low-radiation protocol. These dose reductions were achieved without increasing the volume of contrast media, fluoroscopy time, or rates of procedural complications, and without reducing the productivity of the laboratory. Optimizing the radiation safety protocol effectively reduced radiation exposure in patients and operators during cardiac catheterization procedures

PROPUESTA DE SECUENCIA OPTIMA PARA IMPLEMENTAR TECNOLOGIAS DE INDUSTRIA 4.0 UTLIZANDO ALGORITMO DE BUSQUEDA TABÚ (OPTIMAL SEQUENCE PROPOSAL TO IMPLEMENT 4.0 INDUSTRY TECHNOLOGIES USING TABOO SERCH ALGORITHM )

Resumen El nuevo esquema de producción propuesto por los alemanes a principios de la segunda década del siglo XXI, denominado como industria 4.0, está principalmente influenciado por el avance de tecnologías relacionadas con la interconectividad que permiten una mejor toma de decisiones y así mismo mejorar los sistemas productivos. La implementación de este tipo de tecnologías en los ámbitos industriales puede generar múltiples beneficios, sin embargo, alcanzar el nivel de conectividad y aplicación de estas herramientas resulta en una tarea ardua llegando costos muy elevados. En este artículo se plantea, a través de la aplicación de técnicas metaheurísticas, específicamente el algoritmo de búsqueda tabú, dar orden lógico a la secuencia de implementación de herramientas industria 4.0, de tal manera que se obtenga el máximo beneficio minimizando los costos que estas conllevan. La aportación de este artículo radica en la innovación de la industria 4.0, al no existir actualmente un orden de implementación, pudiéndose utilizar como referencia a futuros proyectos relacionados con el tema. Palabras Clave: búsqueda tabú, conectividad, tecnologías de industria 4.0 Abstract The new production scheme proposed by the Germans at the beginning of the second decade of the 21st century, called as industry 4.0, mainly influenced by the advancement of technologies related to interconnectivity that will allow better decision-making and improve production systems. The implementation of these types of technologies in industrial controls can generate multiple benefits, however, reaching the level of connectivity and application of these tools resulting in an arduous task reaching very high application costs. This article proposes, through the application of metaheuristic techniques, specifically the taboo search algorithm, to give logical order to the implementation sequence of industry 4.0 tools, in such a way that maximum benefit is obtained by minimizing the costs that these imply. The contribution of this article lies in the innovation of industry 4.0, as there is currently no order of implementation, being able to use as reference future projects related to the subject. Keywords: Taboo search, connectivity, industry technologies 4.0

Identification of non-tuberculous mycobacteria isolated from opossum (Didelphis virginiana) lymph nodes and characterisation of lesions

The aim of this study was to investigate the presence of NTM in the lymph nodes of opossums (D. virginiana) and to characterise the microscopic changes in affected tissue. Retropharyngeal and tracheobronchial lymph nodes were collected postmortem from 18 opossums in the state of Colima, Mexico in 2013. The lymph nodes were also cultured for mycobacterial organisms and processed for histopathological examination. Bacteriological cultures yielded 5/18X100 (28%) isolates of NTM, which were subsequently identified as M. terrae, M. szulgai, M. gastri and M. asiaticum. Microscopic examination of the affected nodes revealed a necrotic granulomatous lymphadenitis (3/60%) composed of histiocytes, epithelioid cells and giant cells with intralesional alcoholresistant acid bacteria. An association between the sex of the opossum and the presence of NTM was observed. To our knowledge, this is the first report of NTM isolation in opossums with granulomatous lymphadenitis in Mexico

Inseminação artificial e transferência de embriões em tempo fixo em bovinos de corte sob condições tropicais secas

Gestation percentage was assessed after fixed time embryo transfer (FTET) and artificial insemination (FTAI) programs as a means to evaluate its use to improve reproductive efficiency in beef herds in Tamaulipas, under dry tropic conditions. Two FTET programas were conducted the north part of the state, the first, at the ranch Puesta de Sol (Burgos), where 30 beef crossbred heifers were treated with CIDR plus estradiol benzoate and cipionate for FTET; the second was conducted at the ranch La India (Reynosa), where 95 beef crossbred heifers using the same treatment. Embryos from Beefmaster donors were produced at the Laboratorio FIV of UGRT and transferred fresh, to 23 heifers at Puesta de Sol and 77 heifers at La India, where 8 and 32 gestations were obtained, for 35 and 42%, respectively, for a total gestation rate of 40%. The FTAI program was conducted at the ranch San Isidro (Guémez), where 133 registered Red Brangus (50 cows) and Charolaise (88 cows) were treated with the protocol described for FTET, plus an injection of 330 IU of eCG to nursing cows and cows in poor body condition. A total gestation rate of 41% was obtained, gestation rate was greater for Red Brangus (50%) than for Charolaise (35%) cows; partial gestation rates were 34 and 24% and 23 and 16%, respectively for both programs and breeds. Differences for both FTET and FTAI programs were not significant. Results obtained in this study allow recommending the use of such biotechnological tools to improve reproductive efficiency in beef herds in Tamaulipas.Se determinó el porcentaje de gestación (PG) para transferencia de embriones (TETF) e &nbsp;inseminación artificial (IATF) a tiempo fijo, para bovinos de carne, Tamaulipas, bajo condiciones de trópico seco. Se realizaron dos programas de TETF en el norte de Tamaulipas, el primero en el Rancho Puesta de Sol, Burgos, donde se trataron 30 vaquillas con CIDR + benzoato de estradiol (BE) y cipionato de estradiol (ECP) al retiro del CIDR; el segundo programa se realizó en el Rancho La India (Reynosa), donde se sincronizaron 95 vaquillas cruzadas, con el mismo tratamiento. Se realizaron 23 transferencias en Puesta de Sol y 77 transferencias en La India, con embriones de FIV, de la raza Beefmaster, los cuales fueron producidos en el Laboratorio FIV, de la UGRT. Se obtuvieron 8 y 32 gestaciones, para 35 y 42%, respectivamente, para Puesta de Sol y la India, para un total de 40% de gestación total. El programa de IATF se realizó en el Rancho San Isidro (Guémez), utilizando 133 vacas de registro, 50 Brangus Rojo y 88 Charolais, se utilizó el mismo tratamiento que para TETF, además, las vacas paridas y en condición corporal menor de 2.5, se trataron con 330 UI de eCG, al retiro del CIDR, las vacas se inseminaron entre 55 y 60 horas post-retiro del CIDR; las vacas vacías se re-sincronizaron, utilizando el mismo protocolo. Se obtuvo PG de 41%, y 50% en Brangus Rojo y 35% en Charolais; los parciales para PG fueron de 34 y 24% y de 23 y 16%, respectivamente para ambos programas y ambas razas. Las diferencias no fueron significativas en TETF ni para IATF. Se concluye que se pueden implementar programas de TETF e IATF, para mejorar la eficiencia reproductiva de los hatos en Tamaulipas.A porcentagem de gestação (PG) foi determinada para transferência de embriões (TETF) e inseminação artificial (AITF) em tempo fixo, para bovinos de corte, Tamaulipas, sob condições tropicais secas. Dois programas de TETF foram realizados no norte de Tamaulipas, o primeiro em Rancho Puesta de Sol, Burgos, onde 30 novilhas foram tratadas com CIDR mais benzoato de estradiol (BE) e cipionato de estradiol (ECP) após a retirada do CIDR; O segundo programa foi realizado no Rancho La India (Reynosa), onde foram sincronizadas 95 novilhas cruzadas, com o mesmo tratamento. Foram realizadas 23 transferências em Puesta de Sol e 77 transferências em La India, com embriões FIV da raça Beefmaster, produzidos no Laboratório de FIV da UGRT. Obtiveram-se 8 e 32 gestações, para 35 e 42%, respectivamente, para Puesta de Sol e Índia, para um total de 40% da gestação total. O programa IATF foi realizado no Rancho San Isidro (Guémez), usando 133 vacas registradas, 50 Red Brangus e 88 Charolês, o mesmo tratamento que para TETF foi usado, além disso, vacas paridas e com condição corporal inferior a 2,5, foram tratadas com 330 UI de eCG, na retirada do CIDR, as vacas foram inseminadas entre 55 e 60 horas após a retirada do CIDR; vacas vazias foram ressincronizadas, usando o mesmo protocolo. Obteve-se PG de 41%, sendo 50% no Brangus Vermelho e 35% no Charolês; as parciais para PG foram 34 e 24% e 23 e 16%, respectivamente para ambos os programas e ambas as raças. As diferenças não foram significativas no TETF nem no IATF. Conclui-se que os programas TETF e IATF podem ser implementados para melhorar a eficiência reprodutiva dos rebanhos em Tamaulipas

Thermal biology in river buffalo in the humid tropics: neurophysiological and behavioral responses assessed by infrared thermography

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Escala ABCD-10 como predictor de mortalidad en niños con farmacodermias graves. Reporte de caso

Antecedentes: Las farmacodermias graves se asocian con alta morbilidad y mortalidad. La insuficiencia renal crónica relacionada con diálisis es uno de los principales factores asociados con mortalidad.  Reporte de caso: Paciente pediátrico masculino de 2 años, con antecedente de insuficiencia renal crónica (en tratamiento con diálisis peritoneal) y síndrome de Lennox-Gastaut, que ingresó al hospital por peritonitis aguda y evolucion a síndrome de Stevens-Johnson-necrólisis epidérmica tóxica, debido a la administración de fenitoína. Recibió tratamiento con inmunoglobulina y corticosteroide sistémico sin mejoría; se agregó choque séptico y posteriormente falleció. Conclusiones: La escala ABCD-10 versus SCORTEN es más efectiva para predecir la mortalidad por antecedente de diálisis. En niños con farmacodermias graves no existen escalas predictoras de mortalidad validadas. Las futuras iniciativas deben buscar factores de riesgo de mortalidad en niños con farmacodermias graves para la creación de una escala predictora de mortalidad. Palabras clave: Síndrome Stevens-Johnson; necrólisis epidérmica tóxica; diálisis; mortalidad; escala ABCD-10; niños

The Exceptional Fossil Site of Las Hoyas (SPAIN) from an Educational Perspective

The paleontological heritage of the fossil site of Las Hoyas (Early Cretaceous of Cuenca, Spain) is becoming a relevant part of the sociocultural identity of Castilla-La Mancha autonomous community in general, and of Cuenca province in particular. The most recent scientific advances, including several fossil findings that have had high scientific impact, have made Las Hoyas to regularly be the center of attention in the local, national, and international media, especially since the reinterpretation of the paleoecosystem represented by the locality published in 2010. These results have led to a renaissance of the interest of the society for this unique site. As a consequence, Las Hoyas has been declared Site of Cultural Interest by the regional government, in the form of paleontological zone. This recognition, which grants the locality with the highest level of protection, sets a legal framework for the educative initiatives developed at this locality, which can be categorized as non-formal education, formal education, and Social Paleontology.Unidad de Paleontología, Departamento de Biología, Universidad Autónoma de Madrid, EspañaJURASSICA Museum, SuizaDepartment of Geosciences, University of Fribourg, SuizaMuseo Geominero, Instituto Geológico y Minero de España, EspañaDepartamento de Estratigrafía, Universidad Complutense de Madrid, EspañaGéosciences Rennes Unité Mixte de Recherche, Centre National de la Recherche Scientifique, FranciaDepartamento de Ecología, Universidad Autónoma de Madrid, EspañaLaboratoire de Géologie de Lyon: Terre, Planètes, Environnement, Unité Mixte de Recherche, Centre National de la Recherche Scientifique, Franci

Correlación entre las dosis de vancomicina administradas, el clearance de creatinina y la concentración de la vancomicina en plasma en pacientes críticos

La vancomicina (vanco) sufre variaciones en su concentración plasmática (dosaje) según dosis administradas y clearance de creatinina (ClCr), entre otros factores. Varios autores demostraron que dosis fijas de 2 g/día y sin dosis de carga podrían no ser adecuadas para alcanzar dosaje terapéutico de 20-30 mg/L, por aumento (hiperfiltración) o disminución (insuficiencia renal) del ClCr durante el tratamiento, sugiriendo administrar dosis de carga y de mantenimiento, y medir dosajes para ajustar a una dosis efectiva.Facultad de Ciencias Médica