Development of an In vitro, coupled transcription-to-translation system for analysis of the interactions between the ribosome and RNA polymerase

PhD ThesisThe various properties of actively transcribing RNA polymerase (RNAP) complexes with nucleic acids during different stages involve various types of regulation and different cross-talk with other cellular entities and with RNAP itself. For instance, transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of gene expression, whereas the study of the physical interaction of the ribosome and the RNA polymerase remains obscure due to the lack of a system which allows such observations. In this study we have developed a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing the observation of the outcomes of the interactions between the two machineries at colliding and non-colliding distances; the system also allows the positioning of RNAP in any desired elongation complex such as paused, roadblocked, backtracked, etc. We show the study of the interactions of the ribosome on different aspects of transcription elongation and also the effects on translation caused by RNAP

Development of an in vitro, coupled transcription-to-translation system for analysis of the interactions between the ribosome and RNA polymerase

The various properties of actively transcribing RNA polymerase (RNAP) complexes with nucleic acids during different stages involve various types of regulation and different cross-talk with other cellular entities and with RNAP itself. For instance, transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of gene expression, whereas the study of the physical interaction of the ribosome and the RNA polymerase remains obscure due to the lack of a system which allows such observations. In this study we have developed a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing the observation of the outcomes of the interactions between the two machineries at colliding and non-colliding distances; the system also allows the positioning of RNAP in any desired elongation complex such as paused, roadblocked, backtracked, etc. We show the study of the interactions of the ribosome on different aspects of transcription elongation and also the effects on translation caused by RNAP.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins

Efecto del tiempo de corte y maduración del ensilaje de botón de oro (Tithonia diversifolia) en la digestibilidad in vivo, utilizado como suplemento en ovinos

In the tropical zone of Colombia, nutrition is the most influential factor of productive and reproductive processes of ruminant, therefore, the objective of this work was to evaluate the effect of cut age of Tithonia diversifolia (Td) and the degree of maturity of silage on the nutritional quality and digestibility in vivo in crossbred sheep hair as a supplement to a basal diet of King grass (Pennisetum purpureum) (PP). Leveling pruning was done after three cuts: 45, 60 and 75 days, 25% silages were mixed: 75% molasses (TdS). For digestibility nine sheep were used with average weight 30 kg ± 5kg, were fed adlibitum and PP 400 g dry matter (DM) of TdS. Diets and excreta were evaluated in laboratory Nutrition. Completely randomized design, nine treatments concerning the three cuts and three maturation TdS was used: 30, 45 and 60 days, with three replications. When conducted fieldwork and laboratory values of the nutritional quality of silage were on average: MS (21.7%), protein (12%), and content of neutral detergent fiber (NDF) below 40 %. Apparent digestibility of DM (DAMS) was higher than 60% with an average 68.5 ± 5.5%. Regarding the digestibility of crude protein (CPD), as this is affected increases cutting time, but a longer maturation TdS maintain or increase its digestibility, resulting in average: 62.3 ± 9.9%.En la zona tropical de Colombia, la nutrición constituye el factor de mayor influencia en procesos productivos y reproductivos de rumiantes, por lo tanto, el objetivo de este trabajo fue evaluar el efecto de la edad de corte de botón de oro Tithonia diversifolia, (Td) y el grado de maduración de su ensilaje sobre la calidad nutricional y digestibilidad in vivo en ovinos criollos de pelo, como suplemento de una dieta base de king grass (PP) (Pennisetum purpureum). Se realizó una poda de nivelación, luego tres cortes: 45, 60 y 75 días, los ensilajes se mezclaron melaza 25%: ensilaje de Td (ETd) 75%. Para la digestibilidad se emplearon nueve ovinos con peso promedio 30kg ± 5kg, a los que se les suministró PP a voluntad y 400 gramos de materia seca (MS) de ETd. Las dietas y excretas fueron evaluadas en laboratorio de Nutrición. Se usó un diseño completamente al azar, de nueve tratamientos que conciernen a los tres cortes y tres tiempos de maduración del ETd: 30, 45 y 60 días, con tres repeticiones. Cuando fue realizado el trabajo de campo y de laboratorio los valores de la calidad nutricional del ensilaje, fueron en promedio: MS (21,7%), proteína (12%), y contenidos de fibra detergente neutro (FDN) por debajo de 40%. Las digestibilidades aparente de MS (DAMS) fueron superior al 60% con un promedio 68,5% ± 5,5. Referente a la digestibilidad de la proteína cruda (DPC), esta se ve afectada conforme incrementa el tiempo de corte, pero un mayor tiempo de maduración del ETd mantienen o incrementa su digestibilidad, resultando en promedio: 62,3% ± 9,9

In vitro experimental system for analysis of transcription–translation coupling

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription–translation, both machineries mutually affect each others’ functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription–translation coupling on pausing of transcription

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Congreso Internacional de Responsabilidad Social Apuestas para el desarrollo regional.

Congreso Internacional de Responsabilidad Social: apuestas para el desarrollo regional [Edición 1 / Nov. 6 - 7: 2019 Bogotá D.C.]El Congreso Internacional de Responsabilidad Social “Apuestas para el Desarrollo Regional”, se llevó a cabo los días 6 y 7 de noviembre de 2019 en la ciudad de Bogotá D.C. como un evento académico e investigativo liderado por la Corporación Universitaria Minuto de Dios -UNIMINUTO – Rectoría Cundinamarca cuya pretensión fue el fomento de nuevos paradigmas, la divulgación de conocimiento renovado en torno a la Responsabilidad Social; finalidad adoptada institucionalmente como postura ética y política que impacta la docencia, la investigación y la proyección social, y cuyo propósito central es la promoción de una “sensibilización consciente y crítica ante las situaciones problemáticas, tanto de las comunidades como del país, al igual que la adquisición de unas competencias orientadas a la promoción y al compromiso con el desarrollo humano y social integral”. (UNIMINUTO, 2014). Dicha postura, de conciencia crítica y sensibilización social, sumada a la experiencia adquirida mediante el trabajo articulado con otras instituciones de índole académico y de forma directa con las comunidades, permitió establecer como objetivo central del evento la reflexión de los diferentes grupos de interés, la gestión de sus impactos como elementos puntuales que contribuyeron en la audiencia a la toma de conciencia frente al papel que se debe asumir a favor de la responsabilidad social como aporte seguro al desarrollo regional y a su vez al fortalecimiento de los Objetivos de Desarrollo Sostenible

Congreso Internacional de Responsabilidad Social Apuestas para el desarrollo regional.

Congreso Internacional de Responsabilidad Social: apuestas para el desarrollo regional [Edición 1 / Nov. 6 - 7: 2019 Bogotá D.C.]El Congreso Internacional de Responsabilidad Social “Apuestas para el Desarrollo Regional”, se llevó a cabo los días 6 y 7 de noviembre de 2019 en la ciudad de Bogotá D.C. como un evento académico e investigativo liderado por la Corporación Universitaria Minuto de Dios -UNIMINUTO – Rectoría Cundinamarca cuya pretensión fue el fomento de nuevos paradigmas, la divulgación de conocimiento renovado en torno a la Responsabilidad Social; finalidad adoptada institucionalmente como postura ética y política que impacta la docencia, la investigación y la proyección social, y cuyo propósito central es la promoción de una “sensibilización consciente y crítica ante las situaciones problemáticas, tanto de las comunidades como del país, al igual que la adquisición de unas competencias orientadas a la promoción y al compromiso con el desarrollo humano y social integral”. (UNIMINUTO, 2014). Dicha postura, de conciencia crítica y sensibilización social, sumada a la experiencia adquirida mediante el trabajo articulado con otras instituciones de índole académico y de forma directa con las comunidades, permitió establecer como objetivo central del evento la reflexión de los diferentes grupos de interés, la gestión de sus impactos como elementos puntuales que contribuyeron en la audiencia a la toma de conciencia frente al papel que se debe asumir a favor de la responsabilidad social como aporte seguro al desarrollo regional y a su vez al fortalecimiento de los Objetivos de Desarrollo Sostenible