Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos

<p>Abstract</p> <p>Background</p> <p>The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism.</p> <p>Results</p> <p>Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the <it>in vivo </it>response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-κB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.</p> <p>Conclusion</p> <p>This assay has several advantages compared with the classical <it>in vitro </it>(cell lines) and <it>in vivo </it>(transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.</p

Single-molecule conformational dynamics of viroporin ion channels regulated by lipid-protein interactions

Classic swine fever is a highly contagious and often fatal viral disease that is caused by the classical swine fever virus (CSFV). Protein p7 of CFSV is a prototype of viroporin, a family of small, highly hydrophobic proteins postulated to modulate virus-host interactions during the processes of virus entry, replication and assembly. It has been shown that CSFV p7 displays substantial ion channel activity when incorporated into membrane systems, but a deep rationalization of the size and dynamics of the induced pores is yet to emerge. Here, we use high-resolution conductance measurements and current fluctuation analysis to demonstrate that CSFV p7 channels are ruled by equilibrium conformational dynamics involving protein-lipid interactions. Atomic force microscopy (AFM) confirms the existence of a variety of pore sizes and their tight regulation by solution pH. We conclude that p7 viroporin forms subnanometric channels involved in virus propagation, but also much larger pores (1–10 nm in diameter) with potentially significant roles in virus pathogenicity. Our findings provide new insights into the sources of noise in protein electrochemistry and demonstrate the existence of slow complex dynamics characteristic of crowded systems like biomembrane surfaces

Effects of Sled Towing on Peak Force, the Rate of Force Development and Sprint Performance During the Acceleration Phase

Resisted sprint training is believed to increase strength specific to sprinting. Therefore, the knowledge of force output in these tasks is essential. The aim of this study was to analyze the effect of sled towing (10%, 15% and 20% of body mass (Bm)) on sprint performance and force production during the acceleration phase. Twenty-three young experienced sprinters (17 men and 6 women; men = 17.9 ± 3.3 years, 1.79 ± 0.06 m and 69.4 ± 6.1 kg; women = 17.2 ± 1.7 years, 1.65 ± 0.04 m and 56.6 ± 2.3 kg) performed four 30 m sprints from a crouch start. Sprint times in 20 and 30 m sprint, peak force (Fpeak), a peak rate of force development (RFDpeak) and time to RFD (TRFD) in first step were recorded. Repeated-measures ANOVA showed significant increases (p ≤ 0.001) in sprint times (20 and 30 m sprint) for each resisted condition as compared to the unloaded condition. The RFDpeak increased significantly when a load increased (3129.4 ± 894.6 N·s-1, p ≤ 0.05 and 3892.4 ± 1377.9 N·s-1, p ≤ 0.01). Otherwise, no significant increases were found in Fpeak and TRFD. The RFD determines the force that can be generated in the early phase of muscle contraction, and it has been considered a factor that influences performance of force-velocity tasks. The use of a load up to 20% Bm might provide a training stimulus in young sprinters to improve the RFDpeak during the sprint start, and thus, early acceleration.Actividad Física y Deport

Prognostic implication of TERT promoter mutation and circulating tumor cells in muscle-invasive bladder cancer

Purpose: Current clinical prognostic factors are not accurate enough to identify and monitor those muscle-invasive bladder cancer (MIBC) patients at high risk of progression after radical cystectomy (RC). Here, we determined genetic alterations in the tumor and circulating tumor cell (CTC) enumeration to find biomarkers useful for the management of MIBC after RC. Methods: Thirty-nine MIBC patients undergoing RC were included. Tumoral tissue DNA was analyzed by next generation sequencing. CTCs were isolated from blood collected before RC and one, four and 12 months later. Results: Sixteen (41%) patients progressed in a median time of 8.5 months and 11 (69%) of these patients harbored the TERT c.-124C > T mutation. All progressive patients harboring the TERT c.-124C > T mutation presented a significant increase in CTC number 12 months after RC compared to those without the mutation. Additionally, CTC number at 12 months was identified as an independent prognostic biomarker for tumor progression and cancer specific survival (CSS). Ten (63%) progressive patients showed an increment of CTC number with a median anticipation period of four months compared with imaging techniques. Conclusions: The TERT c.-124C > T mutation could be considered a biomarker of aggressivity. CTC enumeration is a useful tool for identifying MIBC patients at high risk of progression and CSS after RC and for detecting tumor progression earlier than imaging techniques

Differential gene expression profile between progressive and de novo muscle invasive bladder cancer and its prognostic implication

This study aimed to ascertain gene expression profle diferences between progressive muscle-invasive bladder cancer (MIBC) and de novo MIBC, and to identify prognostic biomarkers to improve patients' treatment. Retrospective multicenter study in which 212 MIBC patients who underwent radical cystectomy between 2000 and 2019 were included. Gene expression profles were determined in 26 samples using Illumina microarrays. The expression levels of 94 genes were studied by quantitative PCR in an independent set of 186 MIBC patients. In a median follow-up of 16 months, 46.7% patients developed tumor progression after cystectomy. In our series, progressive MIBC patients show a worse tumor progression (p= 0.024) and cancer-specifc survival (CSS) (p= 0.049) than the de novo group. A total of 480 genes were found to be diferently expressed between both groups. Diferential expression of 24 out of the 94 selected genes was found in an independent cohort. RBPMC2 and DSC3 were found as independent prognostic biomarkers of tumor progression and CALD1 and LCOR were identifed as prognostic biomarkers of CSS between both groups. In conclusion, progressive and de novo MIBC patients show diferent clinical outcome and gene expression profles. Gene expression patterns may contribute to predict high-risk of progression to distant metastasis or CSS

El proceso enseñanza-aprendizaje de la física para la ingeniería/arquitectura

Cuando finalice el curso 2013-14 se producirá la salida de la primera promoción de graduados en la Escuela Politécnica Superior (EPS). Teniendo en cuenta que antes de que transcurran seis años se debe renovar la acreditación de los títulos oficiales de Grado, consideramos adecuado efectuar un análisis crítico del proceso de enseñanza-aprendizaje. En este trabajo presentamos los resultados del seguimiento realizado en el contexto educativo de los fundamentos físicos en los títulos de grado de la EPS. Estos resultados muestran la necesidad de abordar los cambios necesarios para corregir las deficiencias detectadas y mejorar la calidad del aprendizaje. Conviene recordar que el cambio que se pretendía realizar en la enseñanza universitaria era para centrarse en el aprendizaje del alumnado. Además incluimos un estudio de la evolución del aprendizaje de la física analizando las calificaciones de acceso en física y matemáticas y comparándolas con las obtenidas en los fundamentos físicos de los diferentes grados impartidos en la EPS. También presentaremos buena parte de las actividades realizadas para facilitar el aprendizaje continuo de nuestro alumnado mediante el uso de nuevas herramientas informáticas, como blogs, OpenCourseWare, materiales audiovisuales (pUAs), etc

Materiales para el autoaprendizaje y análisis de resultados académicos en dos grupos de física

La preparación previa del alumnado que cursa la asignatura de Fundamentos Físicos en primer curso de grado implantado en la Escuela Politécnica Superior de la Universidad de Alicante, es muy heterogénea. Además, se da la circunstancia que en los últimos cursos alrededor de un 50% del alumnado no ha cursado nunca una asignatura de física anteriormente, aunque esta situación depende del grado concreto. En este trabajo presentamos un análisis de los resultados obtenidos en el curso 14-15 en dos grupos teóricos de Fundamentos Físicos de la Informática a lo largo del curso. Nuestro objetivo es mejorar la metodología de la asignatura en actividades como cursos de iniciación, distribución homogénea del alumnado en los grupos teóricos, o adaptación de la guía docente al contexto particular de cada grupo. Otro aspecto que trabaja la red es el desarrollo de materiales para el autoaprendizaje de la física. Nuestro objetivo es facilitar que el alumnado pueda prepararse la asignatura de Fundamentos Físicos utilizando estos materiales. Entre ellos destacamos la elaboración de libros de resolución de problemas y la elaboración de blogs educativos

Personal Construct Therapy vs Cognitive Behavioral Therapy in the Treatment of Depression in Women with Fibromyalgia: Study Protocol for a Multicenter Randomized Controlled Trial

Background: Fibromyalgia (FM) is a debilitating syndrome, more prevalent in women, which is aggravated by the presence of depressive symptoms. In the last decade, cognitive behavioral therapy (CBT) has demonstrated to reduce such depressive symptoms and pain in these patients, but there are still a considerable number of them who do not respond to interventions. The complexity of the disorder requires the consideration of the unique psychological characteristics of each patient to attain good outcomes. One approach that could accomplish this goal might be personal construct therapy (PCT), an idiographic approach that considers identity features and interpersonal meanings as their main target of intervention. Then, the aim of the study is to test the efficacy of PCT as compared to a well-established treatment in the reduction of depressive symptoms in women with fibromyalgia. Methods and Analysis: This is a multicenter randomized controlled trial. In each condition participants will attend up to eighteen 1-hr weekly therapy sessions and up to three 1-hr booster sessions during the following 3- 5 months after the end of treatment. The depression subscale of the Hospital Anxiety and Depression Scale (HADS-D) will be the primary outcome measure and it will be assessed at baseline, at the end of therapy, and at 6-month follow-up. Other secondary measures will be applied following the same schedule. Participants will be 18- to 70-years-old women with a diagnosis of FM, presenting depressive symptoms evinced by scores above seven in depression items of the HADS-D. Intention-to-treat and complete case analyses will be performed for the main statistical tests. Linear mixed models will be used to analyze and to compare the treatment effects of both conditions

Diagnóstico del posgrado en México. Región Centro Occidente

El estudio tiene como propósito analizar la estructura institucional de la oferta del Posgrado en la Región Centro Occidente, con la finalidad de identificar fortalezas y debilidades y con ello proponer políticas públicas para el impulso de la calidad de los estudios de posgrado en México. Con base en la información nacional se elaboraron seis diagnósticos regionales y un diagnóstico nacional del posgrado. En este volumen se presentan los resultados de la Región Centro Occidente que incluye los estados Jalisco, Colima, Guanajuato, Nayarit, Michoacán y Aguascalientes. Se analizan las características de las Instituciones de Educación Superior que impactan en los posgrados que imparten, tales como origen de financiamiento, grado de desarrollo de su normativa, así como las formas de evaluación, entre otras. Se revisan las características académicas, administrativas y financieras de los programas de posgrado como niveles, orientación, modalidades, formación de competencias. Se da cuenta de las características de los actores del posgrado: estudiantes, coordinadores, egresados. Se presentan las relaciones de los posgrados con el entorno y se ofrece una sistematización y análisis cualitativo respecto a lo que los coordinadores identifican como problemas y perspectivas a futuro y estrategias de fortalecimiento del posgrado.Consejo Mexicano de Estudios de PosgradoConsejo Nacional de Ciencia y Tecnologí

Urine cell-based DNA methylation classifier for monitoring bladder cancer

Background: Current standard methods used to detect and monitor bladder cancer (BC) are invasive or have low sensitivity. This study aimed to develop a urine methylation biomarker classifier for BC monitoring and validate this classifier in patients in follow-up for bladder cancer (PFBC). Methods: Voided urine samples (N = 725) from BC patients, controls, and PFBC were prospectively collected in four centers. Finally, 626 urine samples were available for analysis. DNA was extracted from the urinary cells and bisulfite modificated, and methylation status was analyzed using pyrosequencing. Cytology was available from a subset of patients (N = 399). In the discovery phase, seven selected genes from the literature (CDH13, CFTR, NID2, SALL3, TMEFF2, TWIST1, and VIM2) were studied in 111 BC and 57 control samples. This training set was used to develop a gene classifier by logistic regression and was validated in 458 PFBC samples (173 with recurrence). Results: A three-gene methylation classifier containing CFTR, SALL3, and TWIST1 was developed in the training set (AUC 0.874). The classifier achieved an AUC of 0.741 in the validation series. Cytology results were available for 308 samples from the validation set. Cytology achieved AUC 0.696 whereas the classifier in this subset of patients reached an AUC 0.768. Combining the methylation classifier with cytology results achieved an AUC 0.86 in the validation set, with a sensitivity of 96%, a specificity of 40%, and a positive and negative predictive value of 56 and 92%, respectively. Conclusions: The combination of the three-gene methylation classifier and cytology results has high sensitivity and high negative predictive value in a real clinical scenario (PFBC). The proposed classifier is a useful test for predicting BC recurrence and decrease the number of cystoscopies in the follow-up of BC patients. If only patients with a positive combined classifier result would be cystoscopied, 36% of all cystoscopies can be prevented