CRISPR-ERA for switching off (onco)genes

[EN]Genome editing nucleases like the popular CRISPR/Cas9 allow generate knock - out cell lines and nulls zygotes by inducing site - specific DSB within a genome. In most cases, when a DNA template is not present, the DSB is repaired by non - homologous end joining (NHEJ) resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, for se veral reasons, these mutations do not produce the desired null result in all cases, generating a similar protein with functional activity. That undesirable effect could limit the therapeutic efficiency of gene therapy strategies focused on abrogating oncog ene expression by CRISPR/Cas9 and should be taken in account. This chapter reviews the irruption of CRISPR technology for gene silencing and its application in gene therapy

HyPer biosensor to monitor intracellular hydrogen peroxide in skeletal muscle cells

[EN] Suplemento de la revista Free Radical Biology and Medicine: HyPer biosensor to monitor intracellular hydrogen peroxide in skeletal muscle cells

The embodied typist: Bimanual actions are modulated by words’ implied motility and number of evoked limbs

The planning and execution of manual actions can be influenced by concomitant processing of manual action verbs. However, this phenomenon manifests in varied ways throughout the literature, ranging from facilitation to interference effects. Suggestively, stimuli across studies vary randomly in two potentially relevant variables: verb motility and effector quantity (i.e., the amount of movement and the number of hands implied by the word, respectively). Here we examine the role of these factors during keyboard typing, a strategic bimanual task validated in previous works. Forty-one participants read and typed high and low motility items from four categories: bimanual, unimanual, and non-manual action verbs, as well as minimally motoric verbs. Motor planning and execution were captured by first-letter lag (the lapse between word presentation and first keystroke) and whole-word lag (the lapse between the first and last keystroke). We found that verb motility modulated action planning and execution, both stages being delayed by high (relative to low) motility verbs. Effector quantity also influenced both stages, which were facilitated by bimanual verbs relative to unimanual verbs and non-manual verbs (this effect being confined to high motility items during action execution). Accordingly, motor-language coupling effects seem sensitive to words’ implied motility and number of evoked limbs. These findings refine our understanding of how semantics influences bodily movement.Fil: Rolán, Katia. Universidad de La Laguna; España. Universidad de Vigo; EspañaFil: Sánchez Borges, Iván. Universidad de La Laguna; EspañaFil: Kogan, Boris. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Humanidades. Departamento de Filosofía; Argentina. Centro de Neurociencias Cognitivas ; Rectorado ; Universidad de San Andres;Fil: García Marco, Enrique. Universidad de La Laguna; España. Universidad de Huelva; EspañaFil: Álvarez, Carlos J.. Universidad de La Laguna; EspañaFil: de Vega, Manuel. Universidad de La Laguna; EspañaFil: García, Adolfo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centro de Neurociencias Cognitivas ; Rectorado ; Universidad de San Andres; . University of California; Estados Unidos. Universidad de Santiago de Chile; Chil

Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

[EN]CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons

Aplicaciones de las nuevas herramientas de visualización de modelos 3D y realidad virtual en Internet a la docencia de la conservación y restauración del patrimonio, el diseño, y las bellas artes

Depto. de Diseño e ImagenFac. de Bellas ArtesFALSEsubmitte

The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia

[EN]CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line. CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients

Valor alimenticio comparativo del pasto kikuyo (Pennisetum clandestinum, var. Whittet) en dos estaciones de crecimiento con ryegrass (Lolium multiflorum ) Y sudán (Sorgum sudanense) ofrecido a novillos Holstein

P. 135-139Cuatro novillos Holstein (167kg) con cánulas en rumen y duodeno proximal fueron distribuidos en un diseño Cuadro Latino 4×4 para estimar el valor alimenticio comparativo del pasto kikuyo (Pennisetum clandestinum var. Whittet) cosechado en verano e invierno con el de henos de gramínea de verano (pasto sudán; Sorgum sudanense) e invierno (ryegrass anual; Lolium multiflorum var. Oregon). Las dietas experimentales (88,4% MO, 35,5% FDN y 11,8% PC) fueron formuladas con 70% forraje y 30% suplemento. No hubo efecto de tratamientos (P>0,05) en digestión ruminal de la FDN, N, eficiencia microbial ruminal (g de NM/kg de MO fermentada), ni en eficiencia ruminal del N (N no amoniacal que entra a tracto bajo/N consumido). No hubo efecto (P>0,10) de la estación (cosecha en verano vs invierno) en la digestión ruminal y total de la MO, FDN y N en dietas con kikuyo. La digestión ruminal y total de MO, FDN y N fueron similares (P>0,10) para dietas compuestas por kikuyo y sudán. Sin embargo, la digestión ruminal y total de MO y N fue menor (19, 12 y 9%, respectivamente) para dietas con kikuyo a la observada con ryegrass (P0,05) en la relación acetato-propionato. Se concluye que kikuyo tiene un valor alimenticio similar a sudán y representa una alternativa en la alimentación animal como cultivo perenne con aceptable valor nutricional durante el verano.S

La enseñanza en el máster de abogacía a través del análisis de casos reales de carácter multidisciplinar. Aprendizaje "a partir del estudio de razonamientos parciales temáticos"

Generar blog para publicaciones de alumnos del Máster Abogacía, con "entradas" dirigidas. Habría análisis desde varias disciplinas añadiéndose estudios con enfoque basado en otras áreas de conocimiento, generando interdisciplinariedad

High efficacy of Sofosbuvir plus Simeprevir in a large cohort of Spanish cirrhotic patients infected with genotypes 1 and 4

[Abstract] Background and Aims. Hepatitis C (HCV) therapy with Sofosbuvir (SOF)/Simeprevir (SMV) in clinical trials and real‐world clinical practice, showed high rates of sustained virological response (SVR) in non‐cirrhotic genotype (GT)‐1 and GT‐4 patients. These results were slightly lower in cirrhotic patients. We investigated real‐life effectiveness and safety of SOF/SMV with or without ribavirin (RBV) in a large cohort of cirrhotic patients. Methods. This collaborative multicentre study included data from 968 patients with cirrhosis infected with HCV‐GT1 or 4, treated with SOF/SMV±RBV in 30 centres across Spain between January‐2014 and December‐2015. Demographic, clinical, virological and safety data were analysed. Results. Overall SVR was 92.3%; the majority of patients were treated with RBV (62%) for 12 weeks (92.4%). No significant differences in SVR were observed between genotypes (GT1a:94.3%; GT1b:91.7%; GT4:91.1%). Those patients with more advanced liver disease (Child B/C, MELD≥10) or portal hypertension (platelet count≤100×109/L, transient elastography≥21 Kpa) showed significantly lower SVR rates (84.4%‐91.9%) than patients with less advanced liver disease (93.8%‐95.9%, P<.01 in all cases). In the multivariate analysis, the use of RBV, female gender, baseline albumin≥35 g/L, MELD<10 and lack of exposure to a triple therapy regimen were independent predictors of SVR (P<.05). Serious adverse events (SAEs) and SAE‐associated discontinuation events occurred in 5.9% and 2.6%. Conclusions. In this large cohort of cirrhotic patients managed in the real‐world setting in Spain, SOF/SMV±RBV yielded to excellent SVR rates, especially in patients with compensated liver cirrhosis. In addition, this combination showed to be safe, with low rates of SAEs and early discontinuations.Instituto de Salud Carlos III; PI15/0015