Nuclear functions of prefoldin

Prefoldin is a cochaperone, present in all eukaryotes, that cooperates with the chaperonin CCT. It is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, both canonical and prefoldin-like subunits of this heterohexameric complex have also been found in the nucleus, and are functionally connected with nuclear processes in yeast and metazoa. Plant prefoldin has also been detected in the nucleus and physically associated with a gene regulator. In this review, we summarize the information available on the involvement of prefoldin in nuclear phenomena, place special emphasis on gene transcription, and discuss the possibility of a global coordination between gene regulation and cytoplasmic dynamics mediated by prefoldin.Ministerio de Economía y Competitividad, BFU-2010- 21975-C03-03Junta de Andalucía 08-CVI-03508Andalucía, Junta de Andalucía P12-BIO-1938

Diseño in silico, síntesis y evaluación de una alternativa menos tóxica al octinoxato con propiedades fotoprotectoras adecuadas

La exposición excesiva a los rayos UV provoca diversas patologías cutáneas, como quemaduras solares, fotoenvejecimiento y carcinogénesis. En la actualidad, el uso de filtros solares es el factor más importante para proteger la piel de los daños fotoinducidos. El octinoxato es un filtro UV de uso común, pero su uso se ha vuelto controvertido porque actúa como disruptor endocrino tanto en humanos como en animales marinos. La investigación se ha basado en la biotecnología, los estudios de relación estructura-actividad (SAR) y la química combinatoria para encontrar filtros UV nuevos y menos tóxicos. Sin embargo, no existen ejemplos actuales que describan las posibles aplicaciones de las técnicas in silico para la obtención de estos compuestos. Así pues, en este proyecto se trató de diseñar un análogo del octinoxato que pudiera utilizarse como alternativa fotoprotectora menos tóxica, pero igualmente eficaz, mediante el cribado virtual basado en ligandos (LBVS). Diseñamos 213 moléculas nuevas basadas en la fracción (E)-cinamoil del octinoxato, pero sólo 23 resultaron ser menos tóxicas que el compuesto original. A continuación, se construyó un modelo basado en una red neuronal artificial (RNA) para predecir la absortividad molar de esas 23 moléculas, y se eligió para la síntesis la molécula que presentaba una absortividad molar similar a la del octinoxato (análogo 4, 3-fenilpropil (E)-3-(4-metoxifenil)acrilato). La síntesis del análogo 4 dio como resultado un rendimiento del 90%, y a continuación se evaluaron sus propiedades fotoprotectoras, su lipofilia y su citotoxicidad. El análogo 4 absorbió radiación UV en el rango de 250-340 nm, y presentó una absortividad molar de 36,155 M - 1cm-1. Su lipofilia se evaluó con RP-HPLC, dando como resultado un logkw de 2,49 y su CL50 fue mayor que la del octinoxato (67,41 nM frente a 45,67 nM). Por lo tanto, los resultados mostraron que el cribado virtual basado en ligandos es una estrategia eficaz para el desarrollo de nuevos filtros UV orgánicos, ya que guió el diseño de análogos menos tóxicos y señaló el análogo con más probabilidades de presentar propiedades UV similares a las del octinoxato. En este caso, el análogo 4 es una alternativa prometedora a su compuesto original, ya que demostró ser más eficaz y menos tóxico.Excessive UV exposure leads to several skin pathologies such as sunburns, photoaging and carcinogenesis. Currently, sunscreen use is the most important factor in protecting skin from photoinduced damage. Octinoxate is a commonly used UV filter, but its use has become controversial because it acts as an endocrine disruptor in both humans, and marine animals. Research has relied on biotechnology, structure activity relationship (SAR) studies and combinatorial chemistry to find new and less toxic UV filters. However, there are no current examples that describe the possible applications of in silico techniques for obtaining these compounds. Thus, this project sought to design an octinoxate analog that could be used as a less toxic, but equally effective, photoprotective alternative through ligand based virtual screening (LBVS). We designed 213 novel molecules based on the (E)-cinnamoyl moiety of octinoxate, but only 23 were found to be less toxic than the parent compound. Then, an artificial neural network (ANN) based model was built to predict the molar absorptivity of those 23 molecules, and the molecule that presented a similar molar absorptivity to that of octinoxate was chosen for synthesis (analog 4, 3-phenylpropyl (E)-3-(4-methoxyphenyl)acrylate). Synthesis for analog 4 resulted in a 90% yield, and its photoprotective properties, lipophilicity and cytotoxicity were then evaluated. Analog 4 absorbed UV radiation in the range of 250–340 nm, and it presented a molar absorptivity of 36,155 M − 1cm−1. Its lipophilicity was evaluated with RP-HPLC resulting in a logkw of 2.49 and its LC50 was greater than octinoxate's (67.41 nM vs. 45.67 nM). Therefore, results showed that ligand based virtual screening is an effective strategy for the development of new organic UV filters, because it guided the design of less toxic analogs and pinpointed the most likely analog to exhibit UV properties similar to those of octinoxate. In this case, analog 4 is a promising alternative to its parent compound since it proved to be more effective and less toxic

Monocultural character of the diagnostic assessment of special educational needs in the Mapuche context

El objetivo de este trabajo es analizar críticamente las prácticas de evaluación diagnóstica de Necesidades Educativas Especiales (NEE) en contexto mapuche. Desde esta perspectiva, se sostiene que los procesos de evaluación diagnóstica de NEE que se les realizan a estudiantes mapuches constituyen una práctica pedagógica monocultural. A partir del punto de vista metodológico, se realiza una revisión bibliográfica de investigaciones empíricas y teóricas sobre la evaluación de NEE en contextos de diversidad social y cultural. Para ello se realiza una búsqueda de artículos científicos en las bases de datos Web of Science, Scielo y Eric. Los resultados develan que la política educativa chilena impone un modelo de evaluación diagnóstica de NEE estandarizado, por tanto es aplicado de una misma forma en todos los centros educativos de las distintas regiones del país, sin distinción, convirtiéndose en un dispositivo legal que responde a aspectos teóricos-prácticos, pero no a la diversidad social y cultural presente en el territorio. La evaluación diagnóstica de NEE adopta un carácter monocultural, ya que se basa en la clasificación internacional del funcionamiento de la discapacidad y de salud, y en pruebas estandarizadas para la población chilena no indígena. Se concluye que el diagnóstico de NEE de estudiantes mapuches es consecuencia de la distancia epistemológica entre el conocimiento mapuche y conocimiento escolar-occidental en muchos casos, y no necesariamente a dificultades de aprendizaje y participación a causa de una discapacidad o trastorno.The objective of this work is to critically analyze the diagnosis of Special Educational Needs (SEN) in the Mapuche context. From this perspective, the authors argue that the SEN diagnosis applied to Mapuche students is essentially a monocultural pedagogical practice. The methodology used is a bibliographic review of empirical and theoretical research on the evaluation of SEN in contexts of social and cultural diversity, through a search of scientific articles in the Web of Science, Scielo and Eric databases. The results reveal that Chilean education policy enforces a standardized model of SEN diagnosis, which is applied in the same way in all regions of the country without distinction. It is a legal device that responds to theoretical and practical aspects, but not to the social and cultural diversity present in the territory. The SEN diagnosis applied is monocultural, since it is based on an international classification of disability and health functioning, and on standardized tests designed for the non-indigenous population of Chile. It is concluded that the SEN diagnosis of Mapuche students is a consequence of the epistemological distance between Mapuche knowledge and Western scholarly knowledge, and does not necessarily reveal learning and participation difficulties arising from a disability or disorder

Monocultural character of the diagnostic assessment of special educational needs in the Mapuche context

Agradecimientos a CONICYT-BECA de Doctorado Nacional nº 21170975 y al proyecto FONDECYT 118153El objetivo de este trabajo es analizar críticamente las prácticas de evaluación diagnóstica de Necesidades Educativas Especiales (NEE) en contexto mapuche. Desde esta perspectiva, se sostiene que los procesos de evaluación diagnóstica de NEE que se les realizan a estudiantes mapuches constituyen una práctica pedagógica monocultural. A partir del punto de vista metodológico, se realiza una revisión bibliográfica de investigaciones empíricas y teóricas sobre la evaluación de NEE en contextos de diversidad social y cultural. Para ello se realiza una búsqueda de artículos científicos en las bases de datos Web of Science, Scielo y Eric. Los resultados develan que la política educativa chilena impone un modelo de evaluación diagnóstica de NEE estandarizado, por tanto es aplicado de una misma forma en todos los centros educativos de las distintas regiones del país, sin distinción, convirtiéndose en un dispositivo legal que responde a aspectos teóricos-prácticos, pero no a la diversidad social y cultural presente en el territorio. La evaluación diagnóstica de NEE adopta un carácter monocultural, ya que se basa en la clasificación internacional del funcionamiento de la discapacidad y de salud, y en pruebas estandarizadas para la población chilena no indígena. Se concluye que el diagnóstico de NEE de estudiantes mapuches es consecuencia de la distancia epistemológica entre el conocimiento mapuche y conocimiento escolar-occidental en muchos casos, y no necesariamente a dificultades de aprendizaje y participación a causa de una discapacidad o trastorno

Cytoplasmic 5'-3' exonuclease Xrn1p is also a genome-wide transcription factor in yeast

The 5' to 3' exoribonuclease Xrn1 is a large protein involved in cytoplasmatic mRNA degradation as a critical component of the major decaysome. Its deletion in the yeast Saccharomyces cerevisiae is not lethal, but it has multiple physiological effects. In a previous study, our group showed that deletion of all tested components of the yeast major decaysome, including XRN1, results in a decrease in the synthetic rate and an increase in half-life of most mRNAs in a compensatory manner. Furthermore, the same study showed that the all tested decaysome components are also nuclear proteins that bind to the 5 region of a number of genes. In the present work, we show that disruption of Xrn1 activity preferentially affects both the synthesis and decay of a distinct subpopulation of mRNAs. The most affected mRNAs are the transcripts of the highly transcribed genes, mainly those encoding ribosome biogenesis and translation factors. Previously, we proposed that synthegradases play a key role in regulating both mRNA synthesis and degradation. Evidently, Xrn1 functions as a synthegradase, whose selectivity might help coordinating the expression of the protein synthetic machinery. We propose to name the most affected genes"Xrn1 synthegrado

The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-PJunta de Andalucía P12-BIO1938MO, P08-CVI-03508Comunidad Valenciana 2015/00

Use of Arctium lappa Extract Against Acetaminophen-Induced Hepatotoxicity in Rats

AbstractBackgroundSevere destructive hepatic injuries can be induced by acetaminophen overdose and may lead to acute hepatic failure.ObjectiveTo investigate the ameliorative effects of Arctium lappa root extract on acetaminophen-induced hepatotoxicity.MethodsRats were divided into 4 groups: normal control group, Arctium lappa extract group, acetaminophen-injected group, and acetaminophen treated with Arctium lappa extract group.ResultsThe treatment with Arctium lappa extract reduced serum alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in the acetaminophen group when compared with the control group. DNA fragments in the acetaminophen-injected group were also significantly increased (P < 0.05). The comet assay revealed increased detaching tail length and DNA concentration during the hepatic toxicity in the acetaminophen group. The malondialdehyde content was inhibited by Arctium lappa treatment (12.97±0.89 nmol/mg) when compared with the acetaminophen-treated-only group (12.97±0.89 nmol/mg). Histopathologic examination revealed that acetaminophen administration produced hepatic cell necrosis, infiltrate of lymphocytes, and vacuolation that were associated with the acetaminophen-treated animal group, but the degree of acetaminophen-induced hepatotoxicity was mediated by treatment with Arctium lappa extract.ConclusionsArctium lappa can prevent most of the hepatic tissue damage caused by acetaminophen overdose in rats

One step back before moving forward: regulation of transcription elongation by arrest and backtracking

RNA polymerase II backtracking is a well-known phenomenon, but its involvement in gene regulation is yet to be addressed. Structural studies into the backtracked complex, new reactivation mechanisms and genome-wide approaches are shedding some light on this interesting aspect of gene transcription. In this review, we briefly summarise these new findings, comment about some results recently obtained in our laboratory, and propose a new model for the influence of the chromatin context on RNA polymerase II backtracking.Ministerio de Economía y Competitividad de España. BFU2007-67575-C03-02 y BFU-2010-21975-C03-03Junta de Andalucía. P07-CVI-02623 y P08-CVI-0350

RNA Binding by Histone Methyltransferases Set1 and Set2.

Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity

Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning.

BACKGROUND: TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but introduce noise due to MNase sequence preferences. A systematic way of correcting this bias for massively parallel sequencing experiments is still missing. RESULTS: To investigate the contribution of TFIIS to the chromatin landscape, we developed a refined nucleosome-mapping method in Saccharomyces cerevisiae. Based on partial MNase digestion and a sequence-bias correction derived from naked DNA cleavage, the refined method efficiently mapped nucleosomes in promoter regions rich in MNase-sensitive structures. The naked DNA correction was also important for mapping gene body nucleosomes, particularly in those genes whose core promoters contain a canonical TATA element. With this improved method, we analyzed the global nucleosomal changes caused by lack of TFIIS. We detected a general increase in nucleosomal fuzziness and more restricted changes in nucleosome occupancy, which concentrated in some gene categories. The TATA-containing genes were preferentially associated with decreased occupancy in gene bodies, whereas the TATA-like genes did so with increased fuzziness. The detected chromatin alterations correlated with functional defects in nascent transcription, as revealed by genomic run-on experiments. CONCLUSIONS: The combination of partial MNase digestion and naked DNA correction of the sequence bias is a precise nucleosomal mapping method that does not exclude MNase-sensitive nucleosomes. This method is useful for detecting subtle alterations in nucleosome positioning produced by lack of TFIIS. Their analysis revealed that TFIIS generally contributed to nucleosome positioning in both gene promoters and bodies. The independent effect of lack of TFIIS on nucleosome occupancy and fuzziness supports the existence of alternative chromatin dynamics during transcription elongation