Intronic motif pairs cooperate across exons to promote pre-mRNA splicing

A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions. Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific. The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins

Estudo da estabilidade do éster metilanidroecgonina, marcador de uso do crack, benzoilecgonina e cocaína em urina humana

BACKGROUND: Typically, urine and other biological tissues have been analyzed for cocaine (COC) and/or metabolites to detect COC usage. COC undergoes numerous biotransformation and degradation reactions. Crack smokers are exposed to anhydroecgonine methyl ester (AME), which can be used as an analytical marker for crack smoking. The stability of this analyte in human urine has not been studied. In the body, COC is rapidly converted to metabolites by enzymatic and chemical processes, the major urinary metabolite being benzoylecgonine (BE). OBJECTIVES: This study was carried out in order to determine the effects of time and temperature on the stability of cocaine/crack metabolites in human urine. METHODS : The stability of AME, BE and COC in urine was investigated using samples of urine stored in freezers and refrigerators. The analytes were extracted from urine using a solid-phase extraction technique and analyzed by gas chromatography-flame ionization detection method. RESULTS: COC concentrations decreased while BE concentrations increased. AME concentrations remained stable. CONCLUSIONS: The temperature and the duration of storage are decisive in COC hydrolyzing. This study suggests that AME concentrations are not correlated to either storage duration or with storage temperature and AME is more stable than COC.CONTEXTO: Cocaína (COC) e/ou metabólitos tem sido analisados em urina e outros fluidos biológicos para se determinar o uso de COC. A COC está sujeita a numerosas reações de biotransformação e degradação. Indivíduos que fumam crack estão expostos ao éster metilanidroecgonina (AME), que pode ser empregado como marcador de uso desta droga. Não há referências na literatura a respeito da estabilidade deste analito em urina humana. No organismo a COC é rapidamente biotransformada em outros metabólitos por meio de processos químicos e enzimáticos e o principal metabólito urinário é a benzoilecgonina (BE). OBJETIVOS: O objetivo deste estudo foi determinar o efeito do tempo e da temperatura na estabilidade da COC, BE e do AME em urina humana. MÉTODOS: A estabilidade do AME, BE e COC em urina foi investigada por intermédio do armazenamento da urina em freezer e em geladeira. Os analitos foram extraídos pela técnica de extração em fase sólida e analisados por cromatografia gasosa acoplada ao detector por ionização em chama. RESULTADOS: As concentrações de COC decresceram enquanto as BE aumentaram. As concentrações de AME se mantiveram estáveis. CONCLUSÕES: A temperatura e o tempo de armazenamento são decisivos na hidrólise da COC. Este estudo sugere que as concentrações de AME não estão correlacionadas com o tempo ou a temperatura de armazenamento e o AME é mais estável que a COC.Fundação de Amparo à Pesquisa de São Paulo (FAPESP)Conselho Nacional de Pesquisa (CNPq

Dynamic Regulation of Alternative Splicing by Silencers that Modulate 5′ Splice Site Competition

SummaryAlternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with ∼70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5′ splice site located downstream of a weakened 5′ splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5′ splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5′ splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5′ splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate-limiting step(s) in splicing can lead to dramatic shifts in splice site choice

The Cinderella moment:Exploring consumers’ motivations to engage with renting as collaborative luxury consumption mode

Past literature argued that the purchase of luxury goods is driven by people’s motivation to conform or fit into our economic and social system. In this study, the authors focus on a new aspect of consumption, i.e. renting instead of purchasing luxury goods, backed by the emerging opportunities of sharing economy platforms. Drawing upon the analysis of spontaneous consumers’ online communications (in the form of tweets), this research aims to investigate the motivations to engage with luxury garment renting within a collaborative consumption context. To this end, a series of automatic content analyses, via two studies, were conducted using the tweets posted with respect to the Run the Runway collaborative consumption platform. Results demonstrate consumers’ increased willingness to show their social status through renting rather than owning luxurious apparel based on five main motivators (need to wear new clothes for a special event, inspirations created by the products/brands, possibility to explore a new way of consuming luxury goods, need to make more sustainable choices, and to increase the life cycle of each luxury product). The implications of these findings are discussed, while they pave the way for future research in collaborative consumption of luxury retailing

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins

'Asexual isn't who I am': the politics of asexuality

Some literature on asexuality has claimed that it is inherently radical and contains the potential for resistance. Unfortunately, this literature has tended to be unempirical, has imagined asexuality as a disembodied entity, and has marginalised the multiple identities held by asexual people. This article, inspired by Plummer’s critical humanist approach, seeks to explore how individuals understand their asexuality to encourage forms of political action in the areas of identity, activism, online spaces, and LGBT politics. What we found was a plurality of experiences and attitudes with most adopting a pragmatic position in response to their social situation which saw large-scale political action as irrelevant. We conclude by reflecting on what these results mean for those who see asexuality as potentially radical

Aspects of the methodology validation for light filth in fruit pulp

Methodology validation is an important tool to support the activities of metrology in the quantification analysis. Extraneous materials may be present in the pulps due to the conditions or the practices of production, storage and distribution. Since there is no specific methodology for fruit pulp light filth quantification a research has been carried out to test two methods for isolation of light filth in mango, strawberry, tomato and guava pulps. For guava, mango and tomato pulps the AOAC Official Method 964.23 was used, and for strawberry pulp AOAC Official Method 950.89, with adaptations. The micro-analytic standard for insect fragment and rodent hair was prepared in the laboratory. The study has been conducted on 63 samples of 100 g of pulp analyzed in duplicate after contamination with 5, 15, 30 insect fragments (IF) per 100 g and 5, 10, 15 rodent hair (RH) per 100 g, with blank samples as a control. The validation parameters used were precision and accuracy. Recovery of insect fragment was considered stisfactory in mango, tomato, guava pulps, and the average recovery ranged from 87 to 96%. For strawberry pulp, the recovery rate ranged from 68 to 80%. For rodent hairs, the best recovery rate was observed in mango pulp ranging from 73 to 81%, followed by guava pulp ranging from 65 to 76%, tomato pulp ranging from 50 to 67%, and strawberry pulp ranging from 33 to 35%. It was concluded that the methodologies used to detect light filth in guava, mango, tomato and strawberry pulps can be adopted in the monitoring routine of fruit pulp extraneous material contamination even though they were found not very efficient for rodent hairs. Keywords: Insect fragment, Rodent hair, AOAC determination method, Recovery rate, Fruit pulp

FRECUENCIA DEL USO DEL ANTICONCEPTIVO ORAL DE EMERGENCIA Y SUS REPERCUSIONES EN LA SALUD de las alumnas de la Universidad Alas Peruanas Puerto Maldonado- Marzo-Mayo, 2012

 Objetivo: Conocer como la frecuencia de uso del  anticonceptivo oral de emergencia (AOE), repercute en la salud de las alumnas de la Universidad Alas Peruanas-Puerto Maldonado, marzo-mayo del 2012. Material y métodos:Se trabajó con una población de 60 alumnas usuarias y el muestreo fue intencionado. Es una investigación de tipo Descriptivo, no experimental y  de corte transversal. Resultados:El 33.0% de la muestra tienen 19 años a más  seguidos de los que tienen 18 años (29.6%). Respecto a la religión el 100% son creyentes. Saben lo que es la AOE, el 42.85% de las usuarias obtuvo la información del internet y en menor % la obtuvieron de la pareja y propaganda televisiva. El 71.42% de las usuarias manifiesta que tienen conocimiento de los efectos Secundarios y conocen la dosificación. La frecuencia en el uso de AOE de los últimos 6 meses el 76.20% ha hecho uso de ella,  14 usuarias manifiestan haberla tomado de 1 a 3 veces. La frecuencia del uso de AOE en periodo de un año  el 90.47% manifiesta que si la uso, de estas usuarias el 84.21%  refiere que la uso de 1 a 3 veces.Dentro de las repercusiones en la salud el síntoma que se presentó con  más frecuencia fue el dolor de cabeza (32%) seguido de las náuseas (28%), hinchazón de mamas (12%), secreción blanquecina (12%), y menstruación adelanta un 4%, frente a un 12% que no presento ningún síntoma.Conclusiones:La mayoría de usuarias son adolescentes tardías (18,19 años). En su totalidad son creyentes, y  conocen la dosificación de la AOE. La frecuencia de uso es elevada y  Dentro de las repercusiones en la salud el síntoma que se presentó con  más frecuencia fue el dolor de cabeza, náuseas, hinchazón de mamas, secreción blanquecina, y menstruación adelantada.La frecuencia elevada de AOE ocasiona efectos colaterales no deseados, en las usuarias. Palabras clave: Anticoncepción oral de emergencia, repercusiones en la salud

The politics of in/visibility: carving out queer space in Ul'yanovsk

<p>In spite of a growing interest within sexualities studies in the concept of queer space (Oswin 2008), existing literature focuses almost exclusively on its most visible and territorialised forms, such as the gay scene, thus privileging Western metropolitan areas as hubs of queer consumer culture (Binnie 2004). While the literature has emphasised the political significance of queer space as a site of resistance to hegemonic gender and sexual norms, it has again predominantly focused on overt claims to public space embodied in Pride events, neglecting other less open forms of resistance.</p><p> This article contributes new insights to current debates about the construction and meaning of queer space by considering how city space is appropriated by an informal queer network in Ul’ianovsk. The group routinely occupied very public locations meeting and socialising on the street or in mainstream cafés in central Ul’ianovsk, although claims to these spaces as queer were mostly contingent, precarious or invisible to outsiders. The article considers how provincial location affects tactics used to carve out communal space, foregrounding the importance of local context and collective agency in shaping specific forms of resistance, and questioning ethnocentric assumptions about the empowering potential of visibility.</p&gt