In Vitro Studies on mRNA 3' Processing Using a Herpes Simplex Virus Poly A Site

An in vitro mRNA 3' processing system was established. Nuclear extracts capable of supporting 3' processing in this system were prepared from both HeLa cells and K562 cells; this is the first report of the use of K562 nuclear extracts for in vitro 3' processing. While the processing activities of the K562 extracts appeared to be identical to those of HeLa extracts, their overall levels of activity were significantly lower. HeLa nuclear extracts were used to investigate the properties of a 67bp synthetic poly A site composed of sequences from the HSV-2 equivalent of the HSV-1 UL38 gene. It was established that the synthetic poly A site contained sequences necessary and sufficient to direct 3' processing in vitro in a sequence-specific manner. The efficiency of in vitro 3' processing at the synthetic poly A site was comparable to that obtained with the HSV-2 IE-5 gene poly A site. Experiments using variants of the synthetic poly A site, in which the spatial relationship of the essential sequence elements was altered, indicated that reducing or increasing the spacing between the poly A signal (AAUAAA) and the downstream, GU-rich sequence element caused a diminution of 3' processing activity. The quantitative and qualitative effects on 3' processing of the sequences between the poly A signal and the downstream sequence element were also investigated. Processing of a synthetic poly A site variant, in which the sequence between the poly A signal and the downstream sequence element were inverted, occurred at a different location from that normally observed and also with a slightly higher efficiency. This result indicated that the poly A signal and the downstream signal element are not solely responsible for defining the efficiency and accuracy of cleavage. Gel mobility retardation assays were used to investigate the interactions between nuclear processing factors and precursor RNAs derived from the synthetic poly A site and its variants. The synthetic poly A site formed the same processing-specific complexes as the HSV-2 IE-5 gene poly A site. The variants, in which processing efficiency was lower than that of the synthetic poly A site, did not efficiently form the processing-specific complexes, with one exception. In the latter case, the spacing between the poly A signal and the downstream sequence element was reduced to such an extent that binding of the complex components to the RNA was possible but processing was not. A transient CAT gene expression assay was used to analyse the efficiency of polyadenylation directed by the synthetic poly A site and certain of its variants in vivo. The relative CAT activities observed confirmed the results obtained using the in vitro 3' processing system. The interactions between processing factors and the synthetic poly A site were investigated using complementary RNAs and sense-strand DNA oligonucleotides. By annealing complementary RNAs to the precursor RNA prior to incubation with nuclear extract, it was shown that the presence of double-stranded RNA at either the poly A signal, the cleavage site or the downstream element prevented specific complex assembly and 3' processing. This indicates that processing factors interact with all three elements of the poly A site. Pre-incubation of nuclear extract with the oligonucleotide GGTGTGTT, which corresponds to the GU-rich downstream sequence, inhibited specific complex assembly and 3' processing. This suggested that this oligonucleotide interacted with a processing factor, perhaps that which binds to the downstream sequence element. The implications of such an interaction for 3' processing in vivo are discussed. Other DNA oligonucleotides, corresponding to the poly A signal, the cleavage site and the vector-derived 3' sequence, had no effect on 3' processing in vitro

Optimal Aero-Elastic Design of a Rotor with Bend-Twist Coupling

Passive Bend-Twist Coupling (BTC) can be used in blades to alleviate loads and generate more Annual Energy Production (AEP). However, BTC is inherently aero-elastic, thus difficult to incorporate into the design with sequential design process. Multi-disciplinary Design Optimization (MDO) is an attractive approach for overcoming these challenges. This paper presents the re-design of a 100kW BTC rotor using the MDO rotor design package HAWTOpt2. In the preliminary design phase, MDO was used to assess the differences between elastic BTC (i.e. off-axis fibers) and geometric BTC (i.e. sweep). This work found that aero-elastic design optimization without BTC was able to achieve a 16% improvement, then with sweep a 18% improvement and with material coupling a 17% improvement. Due to the reduced stiffness of off-axis fibers, material coupled designs had more difficulty satisfying the tip deflection constraint. The geometric BTC concept was chosen for the final design. The design optimization was repeated with additional manufacturing constraints. The final design achieved a 12% improvement

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

BackgroundThe use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.ResultsTo develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.ConclusionWe have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.<br /

Studying functions on coral reefs : past perspectives, current conundrums, and future potential

This work was funded by the Australian Research Council (DRB; grant number FL190100062).Function-based studies have opened a new chapter in our understanding of coral reefs. Unfortunately, we are opening this chapter as the world’s reefs rapidly transform. In this context, one of the most important roles of function-based studies is to inform coral reef conservation. At this critical juncture, we have a chance to reflect on where we have come from, and where we are going, in coral reef functional ecology, with specific consideration of what this means for our approaches to conserving reefs. As focal examples, we examine the role of corals on reefs, and the practice of culling crown-of-thorns starfish, from a functional perspective. We also consider how the papers in this special issue build on our current understanding. Ultimately, we highlight how robust scientific investigation, based on an understanding of ecosystem functions, will be key in helping us navigate reefs through the current coral reef crisis.Peer reviewe

Angular momentum redirection phase of vector beams in a non-planar geometry

An electric field propagating along a non-planar path can acquire geometric phases. Previously, geometric phases have been linked to spin redirection and independently to spatial mode transformation, resulting in the rotation of polarisation and intensity profiles, respectively. We investigate the non-planar propagation of scalar and vector light fields and demonstrate that polarisation and intensity profiles rotate by the same angle. The geometric phase acquired is proportional to j = ℓ + σ, where ℓ is the topological charge and σ is the helicity. Radial and azimuthally polarised beams with j = 0 are eigenmodes of the system and are not affected by the geometric path. The effects considered here are relevant for systems relying on photonic spin Hall effects, polarisation and vector microscopy, as well as topological optics in communication systems

Relación entre empoderamiento familiar y calidad de los servicios de atención temprana

Este trabajo pretende estudiar el nivel de empoderamiento de las familias en relación con la Calidad de los Servicios de Atención Temprana en España, donde en la actualidad se está viviendo una transformación en los modelos de intervención. La recogida de datos se esta llevando a cabo a través de la percepción de las familias receptoras de  estos servicios. Se están utilizando las siguientes herramientas: FOS (Early Childhood Outcomes Center, 2010); FES (Paul E. Koen, Neal DeChillo y Barbara J. Fieson, 1992), FSS (Dunst, Trivette y Jenkins, 1984) y Family FINESSE (McWilliam, 2015). Para llevar a cabo este trabajo, hemos desarrollado el Análisis Factorial Exploratorio de Family FINESSE. Así pues, presentamos los factores analizados en dicha escala, así como otros datos psicométricos de la misma

Predictor variables of family empowerment in family-centred early intervention

Objetivo: Determinar algunas de las variables predictoras de empoderamiento familiar en los servicios de atención temprana que implementan prácticas centradas en la familia. Método: Se plantea en una muestra de 431 familias que reciben servicios de atención temprana en Espa˜na, un análisis de regresión lineal múltiple, para definir la relación entre las siguientes variables: tipo de prácticas en atención temprana, nivel de apoyos, estatus socioeconómico y diagnóstico de su hijo/a, como predictoras de empoderamiento familiar. Resultados: Los resultados evidencian diferencias significativas entre el empoderamiento de las familias y los apoyos con los que cuentan, también se observan niveles de empoderamiento diferentes en relación al diagnóstico del hijo o la hija con trastornos del neurodesarrollo. Conclusiones: A partir de los resultados, se sugiere reflexionar sobre las prácticas de atención temprana de profesionales en general y logopedas en particular, para potenciar el impacto sobre el empoderamiento de las familias, mejorando la fidelidad en la implementación de prácticas centradas en la familia, y favoreciendo la movilización de recursos y apoyos por parte de la familia, en ese mismo proceso de intervención.Objective: To determine some of the predictive variables of family empowerment in early intervention services that implement family-centred practices. Method: A multiple linear regression analysis is proposed in a sample of 431 families receiving early care services in Spain, to define the relationship between the following variables: type of early intervention practices, supports, socioeconomic status and diagnosis of their child as predictors of family empowerment. Results: The results show significant differences between the empowerment of the families and the supports they have, and different levels of empowerment are observed in relation to the diagnosis of the son or daughter with neurodevelopmental disorders. Conclusions: Based on the results, we suggest reflecting on professionals early intervention practices and those of speech therapists in particular, to enhance the impact on empowering families, improving fidelity in the implementation of family-centred practices, and favouring the mobilisation of resources and supports by the family, in that same intervention process.Educació

A genomics-informed, SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep

<p>Abstract</p> <p>Background</p> <p>Fleece rot (FR) and body-strike of Merino sheep by the sheep blowfly <it>Lucilia cuprina </it>are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance.</p> <p>Results</p> <p>The majority of the DE genes originated from the fleece rot subtracted libraries and over-representing gene ontology terms included defense response to bacterium and epidermis development, indicating a role of these processes in modulating the sheep's response to fleece rot. We focused on genes that contribute to the physical barrier function of skin, including keratins, collagens, fibulin and lipid proteins, to identify SNPs that were associated to fleece rot scores.</p> <p>Conclusions</p> <p>We identified FBLN1 (fibulin) and FABP4 (fatty acid binding protein 4) as key factors in sheep's resistance to fleece rot. Validation of these markers in other populations could lead to vital tests for marker assisted selection that will ultimately increase the natural fleece rot resistance of Merino sheep.</p

Novel mutations expand the clinical spectrum of DYNC1H1-associated spinal muscular atrophy

OBJECTIVE To expand the clinical phenotype of autosomal dominant congenital spinal muscular atrophy with lower extremity predominance (SMA-LED) due to mutations in the dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) gene. METHODS Patients with a phenotype suggestive of a motor, non-length-dependent neuronopathy predominantly affecting the lower limbs were identified at participating neuromuscular centers and referred for targeted sequencing of DYNC1H1. RESULTS We report a cohort of 30 cases of SMA-LED from 16 families, carrying mutations in the tail and motor domains of DYNC1H1, including 10 novel mutations. These patients are characterized by congenital or childhood-onset lower limb wasting and weakness frequently associated with cognitive impairment. The clinical severity is variable, ranging from generalized arthrogryposis and inability to ambulate to exclusive and mild lower limb weakness. In many individuals with cognitive impairment (9/30 had cognitive impairment) who underwent brain MRI, there was an underlying structural malformation resulting in polymicrogyric appearance. The lower limb muscle MRI shows a distinctive pattern suggestive of denervation characterized by sparing and relative hypertrophy of the adductor longus and semitendinosus muscles at the thigh level, and diffuse involvement with relative sparing of the anterior-medial muscles at the calf level. Proximal muscle histopathology did not always show classic neurogenic features. CONCLUSION Our report expands the clinical spectrum of DYNC1H1-related SMA-LED to include generalized arthrogryposis. In addition, we report that the neurogenic peripheral pathology and the CNS neuronal migration defects are often associated, reinforcing the importance of DYNC1H1 in both central and peripheral neuronal functions