Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro

Trans-splicing, the in vivo joining of two RNA molecules, is well characterized in several groups of simple organisms but was long thought absent from fungi, plants and mammals. However, recent bioinformatic analyses of expressed sequence tag (EST) databases suggested widespread trans-splicing in mammals^1-2^. Splicing, including the characterised trans-splicing systems, involves conserved sequences at the splice junctions. Our analysis of a yeast non-coding RNA revealed that around 30% of the products of reverse transcription lacked an internal region of 117 nt, suggesting that the RNA was spliced. The junction sequences lacked canonical splice-sites but were flanked by direct repeats, and further analyses indicated that the apparent splicing actually arose because reverse transcriptase can switch templates during transcription^3^. Many newly identified, apparently trans-spliced, RNAs lacked canonical splice sites but were flanked by short regions of homology, leading us to question their authenticity. Here we report that all reported categories of non-canonical splicing could be replicated using an in vitro reverse transcription system with highly purified RNA substrates. We observed the reproducible occurrence of ostensible trans-splicing, exon shuffling and sense-antisense fusions. The latter generate apparent antisense non-coding RNAs, which are also reported to be abundant in humans^4^. Different reverse transcriptases can generate different products of template switching, providing a simple diagnostic. Many reported examples of splicing in the absence of canonical splicing signals may be artefacts of cDNA preparation

Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2

SummaryTranscriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5–7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock

Localisation and origin of the bacteriochlorophyll-derived photosensitizer in the retina of the deep-sea dragon fish Malacosteus niger

Most deep-sea fish have a single visual pigment maximally sensitive at short wavelengths, approximately matching the spectrum of both downwelling sunlight and bioluminescence. However, Malcosteus niger produces far-red bioluminescence and its longwave retinal sensitivity is enhanced by red-shifted visual pigments, a longwave reflecting tapetum and, uniquely, a bacteriochlorophyllderived photosensitizer. The origin of the photosensitizer, however, remains unclear. We investigated whether the bacteriochlorophyll was produced by endosymbiotic bacteria within unusual structures adjacent to the photoreceptors that had previously been described in this species. However, microscopy, elemental analysis and SYTOX green staining provided no evidence for such localised retinal bacteria, instead the photosensitizer was shown to be distributed throughout the retina. Furthermore, comparison of mRNA from the retina of Malacosteus to that of the closely related Pachystomias microdon (which does not contain a bacterichlorophyll-derived photosensitzer) revealed no genes of bacterial origin that were specifically up-regulated in Malacosteus. Instead up-regulated Malacosteus genes were associated with photosensitivity and may relate to its unique visual ecology and the chlorophyll-based visual system. We also suggest that the unusual longwave-reflecting, astaxanthin-based, tapetum of Malacosteus may protect the retina from the potential cytotoxicity of such a system

Regulation and Repair of the Alveolar-Capillary Barrier in Acute Lung Injury

Considerable progress has been made in understanding the basic mechanisms that regulate fluid and protein exchange across the endothelial and epithelial barriers of the lung under both normal and pathological conditions. Clinically relevant lung injury occurs most commonly from severe viral and bacterial infections, aspiration syndromes, and severe shock. The mechanisms of lung injury have been identified in both experimental and clinical studies. Recovery from lung injury requires the reestablishment of an intact endothelial barrier and a functional alveolar epithelial barrier capable of secreting surfactant and removing alveolar edema fluid. Repair mechanisms include the participation of endogenous progenitor cells in strategically located niches in the lung. Novel treatment strategies include the possibility of cell-based therapy that may reduce the severity of lung injury and enhance lung repair

Clearance kinetics and matrix binding partners of the receptor for advanced glycation end products

Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE). Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin. Copyright: © 2014 Milutinovic et al

The effectiveness of Stepping stones Triple P: the design of a randomised controlled trial on a parenting programme regarding children with mild intellectual disability and psychosocial problems versus care as usual

<p>Abstract</p> <p>Background</p> <p>Children with an intellectual disability are at increased risk of psychosocial problems. This leads to serious restrictions in the daily functioning of the children and to parental stress. Stepping Stones Triple P aims to prevent severe behavioural, emotional and developmental problems in children with a (intellectual) disability by enhancing parenting knowledge and skills, and the self-confidence of parents. This paper aims to describe the design of a study of the effectiveness of parenting counselling using Stepping Stones Triple P compared to Care as Usual.</p> <p>Methods/Design</p> <p>The effects of Stepping Stones Triple P will be studied in a Randomised Controlled Trial. Parents of children aged 5-12 years with an IQ of 50-85 will be recruited from schools. Prior to randomisation, parents complete a screening questionnaire about their child's psychosocial problems and their parenting skills. Subsequently, parents of children with increased levels of psychosocial problems (score on Strengths and Difficulties Questionnaire ≥ 14) will be invited to participate in the intervention study. After obtaining consent, parents will be randomised either to the experimental group (Stepping Stones Triple P) or to Care as Usual. The primary outcome is a change in the child's psychosocial problems according to parents and teachers. The secondary outcome is a change in parenting skills. Data will be collected before the start of the intervention, immediately after the intervention, and six months after.</p> <p>Discussion</p> <p>This paper presents an outline of the background and design of a randomised controlled trial to investigate the effectiveness of Stepping Stones Triple P, which aims to decrease psychosocial problems in children with a mild intellectual disability. Stepping Stones Triple P seems promising, but evidence on its effectiveness for this population is still lacking. This study provides evidence about the effects of this intervention in a community-based population of children with a mild intellectual disability.</p> <p>Trial registration</p> <p>Netherlands Trial Register (NTR): <a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=NTR2624">NTR2624</a></p

The effect of alcohol advertising, marketing and portrayal on drinking behaviour in young people: systematic review of prospective cohort studies

<p>Abstract</p> <p>Background</p> <p>The effect of alcohol portrayals and advertising on the drinking behaviour of young people is a matter of much debate. We evaluated the relationship between exposure to alcohol advertising, marketing and portrayal on subsequent drinking behaviour in young people by systematic review of cohort (longitudinal) studies.</p> <p>Methods</p> <p>studies were identified in October 2006 by searches of electronic databases, with no date restriction, supplemented with hand searches of reference lists of retrieved articles. Cohort studies that evaluated exposure to advertising or marketing or alcohol portrayals and drinking at baseline and assessed drinking behaviour at follow-up in young people were selected and reviewed.</p> <p>Results</p> <p>seven cohort studies that followed up more than 13,000 young people aged 10 to 26 years old were reviewed. The studies evaluated a range of different alcohol advertisement and marketing exposures including print and broadcast media. Two studies measured the hours of TV and music video viewing. All measured drinking behaviour using a variety of outcome measures. Two studies evaluated drinkers and non-drinkers separately. Baseline non-drinkers were significantly more likely to have become a drinker at follow-up with greater exposure to alcohol advertisements. There was little difference in drinking frequency at follow-up in baseline drinkers. In studies that included drinkers and non-drinkers, increased exposure at baseline led to significant increased risk of drinking at follow-up. The strength of the relationship varied between studies but effect sizes were generally modest. All studies controlled for age and gender, however potential confounding factors adjusted for in analyses varied from study to study. Important risk factors such as peer drinking and parental attitudes and behaviour were not adequately accounted for in some studies.</p> <p>Conclusion</p> <p>data from prospective cohort studies suggest there is an association between exposure to alcohol advertising or promotional activity and subsequent alcohol consumption in young people. Inferences about the modest effect sizes found are limited by the potential influence of residual or unmeasured confounding.</p

Spin canting across core/shell Fe3O4/MnxFe3−xO4 nanoparticles

Magnetic nanoparticles (MNPs) have become increasingly important in biomedical applications like magnetic imaging and hyperthermia based cancer treatment. Understanding their magnetic spin configurations is important for optimizing these applications. The measured magnetization of MNPs can be significantly lower than bulk counterparts, often due to canted spins. This has previously been presumed to be a surface effect, where reduced exchange allows spins closest to the nanoparticle surface to deviate locally from collinear structures. We demonstrate that intraparticle effects can induce spin canting throughout a MNP via the Dzyaloshinskii-Moriya interaction (DMI). We study ~7.4 nm diameter, core/shell Fe3O4/MnxFe3−xO4 MNPs with a 0.5 nm Mn-ferrite shell. Mössbauer spectroscopy, x-ray absorption spectroscopy and x-ray magnetic circular dichroism are used to determine chemical structure of core and shell. Polarized small angle neutron scattering shows parallel and perpendicular magnetic correlations, suggesting multiparticle coherent spin canting in an applied field. Atomistic simulations reveal the underlying mechanism of the observed spin canting. These show that strong DMI can lead to magnetic frustration within the shell and cause canting of the net particle moment. These results illuminate how core/shell nanoparticle systems can be engineered for spin canting across the whole of the particle, rather than solely at the surface

Treatment of post-traumatic degenerative changes of the radio-carpal and distal radio-ulnar joints by combining radius, scaphoid, and lunate (RSL) fusion with ulnar head replacement

Distal radial fractures are a common type of fracture. In the case of intra-articular fractures, they often result in post-traumatic arthrosis. The objective of this study is to describe a novel alternative to the established salvage techniques for the treatment of post-traumatic arthrosis of the radio-carpal and distal radio-ulnar joints (DRUJ). Six patients with radio-carpal and DRUJ arthrosis were treated with a combined radius, scaphoid, and lunate (RSL) arthrodesis and as a Herbert ulnar head prosthesis. Follow-up consisted of both radiographic and functional assessments. Functional measurements were noted both pre- and postoperatively. No non-union or pseudoarthrosis was seen; neither did any of the ulnar head prostheses show loosening. Clinical examination showed an improvement in strength, pain, and range of movement, as well as a decrease in disability. Combining RSL arthrodesis with a Herbert ulnar head prosthesis, which deals with pain while retaining partial wrist movement, can be an alternative to established salvage procedures