A comparison of multiple shRNA expression methods for combinatorial RNAi

RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded

Exercise therapy for prevention of falls in people with Parkinson's disease: A protocol for a randomised controlled trial and economic evaluation

<p>Abstract</p> <p>Background</p> <p>People with Parkinson's disease are twice as likely to be recurrent fallers compared to other older people. As these falls have devastating consequences, there is an urgent need to identify and test innovative interventions with the potential to reduce falls in people with Parkinson's disease. The main objective of this randomised controlled trial is to determine whether fall rates can be reduced in people with Parkinson's disease using exercise targeting three potentially remediable risk factors for falls (reduced balance, reduced leg muscle strength and freezing of gait). In addition we will establish the cost effectiveness of the exercise program from the health provider's perspective.</p> <p>Methods/Design</p> <p>230 community-dwelling participants with idiopathic Parkinson's disease will be recruited. Eligible participants will also have a history of falls or be identified as being at risk of falls on assessment. Participants will be randomly allocated to a usual-care control group or an intervention group which will undertake weight-bearing balance and strengthening exercises and use cueing strategies to address freezing of gait. The intervention group will choose between the home-based or support group-based mode of the program. Participants in both groups will receive standardized falls prevention advice. The primary outcome measure will be fall rates. Participants will record falls and medical interventions in a diary for the duration of the 6-month intervention period. Secondary measures include the Parkinson's Disease Falls Risk Score, maximal leg muscle strength, standing balance, the Short Physical Performance Battery, freezing of gait, health and well being, habitual physical activity and positive and negative affect schedule.</p> <p>Discussion</p> <p>No adequately powered studies have investigated exercise interventions aimed at reducing falls in people with Parkinson's disease. This trial will determine the effectiveness of the exercise intervention in reducing falls and its cost effectiveness. This pragmatic program, if found to be effective, has the potential to be implemented within existing community services.</p> <p>Trial registration</p> <p>The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000303347).</p

Mitochondrial Control Region and microsatellite analyses on harbour porpoise (Phocoena phocoena) unravel population differentiation in the Baltic Sea and adjacent waters

The population status of the harbour porpoise (Phocoena phocoena) in the Baltic area has been a continuous matter of debate. Here we present the by far most comprehensive genetic population structure assessment to date for this region, both with regard to geographic coverage and sample size: 497 porpoise samples from North Sea, Skagerrak, Kattegat, Belt Sea, and Inner Baltic Sea were sequenced at the mitochondrial Control Region and 305 of these specimens were typed at 15 polymorphic microsatellite loci. Samples were stratified according to sample type (stranding vs. by-caught), sex, and season (breeding vs. non-breeding season). Our data provide ample evidence for a population split between the Skagerrak and the Belt Sea, with a transition zone in the Kattegat area. Among other measures, this was particularly visible in significant frequency shifts of the most abundant mitochondrial haplotypes. A particular haplotype almost absent in the North Sea was the most abundant in Belt Sea and Inner Baltic Sea. Microsatellites yielded a similar pattern (i.e., turnover in occurrence of clusters identified by STRUCTURE). Moreover, a highly significant association between microsatellite assignment and unlinked mitochondrial haplotypes further indicates a split between North Sea and Baltic porpoises. For the Inner Baltic Sea, we consistently recovered a small, but significant separation from the Belt Sea population. Despite recent arguments that separation should exceed a predefined threshold before populations shall be managed separately, we argue in favour of precautionary acknowledging the Inner Baltic porpoises as a separate management unit, which should receive particular attention, as it is threatened by various factors, in particular local fishery measures. © Springer Science+Business Media B.V. 2009

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Planning for ex situ conservation in the face of uncertainty

Ex situ conservation strategies for threatened species often require long-term commitment and financial investment to achieve management objectives. We present a framework that considers the decision to adopt ex situ management for a target species as the end point of several linked decisions. We used a decision tree to intuitively represent the logical sequence of decision making. The first decision is to identify the specific management actions most likely to achieve the fundamental objectives of the recovery plan, with or without the use of ex-situ populations. Once this decision has been made, one decides whether to establish an ex situ population, accounting for the probability of success in the initial phase of the recovery plan, for example, the probability of successful breeding in captivity. Approaching these decisions in the reverse order (attempting to establish an ex situ population before its purpose is clearly defined) can lead to a poor allocation of resources, because it may restrict the range of available decisions in the second stage. We applied our decision framework to the recovery program for the threatened spotted tree frog (Litoria spenceri) of southeastern Australia. Across a range of possible management actions, only those including ex situ management were expected to provide \u3e50% probability of the species\u27 persistence, but these actions cost more than use of in situ alternatives only. The expected benefits of ex situ actions were predicted to be offset by additional uncertainty and stochasticity associated with establishing and maintaining ex situ populations. Naïvely implementing ex situ conservation strategies can lead to inefficient management. Our framework may help managers explicitly evaluate objectives, management options, and the probability of success prior to establishing a captive colony of any given species