A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

A second transmissible cancer in Tasmanian devils.

Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.We thank Bill Brown, Phil Iles, Billie Lazenby, Jacinta Marr, Jane McGee, Sarah Peck, Holly Wiersma and Phil Wise for assistance with sample collection and curation. Adrian Baez-Ortega, Andrew Davis, Jo Hanuszewicz, Gina Kalodimos, Amanda Patchett, Narelle Phillips, Elizabeth Reid Swainscoat, Jim Richley, Rachel Stivicic and Jim Taylor assisted with surveying, laboratory analysis, data processing and display. We are grateful for support received from Mike Stratton, the Wellcome Trust Sanger Institute (WTSI) sequencing and informatics teams and the WTSI Cancer Genome Project. This work was supported by a Wellcome Trust Investigator Award (102942/Z/13/Z) and by grants from the Australian Research Council (ARC-DP130100715; ARC-LP130100218). Support was provided by Dr Eric Guiler Tasmanian Devil Research Grants and by the Save the Tasmanian Devil Program. JMCT was partly supported by a Marie Curie Fellowship (FP7-PEOPLE- 2012-IEF, 328364). Sequences associated with this paper have been deposited in Genbank with accession numbers KT188437 and KT188438

Evolution and lineage dynamics of a transmissible cancer in Tasmanian devils.

Devil facial tumour 1 (DFT1) is a transmissible cancer clone endangering the Tasmanian devil. The expansion of DFT1 across Tasmania has been documented, but little is known of its evolutionary history. We analysed genomes of 648 DFT1 tumours collected throughout the disease range between 2003 and 2018. DFT1 diverged early into five clades, three spreading widely and two failing to persist. One clade has replaced others at several sites, and rates of DFT1 co-infection are high. DFT1 gradually accumulates copy number variants (CNVs) and its telomere lengths are short but constant. Recurrent CNVs reveal genes under positive selection, sites of genome instability and repeated loss of a small derived chromosome. Cultured DFT1 cell lines have increased CNV frequency and undergo highly reproducible convergent evolution. Overall, DFT1 is a remarkably stable lineage whose genome illustrates how cancer cells adapt to diverse environments and persist in a parasitic niche.This work was supported by grants from Wellcome (102942/Z/13/A), the National Science Foundation (DEB-1316549), the University of Tasmania Foundation (Eric Guiler Tasmanian Devil Research Grants), the Australian Research Council (DE 170101116) and a Philip Leverhulme Prize from the Leverhulme Trust. YMK was supported by a Herchel Smith Postgraduate Fellowship

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Thrive: Success Strategies for the Modern-Day Faculty Member

The THRIVE collection is intended to help faculty thrive in their roles as educators, scholars, researchers, and clinicians. Each section contains a variety of thought-provoking topics that are designed to be easily digested, guide personal reflection, and put into action. Please use the THRIVE collection to help: Individuals study topics on their own, whenever and wherever they want Peer-mentoring or other learning communities study topics in small groups Leaders and planners strategically insert faculty development into existing meetings Faculty identify campus experts for additional learning, grand rounds, etc. If you have questions or want additional information on a topic, simply contact the article author or email [email protected]://digitalcommons.unmc.edu/facdev_books/1000/thumbnail.jp

IL28B, HLA-C, and KIR Variants Additively Predict Response to Therapy in Chronic Hepatitis C Virus Infection in a European Cohort: A Cross-Sectional Study

Vijayaprakash Suppiah and colleagues show that genotyping hepatitis C patients for the IL28B, HLA-C, and KIR genes improves the ability to predict whether or not patients will respond to antiviral treatment

Specialist physiotherapy for functional motor disorder in England and Scotland (Physio4FMD): a pragmatic, multicentre, phase 3 randomised controlled trial

Summary: Background: Functional motor disorder—the motor variant of functional neurological disorder—is a disabling condition that is commonly associated with poor health outcomes. Pathophysiological models have inspired new treatment approaches such as specialist physiotherapy, although evidence from large randomised controlled trials is absent. We aimed to assess the clinical effectiveness of a specialist physiotherapy intervention for functional motor disorder compared with treatment as usual. Methods: In this pragmatic, multicentre, phase 3 randomised controlled trial at 11 hospitals in England and Scotland, adults with a clinically definite diagnosis of functional motor disorder, diagnosed by a neurologist, were included. Participants were randomly assigned (1:1, stratified by site) using a remote web-based application to either specialist physiotherapy (a protocolised intervention of nine sessions plus follow-up) or treatment as usual (referral to local community neurological physiotherapy). Individuals working on data collection and analysis were masked to treatment allocation. The primary outcome was the physical functioning domain of the 36-item short form health questionnaire (SF36) at 12 months after randomisation. The primary analysis followed a modified intention-to-treat principle, using a complete case approach; participants who were unable to receive their randomised treatment due to the suspension of health-care services during the COVID-19 pandemic were excluded from the primary analysis. This trial is registered with the International Standard Randomised Controlled Trial registry, ISRCTN56136713, and is completed. Findings: Recruitment occurred between Oct 19, 2018, and March 11, 2020, pausing during the COVID-19 lockdown, and resuming from Aug 3, 2021, to Jan 31, 2022. Of 355 participants who were enrolled, 179 were randomly assigned to specialist physiotherapy and 176 to treatment as usual. 89 participants were excluded from the primary analysis due to COVID-19 interruption to treatment (27 were assigned to specialist physiotherapy and 62 to treatment as usual). After accounting for withdrawals (n=11) and loss to follow-up (n=14), the primary analysis included data from 241 participants (138 [91%] assigned specialist physiotherapy and 103 [90%] assigned treatment as usual). Physical functioning, as assessed by SF36, did not differ significantly between groups (adjusted mean difference 3·5, 95% CI –2·3 to 9·3; p=0·23). There were no serious adverse events related to the trial interventions. 35 serious adverse events were recorded in the specialist physiotherapy group by 24 participants (17·0%), and 24 serious adverse events were recorded in the treatment as usual group by 18 participants (17·0%); one death occurred in the specialist physiotherapy group (cause of death was recorded as suicide). All were considered unrelated to specialist physiotherapy. Interpretation: Although more participants who were assigned specialist physiotherapy self-rated their motor symptoms as improved and had better scores on subjective measures of mental health, the intervention did not result in better self-reported physical functioning at 12 months. Both the specialist and community neurological physiotherapy appeared to be a safe and a valued treatment for selected patients with functional motor disorder. Future research should continue to refine interventions for people with functional motor disorder and develop evidence-based methods to guide treatment triage decisions. Funding: National Institute for Health and Care Research and Health Technology Assessment Programme