Design and cloning strategies for constructing shRNA expression vectors

BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 – 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process

The effects of stem length and core placement on shRNA activity

<p>Abstract</p> <p>Background</p> <p>Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined.</p> <p>Results</p> <p>Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and found that stem length does not correspond with predictable changes in suppressive activity. We also detected multiple processed products for all stem lengths tested. However, the entire length of the hairpin stem was not equally processed into active products. As such, the placement of the siRNA core at the base terminus was critical for activity.</p> <p>Conclusion</p> <p>We conclude that there is no fixed correlation between stem length and suppressive activity. Instead, core selection and placement likely have a greater influence on the effectiveness of shRNA-based silencing.</p

Multiple shRNA combinations for near-complete coverage of all HIV-1 strains

<p>Abstract</p> <p>Background</p> <p>Combinatorial RNA interference (co-RNAi) approaches are needed to account for viral variability in treating HIV-1 with RNAi, as single short hairpin RNAs (shRNA) are rapidly rendered ineffective by resistant strains. Current work suggests that 4 simultaneously expressed shRNAs may prevent the emergence of resistant strains.</p> <p>Results</p> <p>In this study we assembled combinations of highly-conserved shRNAs to target as many HIV-1 strains as possible. We analyzed intersecting conservations of 10 shRNAs to find combinations with 4+ matching the maximum number of strains using 1220+ HIV-1 sequences from the Los Alamos National Laboratory (LANL). We built 26 combinations of 2 to 7 shRNAs with up to 87% coverage for all known strains and 100% coverage of clade B subtypes, and characterized their intrinsic suppressive activities in transient expression assays. We found that all combinations had high combined suppressive activities, though there were also large changes in the individual activities of the component shRNAs in our multiple expression cassette configurations.</p> <p>Conclusion</p> <p>By considering the intersecting conservations of shRNA combinations we have shown that it is possible to assemble combinations of 6 and 7 highly active, highly conserved shRNAs such that there is always at least 4 shRNAs within each combination covering all currently known variants of entire HIV-1 subtypes. By extension, it may be possible to combine several combinations for complete global coverage of HIV-1 variants.</p

A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study

Background: A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. Methods an design: There are two principal arms to this study: (i) a cross-sectional study (FACT OBS) of 90 subjects recruited from gastroenterology clinics and; (ii) an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and precancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. Discussion: This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function

Advancing One Health:Updated core competencies

International audienceAbstract One Health recognises the interdependence between the health of humans, animals, plants and the environment. With the increasing inclusion of One Health in multiple global health strategies, the One Health workforce must be prepared to protect and sustain the health and well-being of life on the planet. In this paper, a review of past and currently accepted One Health core competencies was conducted, with competence gaps identified. Here, the Network for Ecohealth and One Health (NEOH) propose updated core competencies designed to simplify what can be a complex area, grouping competencies into three main areas of: Skills; Values and Attitudes; and Knowledge and Awareness; with several layers underlying each. These are intentionally applicable to stakeholders from various sectors and across all levels to support capacity-building efforts within the One Health workforce. The updated competencies from NEOH can be used to evaluate and enhance current curricula, create new ones, or inform professional training programs at all levels, including students, university teaching staff, or government officials as well as continual professional development for frontline health practitioners and policy makers. The competencies are aligned with the new definition of One Health developed by the One Health High-Level Expert Panel (OHHLEP), and when supported by subjectspecific expertise, will deliver the transformation needed to prevent and respond to complex global challenges. One Health Impact Statement Within a rapidly changing global environment, the need for practitioners competent in integrated approaches to health has increased substantially. Narrow approaches may not only limit opportunities for global and local solutions but, initiatives that do not consider other disciplines or social, economic and cultural contexts, may result in unforeseen and detrimental consequences. In keeping with principles of One Health, the Network for Ecohealth and One Health (NEOH) competencies entail a collaborative effort between multiple disciplines and sectors. They focus on enabling practitioners, from any background, at any level or scale of involvement, to promote and support a transformation to integrated health approaches. The updated competencies can be layered with existing disciplinary competencies and used to evaluate and enhance current education curricula, create new ones, or inform professional training programs at all levels-including for students, teachers and government officials as well as continual professional development for frontline health practitioners and policymakers. The competencies outlined here are applicable to all professionals and disciplines who may contribute to One Health, and are complimentary to, not a replacement for, any discipline-specific competencies. We believe the NEOH competencies meet the need outlined by the Quadripartite’s (Food and Agriculture Organisation, United Nations Environment Programme, World Health Organisation, World Organisation for Animal Health) Joint Plan of Action on One Health which calls for cross-sectoral competencies

Aripiprazole in the Maintenance Treatment of Bipolar Disorder: A Critical Review of the Evidence and Its Dissemination into the Scientific Literature

A systematic search of the literature reveals limited evidence to support use of aripiprazole, a second-generation antipsychotic medication, in maintenance therapy of bipolar disorder, despite widespread use

Overview of JET results for optimising ITER operation

The JET 2019–2020 scientific and technological programme exploited the results of years of concerted scientific and engineering work, including the ITER-like wall (ILW: Be wall and W divertor) installed in 2010, improved diagnostic capabilities now fully available, a major neutral beam injection upgrade providing record power in 2019–2020, and tested the technical and procedural preparation for safe operation with tritium. Research along three complementary axes yielded a wealth of new results. Firstly, the JET plasma programme delivered scenarios suitable for high fusion power and alpha particle (α) physics in the coming D–T campaign (DTE2), with record sustained neutron rates, as well as plasmas for clarifying the impact of isotope mass on plasma core, edge and plasma-wall interactions, and for ITER pre-fusion power operation. The efficacy of the newly installed shattered pellet injector for mitigating disruption forces and runaway electrons was demonstrated. Secondly, research on the consequences of long-term exposure to JET-ILW plasma was completed, with emphasis on wall damage and fuel retention, and with analyses of wall materials and dust particles that will help validate assumptions and codes for design and operation of ITER and DEMO. Thirdly, the nuclear technology programme aiming to deliver maximum technological return from operations in D, T and D–T benefited from the highest D–D neutron yield in years, securing results for validating radiation transport and activation codes, and nuclear data for ITER

Overview of JET results for optimising ITER operation

The JET 2019–2020 scientific and technological programme exploited the results of years of concerted scientific and engineering work, including the ITER-like wall (ILW: Be wall and W divertor) installed in 2010, improved diagnostic capabilities now fully available, a major neutral beam injection upgrade providing record power in 2019–2020, and tested the technical and procedural preparation for safe operation with tritium. Research along three complementary axes yielded a wealth of new results. Firstly, the JET plasma programme delivered scenarios suitable for high fusion power and alpha particle (α) physics in the coming D–T campaign (DTE2), with record sustained neutron rates, as well as plasmas for clarifying the impact of isotope mass on plasma core, edge and plasma-wall interactions, and for ITER pre-fusion power operation. The efficacy of the newly installed shattered pellet injector for mitigating disruption forces and runaway electrons was demonstrated. Secondly, research on the consequences of long-term exposure to JET-ILW plasma was completed, with emphasis on wall damage and fuel retention, and with analyses of wall materials and dust particles that will help validate assumptions and codes for design and operation of ITER and DEMO. Thirdly, the nuclear technology programme aiming to deliver maximum technological return from operations in D, T and D–T benefited from the highest D–D neutron yield in years, securing results for validating radiation transport and activation codes, and nuclear data for ITER

Mortality and pulmonary complications in patients undergoing surgery with perioperative sars-cov-2 infection: An international cohort study

Background The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (740%) had emergency surgery and 280 (248%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (261%) patients. 30-day mortality was 238% (268 of 1128). Pulmonary complications occurred in 577 (512%) of 1128 patients; 30-day mortality in these patients was 380% (219 of 577), accounting for 817% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 175 [95% CI 128-240], p&lt;00001), age 70 years or older versus younger than 70 years (230 [165-322], p&lt;00001), American Society of Anesthesiologists grades 3-5 versus grades 1-2 (235 [157-353], p&lt;00001), malignant versus benign or obstetric diagnosis (155 [101-239], p=0046), emergency versus elective surgery (167 [106-263], p=0026), and major versus minor surgery (152 [101-231], p=0047). Interpretation Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research