Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines

Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out

Improving mental health outcomes: achieving equity through quality improvement

Objective. To investigate equity of patient outcomes in a psychological therapy service, following increased access achieved by a quality improvement (QI) initiative. Design. Retrospective service evaluation of health outcomes; data analysed by ANOVA, chi-squared and Statistical Process Control. Setting. A psychological therapy service in Westminster, London, UK. Participants. People living in the Borough of Westminster, London, attending the service (from either healthcare professional or self-referral) between February 2009 and May 2012. Intervention(s). Social marketing interventions were used to increase referrals, including the promotion of the service through local media and through existing social networks. Main Outcome Measure(s). (i) Severity of depression on entry using Patient Health Questionnaire-9 (PHQ9). (ii) Changes to severity of depression following treatment (ΔPHQ9). (iii) Changes in attainment of a meaningful improvement in condition assessed by a key performance indicator. Results. Patients from areas of high deprivation entered the service with more severe depression (M = 15.47, SD = 6.75), com-pared with patients from areas of low (M = 13.20, SD = 6.75) and medium (M = 14.44, SD = 6.64) deprivation. Patients in low

Mandated data archiving greatly improves access to research data

The data underlying scientific papers should be accessible to researchers both now and in the future, but how best can we ensure that these data are available? Here we examine the effectiveness of four approaches to data archiving: no stated archiving policy, recommending (but not requiring) archiving, and two versions of mandating data deposition at acceptance. We control for differences between data types by trying to obtain data from papers that use a single, widespread population genetic analysis, STRUCTURE. At one extreme, we found that mandated data archiving policies that require the inclusion of a data availability statement in the manuscript improve the odds of finding the data online almost a thousand-fold compared to having no policy. However, archiving rates at journals with less stringent policies were only very slightly higher than those with no policy at all. At one extreme, we found that mandated data archiving policies that require the inclusion of a data availability statement in the manuscript improve the odds of finding the data online almost a thousand fold compared to having no policy. However, archiving rates at journals with less stringent policies were only very slightly higher than those with no policy at all. We also assessed the effectiveness of asking for data directly from authors and obtained over half of the requested datasets, albeit with about 8 days delay and some disagreement with authors. Given the long term benefits of data accessibility to the academic community, we believe that journal based mandatory data archiving policies and mandatory data availability statements should be more widely adopted

Herding cats: observing live coding in the wild

After a momentous decade of live coding activities, this paper seeks to explore the practice with the aim of situating it in the history of contemporary arts and music. The article introduces several key points of investigation in live coding research and discusses some examples of how live coding practitioners engage with these points in their system design and performances. In the light of the extremely diverse manifestations of live coding activities, the problem of defining the practice is discussed, and the question raised whether live coding will actually be necessary as an independent category