Tissue-specific expression of Cas9 has no impact on whole-body metabolism in four transgenic mouse lines

Objective:CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly.Methods:In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs.Results:We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass.Conclusions:Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9

Development of a Management Algorithm for Post-operative Pain (MAPP) after total knee and total hip replacement: study rationale and design.

BACKGROUND: Evidence from clinical practice and the extant literature suggests that post-operative pain assessment and treatment is often suboptimal. Poor pain management is likely to persist until pain management practices become consistent with guidelines developed from the best available scientific evidence. This work will address the priority in healthcare of improving the quality of pain management by standardising evidence-based care processes through the incorporation of an algorithm derived from best evidence into clinical practice. In this paper, the methodology for the creation and implementation of such an algorithm that will focus, in the first instance, on patients who have undergone total hip or knee replacement is described. METHODS: In partnership with clinicians, and based on best available evidence, the aim of the Management Algorithm for Post-operative Pain (MAPP) project is to develop, implement, and evaluate an algorithm designed to support pain management decision-making for patients after orthopaedic surgery. The algorithm will provide guidance for the prescription and administration of multimodal analgesics in the post-operative period, and the treatment of breakthrough pain. The MAPP project is a multisite study with one coordinating hospital and two supporting (rollout) hospitals. The design of this project is a pre-implementation-post-implementation evaluation and will be conducted over three phases. The Promoting Action on Research Implementation in Health Services (PARiHS) framework will be used to guide implementation. Outcome measurements will be taken 10 weeks post-implementation of the MAPP. The primary outcomes are: proportion of patients prescribed multimodal analgesics in accordance with the MAPP; and proportion of patients with moderate to severe pain intensity at rest. These data will be compared to the pre-implementation analgesic prescribing practices and pain outcome measures. A secondary outcome, the efficacy of the MAPP, will be measured by comparing pain intensity scores of patients where the MAPP guidelines were or were not followed. DISCUSSION: The outcomes of this study have relevance for nursing and medical professionals as well as informing health service evaluation. In establishing a framework for the sustainable implementation and evaluation of a standardised approach to post-operative pain management, the findings have implications for clinicians and patients within multiple surgical contexts

An intriguing association between dental and mental pathology in addicted and control subjects: A cross-sectional survey

Recent clinical studies suggest that substance use may be associated with an acceleration of the ageing process, possibly related to a deficit of stem cell number or function. As this clinic had access to both medical and drug dependent patients, we tested the hypothesis that there may be an association between previously identified deficits.A cross-sectional survey was performed looking at both dental and mental dysfunction. Both a dental index (DI) and a mental index (MI) were defined as previously described and utilised as summary measures of such pathology.From 249 substance use disorder (SUD) and 134 general medical controls (N-SUD), 248 and 91 patients were selected with ages less than 57 years as the primary focus of analysis. The mean (+/- S.D.) ages (32.59 +/- 7.98 vs 35.65 +/- 15.45 years) were similar. The DI was found to correlate with the MI in a significant manner in SUD (R = 0.14, p = 0.03), N-SUD (R = 0.27, p = 0.009) and in the whole group (R = 0.17, p = 0.001). The (univariate) association of MI with DI (p = 0.019) and DI with MI (p = 0.0037) remained highly significant at multivariate regression after adjustment for psychiatric diagnoses and measures of dose-duration exposure to common addictive drugs. The qualitative appearance of the surfaces of best fit for the relationship between age, DI and MI was different in the two groups.These results suggest that the robust statistical association between dental and mental pathology may be related to common underlying pathophysiological mechanisms such as a progeroid or stem cell deficiency process in clinical addiction