CRISPR-enhanced human adipocyte \u27browning\u27 as cell therapy for metabolic disease [preprint]

Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin [1,2], disturbances in glucose and lipid fluxes [3-5] and comorbidities including steatohepatitis [6] and cardiovascular disease [7,8]. Despite extensive efforts at prevention and treatment [9,10], diabetes afflicts over 400 million people worldwide [11]. Whole body metabolism is regulated by adipose tissue depots [12-14], which include both lipid-storing white adipocytes and less abundant \u27brown\u27 and \u27brite/beige\u27 adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health [15-18]. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing [19,20] to enhance \u27browning\u27 of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP1 [21,22] with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors

Evaluation of variants in the selectin genes in age-related macular degeneration

<p>Abstract</p> <p>Background</p> <p>Age-related macular degeneration (AMD) is a common disease of the elderly that leads to loss of the central visual field due to atrophic or neovascular events. Evidence from human eyes and animal models suggests an important role for macrophages and endothelial cell activation in the pathogenesis of AMD. We sought to determine whether common ancestral variants in genes encoding the selectin family of proteins are associated with AMD.</p> <p>Methods</p> <p>Expression of E-selectin, L-selectin and P-selectin was examined in choroid and retina by quantitative PCR and immunofluorescence. Samples from patients with AMD (n = 341) and controls (n = 400) were genotyped at a total of 34 SNPs in the <it>SELE</it>, <it>SELL </it>and <it>SELP </it>genes. Allele and genotype frequencies at these SNPs were compared between AMD patients and controls as well as between subtypes of AMD (dry, geographic atrophy, and wet) and controls.</p> <p>Results</p> <p>High expression of all three selectin genes was observed in the choroid as compared to the retina. Some selectin labeling of retinal microglia, drusen cores and the choroidal vasculature was observed. In the genetic screen of AMD versus controls, no positive associations were observed for <it>SELE </it>or <it>SELL</it>. One SNP in <it>SELP </it>(rs3917751) produced p-values < 0.05 (uncorrected for multiple measures). In the subtype analyses, 6 SNPs (one in <it>SELE</it>, two in <it>SELL</it>, and three in <it>SELP</it>) produced p-values < 0.05. However, when adjusted for multiple measures with a Bonferroni correction, only one SNP in <it>SELP </it>(rs3917751) produced a statistically significant p-value (p = 0.0029).</p> <p>Conclusions</p> <p>This genetic screen did not detect any SNPs that were highly associated with AMD affection status overall. However, subtype analysis showed that a single SNP located within an intron of <it>SELP </it>(rs3917751) is statistically associated with dry AMD in our cohort. Future studies with additional cohorts and functional assays will clarify the biological significance of this discovery. Based on our findings, it is unlikely that common ancestral variants in the other selectin genes (<it>SELE </it>and <it>SELL</it>) are risk factors for AMD. Finally, it remains possible that sporadic or rare mutations in <it>SELE</it>, <it>SELL</it>, or <it>SELP </it>have a role in the pathogenesis of AMD.</p

Cardiovascular risk and level of statin use among women with breast cancer in a cardio-oncology clinic

Background: Because of the improvements in survival rates, patients with breast cancer are now more likely to die from cardiovascular disease than from cancer. Thus, providing appropriate preventive cardiovascular care to patients with cancer is of the utmost importance. Methods: We retrospectively compared the cardiovascular risk and management of 146 women treated at the Cardio-Oncology (Cardio-Onc) and the Obstetrics and Gynecology (Ob-Gyn) clinics. We calculated cardiovascular risk using the American College of Cardiology (ACC)/American Heart Association (AHA) atherosclerotic cardiovascular disease (ASCVD) risk calculator and the Framingham Risk Score Calculator. We also determined the prevalence of appropriate statin use according to both the 2013 ACC/AHA and the 2002 Adult Treatment Panel (ATP) III lipid guidelines. Results: The 10-year ASCVD risk score was not significantly different between the 2 cohorts. More patients in the Ob-Gyn cohort with an ASCVD risk score >7.5% were already appropriately on statins compared to patients in the Cardio-Onc cohort (60.9% vs 31.0%, respectively, P=0.003), but after the first Cardio-Onc visit, 4 additional patients with breast cancer were prescribed statins (44.8% total). Fourteen (19.2%) Cardio-Onc patients had a high Framingham Risk Score compared to 6 (8.2%) Ob-Gyn patients. Conclusion: We demonstrated that the ASCVD risk is similar between women with breast cancer attending the Cardio-Onc clinic and the women without breast cancer attending the Ob-Gyn clinic, but the Cardio-Onc cohort had significantly more patients with a high Framingham Risk Score. Both clinics had similarly poor rates of appropriate statin prescribing rates according to the ATP III guidelines

An Assessment of Iterative Reconstruction Methods for Sparse Ultrasound Imaging

Ultrasonic image reconstruction using inverse problems has recently appeared as an alternative to enhance ultrasound imaging over beamforming methods. This approach depends on the accuracy of the acquisition model used to represent transducers, reflectivity, and medium physics. Iterative methods, well known in general sparse signal reconstruction, are also suited for imaging. In this paper, a discrete acquisition model is assessed by solving a linear system of equations by an ℓ 1 -regularized least-squares minimization, where the solution sparsity may be adjusted as desired. The paper surveys 11 variants of four well-known algorithms for sparse reconstruction, and assesses their optimization parameters with the goal of finding the best approach for iterative ultrasound imaging. The strategy for the model evaluation consists of using two distinct datasets. We first generate data from a synthetic phantom that mimics real targets inside a professional ultrasound phantom device. This dataset is contaminated with Gaussian noise with an estimated SNR, and all methods are assessed by their resulting images and performances. The model and methods are then assessed with real data collected by a research ultrasound platform when scanning the same phantom device, and results are compared with beamforming. A distinct real dataset is finally used to further validate the proposed modeling. Although high computational effort is required by iterative methods, results show that the discrete model may lead to images closer to ground-truth than traditional beamforming. However, computing capabilities of current platforms need to evolve before frame rates currently delivered by ultrasound equipments are achievable