Cas9-triggered chain ablation of cas9 as a gene drive brake

With the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology, researchers can construct gene drives that can bias the inheritance of edited alleles to alter entire populations. As demonstrated with the mutagenic chain reaction in Drosophila4, the CRISPR-Cas9 system can propagate genomic modification together with the genome-editing machinery itself. Although gene drives might have the potential to control insect-borne diseases and agricultural pests, substantial concerns have been raised over unanticipated ecological consequences as a result of drive use. Here we report the development of a potential Cas9-based gene drive 'brake' that remains inert in a wild-type genome but is activated by Cas9 to both cleave the genomic cas9 sequence and to convert an incoming cas9 allele into a brake. This means that the propagation of the brake is favored in a cas9-carrying population

Markers of Myocardial Damage Predict Mortality in Patients With Aortic Stenosis

Background: Cardiovascular magnetic resonance (CMR) is increasingly used for risk stratification in aortic stenosis (AS). However, the relative prognostic power of CMR markers and their respective thresholds remains undefined. Objectives: Using machine learning, the study aimed to identify prognostically important CMR markers in AS and their thresholds of mortality. Methods: Patients with severe AS undergoing AVR (n = 440, derivation; n = 359, validation cohort) were prospectively enrolled across 13 international sites (median 3.8 years’ follow-up). CMR was performed shortly before surgical or transcatheter AVR. A random survival forest model was built using 29 variables (13 CMR) with post-AVR death as the outcome. Results: There were 52 deaths in the derivation cohort and 51 deaths in the validation cohort. The 4 most predictive CMR markers were extracellular volume fraction, late gadolinium enhancement, indexed left ventricular end-diastolic volume (LVEDVi), and right ventricular ejection fraction. Across the whole cohort and in asymptomatic patients, risk-adjusted predicted mortality increased strongly once extracellular volume fraction exceeded 27%, while late gadolinium enhancement >2% showed persistent high risk. Increased mortality was also observed with both large (LVEDVi >80 mL/m2) and small (LVEDVi ≤55 mL/m2) ventricles, and with high (>80%) and low (≤50%) right ventricular ejection fraction. The predictability was improved when these 4 markers were added to clinical factors (3-year C-index: 0.778 vs 0.739). The prognostic thresholds and risk stratification by CMR variables were reproduced in the validation cohort. Conclusions: Machine learning identified myocardial fibrosis and biventricular remodeling markers as the top predictors of survival in AS and highlighted their nonlinear association with mortality. These markers may have potential in optimizing the decision of AVR

Wnt expression is not correlated with β-catenin dysregulation in Dupuytren's Disease

BACKGROUND: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures. RESULTS: The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis. CONCLUSION: These data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of β-catenin expression in DD

Type-1 Collagen differentially alters β-catenin accumulation in primary Dupuytren's Disease cord and adjacent palmar fascia cells

<p>Abstract</p> <p>Background</p> <p>Dupuytren's Disease (DD) is a debilitating contractile fibrosis of the palmar fascia characterised by excess collagen deposition, contractile myofibroblast development, increased Transforming Growth Factor-β levels and β-catenin accumulation. The aim of this study was to determine if a collagen-enriched environment, similar to <it>in vivo </it>conditions, altered β-catenin accumulation by primary DD cells in the presence or absence of Transforming Growth Factor-β.</p> <p>Methods</p> <p>Primary DD and patient matched, phenotypically normal palmar fascia (PF) cells were cultured in the presence or absence of type-1 collagen and Transforming Growth Factor-β1. β-catenin and α-smooth muscle actin levels were assessed by western immunoblotting and immunofluorescence microscopy.</p> <p>Results</p> <p>DD cells display a rapid depletion of cellular β-catenin not evident in patient-matched PF cells. This effect was not evident in either cell type when cultured in the absence of type-1 collagen. Exogenous addition of Transforming Growth Factor-β1 to DD cells in collagen culture negates the loss of β-catenin accumulation. Transforming Growth Factor-β1-induced α-smooth muscle actin, a marker of myofibroblast differentiation, is attenuated by the inclusion of type-1 collagen in cultures of DD and PF cells.</p> <p>Conclusion</p> <p>Our findings implicate type-1 collagen as a previously unrecognized regulator of β-catenin accumulation and a modifier of TGF-β1 signaling specifically in primary DD cells. These data have implications for current treatment modalities as well as the design of <it>in vitro </it>models for research into the molecular mechanisms of DD.</p

A Novel CCR5 Mutation Common in Sooty Mangabeys Reveals SIVsmm Infection of CCR5-Null Natural Hosts and Efficient Alternative Coreceptor Use In Vivo

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species

Genome-wide scan reveals association of psoriasis with IL-23 and NF-κB pathways

Psoriasis is a common immune-mediated disorder that affects the skin, nails and joints. To identify psoriasis susceptibility loci, we genotyped 438,670 SNPs in 1,409 psoriasis cases and 1,436 controls of European ancestry. We followed up 21 promising SNPs in 5,048 psoriasis cases and 5,041 controls. Our results provide strong support for the association of at least seven genetic loci and psoriasis (each with combined P less than 5 × 10−8). Loci with confirmed association include HLA-C, three genes involved in IL-23 signaling (IL23A, IL23R, IL12B), two genes that act downstream of TNF-α and regulate NF-κB signaling (TNIP1, TNFAIP3) and two genes involved in the modulation of Th2 immune responses (IL4, IL13). Although the proteins encoded in these loci are known to interact biologically, we found no evidence for epistasis between associated SNPs. Our results expand the catalog of genetic loci implicated in psoriasis susceptibility and suggest priority targets for study in other auto-immune disorders

Genetic architecture of human plasma lipidome and its link to cardiovascular disease

Understanding genetic architecture of plasma lipidome could provide better insights into lipid metabolism and its link to cardiovascular diseases (CVDs). Here, we perform genome-wide association analyses of 141 lipid species (n = 2,181 individuals), followed by phenome-wide scans with 25 CVD related phenotypes (n = 511,700 individuals). We identify 35 lipid-species-associated loci (P <5 x10(-8)), 10 of which associate with CVD risk including five new loci-COL5A1, GLTPD2, SPTLC3, MBOAT7 and GALNT16 (false discovery rate<0.05). We identify loci for lipid species that are shown to predict CVD e.g., SPTLC3 for CER(d18:1/24:1). We show that lipoprotein lipase (LPL) may more efficiently hydrolyze medium length triacylglycerides (TAGs) than others. Polyunsaturated lipids have highest heritability and genetic correlations, suggesting considerable genetic regulation at fatty acids levels. We find low genetic correlations between traditional lipids and lipid species. Our results show that lipidomic profiles capture information beyond traditional lipids and identify genetic variants modifying lipid levels and risk of CVD