The rockefeller university

Cyclic GMP-AMP synthase is essential for innate immunity against infection and cellular damage, serving as a sensor of DNA from pathogens or mislocalized self-DNA. Upon binding double-stranded DNA, cyclic GMP-AMP synthase synthesizes a cyclic dinucleotide that initiates an inflammatory cellular response. Mouse studies that recapitulate causative mutations in the autoimmune disease Aicardi-Goutieres syndrome demonstrate that ablating the cyclic GMP-AMP synthase gene abolishes the deleterious phenotype. Here, we report the discovery of a class of cyclic GMP-AMP synthase inhibitors identified by a high-throughput screen. These compounds possess defined structure-activity relationships and we present crystal structures of cyclic GMP-AMP synthase, double-stranded DNA, and inhibitors within the enzymatic active site. We find that a chemically improved member, RU. 521, is active and selective in cellular assays of cyclic GMP-AMP synthase-mediated signaling and reduces constitutive expression of interferon in macrophages from a mouse model of Aicardi-Goutieres syndrome. RU. 521 will be useful toward understanding the biological roles of cyclic GMP-AMP synthase and can serve as a molecular scaffold for development of future autoimmune therapies.

[No abstract available]

Induced pluripotent stem cells (iPSCs), which are used to produce transplantable tissues, may particularly benefit older patients, who are more likely to suffer from degenerative diseases. However, iPSCs generated from aged donors (A-iPSCs) exhibit higher genomic instability, defects in apoptosis and a blunted DNA damage response compared with iPSCs generated from younger donors. We demonstrated that A-iPSCs exhibit excessive glutathione-mediated reactive oxygen species (ROS) scavenging activity, which blocks the DNA damage response and apoptosis and permits survival of cells with genomic instability. We found that the pluripotency factor ZSCAN10 is poorly expressed in A-iPSCs and addition of ZSCAN10 to the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) during A-iPSC reprogramming normalizes ROS-glutathione homeostasis and the DNA damage response, and recovers genomic stability. Correcting the genomic instability of A-iPSCs will ultimately enhance our ability to produce histocompatible functional tissues from older patients' own cells that are safe for transplantation.

Numerous genetic polymorphisms have been identified as associated with disease or treatment outcome, but the routine implementation of genotyping into actionable medical care remains limited. Point-of-care (PoC) technologies enable rapid and real-time treatment decisions, with great potential for extending molecular diagnostic approaches to settings with limited medical infrastructure (e.g., CLIA certified diagnostic laboratories). With respect to resource-limited settings, there is a need for simple devices to implement biomarker guided treatment strategies. One relevant example is chronic hepatitis C infection, for which several treatment options are now approved. Single nucleotide polymorphisms (SNPs) in the IL-28B / IFNL3 locus have been well described to predict both spontaneous clearance and response to interferon based therapies. We utilized the Genedrive (R) platform to develop an assay for the SNP rs12979860 variants (CC, CT and TT). The assay utilizes a hybrid thermal engine, permitting rapid heating and cooling, enabling an amplification based assay with genetic variants reported using endpoint differential melting cure analysis in less than 60 minutes. We validated this assay using non-invasive buccal swab sampling in a prospective study of 246 chronic HCV patients, achieving 100% sensitivity and 100% specificity (95% exact CI: 98.8-100%)) in 50 minutes as compared to conventional lab based PCR testing. Our results provide proof of concept that precision medicine is feasible in resource-limited settings, offering the first CE-IVD (in vitro diagnostics) validated PoC SNP test. We propose that IL-28B genotyping may be useful for directing patients towards lower cost therapies, and rationing use of costly direct antivirals for use in those individuals showing genetic risk.

X-linked recessive ectodermal dysplasia with immunodeficiency is a rare primary immunodeficiency caused by hypomorphic mutations of the IKBKG gene encoding the nuclear factor kappa B essential modulator (NEMO) protein. This condition displays enormous allelic, immunological, and clinical heterogeneity, and therapeutic decisions are difficult because NEMO operates in both hematopoietic and nonhematopoietic cells. Hematopoietic stem cell transplantation (HSCT) is potentially life-saving, but the small number of case reports available suggests it has been reserved for only the most severe cases. Here, we report the health status before HSCT, transplantation outcome, and clinical follow-up for a series of 29 patients from unrelated kindreds from 11 countries. Between them, these patients carry 23 different hypomorphic IKBKG mutations. HSCT was performed from HLA-identical related donors (n = 7), HLA-matched unrelated donors (n = 12), HLA-mismatched unrelated donors (n = 8), and HLA-haploidentical related donors (n = 2). Engraftmentwas documented in 24 patients, and graft-versus-host disease in 13 patients. Up to 7 patients died 0.2 to 12 months after HSCT. The global survival rate after HSCT among NEMO-deficient children was 74% at a median follow-up after HSCT of 57months (range, 4-108 months). Preexisting mycobacterial infection and colitis were associated with poor HSCT outcome. The underlyingmutation does not appear to have any influence, as patients with the same mutation had different outcomes. Transplantation did not appear to cure colitis, possibly as a result of cell-intrinsic disorders of the epithelial barrier. Overall, HSCT can cure most clinical features of patients with a variety of IKBKG mutations.

This month: relating single cells to populations (Cao/Packer, Wu/Altschuler, O'Brien, Friedman), an excess of ribosomes (Barkai), human pathology atlas (Uhlen), signatures of feedback (Rahi), and major genome redesign (Baumgart).

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

Although human blood is believed to be a sterile environment, recent studies suggest that pleomorphic bacteria exist in the blood of healthy humans. These studies have led to the development of "liveblood analysis," a technique used by alternative medicine practitioners to diagnose various human conditions, including allergies, cancer, cardiovascular disease and septicemia. We show here that bacteria-like vesicles and refringent particles form in healthy human blood observed under dark-field microscopy. These structures gradually increase in number during incubation and show morphologies reminiscent of cells undergoing division. Based on lipid analysis and Western blotting, we show that the bacteria-like entities consist of membrane vesicles containing serum and exosome proteins, including albumin, fetuin-A, apolipoprotein-A1, alkaline phosphatase, TNFR1 and CD63. In contrast, the refringent particles represent protein aggregates that contain several blood proteins. 16S rDNA PCR analysis reveals the presence of bacterial DNA in incubated blood samples but also in negative controls, indicating that the amplified sequences represent contaminants. These results suggest that the bacteria-like vesicles and refringent particles observed in human blood represent non-living membrane vesicles and protein aggregates derived from blood. The phenomena observed during live-blood analysis are therefore consistent with time-dependent decay of cells and body fluids during incubation ex vivo.

The immense and growing repositories of transcriptional data may contain critical insights for developing new therapies. Current approaches to mining these data largely rely on binary classifications of disease vs. control, and are not able to incorporate measures of disease severity. We report an analytical approach to integrate ordinal clinical information with transcriptomics. We apply this method to public data for a large cohort of Huntington's disease patients and controls, identifying and prioritizing phenotype-associated genes. We verify the role of a high-ranked gene in dysregulation of sphingolipid metabolism in the disease and demonstrate that inhibiting the enzyme, sphingosine-1-phosphate lyase 1 (SPL), has neuroprotective effects in Huntington's disease models. Finally, we show that one consequence of inhibiting SPL is intracellular inhibition of histone deacetylases, thus linking our observations in sphingolipid metabolism to a well-characterized Huntington's disease pathway. Our approach is easily applied to any data with ordinal clinical measurements, and may deepen our understanding of disease processes.

A search for heavy resonances with masses above 1 TeV, decaying to final states containing a vector boson and a Higgs boson, is presented. The search considers hadronic decays of the vector boson, and Higgs boson decays to b quarks. The decay products are highly boosted, and each collimated pair of quarks is reconstructed as a single, massive jet. The analysis is performed using a data sample collected in 2016 by the CMS experiment at the LHC in proton-proton collisions at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1). The data are consistent with the background expectation and are used to place limits on the parameters of a theoretical model with a heavy vector triplet. In the benchmark scenario with mass-degenerate W' and Z' bosons decaying predominantly to pairs of standard model bosons, for the first time heavy resonances for masses as high as 3.3 TeV are excluded at 95% confidence level, setting the most stringent constraints to date on such states decaying into a vector boson and a Higgs boson.