Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9

2-Oxoesters: A Novel Class of Potent and Selective Inhibitors of Cytosolic Group IVA Phospholipase A2.

Cytosolic phospholipase A2 (GIVA cPLA2) is the only PLA2 that exhibits a marked preference for hydrolysis of arachidonic acid containing phospholipid substrates releasing free arachidonic acid and lysophospholipids and giving rise to the generation of diverse lipid mediators involved in inflammatory conditions. Thus, the development of potent and selective GIVA cPLA2 inhibitors is of great importance. We have developed a novel class of such inhibitors based on the 2-oxoester functionality. This functionality in combination with a long aliphatic chain or a chain carrying an appropriate aromatic system, such as the biphenyl system, and a free carboxyl group leads to highly potent and selective GIVA cPLA2 inhibitors (X I(50) values 0.00007-0.00008) and docking studies aid in understanding this selectivity. A methyl 2-oxoester, with a short chain carrying a naphthalene ring, was found to preferentially inhibit the other major intracellular PLA2, the calcium-independent PLA2. In RAW264.7 macrophages, treatment with the most potent 2-oxoester GIVA cPLA2 inhibitor resulted in over 50% decrease in KLA-elicited prostaglandin D2 production. The novel, highly potent and selective GIVA cPLA2 inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of inflammatory diseases

Passive Decisions and Potent Defaults

Default options have an enormous impact on household choices.' Defaults matter because opting out of a default is costly and these costs change over time, generating an option value of waiting. In addition, people have a tendency to procrastinate. We develop a theory of optimal defaults based on these considerations. We find that it is sometimes optimal to set extreme defaults, which are far away from the mean optimal savings rate. A default that is far away from a consumer's optimal savings rate may make that consumer better off since such a bad' default will lead procrastinating consumers to more quickly opt out of the default. We calibrate our model and use it to calculate optimal defaults for employees at four different companies. Our work suggests that optimal defaults are likely to be at one of three savings rates: the minimum savings rate (i.e., 0%), the match threshold (typically 5% or 6%), or the maximal savings rate.

A Potent and Effective Suicidal Listeria Vaccine Platform.

Live-attenuated Listeria monocytogenes has shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenes recombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens. Lm-RIID grows normally in broth but commits suicide inside host cells by inducing Cre recombinase and deleting essential genes flanked by loxP sites, resulting in a self-limiting infection even in immunocompromised mice. Lm-RIID vaccination of mice induces potent CD8+ T cells and protects against virulent challenges, similar to live L. monocytogenes vaccines. When combined with α-PD-1, Lm-RIID is as effective as live-attenuated L. monocytogenes in a therapeutic tumor model. This impressive efficacy, together with the increased clearance rate, makes Lm-RIID ideal for prophylactic immunization against diseases that require T cells for protection

Products of Vitamin D3 or 7-Dehydrocholesterol Metabolism by Cytochrome P450scc Show Anti-Leukemia Effects, Having Low or Absent Calcemic Activity

BACKGROUND. Cytochrome P450scc metabolizes vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,23(OH)2D3, as well as 1-hydroxyvitamin D3 to 1a,20-dihydroxyvitamin D3 (1,20(OH)2D3). It also cleaves the side chain of 7-dehydrocholesterol producing 7-dehydropregnenolone (7DHP), which can be transformed to 20(OH)7DHP. UVB induces transformation of the steroidal 5,7-dienes to pregnacalciferol (pD) and a lumisterol-like compounds (pL). METHODS AND FINDINGS. To define the biological significance of these P450scc-initiated pathways, we tested the effects of their 5,7-diene precursors and secosteroidal products on leukemia cell differentiation and proliferation in comparison to 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3). These secosteroids inhibited proliferation and induced erythroid differentiation of K562 human chronic myeloid and MEL mouse leukemia cells with 20(OH)D3 and 20,23(OH)2D3 being either equipotent or slightly less potent than 1,25(OH)2D3, while 1,20(OH)2D3, pD and pL compounds were slightly or moderately less potent. The compounds also inhibited proliferation and induced monocytic differentiation of HL-60 promyelocytic and U937 promonocytic human leukemia cells. Among them 1,25(OH)2D3 was the most potent, 20(OH)D3, 20,23(OH)2D3 and 1,20(OH)2D3 were less active, and pD and pL compounds were the least potent. Since it had been previously proven that secosteroids without the side chain (pD) have no effect on systemic calcium levels we performed additional testing in rats and found that 20(OH)D3 had no calcemic activity at concentration as high as 1 µg/kg, whereas, 1,20(OH)2D3 was slightly to moderately calcemic and 1,25(OH)2D3 had strong calcemic activity. CONCLUSIONS. We identified novel secosteroids that are excellent candidates for anti-leukemia therapy with 20(OH)D3 deserving special attention because of its relatively high potency and lack of calcemic activity.National Institutes of Health (R01A052190

OMEGA AND BIASING FROM OPTICAL GALAXIES VERSUS POTENT MASS

The mass density field in the local universe, recovered by the POTENT method from peculiar velocities of $\sim$3000 galaxies, is compared with the density field of optically-selected galaxies. Both density fields are smoothed with a Gaussian filter of radius 12 $h^{-1}$ Mpc. Under the assumptions of gravitational instability and a linear biasing parameter b\sbo between optical galaxies and mass, we obtain \beta\sbo \equiv \om^{0.6}/b\sbo = 0.74 \pm 0.13. This result is obtained from a regression of POTENT mass density on optical density after correcting the mass density field for systematic biases in the velocity data and POTENT method. The error quoted is just the $1\sigma$ formal error estimated from the observed scatter in the density--density scatterplot; it does not include the uncertainty due to cosmic scatter in the mean density or in the biasing relation. We do not attempt a formal analysis of the goodness of fit, but the scatter about the fit is consistent with our estimates of the uncertainties.Comment: Final revised version (minor typos corrected). 13 pages, gzipped tar file containing LaTeX and figures. The Postscript file is available at ftp://dust0.dur.ac.uk/pub/mjh/potopt/potopt.ps.Z or (gzipped) at ftp://xxx.lanl.gov/astro-ph/ps/9501/9501074.ps.gz or via WWW at http://xxx.lanl.gov/ps/astro-ph/9501074 or as separate LaTeX text and encapsulated Postscript figures in a compressed tar'd file at ftp://dust0.dur.ac.uk/pub/mjh/potopt/latex/potopt.tar.