The simplicity of programming the CRISPR-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Our highest-ranking candidates include previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

Obesity is a major risk factor for coronary artery disease, but its impact on anesthetic-induced cardioprotective actions is unexplored. We tested whether obesity inhibits anesthetic sevoflurane-induced preconditioning and whether this effect is mediated via the AMP-activated protein kinase (AMPK) signaling pathway. Sprague-Dawley rats were fed a high-fat (HF, 45% kcal as fat) or low-fat (LF, 10% kcal as fat) diet for 12 weeks. HF-fed rats developed metabolic disturbances including visceral obesity, hyperinsulinemia, hyperleptinemia and dyslipidemia. HF- or LF-fed rats subjected to 25 min of myocardial ischemia followed by 120 min of reperfusion were assigned to the following groups: control, sevoflurane preconditioning, sevoflurane plus AMPK inhibitor ara-A or AMPK activator A769662 alone. Infarct size was similar between the two control groups. Sevoflurane preconditioning significantly reduced infarct size in LF-fed rats but failed to induce cardioprotection in HF-fed rats. Phosphorylation of AMPK and endothelial nitric oxide synthase, as well as myocardial nitrite and nitrate, were also increased by sevoflurane preconditioning in LF-fed rats but not in HF-fed rats. Pretreatment with ara-A inhibited phosphorylation of AMPK and reversed sevoflurane preconditioning-induced cardioprotection in LF-fed rats, whereas it had no effects in HF-fed rats. In addition, sevoflurane preconditioning failed to enhance reactive oxygen species (ROS) generation in the myocardium of HF-fed rats compared with LF-fed rats. Direct activation of AMPK with A769662 equally increased phosphorylation of AMPK and reduced infarct size in both LF- and HF-fed rats. The results suggest that diet-induced obesity suppresses sevoflurane preconditioning-induced cardioprotective action, probably due to a diminished effect of sevoflurane preconditioning on activation of the ROS-mediated AMPK signaling pathway.

Nutrient additions can increase carbon (C) inputs to soil, but there is no consensus about the response of soil organic C (SOC) storage and C sequestration. For the Tibetan alpine meadows, little is known about the effects and mechanisms of nitrogen (N) and phosphorus (P) addition on SOC stocks. In this study, we applied N and/or P fertilization for 7 years and analyzed soil changes in bulk density, pH, SOC, soil inorganic C (SIC), δ13C, and microbial biomass C (MBC), as well as stocks of SOC, SIC, and MBC for soil to a depth of 40 cm. We found that C:N decreased in 0–20 cm, and pH decreased at both 0–20 cm and 20–40 cm after fertilization. Fertilization with N and/or P decreased SOC stocks in 0–20 cm by 5–12% and SOC stocks from 0 to 40 cm by 3–5%. This SOC stock decline was associated with changes in SOC concentration but not with changes in bulk density. The SIC stock was 18% of total soil C, and was not influenced by either N or P fertilization. Soil δ13C in the 0–20 cm layer was depleted by fertilization with N or N + P, whereas P enriched soil δ13C. Soil MBC was positively correlated with SOC concentration, whereas soil δ13C was negatively correlated with SOC concentration. Soil δ13C, as a proxy of decomposition rate, indicated potentially higher SOC decomposition under N fertilization. These findings suggest that fertilization with N and/or P lowered SOC sequestration in Tibetan alpine meadows.

The catalytic asymmetric Diels-Alder reaction (DAR) [1] is among the most powerful protocols for the stereoselective construction of six-membered functionalized cyclic frameworks. Its versatility in the synthesis of diverse natural products provides organic chemists with a prodigious starting point to discover new reaction modes for this cycloaddition. In the wake of the emergence of the first metal complexes for the Lewis acid catalyzed asymmetric inverse-electrondemand Diels-Alder reactions (IEDDAR) through the LUMO-lowering strategy reported by the group of Kobayashi, [2] several remarkable studies have been presented involving the activation of dienes through lowering of the LUMO energy by Lewis acidic metal complexes [3] or organic molecules [4] [Eq.(1), Scheme 1]. Recently, amine organocatalysis has been attracting considerable interest since the development of the highly enantioselective organocatalytic DAR by MacMillan and co-workers.[5] Alternatively, Jørgensen and co-workers [6] reported the first organocatalytic asymmetric IEDDAR with dienophiles whose HOMO energy has been raised by an enamine activation [Eq. (2)], and considerable advances in this field have recently been achieved by the Chen group. [7] Bifunctional organocatalysis has emerged as a potentially powerful tool in catalytic asymmetric synthesis. [8] This concept aims to efficiently achieve asymmetric transformations that cannot be approached by using either a Lewis acid or base catalyst alone. To the best of our knowledge, there is no report to date of an asymmetric IEDDAR that is controlled with a single reactive catalyst through a bifunctional activation strategy; that is simultaneous activation of the HOMO of the dienophile and the LUMO of the diene [Eq. (3)]. There is a report on the use of a combination of an enamine with metal Lewis acid activation that proved to have potential for this transformation.[9] Herein, we introduce a bifunctional catalyst for an in situ substrate generation/ activation strategy as a new platform for the design of organocatalytic intermolecular cycoaddition processes. In this context, we document the first highly enantioselective bifunctional catalytic IEDDAR that involves dual control of the HOMO dienophiles and LUMO dienes energies of the substrates.The spirocyclic core is a privileged structural element that is featured in a large number of naturally occurring bioactive alkaloids. Although the significant bioactivity and preparation methods of such motifs attract the interest of chemists, as reported in some elegant works including intramolecular alkylation, metal-based cyclization, intermolecular cycloaddition, rearrangements, and other reactions, [10] the enantioselective catalytic approach to access chiral spiro architectures containing all-carbon quaternary stereocenters still remains challenging. As an important skeleton in the larger spirocycle family, the spirolactam scaffold not only often shows interesting biological activities, but it can also act as an intermediate in the synthesis of more s...