The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and wild boars is caused by African swine fever virus (ASFV), can reach 100%. Since the first confirmed ASF outbreak in China on 3 August 2018, 156 ASF outbreaks were detected in 32 provinces. About 1,170,000 pigs were culled in order to halt further spread. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12abased On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout and the assay displayed no crossreactivity to other 13 swine viruses including classical swine fever (CSF). CORDS could identify the ASFV DNA target at femtomolar sensitivity in an hour at 37 • C, and only requires an incubator. For ease of use, the reagents of CORDS were lyophilized to three tubes and remained the same sensitivity when stored at 4 • C for at least 7 days. Thus, CORDS provide a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field-based applications.

13The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and is caused 14 by African swine fever virus (ASFV), can reach 100%. ASF has been reported in 25 Chinese provinces 15 since August 2018. There is no effective treatment or vaccine for it and the present molecular diagnosis 16 technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly 17 to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low 18 cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-19 Cas12a-based system that combines recombinase-aided amplification (RAA) and CRISPR/Cas12a for 20 ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as 21 low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in 22 the RAA-Cas12a based system (named CORDS, Cas12a-based On-site and Rapid Detection System). 23We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout. CORDS 24 could identify the p72 gene at femtomolar sensitivity in an hour at 37°C, and only requires an incubator. 25For ease of use, the regents of CORDS was lyophilized to three tubes and remained the same sensitivity 26 when stored at 4 °C for at least 7 days. Thus, CORDS provides a rapid, sensitive and easily operable 27 method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and 28 storage, and is ready for field applications. 29 30

No abstract

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

Reporter cell lines are a particularly useful tool to screen for the skin sensitization potential of chemicals. Current cell models based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids. However, plasmid-based reporters may ignore comprehensive aspects of induction, thus affecting the accuracy of hazard identification. Herein, we developed a novel HaCaT-based reporter system, EndoSens, whereby luciferase was specifically inserted into the cassette for heme oxygenase (decycling) 1 (HMOX1, the most consistent marker induced by skin sensitizers) by CRISPR/Cas9. Testing data from 20 coded substances showed an accuracy of 90%, sensitivity of 91.7%, and specificity of 87.5%, which exceeded the OECD requirement. Among the 35 chemicals examined, predictivity was better than reported for the validated KeratinoSens™. These results indicate that the EndoSens assay could advance the predictivity of skin sensitization, thus making it a promising tool for in vitro skin sensitization testing.

Genomic stability is essential for organismal development, cellular homeostasis, and survival. The DNA double-strand breaks are particularly deleterious, creating an environment prone to cellular transformation and oncogenic activation. The histone variant H2AX is an essential component of the nucleosome responsible for initiating the early steps of the DNA repair process. H2AX maintains genomic stability by initiating a signaling cascade that collectively functions to promote DNA double-strand breaks repair. Recent advances have linked genomic stability to energetic metabolism, and alterations in metabolism were found to interfere with genome maintenance. Utilizing genome-wide transcripts profiling to identify differentially-expressed genes involved in energetic metabolism, we compared control and H2AX-deficient metastatic breast cancer cell lines, and found that H2AX loss leads to the repression of key genes regulating glycolysis, with a prominent effect on hexokinase-2 (HK2). These observations are substantiated by evidence that H2AX loss compromises glycolysis, effect which was reversed by ectopic expression of HK2. Utilizing models of experimental metastasis, we found that H2AX silencing halts progression of metastatic breast cancer cells MDA-MB-231. Most interestingly, ectopic expression of HK2 in H2AX-deficient cells restores their metastatic potential. Using multiple publicly available datasets, we found a significantly strong positive correlation between H2AX expression levels in patients with invasive breast cancer, and levels of glycolysis genes, particularly HK2. These observations are consistent with the evidence that high H2AX expression is associated with shorther distant metastasis-free survival. Our findings reveal a role for histone H2AX in controlling the metastatic ability of breast cancer cells via maintenance of HK2-driven glycolysis.

2015, 2018a,b). The KEs in the AOP include the molecular initiating event (covalent binding to skin proteins) and the cellular response (activation of keratinocytes and dendritic cells) to the sensitizers. To evaluate these KEs, in vitro methods such as the direct peptide reactivity assay (DPRA), KeratinoSens™, and h-CLAT have been developed and accepted by the Organisation for Economic Co-operation and Development (OECD) (Emter et al., 2010;Gerberick et al., 2004;Sakaguchi et al., 2006). However, no single in vitro method can comprehensively represent the complexity of the processes involved in skin sensitization (Osamu et al., 2015). Therefore, as the components of the integrated approach to testing and assessment (IATA), many defined approaches (DA), which cover complementary characteristics of the in vitro methods and further take physicochemical properties and structure into consideration, have greatly improved the predictivity of skin sensitization (Worth and Patlewicz, 2016).

Genomic stability is essential for organismal development, cellular homeostasis, and survival. The DNA double-strand breaks are particularly deleterious, creating an environment prone to cellular transformation and oncogenic activation. The histone variant H2AX is an essential component of the nucleosome responsible for initiating the early steps of the DNA repair process. H2AX maintains genomic stability by initiating a signaling cascade that collectively functions to promote DNA double-strand breaks repair. Recent advances have linked genomic stability to energetic metabolism, and alterations in metabolism were found to interfere with genome maintenance. Utilizing genome-wide transcripts profiling to identify differentially-expressed genes involved in energetic metabolism, we compared control and H2AX-deficient metastatic breast cancer cell lines, and found that H2AX loss leads to the repression of key genes regulating glycolysis, with a prominent effect on hexokinase-2 (HK2). These observations are substantiated by evidence that H2AX loss compromises glycolysis, effect which was reversed by ectopic expression of HK2. Utilizing models of experimental metastasis, we found that H2AX silencing halts progression of metastatic breast cancer cells MDA-MB-231. Most interestingly, ectopic expression of HK2 in H2AX-deficient cells restores their metastatic potential. Using multiple publicly available datasets, we found a significantly strong positive correlation between H2AX expression levels in patients with invasive breast cancer, and levels of glycolysis genes, particularly HK2. These observations are consistent with the evidence that high H2AX expression is associated with shorter distant metastasis-free survival. Our findings reveal a role for histone H2AX in controlling the metastatic ability of breast cancer cells via maintenance of HK2-driven glycolysis.