RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel ''all-in-one'' lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an ''all-in-one'' system to track disrupted Trp53 in chemoresistant lymphomas in the Em-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

AimsTo assess associations between hypoglycaemia and risk of accidents resulting in hospital visits among people with type 2 diabetes receiving antidiabetes drugs without insulin.MethodsPeople with type 2 diabetes who were not treated with insulin were identified from a US-based employer claims database (1998–2010). Following initiation of an antidiabetes drug, the occurrence of accidents resulting in hospital visits was compared between people with, and without, claims for hypoglycaemia using multivariable Cox proportional hazard models adjusted for demographics, comorbidities, prior treatments and prior medical service use. Additional analyses were stratified by age 65 years or older.ResultsA total of N = 5582 people with claims for hypoglycaemia and N = 27 910 with no such claims were included. Accidents resulting in hospital visits occurred in 5.5 and 2.8% of people with, and without, hypoglycaemia, respectively. After adjusting for baseline characteristics, hypoglycaemia was associated with significantly increased hazards for any accident [hazard ratio (HR) 1.39, 95% CI 1.21–1.59, p < 0.001], accidental falls (HR 1.36, 95% CI 1.13–1.65, p < 0.001) and motor vehicle accidents (HR 1.82, 95% CI 1.18–2.80, p = 0.007). In age-stratified analyses, hypoglycaemia was associated with greater hazards of driving-related accidents in people younger than age 65 and falls in people aged 65 or older.ConclusionsIn people with type 2 diabetes receiving antidiabetes drugs without insulin, hypoglycaemia was associated with a significantly higher risk of accidents resulting in hospital visits, including accidents related to driving and falls.

No abstract

Ribavirin is a guanosine ribonucleoside analog that displays broad-spectrum anti-viral activity and is currently used for the treatment of some viral infections. Ribavirin has recently been proposed to also be a mimic of the 7-methyl guanosine cap found at the 5 0 end of mRNAs. To obtain supporting functional data for this hypothesis, we assessed the ability of ribavirin triphosphate to interfere with the interaction between eIF4E and 7-methyl guanosine capped mRNA. In chemical cross-linking assays, cap-affinity chromatography, and cap-dependent translation assays, ribavirin was unable to function as a cap analog.

No abstract