CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.

OBJECTIVE: The purpose of this ancillary study is to determine the quality of diets in patients with documented coronary heart disease (CHD). DESIGN: Dietary data were originally collected using a 24-hour dietary recall in 555 patients with CHD, 1 year after a diagnostic coronary angiography. Data used for this investigation were collected between March 2001 and November 2003. SUBJECTS/SETTING: Patients were participants in a clinical trial to improve adherence to lipid-lowering medications. The Alternate Healthy Eating Index, an instrument designed to evaluate the degree to which a diet has the potential to prevent cardiovascular disease, measured dietary quality. MAIN OUTCOME MEASURES: Linear regression models were used to assess the association of dietary quality with patients' sociodemographic and clinical characteristics. RESULTS: Mean age of participants was 61 years, with an average body mass index of 30 (calculated as kg/m(2)). Sixty percent were men. Average daily caloric intake was 1,775 kcal, with 50% of calories derived from carbohydrates, 18% from protein, and 32% from total fat. Average Alternate Healthy Eating Index score was 30.8 out of a possible maximum score of 80. Only 12.4% of subjects met the recommended consumption of vegetables, 7.8% for fruit, 8% for cereal fiber, and 5.2% for trans-fat intake. Lower dietary quality was associated with lower total caloric intake, as well as with smoking, obesity, and lower educational level. CONCLUSIONS: A high proportion of patients reported poor dietary quality 1 year after experiencing a coronary event. Our data support continued efforts to enhance healthful dietary changes over time for secondary prevention of CHD. Dietary change should be emphasized with CHD patients who are less educated, smokers, or obese.

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.