.were taken every week. After 3 or 4 weeks, when the primary tumors approached 20 mm, the lungs were excised and lung luminescence was compared.All luminescent and fluorescent images were acquired and analyzed using Living Image software (Caliper Life Sciences).Statistics. Student's t tests, computed using Microsoft Excel, were used to analyze the significance between the treated samples and the controls where the test type was set to 1-tail distribution and 2-sample equal variance.

No abstract

Apoptosis is a tightly coordinated cell death program that damages mitochondria, DNA, proteins, and membrane lipids. Little is known about the fate of RNA as cells die. Here, we show that mRNAs, but not noncoding RNAs, are rapidly and globally degraded during apoptosis. mRNA decay is triggered early in apoptosis, preceding membrane lipid scrambling, genomic DNA fragmentation, and apoptotic changes to translation initiation factors. mRNA decay depends on mitochondrial outer membrane permeabilization and is amplified by caspase activation. 3′ truncated mRNA decay intermediates with nontemplated uridylate-rich tails are generated during apoptosis. These tails are added by the terminal uridylyl transferases (TUTases) ZCCHC6 and ZCCHC11, and the uridylated transcript intermediates are degraded by the 3′ to 5′ exonuclease DIS3L2. Knockdown of DIS3L2 or the TUTases inhibits apoptotic mRNA decay, translation arrest, and cell death, whereas DIS3L2 overexpression enhances cell death. Our results suggest that global mRNA decay is an overlooked hallmark of apoptosis.

Summary A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived and the transfection lipid on its own unacceptably enhances transmission. Here we show that cholesterol-conjugated (chol)-siRNAs without lipid silence gene expression in the vagina without causing inflammation or inducing interferons. A viral siRNA prevents transmission within a day of challenge, whereas an siRNA targeting nectin-1, an HSV-2 receptor, protects for a week, but protection is delayed for a few days until the receptor is down-modulated. Combining siRNAs targeting a viral and host gene protects mice from HSV-2 for a week, irrespective of the time of challenge. Therefore, intravaginal siRNAs could provide sustained protection against viral transmission.

Protective antigen (PA) is a central component of anthrax toxin and a major antigen in anthrax vaccines. However, the use of native PA as a vaccine is not optimal. If administered to people who have been freshly exposed to anthrax, PA may actually aid in anthrax toxin formation and thus may pose a serious safety concern for postexposure vaccination applications. A non-functional PA mutant may be much safer alternative. To identify an improved anthrax vaccine antigen, we examined four non-functional mutants of PA, each being impaired in a critical step of the cellular intoxication pathway of PA. These mutants were Rec -(unable to bind PA-receptors), SSSR (resistant to activation by furin), Oligo -(unable to form oligomers), and DNI (unable to form endosomal transmembrane pores). When tested in mice and after three doses of immunization, all four mutants were highly potent in eliciting PA-specific, toxin-neutralizing antibodies, with immunogenicity increasing in the order of PA < Rec -< SSSR < Oligo -< DNI. While the differences between Rec -or SSSR and PA were small and not statistically significant, DNI and Oligo -were significantly more immunogenic than wild-type PA. One year after immunization and compared with PA-immunized mice, DNIimmunized mice maintained significantly higher levels of anti-PA IgG with correspondingly higher titers of toxin-neutralizing activity. In contrast, Oligo --immunized mice had high levels of anti-PA IgG but lower titers of toxin-neutralizing activity, suggesting that Oligo -mutation sites may overlap with critical protective epitopes of PA. Our study demonstrates that PA-based vaccines could be improved both in terms of safety and efficacy by strategic mutations that not only render PA nonfunctional but simultaneously enhance its immunogenic potency. Recombinant PA mutants, particularly DNI, hold great promise as better and safer antigens than wild-type PA for use in postexposure vaccination.