Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Synthesis of Non-natural, Frame-Shifted Isoprenoid Diphosphate Analogues

A set of synthetic approaches was developed and applied to the synthesis of eight frame-shifted isoprenoid diphosphate analogues. These analogues were designed to increase or decrease the methylene units between the double bonds and/or the pyrophosphate moieties of the isoprenoid structure. Evaluation of mammalian GGTase-I and FTase revealed that small structural changes can result in substantial changes in substrate activity

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)‑1<i>H</i>‑indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ‑Thrombin

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based <b>3</b>. Of these, <b>9j</b> (PAR4 IC₅₀ = 445 nM, PAR1 response IC₅₀ > 30 μM) and <b>10h</b> (PAR4 IC₅₀ = 179 nM, PAR1 response IC₅₀ > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)‑1<i>H</i>‑indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ‑Thrombin

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based <b>3</b>. Of these, <b>9j</b> (PAR4 IC₅₀ = 445 nM, PAR1 response IC₅₀ > 30 μM) and <b>10h</b> (PAR4 IC₅₀ = 179 nM, PAR1 response IC₅₀ > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin