Simultaneous identification of host, ectoparasite and pathogen DNA via in-solution capture.

Ectoparasites frequently vector pathogens from often unknown pathogen reservoirs to both human and animal populations. Simultaneous identification of the ectoparasite species, the wildlife host that provided their most recent blood meal(s), and their pathogen load would greatly facilitate the understanding of the complex transmission dynamics of vector-borne diseases. Currently, these identifications are principally performed using multiple polymerase chain reaction (PCR) assays. We developed an assay (EctoBaits) based on in-solution capture paired with high-throughput sequencing to simultaneously identify ectoparasites, host blood meals and pathogens. We validated our in-solution capture results using double-blind PCR assays, morphology and collection data. The EctoBaits assay effectively and efficiently identifies ectoparasites, blood meals, and pathogens in a single capture experiment, allowing for high-resolution taxonomic identification while preserving the DNA sample for future analyses.

Broadening the spectrum of actin-based protrusive activity mediated by Arp2/3 complex-facilitated polymerization: motility of cytoplasmic ridges and tubular projections.

Arp2/3 complex-facilitated actin polymerization plays an essential role in a variety of cellular functions including motility, adherence, endocytosis, and trafficking. In the present study, we employ the sea urchin coelomocyte experimental model system to test the hypotheses that Arp2/3 complex-nucleated actin assembly mediates the motility of two unusual cellular protrusions; the cytoplasmic ridges present during coelomocyte spreading, and inducible, tubular-shaped, and neurite-like projections. Our investigations couple pharmacological manipulation employing inhibitors of actin polymerization and the Arp2/3 complex with a wide array of imaging methods including digitally enhanced phase contrast, DIC, and polarization light microscopy of live cells; conventional, confocal and super-resolution light microscopy of fluorescently labeled cells; and scanning and transmission electron microscopy. Taken together, the results of this study indicate that Arp2/3 complex-facilitated actin polymerization underlies the motility of coelomocyte cytoplasmic ridges and tubular projections, that these processes are related to each other, and that they have been preliminarily identified in other cell types. The results also highlight the broad spectrum of actin-based protrusive activities dependent on the Arp2/3 complex and provide additional insights into the pervasive nature of this ubiquitous actin nucleator. Furthermore, we provide the first evidence of a possible mechanistic difference between the impacts of the small molecule drugs BDM and CK666 on the Arp2/3 complex.

Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity.

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.

Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes.

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.