Prevalence of antibodies to Toxoplasma gondii in raccoons (Procyon lotor) from an urban area of Northern Virginia

Raccoons (Procyon lotor) are intermediate hosts for Toxoplasma gondii, and clinical toxoplasmosis in raccoons has been reported. A 2-yr serological survey was conducted to determine the prevalence of antibodies to T. gondii in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 256 raccoons were examined for T. gondii antibodies at a 1:50 dilution using the modified direct agglutination test. Results indicated that 216 (84.4%) of the raccoons had been exposed to T. gondii. Our results indicate that raccoons in this area of Virginia are frequently exposed to T. gondii. Domestic cats were common in the study area and may have served as a source of oocysts for raccoons and the food items of raccoons.     

Phylogenetic analyses of mtDNA sequences corroborate taxonomic designations based on cuticular hydrocarbons in subterranean termites

Cuticular hydrocarbons (CHCs) are valuable characters for the analysis of cryptic insect species with few discernible morphological characters. Yet, their use in insect systematics, specifically in subterranean termites in the genus Reticulitermes (Isoptera: Rhinotermitidae), remains controversial. In this paper, we show that taxonomic designations in Reticulitermes from California (USA) suggested in light of differences among CHC phenotypes are corroborated by phylogenetic analyses using mtDNA sequences. Analyses based on CHC phenotypes and supported, in part, by behavioral and ecological differences have suggested the presence of more species than the two currently recognized: R. hesperus Banks and R. tibialis Banks. We analyze a 680 base pair fragment of the mitochondrial DNA cytochrome oxidase (COII) gene from 45 new (21 collection localities) and two previously recorded samples of Reticulitermes from California using parsimony and maximum likelihood methods. Both methods result in trees with highly similar topologies. Bootstrapping indicates support for six clades of Reticulitermes, and corroborates groupings based on cuticular hydrocarbons. One of the clades, R. hesperus, is already recognized in California, while four clades appear to be previously undescribed taxa. Although identification of the final clade is inconclusive, it includes a sample putatively identified as R. tibialis. Therefore, using phylogenetic analyses we corroborate chemical characters used to identify taxa, associate a chemical phenotype with a previously described species, and provide additional support for undescribed taxa of Reticulitermes.     

Nm23H2 facilitates coat protein complex II assembly and endoplasmic reticulum export in mammalian cells

The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.     

Weapons of mass retraction

Twitching motility is a unique form of bacterial propulsion on solid surfaces associated with cycles of extension, tethering and retraction of type IV pili (T4P). Although investigations over the last two decades in a number of species have identified the majority of the genes involved in this process, we are still learning how these pili are assembled and the mechanics by which bacteria use T4P to drag themselves from one place to another. Among the puzzles that remain to be solved is the mechanism by which hydrolysis of ATP is coupled to pilus assembly and disassembly, and how the cell envelope structure is modified to accommodate the passage of the pilus through the periplasm. Unravelling of these and other enigmas in the T4P system will not only teach us more about these important colonization and virulence factors, but also help us to understand related processes such as type II secretion, which relies on a set of proteins homologous to those in the T4P system, and bacterial conjugation, involving retractable pili belonging to the F-like subgroup of the type IV secretion family. This review focuses on recent discoveries relating to the assembly and function of T4P in generation of twitching motility.     

Integration and excision by the large serine recombinase phiRv1 integrase

The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.     

Suitability of aquaculture effluent solids mixed with cardboard as a feedstock for vermicomposting

Recirculating aquaculture systems are highly intensive culture systems that actively filter and reuse water, thus minimizing water requirements and creating relatively small volumes of concentrated waste (compared to flow-through aquaculture systems). Vermicomposting, which uses earthworms to stabilize and transform organic wastes into valuable end-products, has been proposed as an alternative treatment technology for high-moisture-content organic wastes from agricultural, industrial and municipal sources. This study was conducted to determine if the effluent solids from a large recirculating aquaculture facility (Blue Ridge Aquaculture, Martinsville, Virginia) were suitable for vermicomposting using the earthworm Eisenia fetida. In two separate experiments, worms were fed mixtures of solids removed from aquaculture effluent (sludge) and shredded. Mixtures containing 0%, 5%, 10%, 15%, 20%, 25%, and 50% aquaculture sludge (dry weight basis) were fed to the worms over a 12-week period and their growth (biomass) was measured. Worm mortality, which occurred only in the first experiment, was not influenced by feedstock sludge concentration. In both experiments worm growth rates tended to increase with increasing sludge concentration, with the highest growth rate occurring with feedstocks containing 50% aquaculture sludge. Effluent solids from recirculating aquaculture systems mixed with shredded cardboard appear to be suitable feedstocks for vermicomposting.     

Potent pyrimidinetrione-based inhibitors of MMP-13 with enhanced selectivity over MMP-14

Through the use of computational modeling, a series of pyrimidinetrione-based inhibitors of MMP-13 was designed based on a lead inhibitor identified through file screening. Incorporation of a biaryl ether moiety at the C-5 position of the pyrimidinetrione ring resulted in a dramatic enhancement of MMP-13 potency. Protein crystallography revealed that this moiety binds in the S(1)(') pocket of the enzyme. Optimization of the C-4 substituent of the terminal aromatic ring led to incorporation of selectivity versus MMP-14 (MT-1 MMP). Structure activity relationships of the biaryl ether substituent are presented as is pharmacokinetic data for a compound that meets our in vitro potency and selectivity goals.     

A series of 5-(5,6)-dihydrouracil substituted 8-hydroxy-[1,6]naphthyridine-7-carboxylic acid 4-fluorobenzylamide inhibitors of HIV-1 integrase and viral replication in cells

Introduction of a 5,6-dihydrouracil functionality in the 5-position of N-(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent antiviral agent than 1, with a CIC(95) of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats and dogs.     

Orally bioavailable highly potent HIV protease inhibitors against PI-resistant virus

Efforts directed to identifying potent HIV protease inhibitors (PI) have yielded a class of compounds that are not only very active against wild-type (NL4-3) HIV virus but also very potent against a panel of PI-resistant viral isolates. Chemistry and biology are described.