Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages (M) derived from certain inbred strains. We used nine inbred strains and two inducible nitric oxide synthase (iNOS) knockout C57BL/6J strains polymorphic for the LT M sensitivity Kif1C locus to analyze the role of M sensitivity (to lysis) in LT-mediated cytokine responses and lethality. LT-mediated induction of cytokines KC, MCP-1/JE, MIP-2, eotaxin, and interleukin-1␤ occurred only in mice having LT-sensitive M. However, while iNOS knockout C57BL/6J mice having LT-sensitive M were much more susceptible to LT than the knockout mice with LT-resistant M, a comparison of susceptibilities to LT in the larger set of inbred mouse strains showed a lack of correlation between M sensitivity and animal susceptibility to toxin. For example, C3H/HeJ mice, harboring LT-sensitive M and having the associated LT-mediated cytokine response, were more resistant than mice with LT-resistant M and no cytokine burst. Toll-like receptor 4 (Tlr4)-deficient, lipopolysaccharide-nonresponsive mice were not more resistant to LT. We also found that CAST/Ei mice are uniquely sensitive to LT and may provide an economical bioassay for toxindirected therapeutics. The data indicate that while the cytokine response to LT in mice requires M lysis and while M sensitivity in the C57BL/6J background is sufficient for BALB/cJ-like mortality of that strain, the contribution of M sensitivity and cytokine response to animal susceptibility to LT differs among other inbred strains. Thus, LT-mediated lethality in mice is influenced by genetic factors in addition to those controlling M lysis and cytokine response and is independent of Tlr4 function.Anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, consists of two polypeptides, lethal factor (LF) and protective antigen (PA). PA binds to receptors and translocates LF (a protease) into the cytosol (10). LT injection into animals is sufficient to induce symptoms of anthrax (1-3, 7, 8). We recently showed that LT injection in mice results in death through cytokine-independent mechanisms involving vascular collapse and resultant hypoxia (12).Macrophages (M) from certain inbred strains of mice are uniquely sensitive to rapid lysis by LT, while those from other strains are resistant (4). Three polymorphisms in the LT susceptibility locus (kinesin gene Kif1C on mouse chromosome 11) correlate with M sensitivity to LT (17). Early studies showing that LT-induced death was more rapid in inbred CBA/J mice harboring LT-sensitive (LT s ) M than in A/J mice harboring LT-resistant (LT r ) M suggested a correlation between M sensitivity and animal susceptibility (18). Studies by Hanna et al. implicated M sensitivity in LT toxicity by showing that depleting BALB/cJ mice of LT s M made them LT r (5). However, new studies comparing BALB/c and DBA/2 mice showed that two additional loci on mouse chromosome 11 contribute to LT susceptibility (11). Recent studies suggest that M sensitivity to LT ma...

Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.

Microfluidic paper-based analytical devices (microPADs) are emerging as cost-effective and portable platforms for point-of-care assays. A fundamental limitation of microPAD fabrication is the imprecise nature of most methods for patterning paper. The present work demonstrates that paper patterned via wax printing can be miniaturized by treating it with periodate to produce higher-resolution, high-fidelity microPADs. The optimal miniaturization parameters were determined by immersing microPADs in various concentrations of aqueous sodium periodate (NaIO4) for varying lengths of time. This treatment miniaturized microPADs by up to 80% in surface area, depending on the concentration of periodate and length of the reaction time. By immersing microPADs in 0.5-M NaIO4 for 48 hours, devices were miniaturized by 78% in surface area, and this treatment allowed for the fabrication of functional channels with widths as small as 301 µm and hydrophobic barriers with widths as small as 387 µm. The miniaturized devices were shown to be compatible with redox-based colorimetric assays and enzymatic reactions. This miniaturization technique provides a new option for fabricating sub-millimeter-sized features in paper-based fluidic devices without requiring specialized equipment and could enable new capabilities and applications for microPADs.

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment.