Lab-on-a-bubble: synthesis, characterization, and evaluation of buoyant gold nanoparticle-coated silica spheres.

This paper describes the development and preparation of a new class of materials for surface-enhanced Raman scattering (SERS) consisting of gold nanoparticles coated onto hollow, buoyant silica microspheres. These materials allow for a new type of molecular assay designated as a lab-on-a-bubble (LoB). LoB materials serve as a convenient platform for the detection of analytes in solution and offer several advantages over traditional colloidal gold and planar SERS substrates, such as the ability to localize and concentrate analytes for detection. An example assay is presented using the LoB method and cyanide detection. Cyanide binds to SERS-active, gold-coated LoBs and is detected directly from the corresponding SERS signal. The abilities of LoBs and a gold colloid to detect cyanide are compared, and in both cases, a detection limit of ~170 ppt was determined. Differences in measurement error using LoBs versus gold colloid are also described, as well as an assay for 5,5'-dithiobis(2-nitrobenzoic acid) that shows the benefit of using LoBs over SERS analyses in colloids, which are often plagued by particle aggregation.

Wild felids as hosts for human plague, Western United States.

Plague seroprevalence was estimated in populations of pumas and bobcats in the western United States. High levels of exposure in plague-endemic regions indicate the need to consider the ecology and pathobiology of plague in nondomestic felid hosts to better understand the role of these species in disease persistence and transmission.

Global transcriptome analysis of Borrelia burgdorferi during association with human neuroglial cells.

As adherence and entry of a pathogen into a host cell are key components to an infection, identifying the molecular mechanisms responsible for cellular association will provide a better understanding of a microbe's pathogenesis. We previously established an in vitro model for Borrelia burgdorferi infection of human neuroglial cells. To expand on our earlier study, we performed B. burgdorferi whole-genome expression analysis following a 20-hour infection of human neuroglial cells to identify borrelial genes that were differentially regulated during host-cell association compared with cultured Borrelia in cell-free medium. This study identifies several regulated genes, the products of which may be important mediators of cellular pathogenesis.

Borrelia burgdorferi surface-localized proteins expressed during persistent murine infection are conserved among diverse Borrelia spp.

Borrelia burgdorferi, the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the sigma(N)-sigma(S) regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.

Analysis of Borrelia burgdorferi Surface Proteins as Determinants in Establishing Host Cell Interactions.

Borrelia burgdorferi infection causes Lyme borreliosis in humans, a condition which can involve a systemic spread of the organism to colonize various tissues and organs. If the infection is left untreated by antimicrobials, it can lead to manifestations including, arthritis, carditis, and/or neurological problems. Identification and characterization of B. burgdorferi outer membrane proteins that facilitate cellular attachment and invasion to establish infection continue to be investigated. In this study, we sought to further define putative cell binding properties of surface-exposed B. burgdorferi proteins by observing whether cellular adherence could be blocked by antibodies. B. burgdorferi mixed separately with monoclonal antibodies (mAbs) against outer surface protein (Osp) A, OspC, decorin-binding protein (Dbp) A, BBA64, and RevA antigens were incubated with human umbilical vein endothelial cells (HUVEC) and human neuroglial cells (H4). B. burgdorferi treated with anti-OspA, -DbpA, and -BBA64 mAbs showed a significant decrease in cellular association compared to controls, whereas B. burgdorferi treated with anti-OspC and anti-RevA showed no reduction in cellular attachment. Additionally, temporal transcriptional analyses revealed upregulated expression of bba64, ospA, and dbpA during coincubation with cells. Together, the data provide evidence that OspA, DbpA, and BBA64 function in host cell adherence and infection mechanisms.

Borrelia burgdorferi expression of the bba64, bba65, bba66, and bba73 genes in tissues during persistent infection in mice.

Borrelia burgdorferi, the etiological agent of Lyme disease in humans, is vectored between mammalian hosts in nature by Ixodes ticks. The organism adapts to diverse environments encountered throughout the enzootic cycle by differentially expressing essential gene products to survive the specialized conditions, whether in ticks or warm-blooded hosts. However, little is known regarding the identity and/or function of B. burgdorferi genes expressed during colonization of tissues during mammalian infection. Experimental evidence has shown that a group of genes (formerly classified as paralogous gene family 54) contiguously localized on the 54-kilobase linear plasmid of B. burgdorferi, are among the most highly regulated by in vitro conditions resembling mammalian infection. In this study, we employed quantitative reverse transcription-PCR to measure temporal gene expression of a subset of this B. burgdorferi gene family (bba64, bba65, bba66, and bba73) in tissues during chronic murine infection. The goal was to gain insight into the role of these genes in infectivity and pathogenesis by identifying when the genes are induced and whether they are expressed in specific target tissues. B. burgdorferi bba64, bba65, bba66, and bba73 expression was measured from infected mouse tissues relative to expression in in vitro culture conditions at specific times post-infection. bba64 expression was highly upregulated in bladder, heart, and spleen tissues throughout the infection period, contrasting with the sharp downregulation previously observed in ear tissues. bba65, bba66, and bba73 demonstrated upregulated differential expression in various tissues over 1 year post-infection. These results suggest an essential role for these genes in borrelial survival, persistence, and/or pathogenesis.

Temporal expression analysis of the Borrelia burgdorferi paralogous gene family 54 genes BBA64, BBA65, and BBA66 during persistent infection in mice.

Members of the Borrelia burgdorferi paralogous gene family 54 (pgf 54) are regulated by conditions simulating mammalian infection and are thought to be instrumental in borrelial host survival and pathogenesis. To explore the activities of these genes in vivo, a comprehensive analysis of pgf 54 genes BBA64, BBA65, and BBA66 was performed to assess the genetic stability, host antibody responses, and kinetics of gene expression in the murine model of persistent infection. DNA sequencing of pgf 54 genes obtained from re-isolates at 1 year postinfection demonstrated that all genes of this family are stable and do not undergo recombination to generate variant antigens during persistent infection. Antibodies against BBA64 and BBA66 appeared soon after infection and were detectable throughout the infection, suggesting that there was gene expression during infection. However, quantitative reverse transcription-PCR revealed that BBA64 gene expression was considerably decreased in Borrelia residing in the mouse ear tissue compared to the expression in cultured spirochetes by 20 days postinfection and that the levels of expression remained low throughout the infection. Conversely, transcription of the BBA65 and BBA66 genes was increased, and both of these genes were continuously expressed until 100 days postinfection; this was followed by periods of differential expression late in infection. The expression profile of the BBA64 gene suggests that this gene has an important role during tick-to-host transmission and early infection, whereas the expression profile of the BBA65 and BBA66 genes suggests that these genes have a role in persistent infection. The differential regulation of pgf 54 genes observed during infection may help confer a survival advantage during persistent infection, influencing mechanisms for B. burgdorferi dissemination, tissue tropism, or evasion of the adaptive immune response.