Publications

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To address challenges in early detection of pond pests, we have extended a spectroradiometric monitoring method, initially demonstrated for measurement of pigment optical activity and biomass, to the detection of algal competitors and grazers. The method relies upon measurement and interpretation of pond reflectance spectra spanning from the visible into the near-infrared. Reflectance spectra are acquired every 5 min with a multi-channel, fiber-coupled spectroradiometer, providing monitoring of algal pond conditions with high temporal frequency. The spectra are interpreted via numerical inversion of a reflectance model, in which the above-water reflectance is expressed in terms of the absorption and backscatter coefficients of the cultured species, with additional terms accounting for the pigment fluorescence features and for the water-surface reflection of sunlight and skylight. With this method we demonstrate detection of diatoms and the predator Poteriochromonas in outdoor cultures of Nannochloropsis oceanica and Chlorella vulgaris, respectively. The relative strength of these signatures is compared to microscopy and sequencing analysis. Spectroradiometric detection of diatoms is then further assessed on beaker-contained mixtures of Microchloropsis salina with Phaeodactylum tricornutum, Thalassiosira weissflogii, and Thalassiosira pseudonana, respectively, providing an initial evaluation of the sensitivity and specificity of detecting pond competitors.

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Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocoliticabiovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

Monitoring infections in vectors such as mosquitoes,sand flies, tsetse flies, and ticks to identify human pathogens may serve as an early warning detection system to direct local government disease preventive measures. One major hurdle in detection is the ability to screen large numbers of vectors for human pathogens without the use of genotype-specific molecular techniques. Next generation sequencing (NGS) provides an unbiased platform capable of identifying known and unknown pathogens circulating within a vector population, but utilizing this technology is time-consuming and costly for vector-borne disease surveillance programs. To address this we developed cost-effective Ilumina® RNA-Seq library preparation methodologiesin conjunction with an automated computational analysis pipeline to characterize the microbial populations circulating in Culex mosquitoes (Culex quinquefasciatus, Culex quinquefasciatus/pipiens complex hybrids, and Culex tarsalis) throughout California. We assembled 20 novel and well-documented arboviruses representing members of Bunyaviridae, Flaviviridae, Ifaviridae, Mesoniviridae, Nidoviridae, Orthomyxoviridae, Parvoviridae, Reoviridae, Rhabdoviridae, Tymoviridae, as well as several unassigned viruses. In addition, we mapped mRNA species to divergent species of trypanosoma and plasmodium eukaryotic parasites and characterized the prokaryotic microbial composition to identify bacterial transcripts derived from wolbachia, clostridium, mycoplasma, fusobacterium and campylobacter bacterial species. We utilized these microbial transcriptomes present in geographically defined Culex populations to define spatial and mosquito species-specific barriers of infection. The virome and microbiome composition identified in each mosquito pool provided sufficient resolution to determine both the mosquito species and the geographic region in California where the mosquito pool originated. This data provides insight into the complexity of microbial species circulating in medically important Culex mosquitoes and their potential impact on the transmission of vector-borne human/veterinary pathogens in California.

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Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows. © 2013 Landes Bioscience.

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