The goal of this proposal is to develop novel biochemical methods and devices for the attomolar detection of all seven serotypes and subtypes of botulinum neurotoxin (BoNT). Based on our preliminary data, we hypothesize that it is feasible to generate both fluorescent and bioluminescent substrates that are resistant to non-BoNT proteolytic cleavage, but can rapidly be activated by BoNTs.
The specific aims of the proposed research are: 1) Development of novel fluoroaenic substrates for BoNT serotypes A to G. These substrates will be designed through peptide libraries with proteinogenic and non-proteinogenic amino acids and will be selected for resistance to non-BoNT proteases and highly efficient cleavage by BoNTs. We will test ALISSAs with those novel substrates in various BoNT-spiked samples of serum, solid organ extracts, enema and stool specimens as well as a variety of foods. 2) Development of a luminogenic protein substrate for botulinum toxin. We are proposing to genetically engineer variants of recombinant luciferase proteins that become activated by specific cleavage reactions mediated by the metalloprotease activity of botulinum toxin. Luminescent substrates will be created for all seven serotypes (A to G) of botulinum toxin. We will also bioluminescent assays that emit light at multiple wavelenght for multiplexed simultaneous detection of more than one serotype per sample. 3) Application of existing and novel BoNT ALISSAs to measure toxin distribution in animal sera and organs. In collaboration with Dr. Luisa Cheng at the U.S. Department for Agriculture in Albany, CA, we will utilize BoNT ALISSA to measure the systemic content and tissue distribution in sub-lethally BoNT-intoxicated mice. Our goal is to determine pharmacokinetics and serotypedependent toxin distributions in organs following parenteral or oral intoxication. Furthermore, in collaboration with Dr. Nedelkov at Intrinsic Bioprobes and with Dr. Chris Myatt at Precision Photonics we will develop and test fully automated devices that will utilize our novel methodology for the quantitative detection of BoNTs at attomolar sensitivity. The BoNT ALISSA technology will be adapted into into a monolithic microcolumn format to be utilized by common liquid handling robots. The resulting benchtop instrumentation will also incorporate a highly sensitive, and inexpensive wave-guide-based optical readout system. Our assay system will be tested and validated with complex biological matrices such as serum, solid organ and food extracts as well as clinical samples (at Dr. Stephen Arnon's laboratory at the California Department for Public Health).
|Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha et al. (2016) Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses. Emerg Infect Dis 22:1295-7|
|Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A et al. (2016) The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles. PLoS Pathog 12:e1005501|
|Barbour, Alan G (2016) Infection resistance and tolerance in Peromyscus spp., natural reservoirs of microbes that are virulent for humans. Semin Cell Dev Biol :|
|Park, Arnold; Yun, Tatyana; Hill, Terence E et al. (2016) Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality. J Gen Virol 97:839-43|
|Levin, Mattias; King, Jasmine J; Glanville, Jacob et al. (2016) Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy. J Allergy Clin Immunol 137:1535-44|
|Chomel, Bruno B; Molia, Sophie; Kasten, Rickie W et al. (2016) Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats. PLoS One 11:e0148299|
|Kern, Aurelie; Zhou, Chensheng W; Jia, Feng et al. (2016) Live-vaccinia virus encapsulation in pH-sensitive polymer increases safety of a reservoir-targeted Lyme disease vaccine by targeting gastrointestinal release. Vaccine 34:4507-13|
|Zeltina, Antra; Bowden, Thomas A; Lee, Benhur (2016) Emerging Paramyxoviruses: Receptor Tropism and Zoonotic Potential. PLoS Pathog 12:e1005390|
|Waggoner, Jesse J; Ballesteros, Gabriela; Gresh, Lionel et al. (2016) Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection. J Clin Virol 78:57-61|
|Sanman, Laura E; Qian, Yu; Eisele, Nicholas A et al. (2016) Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death. Elife 5:e13663|
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