A novel approach to interrogating the effects of chemical warfare agent exposure using organ-on-a-chip technology and multiomic analysis.

Organ-on-a-chip platforms are utilized in global bioanalytical and toxicological studies as a way to reduce materials and increase throughput as compared to in vivo based experiments. These platforms bridge the infrastructure and regulatory gaps between in vivo animal work and human systems, with models that exemplify active biological pathways. In conjunction with the advent of increased capabilities associated with next generation sequencing and mass spectrometry based '-omic' technologies, organ-on-a-chip platforms provide an excellent opportunity to investigate the global changes at multiple biological levels, including the transcriptome, proteome and metabolome. When investigated concurrently, a complete profile of cellular and regulatory perturbations can be characterized following treatment with specific agonists. In this study, global effects were observed and analyzed following liver chip exposure to the chemical warfare agent, VX. Even though the primary mechanism of action of VX (i.e. acetylcholinesterase inhibition) is well characterized, recent in vivo studies suggest additional protein binding partners that are implicated in metabolism and cellular energetic pathways. In addition, secondary toxicity associated with peripheral organ systems, especially in human tissues, is not well defined. Our results demonstrate the potential of utilizing an organ-on-a-chip platform as a surrogate system to traditional in vivo studies. This is realized by specifically indicating significant dysregulation of several cellular processes in response to VX exposure including but not limited to amino acid synthesis, drug metabolism, and energetics pathways.

barCoder: a tool to generate unique, orthogonal genetic tags for qPCR detection.

BACKGROUND: Tracking dispersal of microbial populations in the environment requires specific detection methods that discriminate between the target strain and all potential natural and artificial interferents, including previously utilized tester strains. Recent work has shown that genomic insertion of short identification tags, called "barcodes" here, allows detection of chromosomally tagged strains by real-time PCR. Manual design of these barcodes is feasible for small sets, but expansion of the technique to larger pools of distinct and well-functioning assays would be significantly aided by software-guided design. RESULTS: Here we introduce barCoder, a bioinformatics tool that facilitates the process of creating sets of uniquely identifiable barcoded strains. barCoder utilizes the genomic sequence of the target strain and a set of user-specified PCR parameters to generate a list of suggested barcode "modules" that consist of binding sites for primers and probes, and appropriate spacer sequences. Each module is designed to yield optimal PCR amplification and unique identification. Optimal amplification includes metrics such as ideal melting temperature and G+C content, appropriate spacing, and minimal stem-loop formation; unique identification includes low BLAST hits against the target organism, previously generated barcode modules, and databases (such as NCBI). We tested the ability of our algorithm to suggest appropriate barcodes by generating 12 modules for Bacillus thuringiensis serovar kurstaki-a simulant for the potential biowarfare agent Bacillus anthracis-and three each for other potential target organisms with variable G+C content. Real-time PCR detection assays directed at barcodes were specific and yielded minimal cross-reactivity with a panel of near-neighbor and potential contaminant materials. CONCLUSIONS: The barCoder algorithm facilitates the generation of synthetically barcoded biological simulants by (a) eliminating the task of creating modules by hand, (b) minimizing optimization of PCR assays, and (c) reducing effort wasted on non-unique barcode modules.

Synthesis and µ-Opioid Activity of the Primary Metabolites of Carfentanil.

Carfentanil is a synthetic opioid significantly more potent than clinically prescribed fentanyl. The primary metabolites of carfentanil, generated from human liver microsomes, were structurally confirmed through chemical synthesis. The synthesized compounds were evaluated for µ-opioid receptor (MOR) functional activity. Of the six metabolites assayed, a major metabolite showed comparable activity to the parent opioid. Three other metabolites showed significant MOR functional activity. The availability of the metabolites could aid improvements in the analysis of biomedical samples obtained from suspected human exposures to carfentanil and development of treatment protocols.

Bioresponsive peptide-polysaccharide nanogels - A versatile delivery system to augment the utility of bioactive cargo.

We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or 'nanogel', prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.

A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes.

Bacterial ribosomes frequently translate to the 3' end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these "nonstop" ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type (wt) cells but not in cells that lack trans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues nonstop ribosomes in vivo Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.IMPORTANCEFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identified a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

Tetrazole-Based trans-Translation Inhibitors Kill Bacillus anthracis Spores To Protect Host Cells.

Bacillus anthracis, the causative agent of anthrax, remains a significant threat to humans, including potential use in bioterrorism and biowarfare. The capacity to engineer strains with increased pathogenicity coupled with the ease of disseminating lethal doses of B. anthracis spores makes it necessary to identify chemical agents that target and kill spores. Here, we demonstrate that a tetrazole-based trans-translation inhibitor, KKL-55, is bactericidal against vegetative cells of B. anthracis in culture. Using a fluorescent analog, we show that this class of compounds colocalizes with developing endospores and bind purified spores in vitro KKL-55 was effective against spores at concentrations close to its MIC for vegetative cells. Spore germination was inhibited at 1.2× MIC, and spores were killed at 2× MIC. In contrast, ciprofloxacin killed germinants at concentrations close to its MIC but did not prevent germination even at 32× MIC. Because toxins are released by germinants, macrophages infected by B. anthracis spores are killed early in the germination process. At ≥2× MIC, KKL-55 protected macrophages from death after infection with B. anthracis spores. Ciprofloxacin required concentrations of ≥8× MIC to exhibit a similar effect. Taken together, these data indicate that KKL-55 and related tetrazoles are good lead candidates for therapeutics targeting B. anthracis spores and suggest that there is an early requirement for trans-translation in germinating spores.

Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication.

Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development.