Function-based classification of hazardous biological sequences: Demonstration of a new paradigm for biohazard assessments.

Bioengineering applies analytical and engineering principles to identify functional biological building blocks for biotechnology applications. While these building blocks are leveraged to improve the human condition, the lack of simplistic, machine-readable definition of biohazards at the function level is creating a gap for biosafety practices. More specifically, traditional safety practices focus on the biohazards of known pathogens at the organism-level and may not accurately consider novel biodesigns with engineered functionalities at the genetic component-level. This gap is motivating the need for a paradigm shift from organism-centric procedures to function-centric biohazard identification and classification practices. To address this challenge, we present a novel methodology for classifying biohazards at the individual sequence level, which we then compiled to distinguish the biohazardous property of pathogenicity at the whole genome level. Our methodology is rooted in compilation of hazardous functions, defined as a set of sequences and associated metadata that describe coarse-level functions associated with pathogens (e.g., adherence, immune subversion). We demonstrate that the resulting database can be used to develop hazardous "fingerprints" based on the functional metadata categories. We verified that these hazardous functions are found at higher levels in pathogens compared to non-pathogens, and hierarchical clustering of the fingerprints can distinguish between these two groups. The methodology presented here defines the hazardous functions associated with bioengineering functional building blocks at the sequence level, which provide a foundational framework for classifying biological hazards at the organism level, thus leading to the improvement and standardization of current biosecurity and biosafety practices.

Biochemical and aerosol characterization of ricin for use in non-clinical efficacy studies.

Ricin toxin may be used as a biological warfare agent and no medical countermeasures are currently available. Here, a well-characterized lot of ricin was aerosolized to determine the delivered dose for future pre-clinical efficacy studies.  Mouse intraperitoneal (IP) median lethal dose (LD50 ) bioassay measured potency at 5.62 and 7.35 µg/kg on Days 0 and 365, respectively. Additional analyses included total protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and rabbit reticulocyte lysate activity assay. The nebulizer aerosol produced consistent concentrations (2.5 × 103 , 5.0 × 103 , 1.0 × 104 , and 1.5 × 104  µg/mL) and spray factor values. The aerosol particle size distribution was of sufficient size to deposit in lung alveoli (1.12-1.43 µm). Ricinus communis Agglutinin II (RCA 60), prepared at 19 mg/mL in phosphate-buffered saline, pH 7.8, and stored at -70°C, maintained attributes for toxicity following 1-year storage and aerosolized consistently.

Stability Characterization of a Polysorbate 80-Dimethyl Trisulfide Formulation, a Cyanide Antidote Candidate.

Novel cyanide countermeasures are needed for cases of a mass-exposure cyanide emergency. A lead candidate compound is dimethyl trisulfide (DMTS), which acts as a sulfur donor for rhodanese, thereby assisting the conversion of cyanide into thiocyanate. DMTS is a safe compound for consumption and, in a 15% polysorbate 80 (DMTS-PS80) formulation, has demonstrated good efficacy against cyanide poisoning in several animal models. We performed a stability study that investigated the effect of temperature, location of formulation preparation, and pH under buffered conditions. We found that while the stability of the DMTS component was fairly independent of which laboratory prepared the formulation, the concentration of DMTS in the formulation was reduced 36-58% over the course of 29 weeks when stored at room temperature. This loss typically increased with increasing temperatures, although we did not find statistical differences between the stability at different storage temperatures in all formulations. Further, we found that addition of a light buffer negatively impacted the stability, whereas the pH of that buffer did not impact stability. We investigated the factors behind the reduction of DMTS over time using various techniques, and we suggest that the instability of the formulation is governed at least partially by precipitation and evaporation, although a combination of factors is likely involved.