Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1.

Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue (NH2NAE…IEKCOOH) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-KCOOH) and a double mutant (KD1-Y11T/L17R- KCOOH) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-KCOOH was also expressed in Pichia pastoris. KD1-Y11T/L17R-KCOOH inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with Kd of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-KCOOH and KD1-Y11T/L17R-KCOOH did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-KCOOH was better than that of KD1-L17R-KCOOH and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-KCOOH was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-KCOOH did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-KCOOH is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia.

Cyberbiosecurity: An Emerging New Discipline to Help Safeguard the Bioeconomy.

Cyberbiosecurity is being proposed as a formal new enterprise which encompasses cybersecurity, cyber-physical security and biosecurity as applied to biological and biomedical-based systems. In recent years, an array of important meetings and public discussions, commentaries and publications have occurred that highlight numerous vulnerabilities. While necessary first steps, they do not provide a systematized structure for effectively promoting communication, education and training, elucidation and prioritization for analysis, research, development, test and evaluation and implementation of scientific, technological, standards of practice, policy, or even regulatory or legal considerations for protecting the bioeconomy. Further, experts in biosecurity and cybersecurity are generally not aware of each other's domains, expertise, perspectives, priorities, or where mutually supported opportunities exist for which positive outcomes could result. Creating, promoting and advancing a new discipline can assist with formal, beneficial and continuing engagements. Recent key activities and publications that inform the creation of Cyberbiosecurity are briefly reviewed, as is the expansion of Cyberbiosecurity to include biomanufacturing which is supported by a rigorous analysis of a biomanufacturing facility. Recommendations are provided to initialize Cyberbiosecurity and place it on a trajectory to establish a structured and sustainable discipline, forum and enterprise.

Cyberbiosecurity: From Naive Trust to Risk Awareness.

The cyber-physical nature of biotechnology raises unprecedented security concerns. Computers can be compromised by encoding malware in DNA sequences, and biological threats can be synthesized using publicly available data. Trust within the biotechnology community creates vulnerabilities at the interface between cyberspace and biology. Awareness is a prerequisite to managing these risks.

Production of Recombinant Chemokines and Validation of Refolding.

The diverse roles of chemokines in normal immune function and many human diseases have motivated numerous investigations into the structure and function of this family of proteins. Recombinant chemokines are often used to study how chemokines coordinate the trafficking of immune cells in various biological contexts. A reliable source of biologically active protein is vital for any in vitro or in vivo functional analysis. In this chapter, we describe a general method for the production of recombinant chemokines and robust techniques for efficient refolding that ensure consistently high biological activity. Considerations for initiating development of protocols consistent with Current Good Manufacturing Practices (cGMPs) to produce biologically active chemokines suitable for use in clinical trials are also discussed.

Workshop report: modeling the molecular mechanism of bacterial spore germination and elucidating reasons for germination heterogeneity.

Over the course of 2 days, top researchers in the fields of bacterial spore biology and computational biology discussed approaches to determine the cause of spore germination heterogeneity. Biological and mathematical data gaps were identified, and experimental approaches and computational strategies for modeling spore germination were presented and evaluated. As a result of these interactions, future research directions were defined, the outcome of which should result in a robust model to help define the molecular mechanism(s) of spore germination. Mechanistic understanding of germination will be instrumental for developing novel sterilization, treatment, and decontamination strategies to mitigate threats posed by spores.