Yes / Matt Meselson, Professor of Molecular and Cellular Biology, Harvard University, 'Biotechnology and Weapons of Mass Destruction - the Future? ' November 2002.

Cholera, caused by the secretion of cholera toxin (CT) by Vibrio cholerae within the intestinal lumen, triggers massive secretory diarrhea which may lead to life-threatening dehydration. CT is an AB5-type protein toxin that is comprised of an enzymatically active A1 chain, an A2 linker, and a cell-binding B pentamer. Once secreted, the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) of a host target cell. To cause intoxication, CTA1 must be displaced from CTA2/CTB5 in the ER and is then transferred to the cytosol where it induces a diarrheal response by stimulating the efflux of chloride ions into the intestinal lumen. Protein disulfide isomerase (PDI), a resident ER oxidoreductase and chaperone, is involved in detaching CTA1 from the holotoxin. The PDI domain(s) that binds to CTA1 and precisely how this interaction is involved in CTA1 dissociation from the holotoxin are unknown. The goal of this project is to identify which domain(s) of PDI is responsible for binding to and dislodging CTA1 from the CT holotoxin. Through incorporation of ELISA, surface plasmon resonance (SPR), and Fourier transform infrared (FTIR) spectroscopy techniques in conjunction with a panel of purified PDI deletion constructs, this project aims to provide important molecular insight into a crucial interaction of the CT intoxication process.

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Cytolysin A (ClyA) is a member of a class of proteins called pore-forming toxins (PFTs). ClyA is secreted by Gram-negative bacteria, and it attacks a number of mammalian cells by inserting into and forming channels within the cell membrane (Oscarsson J et al., 1999). It has been suggested that ClyA binds to cholesterol (Oscarsson J et al., 1999) and thus can insert into the membranes of many different cell types of eukaryotic origin. In our studies we propose to engineer a ClyA protein that can only attack a small subset of cell types. We propose to engineer ClyA that can be only activated when exposed to specific cell-surface proteases produced by a specific cell type. We ultimately want to target breast cancer cells that differentially secrete or express proteases such as matrix-metalloproteases (Stautz D et al., 2012; Zhang, M et al. 2013). To engineer this protein we took advantage of the N-terminus of ClyA. The N-terminus of ClyA, which is highly hydrophobic (Oscarsson J et al), undergoes a conformational change to insert into the target cell membrane (Oscarsson J et al). This conformational change allows ClyA to penetrate the target membrane to form a transmembrane domain of ClyA. The hydrophobic nature of lipid membranes makes it highly unfavorable for any charged residues to cross the membrane (Hunt J 1997). With this in mind, we hypothesize that negative charges inserted into the N-terminus of ClyA will inhibit it from inserting into the membrane. Thus, we mutated the N-terminus of the ClyA protein by inserting an inactivation site composed of negatively charged amino acids that we hypothesize would prevent insertion into the plasma membrane of the target cell. Once we confirmed that this construct was an inactive ClyA mutant, we inserted a thrombin cleavage site right after the inserted negative charges. This site should allow us to remove the negative charges once the protein is exposed to thrombin. Once the negative charges are removed, the protein should recover its activity. This approach will allow us to create a version of ClyA that is protease-switchable.

Cell biology is a branch of science with a seemingly infinite abundance of interesting phenomena which are essential to our understanding of life and which may potentially drive the development of technology that improves our lives. Among the open ended questions within the field, an understanding of how gene networks are affected by limited cellular components is both broad and rich with interest. Common to all cellular systems are enzymes which perform many tasks within cells without which organisms could not remain healthy. Here are presented several explorations of enzymatic processing as well as a tool constructed for this purpose. More specifically, these works consider the effect of coupling of gene networks via competition for enzymes found within the cell. It is shown that a limitation on the number of available enzymes permits the formation of bottlenecks which drastically affect molecular dynamics within cells. These effects potentially afford cell behaviors that in part explain the impressive robustness of life to constantly fluctuating environments. / Ph. D.

Benign masseter hypertrophy causes swelling at the angulus mandibulae and may be associated with masticatory myofascial pain due to hyperfunction from bruxism. The aim of this research was to use the systematic review process to investigate the true or reliable scientific evidence contained in four major databases pertaining to the efficacy and safety of intra-muscular injections of botulinum toxin A (BTX-A) for the treatment of masticatory myofascial pain and benign masseter hypertrophy associated with bruxism, compared with placebo or other traditional treatments prescribed for bruxism such as occlusal splints, pharmacotherapy, or lifestyle modification. Using the PICO format, a research question was formulated, MeSH terms were derived, and an electronic literature search was conducted in PubMed, Embase, Web of Science, and Cochrane. This sequence was followed by a screening and selection of articles by two independent reviewers according to defined inclusion and exclusion criteria. The selected studies were then evaluated and assessed based on study quality and identification of biases, and the results were summarized and reported. This review highlighted the lack of well-designed, randomized controlled trials to evaluate the efficacy and safety of botulinum toxin A for reducing the size/volume of the masseter muscles and for improving masticatory myofascial pain in patients who present with bruxism. Thus, the results were inconclusive.

Yes