Evaluation of Salmon (<i>Salmo salar</i>) and Rainbow Trout (<i>Oncorhynchus mykiss</i>) pin bones using textural analysis and micro X-ray computational tomography

Industrially, common problems arise with the deboning pin bone process, where Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss) fillets, post rigor, are subjected to a pulling process to remove the pin bones from the fillet. This study measured the length of pin bones from two species of fish and two different industrial graded weights, and then used a texture analyser and lCT X-ray to measure the pulling force, break point and volume of the pin bones of both species of fish. Results showed that salmon pin bones required significantly higher pulling force to remove pin bones from the fish fillet when compared with Trout pin bones. Interestingly Trout pin bones were significantly longer and stronger than Salmon pin bones, but had significantly lower volume. This research has progressed the issues surrounding pin boning industrially, however, more studies are required in order to understand if these differences affect the overall deboning pin bone process

Molecular modeling and simulation of bacterial chemosensory arrays

The movement of an organism in response to environmental chemical cues is known as chemotaxis. Motile bacteria use chemotaxis to navigate through their environments, enabling cells to efficiently locate favorable growing conditions while avoiding harmful ones. Central to this ability, bacteria posses a universally conserved sensory apparatus, known as the chemosensory array, which involves the clustering of thousands of proteins into a highly cooperative signaling network. The present dissertation will present my work using techniques in computational modeling and simulation to investigate the molecular structure and function of the bacterial chemosensory array. A brief overview of each chapter follows. Chapter 1 provides an introduction to the systems-level features of chemotaxis in the model organism Escherichia coli as well as an overview of the molecular organization and function of the chemosensory array. Chapter 2 gives an outline of the core methodologies used in my work, specifically all-atom molecular dynamics (MD) simulation and Molecular Dynamics Flexible Fitting (MDFF). In addition, two of the primary techniques used to analyze the MD simulations presented in this dissertation are sketched out, namely structural clustering based on root- mean-square displacement (RMSD) and Principal Component Analysis (PCA). Chapter 3 reports my work, in collaboration with Peijun Zhang’s Lab, to investigate the structural and dynamical features of the extended chemosensory array. Using computational techniques to synthesize multi-scale structural data from X-ray crystallography and cryo-electron tomography (cryo-ET), an atomic model of the cytoplasmic portion of the chemosensory array from Thermotoga maritima is constructed and refined. Through the use of large-scale MD simulations, a novel conformational change in a key signaling protein is identified and subsequently shown to be critical for chemotaxis signaling in live E. coli cells. Chapter 4 details the construction of an atomic model of a complete, transmembrane (TM) chemoreceptor. In particular, I use homology modeling and MD simulations, in- formed by biochemical and X-ray crystallographic data, to derive a model of the E. coli serine receptor (Tsr), including the previously uncharacterized TM four-helix bundle and HAMP domains. In addition, I report a series of MD simulations of a fragment of the resulting Tsr model, investigating the structural and dynamical effects of mutations on a key control cable residue. Preliminary MD simulations of the intact Tsr model are also presented. Chapter 5 reports work in collaboration with Michael Eisenbach’s Lab at the Weizmann Institute, exploring the role of acetylation on CheY activation and the generation of clockwise (CW) flagellar motor rotation. Specifically, I present a series of MD simulations that investigate the effect of a hyperactivating mutation at a key acetylation site and offer a molecular explanation of acetylation-dependent generation of CW flagellar motor rotation. I conclude with a brief description of recent work, expanding upon the results of the previous chapters, which has resulted in the first atomically resolved model of the E. coli transmembrane chemosensory array

A new self-assessment teaching assistant survey for growth and development

During their time as Teaching Assistants (TAs), graduate students develop a variety of skills, knowledge, and attitudes, based on teaching and related facilitation experiences. As TAs move on to future opportunities, their prior experiences form a foundation upon which additional teaching experience builds. Presently, there are few tools to gauge pedagogical growth during graduate student involvement as TAs in a specific post-secondary course, or as a consequence of their participation in a specialized TA training or teaching program.&nbsp; We created a model for TA development in SCIE 113 (First-year Seminar in Science) at the University of British Columbia. Based on this model, we designed a new survey for TAs to self-assess skills, knowledge and attitudes that they bring with them from prior experience, and those that they develop or further during their time as a TA in SCIE113. We administered the survey to 18 current and past SCIE 113 TAs as of December 2015, representing the complete population of TAs. The results showed that SCIE 113 TAs with similar levels of experience shared similar skills, knowledge, and attitudes as assessed by this survey. Those TAs with the most experience had greater abilities in roles previously identified as unique to the course. Others working with graduate students can use or adapt the survey questions to investigate and stimulate the growth of TAs in their course or program

Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations

To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism

The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis in Bacillus subtilis

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis. Ethanol and other short-chain alcohols are attractants for B. subtilis. Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino-acid residue Ala431 on the cytoplasmic signaling domain of McpB, that when mutated to a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol binds McpB near residue Ala431 and mutation of this residue to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely alcohol-producing microorganisms

Alternative architecture of the E. coli chemosensory array

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation

The reactive oxygen species produced during inflammation through the neutrophilic respiratory burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. Thus, HOCl can be viewed as a host-specific cue that conveys important information about what bacterial physiology and lifestyle programs may be required for successful colonization. Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly selective detection of HOCl due to its reactive and transient nature and chemical similarity to more benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in response to increasing HOCl levels E. coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state alters the delicate equilibrium of competition for Zn++ between the CZB domain and other zinc binders to relay the presence of HOCl through activating the associated GGDEF domain to catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent and are possessed by pathogens that manipulate host inflammation as part of their colonization strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases of chronic inflammation and gut dysbiosis