Novel methods to characterise texture changes during food breakdown : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosphy in Food Technology at Massey University, New Zealand

Figures 2-6 (=Ogawa et al., 2001 Fig 2) & 2-12 (=Hutchings & Lillford, 1998 Fig 1) were removed for copyright reasons. Some possibly copyrighted Figures remain for the sake of clarity, while other Figures are in the public domain.The purpose of the mastication process is to break down food for bolus formation so that it can be swallowed safely. Although light has been shed on the criterion for a swallow safe bolus, quantifying these in terms of the bolus properties is not fully understood. There is a lack of suitable measurement techniques to quantify these identified bolus properties. Thus, the purpose of this work was to develop novel techniques that would be useful in in-vitro studies of food breakdown for the characterisation of bolus properties. A mastication robot (MR) had been previously developed to enable the reproducible mastication of food so that masticatory efficiency and food breakdown dynamics can be assessed quantitatively. To evaluate if the MR could be a controllable and reproducible alternative to subjects for food break down studies, a series of experiments involving the mastication of peanuts using a range of machine parameters was conducted. The bolus particle size distributions were used to characterise the breakdown of the peanuts. There were significant differences in the average particle size of the particles chewed by the different chewing trajectories during the initial chews. The performance of the mastication robot was validated against human subjects (n=5) by comparing the particle size distribution (PSD) of peanut boluses collected from subjects and the MR. Although the MR was unable to achieve similar breakdown capability as that for the human subjects, the MR proved to have good reproducibility in bolus preparation. Two novel techniques were developed to characterise bolus properties. The slip extrusion test was developed to characterise two determinant properties for safe swallowing, the bolus deformation and slippage properties. The test measures the force needed to extrude a bolus through a test bag imitating the swallowing action of a bolus. The multiple pin penetrometer was previously developed to measure the spatial distribution of texture in foods exhibiting heterogenous structures. The forces experienced by each pin is measured independently as they pushed through the food, providing a pressure distribution for each food. This allowed the characterisation of fibrous (non-fracturable) foods in a similar way to PSD analysis, offering a method to characterise boluses that do not form discrete particles. The variability in the structure of the boluses was also characterised using the grey level co-occurrence matrix through the image textural features: contrast, energy and homogeneity. Finally, these developed novel techniques were applied to five real foods with varying textures to show how the MR and these techniques may be used to characterise the changes in bolus properties across the mastication stages. Subjects (n=5) were asked to masticate the foods to determine their chewing behaviour and the bolus properties (deformation and slip properties) at swallow point. The chewing parameters from the median subject (subject A) was used to establish the parameters for the mastication robot’s set up for the factorial design of experiments. The developed models from the factorial study were used to optimize the conditions needed for the MR to achieve boluses with similar DR and SR properties as subject A. The five foods were then broken down using the MR configured in this way, and bolus properties were evaluated at various stages of the mastication process through the application of the slip extrusion test, textural mapping using the multiple pin penetrometer, and the back-extrusion test. Factor analysis was applied to the various data collected, which showed that the properties related to the hardness, swallowability and homogeneity attributes were best at describing the changes in the boluses as they were masticated to swallow point. In conclusion, the mastication robot could be used to replicate human chewing trajectories to consistently produce boluses in a controlled trajectory with controlled “simulated saliva” rates throughout the various stages of mastication. Thus, it is relevant as a tool to produce boluses for comparative analysis especially for studies investigating the properties of boluses collected from various stages of the mastication process. In addition, the developed characterisation techniques could be used to track the dynamic changes in the bolus properties for most of the mastication stages from initial chews to the swallow point and beyond that

Cellular trafficking of Sn-2 phosphatidylcholine prodrugs studied with fluorescence lifetime imaging and super-resolution microscopy

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Conversational ontology operator: Patient-centric vaccine dialogue management engine for spoken conversational agents

BACKGROUND: Previously, we introduced our Patient Health Information Dialogue Ontology (PHIDO) that manages the dialogue and contextual information of the session between an agent and a health consumer. In this study, we take the next step and introduce the Conversational Ontology Operator (COO), the software engine harnessing PHIDO. We also developed a question-answering subsystem called Frankenstein Ontology Question-Answering for User-centric Systems (FOQUS) to support the dialogue interaction. METHODS: We tested both the dialogue engine and the question-answering system using application-based competency questions and questions furnished from our previous Wizard of OZ simulation trials. RESULTS: Our results revealed that the dialogue engine is able to perform the core tasks of communicating health information and conversational flow. Inter-rater agreement and accuracy scores among four reviewers indicated perceived, acceptable responses to the questions asked by participants from the simulation studies, yet the composition of the responses was deemed mediocre by our evaluators. CONCLUSIONS: Overall, we present some preliminary evidence of a functioning ontology-based system to manage dialogue and consumer questions. Future plans for this work will involve deploying this system in a speech-enabled agent to assess its usage with potential health consumer users

Loss-of-Function K\u3csub\u3eCa\u3c/sub\u3e2.2 Mutations Abolish Channel Activity

Small-conductance Ca2+-activated potassium channels subtype 2 (KCa2.2, also called SK2) are operated exclusively by a Ca2+-calmodulin gating mechanism. Heterozygous genetic mutations of KCa2.2 channels have been associated with autosomal dominant neurodevelopmental disorders including cerebellar ataxia and tremor in humans and rodents. Taking advantage of these pathogenic mutations, we performed structure-function studies of the rat KCa2.2 channel. No measurable current was detected from HEK293 cells heterologously expressing these pathogenic KCa2.2 mutants. When co-expressed with the KCa2.2_WT channel, mutations of the pore-lining amino acid residues (I360M, Y362C, G363S and I389V) and two proline substitutions (L174P and L433P) dominant negatively suppressed and completely abolished the activity of the co-expressed KCa2.2_WT channel. Co-expression of the KCa2.2_I289N and the KCa2.2_WT channels reduced the apparent Ca2+ sensitivity compared with the KCa2.2_WT channel, which was rescued by a KCa2.2 positive modulator

The Strong Cell-based Hydrogen Peroxide Generation Triggered by Cold Atmospheric Plasma.

Hydrogen peroxide (H2O2) is an important signaling molecule in cancer cells. However, the significant secretion of H2O2 by cancer cells have been rarely observed. Cold atmospheric plasma (CAP) is a near room temperature ionized gas composed of neutral particles, charged particles, reactive species, and electrons. Here, we first demonstrated that breast cancer cells and pancreatic adenocarcinoma cells generated micromolar level H2O2 during just 1 min of direct CAP treatment on these cells. The cell-based H2O2 generation is affected by the medium volume, the cell confluence, as well as the discharge voltage. The application of cold atmospheric plasma (CAP) in cancer treatment has been intensively investigated over the past decade. Several cellular responses to CAP treatment have been observed including the consumption of the CAP-originated reactive species, the rise of intracellular reactive oxygen species, the damage on DNA and mitochondria, as well as the activation of apoptotic events. This is a new previously unknown cellular response to CAP, which provides a new prospective to understand the interaction between CAP and cells in vitro and in vivo. The short-lived reactive species in CAP may activate cells in vivo to generate long-lived reactive species such as H2O2, which may trigger immune attack on tumorous tissues via the H2O2-mediated lymphocyte activation

Channelopathy-Causing Mutations in the S\u3csub\u3e45\u3c/sub\u3eA/S\u3csub\u3e45\u3c/sub\u3eB and HA/HB Helices of K\u3csub\u3eCa\u3c/sub\u3e2.3 and K\u3csub\u3eCa\u3c/sub\u3e3.1 Channels Alter Their Apparent Ca\u3csup\u3e2+\u3c/sup\u3e Sensitivity

Small- and intermediate-conductance Ca2+-activated potassium (KCa2.x and KCa3.1, also called SK and IK) channels are activated exclusively by a Ca2+-calmodulin gating mechanism. Wild-type KCa2.3 channels have a Ca2+ EC50 value of ∼0.3 μM, while the apparent Ca2+ sensitivity of wild-type KCa3.1 channels is ∼0.27 μM. Heterozygous genetic mutations of KCa2.3 channels have been associated with Zimmermann-Laband syndrome and idiopathic noncirrhotic portal hypertension, while KCa3.1 channel mutations were reported in hereditary xerocytosis patients. KCa2.3_S436C and KCa2.3_V450L channels with mutations in the S45A/S45B helices exhibited hypersensitivity to Ca2+. The corresponding mutations in KCa3.1 channels also elevated the apparent Ca2+ sensitivity. KCa3.1_S314P, KCa3.1_A322V and KCa3.1_R352H channels with mutations in the HA/HB helices are hypersensitive to Ca2+, whereas KCa2.3 channels with the equivalent mutations are not. The different effects of the equivalent mutations in the HA/HB helices on the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels may imply distinct modulation of the two channel subtypes by the HA/HB helices. AP14145 reduced the apparent Ca2+ sensitivity of the hypersensitive mutant KCa2.3 channels, suggesting the potential therapeutic usefulness of negative gating modulators

Subtype-selective Positive Modulation of K\u3csub\u3eCa\u3c/sub\u3e 2 Channels Depends on the HA/HB Helices

Background and Purpose In the activated state of small-conductance Ca2+-activated potassium (KCa 2) channels, calmodulin interacts with the HA/HB helices and the S4-S5 linker. CyPPA potentiates KCa 2.2a and KCa 2.3 channel activity but not the KCa 2.1 and KCa 3.1 subtypes. Experimental Approach Site-directed mutagenesis, patch-clamp recordings and in silico modeling were utilized to explore the structural determinants for the subtype-selective modulation of KCa 2 channels by CyPPA. Key Results Mutating residues in the HA (V420) and HB (K467) helices of KCa 2.2a channels to their equivalent residues in KCa 3.1 channels diminished the potency of CyPPA. CyPPA elicited prominent responses on mutant KCa 3.1 channels with an arginine residue in the HB helix substituted for its equivalent lysine residue in the KCa 2.2a channels (R355K). KCa 2.1 channels harboring a three-amino-acid insertion upstream of the cognate R438 residues in the HB helix showed no response to CyPPA, whereas the deletion mutant (KCa 2.1_ΔA434/Q435/K436) became sensitive to CyPPA. In molecular dynamics simulations, CyPPA docked between calmodulin C-lobe and the HA/HB helices widens the cytoplasmic gate of KCa 2.2a channels. Conclusion and Implications Selectivity of CyPPA among KCa 2 and KCa 3.1 channel subtypes relies on the HA/HB helices

Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (–)-epigallocatechin-3-gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD