Development and application of an oxygen electrochemical sensor

In this project, we developed a real-time electrochemical mediator assay to enable the assessment of cell numbers and cell viability. It allows us to monitor metabolism calculable down to a single cell in a low cost easy to use rapid assay, as yet not possible with current technology. The developed assay is based on the determination of oxygen. This was made possible via the use of electrochemical mediator ferrocene carboxylic acid (FcA). The FcA showed distinctive catalytic properties in interacting with reactive oxygen species generated from oxygen when compared to ferrocene methanol (FcMeOH). A deeper insight into the chemistry controlling this behaviour is provided. The behaviour is then taken advantage of to develop a cellular aerobic respiration assay. We describe the properties of the FcA system to detect, in real-time, the oxygen consumption of Escherichia coli DH5α (E. coli). We demonstrated that the FcA-based oxygen assay is highly sensitive, and using a population of cells, oxygen consumption rates could be calculated down to a single cell level. More importantly, the results can be accomplished in minutes, considerably outperforming current commercially available biooxygen demand assays. The developed assay is expected to have a significant impact in diverse fields and industries, ranging from environmental toxicology through to pharmaceutical and agrochemical industries. A significant start has been made into optimisation of the system into a commercially marketable product, including much work on mediator immobilisation and electrode synthesis

Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway

Real-time electrocatalytic sensing of cellular respiration

In the present work we develop a real-time electrochemical mediator assay to enable the assessment of cell numbers and chemical toxicity. This allowed us to monitor metabolism down to a single cell in a low cost easy to use rapid assay which is not possible with current technology. The developed assay was based on the determination of oxygen. This was made possible via the use of electrochemical mediator ferrocene carboxylic acid (FcA). The FcA showed distinctive catalytic properties in interacting with reactive oxygen species generated from oxygen when compared to ferrocene methanol (FcMeOH). A deeper insight into the chemistry controlling this behaviour is provided. The behaviour is then taken advantage of to develop a cellular aerobic respiration assay. We describe the properties of the FcA system to detect, in real-time, the oxygen consumption of Escherichia coli DH5-α (E. coli). We demonstrated that the FcA-based oxygen assay is highly sensitive, and using a population of cells, oxygen consumption rates could be calculated down to a single cell level. More importantly, the results can be accomplished in minutes, considerably outperforming current commercially available biooxygen demand assays. The developed assay is expected to have a significant impact in diverse fields and industries, ranging from environmental toxicology through to pharmaceutical and agrochemical industries

Fast, ultrasensitive detection of reactive oxygen species using a carbon nanotube based-electrocatalytic intracellular sensor

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular “pulse” of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection

Electronic communication of cells with a surface mediated by boronic acid saccharide interactions

The fabrication of a molecularly tailored surface functionalised with a saccharide binding motif, a phenyl boronic acid derivative is reported.The functionalised surface facilitated the transfer of electrons, via unique electronic interactions mediated by the presence of the boronic acid, from a macrophage cell line. This is the first example of eukaryotic cellular-electrical communication mediated by the binding of cells via their cell–surface saccharide units

Specific Binding and Mineralization of Calcified Surfaces by Small Peptides

Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)n repeats, and though similar repeated sequences—(NTT)n, (DTT)n, (ETT)n, (NSS)n, (ESS)n, (DAA)n, (ASS)n, and (NAA)n—also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)n peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed

The impact of compassion from others and self-compassion on psychological distress, flourishing, and meaning in life among university students

Objectives: Research shows that compassion from others and from the self may enable university students to face, overcome, and bounce back from adversity and generate a greater sense of thriving and meaning in life. However, the underlying processes are largely unknown. The present study aimed to examine the associations of compassion with psychological distress, flourishing, and meaning in life among university students and explore the mechanisms underlying these associations. Methods: A total of 536 Hong Kong university students completed questionnaires measuring their experiences of compassion from others, self-compassion, resilience, psychological distress, flourishing, and meaning in life. Results: Serial mediation analyses showed that compassion from others was associated positively with self-compassion, which was, in turn, linked to greater resilience and consequently lower levels of psychological distress and higher levels of flourishing and meaning in life. Conclusions: Our findings reveal the associations of compassion from others and self-compassion with the well-being and life meaning of university students. The findings highlight the importance of being open and receptive to love and kindness from others. The findings also point to the importance of developing a caring attitude toward oneself

Regucalcin (RGN/SMP30) alters agonist- and thapsigargin- induced cytosolic [Ca2+] transients in cells by increasing SERCA Ca(2+)ATPase levels

AbstractRegucalcin (RGN), also reported as senescence marker protein-30 (SMP30), plays a role in Ca2+ homeostasis by modulating a number of Ca2+-dependent proteins. RGN also plays a cyto-protective role and its decrease is linked to age-related diseases and cell death. This study shows that RGN reduces agonist (histamine)-induced Ca2+ transients in RGN+ transfected COS-7 cells (RGN+) and also increases their Ca2+ storage capacity. These observations are explained by RGN+ cells having increased mRNA and protein expression levels of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Therefore down-regulation of RGN expression may contribute to characteristics of age-dependent Ca2+ homeostasis dis-regulation, by decreasing SERCA levels