Development and application of a self-referencing glucose microsensor for the measurement of glucose consumption by pancreatic ?-cells

Glucose gradients generated by an artificial source and ?-cells were measured using an enzyme-based glucose microsensor, 8-?m tip diameter, as a self-referencing electrode. The technique is based on a difference measurement between two locations in a gradient and thus allows us to obtain real-time flux values with minimal impact of sensor drift or noise. Flux values were derived by incorporation of the measured differential current into Fick's first equation. In an artificial glucose gradient, a flux detection limit of 8.2 ± 0.4 pmol·cm-2·s-1 (mean ± SEM, n = 7) with a sensor sensitivity of 7.0 ± 0.4 pA/mM (mean ± SEM, n = 16) was demonstrated. Under biological conditions, the glucose sensor showed no oxygen dependence with 5 mM glucose in the bulk medium. The addition of catalase to the bulk medium was shown to ameliorate surface-dependent flux distortion close to specimens, suggesting an underlying local accumulation of hydrogen peroxide. Glucose flux from ?-cell clusters, measured in the presence of 5 mM glucose, was 61.7 ± 9.5 fmol·nL-1·s-1 (mean ± SEM, n = 9) and could be pharmacologically modulated. Glucose consumption in response to FCCP (1 ?M) transiently increased, subsequently decreasing to below basal by 93 ± 16 and 56 ± 6%, respectively (mean ± SEM, n = 5). Consumption was decreased after the application of 10 ?M rotenone by 74 ± 5% (mean ± SEM, n = 4). These results demonstrate that an enzyme-based amperometric microsensor can be applied in the self-referencing mode. Further, in obtaining glucose flux measurements from small clusters of cells, these are the first recordings of the real-time dynamic of glucose movements in a biological microenvironment. <br/

A non-invasive method for measuring preimplantation embryo physiology

Author Posting. © Cambridge University Press, 2000. This article is posted here by permission of Cambridge University Press for personal use, not for redistribution. The definitive version was published in Zygote 8 (2000): 15-24, doi:10.1017/S0967199400000782.The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration ([similar]0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 [mu]m from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocysts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.A portion of this work was funded by an NIH R21 #RR 12718–02 to D.L.K. and P.J.S.S., KO81099 to D.L.K. and NIH P41 RR01395 to P.J.S.S

A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes

Second asymptomatic carotid surgery trial (ACST-2): a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86–1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91–1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable. Funding: UK Medical Research Council and Health Technology Assessment Programme

Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos

Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 microM H(2)O(2) exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 +/- 0.1 microM. Pretreatment with tetraethylammonium inhibited this increase (0.2 +/- 0.1 microM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death

Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation

Cell shrinkage is an incipient hallmark of apoptosis and is accompanied by potassium release that decreases the concentration of intracellular potassium and regulates apoptotic progression. The plasma membrane K+ channel recruited during apoptosis has not been characterized despite its importance as a potential therapeutic target. Here we provide evidence that two-pore domain K+ (K(2P)) channels underlie K+ efflux during apoptotic volume decreases (AVD) in mouse embryos. These K(2P) channels are inhibited by quinine but are not blocked by an array of pharmacological agents that antagonize other K+ channels. The K(2P) channels are uniquely suited to participate in the early phases of apoptosis because they are not modulated by common intracellular messengers such as calcium, ATP, and arachidonic acid, transmembrane voltage, or the cytoskeleton. A K+ channel with similar biophysical properties coordinates regulatory volume decreases (RVD) triggered by changing osmotic conditions. We propose that K(2P) channels are the pathway by which K+ effluxes during AVD and RVD and that apoptosis co-opts mechanisms more routinely employed for homeostatic cell volume regulatio

Mitochondrial dysfunction leads to telomere attrition and genomic instability. Aging Cell 2002

Summary Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging-associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging-associated pathologies