Measuring glucose at the site of insulin delivery with a redox-mediated sensor.

Automated insulin delivery systems for people with type 1 diabetes rely on an accurate subcutaneous glucose sensor and an infusion cannula that delivers insulin in response to measured glucose. Integrating the sensor with the infusion cannula would provide substantial benefit by reducing the number of devices inserted into subcutaneous tissue. We describe the sensor chemistry and a calibration algorithm to minimize impact of insulin delivery artifacts in a new glucose sensing cannula. Seven people with type 1 diabetes undergoing automated insulin delivery used two sensing cannulae whereby one delivered a rapidly-acting insulin analog and the other delivered a control phosphate buffered saline (PBS) solution with no insulin. While there was a small artifact in both conditions that increased for larger volumes, there was no difference between the artifacts in the sensing cannula delivering insulin compared with the sensing cannula delivering PBS as determined by integrating the area-under-the-curve of the sensor values following delivery of larger amounts of fluid (P = 0.7). The time for the sensor to recover from the artifact was found to be longer for larger fluid amounts compared with smaller fluid amounts (10.3 ± 8.5 min vs. 41.2 ± 78.3 s, P < 0.05). Using a smart-sampling Kalman filtering smoothing algorithm improved sensor accuracy. When using an all-point calibration on all sensors, the smart-sampling Kalman filter reduced the mean absolute relative difference from 10.9% to 9.5% and resulted in 96.7% of the data points falling within the A and B regions of the Clarke error grid. Despite a small artifact, which is likely due to dilution by fluid delivery, it is possible to continuously measure glucose in a cannula that simultaneously delivers insulin.

An Amperometric Glucose Sensor Integrated into an Insulin Delivery Cannula: In Vitro and In Vivo Evaluation.

BACKGROUND: Labeling prohibits delivery of insulin at the site of subcutaneous continuous glucose monitoring (CGM). Integration of the sensing and insulin delivery functions into a single device would likely increase the usage of CGM in persons with type 1 diabetes. METHODS: To understand the nature of such interference, we measured glucose at the site of bolus insulin delivery in swine using a flexible electrode strip that was laminated to the outer wall of an insulin delivery cannula. In terms of sensing design, we compared H2O2-measuring sensors biased at 600 mV with redox mediator-type sensors biased at 175 mV. RESULTS: In H2O2-measuring sensors, but not in sensors with redox-mediated chemistry, a spurious rise in current was seen after insulin lis-pro boluses. This prolonged artifact was accompanied by electrode poisoning. In redox-mediated sensors, the patterns of sensor signals acquired during delivery of saline and without any liquid delivery were similar to those acquired during insulin delivery. CONCLUSION: Considering in vitro and in vivo findings together, it became clear that the mechanism of interference is the oxidation, at high bias potentials, of phenolic preservatives present in insulin formulations. This effect can be avoided by the use of redox mediator chemistry using a low bias potential.

Role for Rab10 in Methamphetamine-Induced Behavior.

Lipid rafts are specialized, cholesterol-rich membrane compartments that help to organize transmembrane signaling by restricting or promoting interactions with subsets of the cellular proteome. The hypothesis driving this study was that identifying proteins whose relative abundance in rafts is altered by the abused psychostimulant methamphetamine would contribute to fully describing the pathways involved in acute and chronic effects of the drug. Using a detergent-free method for preparing rafts from rat brain striatal membranes, we identified density gradient fractions enriched in the raft protein flotillin but deficient in calnexin and the transferrin receptor, markers of non-raft membranes. Dopamine D1- and D2-like receptor binding activity was highly enriched in the raft fractions, but pretreating rats with methamphetamine (2 mg/kg) once or repeatedly for 11 days did not alter the distribution of the receptors. LC-MS analysis of the protein composition of raft fractions from rats treated once with methamphetamine or saline identified methamphetamine-induced changes in the relative abundance of 23 raft proteins, including the monomeric GTP-binding protein Rab10, whose abundance in rafts was decreased 2.1-fold by acute methamphetamine treatment. Decreased raft localization was associated with a selective decrease in the abundance of Rab10 in a membrane fraction that includes synaptic vesicles and endosomes. Inhibiting Rab10 activity by pan-neuronal expression of a dominant-negative Rab10 mutant in Drosophila melanogaster decreased methamphetamine-induced activity and mortality and decreased caffeine-stimulated activity but not mortality, whereas inhibiting Rab10 activity selectively in cholinergic neurons had no effect. These results suggest that activation and redistribution of Rab10 is critical for some of the behavioral effects of psychostimulants.

TLR8-dependent TNF-(alpha) overexpression in Fanconi anemia group C cells.

Tumor necrosis factor alpha (TNF-alpha) production is abnormally high in Fanconi anemia (FA) cells and contributes to the hematopoietic defects seen in FA complementation group C-deficient (Fancc(-/-)) mice. Applying gene expression microarray and proteomic methods to studies on FANCC-deficient cells we found that genes encoding proteins directly involved in ubiquitinylation are overrepresented in the signature of FA bone marrow cells and that ubiquitinylation profiles of FA-C and complemented cells were substantially different. Finding that Toll-like receptor 8 (TLR8) was one of the proteins ubiquitinylated only in mutant cells, we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells and that TNF-alpha production in mutant cells depended upon TLR8 and the canonical downstream signaling intermediates interleukin 1 receptor-associated kinase (IRAK) and IkappaB kinase-alpha/beta. FANCC-deficient THP-1 cells and macrophages from Fancc(-/-) mice overexpressed TNF-alpha in response to TLR8 agonists but not other TLR agonists. Ectopically expressed FANCC point mutants were capable of fully complementing the mitomycin-C hypersensitivity phenotype of FA-C cells but did not suppress TNF-alpha overproduction. In conclusion, FANCC suppresses TNF-alpha production in mononuclear phagocytes by suppressing TLR8 activity and this particular function of FANCC is independent of its function in protecting the genome from cross-linking agents.

Consequences of copper accumulation in the livers of the Atp7b-/- (Wilson disease gene) knockout mice.

Wilson disease is a severe genetic disorder associated with intracellular copper overload. The affected gene, ATP7B, has been identified, but the molecular events leading to Wilson disease remain poorly understood. Here, we demonstrate that genetically engineered Atp7b-/- mice represent a valuable model for dissecting the disease mechanisms. These mice, like Wilson disease patients, have intracellular copper accumulation, low-serum oxidase activity, and increased copper excretion in urine. Their liver pathology developed in stages and was determined by the time of exposure to elevated copper rather than copper concentration per se. The disease progressed from mild necrosis and inflammation to extreme hepatocellular injury, nodular regeneration, and bile duct proliferation. Remarkably, all animals older than 9 months showed regeneration of large portions of the liver accompanied by the localized occurrence of cholangiocarcinoma arising from the proliferating bile ducts. The biochemical characterization of Atp7b-/- livers revealed copper accumulation in several cell compartments, particularly in the cytosol and nuclei. The increase in nuclear copper is accompanied by marked enlargement of the nuclei and enhanced DNA synthesis, with these changes occurring before pathology development. Our results suggest that the early effects of copper on cell genetic material contribute significantly to pathology associated with Atp7b inactivation.

Induction of apoptosis by aryl-substituted diamines: role of aromatic group substituents and distance between nitrogens.

A series of aromatic substituted diamines was synthesized and characterized for their cytotoxic profiles against human breast and prostate tumor cell lines. Following a structure function analysis of the effects of changes of the benzyl substituents and the distance between amino groups the most potent analogues were analyzed biologically and were shown to induce apoptosis. These compounds do not induce the enzyme SSAT or deplete intracellular polyamine levels, mechanisms demonstrated by other cytotoxic polyamine analogues.

Synthesis of bis-spermine dimers that are potent polyamine transport inhibitors.

A series of novel spermine dimer analogues was synthesized and assessed for their ability to inhibit spermidine transport into MDA-MB-231 breast carcinoma cells. Two spermine molecules were tethered via their N(1) primary amines with naphthalenedisulfonic acid, adamantanedicarboxylic acid and a series of aliphatic dicarboxylic acids. The linked spermine analogues were potent polyamine transport inhibitors and inhibited cell growth cytostatically in combination with a polyamine synthesis inhibitor. Variation in the linker length did not alter polyamine transport inhibition. The amount of charge on the molecule may influence the molecular interaction with the transporter since the most potent spermidine transport inhibitors contained 5-6 positive charges.