Bare and Polymer-Coated Indium Tin Oxide as Working Electrodes for Manganese Cathodic Stripping Voltammetry.

Though an essential metal in the body, manganese (Mn) has a number of health implications when found in excess that are magnified by chronic exposure. These health complications include neurotoxicity, memory loss, infertility in males, and development of a neurologic psychiatric disorder, manganism. Thus, trace detection in environmental samples is increasingly important. Few electrode materials are able to reach the negative reductive potential of Mn required for anodic stripping voltammetry (ASV), so cathodic stripping voltammetry (CSV) has been shown to be a viable alternative. We demonstrate Mn CSV using an indium tin oxide (ITO) working electrode both bare and coated with a sulfonated charge selective polymer film, polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene-sulfonate (SSEBS). ITO itself proved to be an excellent electrode material for Mn CSV, achieving a calculated detection limit of 5 nM (0.3 ppb) with a deposition time of 3 min. Coating the ITO with the SSEBS polymer was found to increase the sensitivity and lower the detection limit to 1 nM (0.06 ppb). This polymer modified electrode offers excellent selectivity for Mn as no interferences were observed from other metal ions tested (Zn(2+), Cd(2+), Pb(2+), In(3+), Sb(3+), Al(3+), Ba(2+), Co(2+), Cu(2+), Ni(3+), Bi(3+), and Sn(2+)) except Fe(2+), which was found to interfere with the analytical signal for Mn(2+) at a ratio 20:1 (Fe(2+)/Mn(2+)). The applicability of this procedure to the analysis of tap, river, and pond water samples was demonstrated. This simple, sensitive analytical method using ITO and SSEBS-ITO could be applied to a number of electroactive transition metals detectable by CSV.

Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection.

Clostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase-locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.

A method for selecting structure-switching aptamers applied to a colorimetric gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

Insulin resistance and vascular dysfunction in nondiabetic Asian Indians.

Asian Indians are at higher risk for diabetes and cardiovascular disease than European Caucasians. To examine the pathophysiology of this increased risk, we measured insulin sensitivity, cardiovascular risk factors, fat distribution, and endothelium-dependent (reactive hyperemia) and -independent (nitroglycerin) vasodilation before and after a 2-h hyperinsulinemic clamp (40 mU/m(2).min) in 25 nondiabetic Asian Indians and 15 Caucasians with similar age and body mass index. Asian Indians had higher fasting insulin than Caucasians (6.7 +/- 0.8 vs. 3.7 +/- 0.3 microU/ml, P = 0.007) but similar FPG (90 +/- 2 vs. 88 +/- 2 mg/dl). Glucose uptake during the clamp was markedly reduced in Asian Indians vs. Caucasians (4.5 +/- 0.3 vs. 7.5 +/- 0.4 mg/kg x min, P < 0.0001). During the clamp, basal brachial artery diameter increased less in Asian Indians vs. Caucasians (2.6 +/- 1.0 vs. 5.7 +/- 1.0%, P = 0.04), and the reduction was correlated with the impairment in insulin sensitivity (r = 0.38, P = 0.04). In contrast, vasodilatory responses to reactive hyperemia and nitroglycerin were similar in Asian Indians and Caucasians both before and during hyperinsulinemia. Plasminogen activator inhibitor-1 and FFA were significantly elevated and adiponectin was significantly lower in Asian Indians vs. Caucasians, and there were trends toward higher low-density lipoprotein and triglycerides, lower high-density lipoprotein, and increased total, sc, and visceral fat. These risk factors were all significantly correlated with insulin sensitivity. Thus, apparently healthy Asian Indians have severe insulin resistance, dyslipidemia, elevated plasminogen activator inhibitor-1, impaired insulin-mediated vasodilation, and trends toward altered body fat distribution. These abnormalities may contribute to the increased risk of diabetes and cardiovascular disease in this population.