Mode of nitric oxide delivery affects antibacterial action.

Nitric oxide (NO) is a broad-spectrum antibacterial agent, making it an attractive alternative to traditional antibiotics for treating infections. To date, a direct comparison of the antibacterial activity of gaseous NO (gNO) versus water-soluble NO-releasing biopolymers has not been reported. In this study, the bactericidal action of NO-releasing chitosan oligosaccharides was compared to gNO treatment against cystic fibrosis-relevant Gram-positive and Gram-negative bacteria. A NO exposure chamber was constructed to enable the dosing of bacteria with gNO at concentrations up to 800 ppm under both aerobic and anaerobic conditions. Bacteria viability, solution properties (i.e., pH, NO concentration), and toxicity to mammalian cells were monitored to ensure a thorough understanding of bactericidal action and reproducibility for each delivery method. The NO-releasing chitosan oligosaccharides required significantly lower NO doses relative to gNO therapy to elicit antibacterial action against Pseudomonas aeruginosa and Staphylococcus aureus under both aerobic and anaerobic conditions. Reduced NO doses required for bacteria eradication using water-soluble NO-releasing chitosan were attributed to the release of NO in solution, removing the need to transfer from gas to liquid phase and the associated long diffusion distances of gNO treatment.

Electrochemical investigations of metal nanostructure growth with single crystals.

Control over the nanoscopic structure of a material allows one to tune its properties for a wide variety of applications. Colloidal synthesis has become a convenient way to produce anisotropic metal nanostructures with a desired set of properties, but in most syntheses, the facet-selective surface chemistry causing anisotropic growth is not well-understood. This review highlights the recent use of electrochemical methods and single-crystal electrodes to investigate the roles of organic and inorganic additives in modulating the rate of atomic addition to different crystal facets. Differential capacitance and chronocoulometric techniques can be used to extract thermodynamic data on how additives selectively adsorb, while mixed potential theory can be used to observe the effect of additives on the rate of atomic addition to a specific facet. Results to date indicate that these experimental methods can provide new insights into the role capping agents and halides play in controlling anisotropic growth.

Electrochemical Nitric Oxide Sensors: Principles of Design and Characterization.

Nitric oxide (NO) is a molecule of vast physiological significance, but much remains unknown about the in vivo concentration dependence of its activity, its basal level concentrations, and how levels fluctuate in the course of certain disease states. Although electrochemical methods are best suited to real-time, continuous monitoring of NO, sensors must be appropriately modified to ensure adequate selectivity, sensitivity, sensocompatibility, and biocompatibility in challenging biological environments. Herein, we provide a critical overview of recent advances in the field of electrochemical NO sensors designed to operate in physiological milieu. Unique to this review, we have opted to highlight research efforts undertaking meticulous characterization of the sensor's analytical performance. Furthermore, we compile basic recommendations to inform future electrochemical NO sensor development and facilitate cross-comparison of proposed sensor designs.

Selective and Sensocompatible Electrochemical Nitric Oxide Sensor with a Bilaminar Design.

Macrophages mediate mammalian inflammation in part by the release of the gasotransmitter, nitric oxide (NO). Electrochemical methods represent the best means of direct, continuous measurement of NO, but monitoring continuous release from immunostimulated macrophages remains analytically challenging. Long release durations necessitate consistent sensor performance (i.e., sensitivity and selectivity for NO) in proteinaceous media. Herein, we describe the fabrication of an electrochemical sensor modified by an electropolymerized 5-amino-1-naphthol (poly(5A1N)) film in conjunction with a fluorinated xerogel topcoat. The unique combination of these membranes ensures selective detection of NO that is maintained over extended periods of use (>24 h) in biological media without performance deterioration. The hydrophobic xerogel topcoat protects the underlying NO-selective poly(5A1N) film from hydration-induced desorption. The bilaminar sensor is then readily adapted for measurement of the temporal NO-release profiles from immunostimulated macrophages.

A direct and selective electrochemical hydrogen sulfide sensor.

Continuous, in situ detection of hydrogen sulfide (H2S) in biological milieu is made possible with electrochemical methods, but direct amperometry is constrained by the generation of elemental sulfur as an oxidative byproduct. Deposition of a sulfur layer passivates the working electrode, reducing sensitivity and causing performance variability. Herein, we report on the use of a surface preconditioning procedure to deposit elemental sulfur on a glassy carbon electrode prior to measurement and evaluate performance with common analytical metrics. The lack of traditional anti-poisoning techniques (e.g. redox mediators, cleaning pulses) also allowed for facile surface modification with electropolymerized films. For the first time, a series of electropolymerized films were characterized for their H2S permselective behavior against common biological interferents. Highly selective, film-modified electrodes were then evaluated for their anti-biofouling ability in simulated wound fluid. The final optimized electrode was capable of measuring H2S with a low detection limit (i.e., <100 nM) and ∼80% of its initial sensitivity in proteinaceous media.

Catalytic selectivity of metallophthalocyanines for electrochemical nitric oxide sensing.

The catalytic properties of metallophthalocyanine (MPc) complexes have long been applied to electrochemical sensing of nitric oxide (NO) to amplify sensitivity and reduce the substantial overpotential required for NO oxidation. The latter point has significant ramifications for in situ amperometric detection, as large working potentials oxidize biological interferents (e.g., nitrite, L-ascorbate, and carbon monoxide). Herein, we sought to isolate and quantify, for the first time, the selectivity benefits of MPc modification of glassy carbon electrodes. A series of the most catalytically active MPc complexes towards NO, including Fe(II)Pc, Co(II)Pc, Ni(II)Pc, and Zn(II)Pc, was selected and probed for NO sensing ability under both differential pulse voltammetry (DPV) and constant potential amperometry (CPA). Data from DPV measurements provided information with respect to MPc signal sensitivity amplification (~1.5×) and peak shifting (100-200 mV). Iron-Pc exerted the most specific catalytic activity towards NO over nitrite. Catalyst-enabled reduction of the working potential under CPA was found to improve selectivity for NO over high potential interferents, regardless of MPc. However, impaired selectivity against low potential interferents was also noted.

Nitric oxide permselectivity in electropolymerized films for sensing applications.

The presence of biological interferents in physiological media necessitates chemical modification of the working electrode to facilitate accurate electrochemical measurement of nitric oxide (NO). In this study, we evaluated a series of self-terminating electropolymerized films prepared from one of three isomers of phenylenediamine (PD), phenol, eugenol, or 5-amino-1-naphthol (5A1N) to improve the NO selectivity of a platinum working electrode. The electrodeposition procedure for each monomer was individually optimized using cyclic voltammetry (CV) or constant potential amperometry (CPA). Cyclic voltammetry deposition parameters favoring slower film formation generally yielded films with improved selectivity for NO over nitrite and l-ascorbate. Nitric oxide sensors were fabricated and compared using the optimized deposition procedure for each monomer. Sensors prepared using poly-phenol and poly-5A1N film-modified platinum working electrodes demonstrated the most ideal analytical performance, with the former demonstrating the best selectivity. In simulated wound fluid, platinum electrodes modified with poly-5A1N films proved superior with respect to the NO sensitivity and detection limit.

Programmed evolution for optimization of orthogonal metabolic output in bacteria.

Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields - evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in energy, pharmaceuticals, chemical commodities, biomining, and bioremediation.

Macrocyclization of organo-peptide hybrids through a dual bio-orthogonal ligation: insights from structure-reactivity studies.

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo-biosynthetic strategy for generating macrocyclic organo-peptide hybrids (MOrPHs) through a dual oxime-/intein-mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3-amino-thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2-amino- mercaptomethyl-aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the "i-1" site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side-chain→C-end ligation versus C-end→side-chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo-peptide macrocycles and provide valuable structure-reactivity insights to inform the construction of macrocycle libraries through this chemo-biosynthetic strategy.

The underrecognized epilepsy spectrum: the effects of levetiracetam on neuropsychological functioning in relation to subclinical spike production.

The purpose of this prospective, open-label pilot study was to determine whether treatment with levetiracetam improves neuropsychological functioning in children and adolescents who have evidence of subclinical spike production associated with attention and learning difficulties. Six participants (mean age 9.8 years) were treated with levetiracetam up to 40 mg/kg per day and evaluated using neuropsychological (Wide Range Assessment of Memory and Learning, Second Edition), academic (Wechsler Individual Achievement Test, Second Edition, Abbreviated), and electroencephalographic assessments at baseline and after 10 weeks of treatment. Statistically significant improvements on indexes of the Wide Range Assessment of Memory and Learning, Second Edition were observed in 4 participants after 10 weeks. No statistically significant differences were observed for the Wechsler Individual Achievement Test, Second Edition, Abbreviated. Concomitant spike suppression was observed. Levetiracetam was generally well tolerated. A subset of patients exists with attention and learning problems that have associated aberrant cortical electrical activity without clinical seizures and associated neuropsychological deficits that may improve after treatment with levetiracetam.

Localization of extratemporal seizure with noninvasive dense-array EEG. Comparison with intracranial recordings.

A 13-year-old girl presented with refractory seizures since the age of 5 years. Clinical exam and MRI studies were normal. Ictal EEG discharges suggested possible left posterior quadrant distribution but were not well localized with standard methods. A seizure was recorded during 128-channel EEG video long-term monitoring prior to invasive recordings. Applying a source analysis method, seizure onset and propagation patterns were calculated and displayed on an MRI model. The onset was localized to the left inferior posterior occipital cortex, followed by propagation to the right, then left, posterior cerebral hemispheres, and finally to the left superior-medial parietal lobe. These patterns were replicated closely on subsequent invasive recordings. Surgery was based on intracranial findings and she is seizure-free 30 months after resection. Noninvasive dense-array EEG, used in conjunction with realistic source analysis methods, may have the potential to assist in localizing seizure onsets when standard methods fail.

Discharges in ventromedial frontal cortex during absence spells.

Neural mechanisms of conscious attention require thalamic control of widespread cortical networks. Absence spells involve a momentary loss of voluntary control of attention, during which the person is inactive and unresponsive. The spike-wave seizure discharges of these spells rapidly engage both cerebral hemispheres in the classic sign of a "generalized" seizure. Animal evidence suggests that spike-wave seizures are caused by a disruption of thalamic circuitry, with extensive spread to cortex through thalamocortical propagation. We applied advanced methods of electrical source analysis to dense array (256-channel) electroencephalographic recordings of spike-wave discharges of absence spells. Neither the onset nor the spread of these seizures is generalized. Rather, the slow waves of the discharges are restricted to frontotemporal networks, and the spikes represent a highly localized and stereotyped progression of electrophysiological activity in ventromedial frontal networks. Given the current knowledge of the neurophysiology of absence seizures, this specificity of the frontal cortical discharges suggests the hypothesis that absence spells are associated with pathology in a circuit comprising ventromedial frontal cortex, rostral thalamic reticular nucleus, and limbic nuclei of the thalamus. Disrupted in absence, this circuit appears to regulate important aspects of the voluntary control of conscious attention.

Are "generalized" seizures truly generalized? Evidence of localized mesial frontal and frontopolar discharges in absence.

PURPOSE: To determine whether specific regions of cerebral cortex are activated at the onset and during the propagation of absence seizures. METHODS: Twenty-five absence seizures were recorded in five subjects (all women; age 19-58 years) with primary generalized epilepsy. To improve spatial resolution, all studies were performed with dense-array, 256-channel scalp EEG. Source analysis was conducted with equivalent dipole (BESA) and smoothed linear inverse (LORETA) methods. Analyses were applied to the spike components of each spike-wave burst in each seizure, with sources visualized with standard brain models. RESULTS: For each patient, the major findings were apparent on inspection of the scalp EEG maps and waveforms, and the two methods of source analysis gave generally convergent results. The onset of seizures was typically associated with activation of discrete, often unilateral areas of dorsolateral frontal or orbital frontal lobe. Consistently across all seizures, the negative slow wave was maximal over frontal cortex, and the spike that appeared to follow the slow wave was highly localized over frontopolar regions of orbital frontal lobe. In addition, sources in dorsomedial frontal cortex were engaged for each spike-wave cycle. Although each patient showed unique features, the absence seizures of all patients showed rapid, stereotyped evolution to engage both mesial frontal and orbital frontal cortex sources during the repeating cycles of spike-wave activity. CONCLUSIONS: These data suggest that absence seizures are not truly "generalized," with immediate global cortical involvement, but rather involve selective cortical networks, including orbital frontal and mesial frontal regions, in the propagation of ictal discharges.