Modeling pulmonary nitric oxide exchange.

Nitric oxide (NO) was first detected in the exhaled breath more than a decade ago and has since been investigated as a noninvasive means of assessing lung inflammation. Exhaled NO arises from the airway and alveolar compartments, and new analytical methods have been developed to characterize these sources. A simple two-compartment model can adequately represent many of the observed experimental observations of exhaled concentration, including the marked dependence on exhalation flow rate. The model characterizes NO exchange by using three flow-independent exchange parameters. Two of the parameters describe the airway compartment (airway NO diffusing capacity and either the maximum airway wall NO flux or the airway wall NO concentration), and the third parameter describes the alveolar region (steady-state alveolar NO concentration). A potential advantage of the two-compartment model is the ability to partition exhaled NO into an airway and alveolar source and thus improve the specificity of detecting altered NO exchange dynamics that differentially impact these regions of the lungs. Several analytical techniques have been developed to estimate the flow-independent parameters in both health and disease. Future studies will focus on improving our fundamental understanding of NO exchange dynamics, the analytical techniques used to characterize NO exchange dynamics, as well as the physiological interpretation and the clinical relevance of the flow-independent parameters.     

Analytical method for determination of nitric oxide in zebrafish larvae: toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coli lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae.     

Progress in research on ligands,nuclides and techniques for labeling monoclonal antibodies

This paper reviews the current methods for radiolabelling monoclonal antibodies with particular emphasis on radiometals useful for radioimmunoscintigraphy. The discussion, however, is equally applicable to therapeutic radionuclides. The advantages and the pitfalls of the various techniques are critically evaluated. Both direct labeling methods, as well as indirect methods using the bifunctional chelating agent approach, are covered. Recent work on the development and synthesis of new and more specific chelating agents, including the approach of utilizing rigid polyaminocarboxylates, is described. Preliminary promising results with these newer generation chelating agents are presented.     

Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment

Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chemical and biological methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions versus transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended average frequency of induced mutations can be adjusted to a desirable level.     

The use of killer sensitivity patterns for biotyping yeast strains: the state of the art, potentialities and limitations

In recent years molecular techniques have been the most useful tools for the unequivocal identification of undetermined strains at the species level. In many instances, however, a further discrimination at the strain level (biotyping) is required, such as during epidemiological investigations, in which the distribution of pathogenic microorganisms is studied, and for patent protection purposes. Although molecular methods are routinely used also for yeast biotyping, several nonmolecular techniques have been proposed. One of these, the determination of the killer sensitivity pattern (KSP) towards a panel of selected killer toxins has proven to be a good auxiliary method. Despite the plethora of studies published, the potential and limitations of the determination of KSPs have never been critically evaluated. In this review the use of this nonmolecular technique as a biotyping tool is discussed and compared with some currently used DNA-based procedures. In addition, methodological, mechanistic and ecological implications are evaluated.     

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Bioanalytical profile of the L-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

This review briefly summarizes recent progress in fundamental understanding and analytical profiling of the L-arginine/nitric oxide (NO) pathway. It focuses on key analytical references of NO actions and the experimental acquisition of these references in vivo, with capillary electrophoresis (CE) and high-performance capillary electrophoresis (HPCE) comprising one of the most flexible and technologically promising analytical platform for comprehensive high-resolution profiling of NO-related metabolites. Another aim of this review is to express demands and bridge efforts of experimental biologists, medical professionals and chemical analysis-oriented scientists who strive to understand evolution and physiological roles of NO and to develop analytical methods for use in biology and medicine.     

Recent methodological advances in the mass spectrometric analysis of free and protein-associated 3-nitrotyrosine in human plasma

L-Tyrosine and L-tyrosine residues in proteins are attacked by various reactive-nitrogen species (RNS) including peroxynitrite to form 3-nitrotyrosine (NO(2)Tyr) and protein-associated 3-nitrotyrosine (NO(2)TyrProt). Circulating NO(2)Tyr and NO(2)TyrProt have been suggested and are widely used as biomarkers of oxidative stress in humans. In this article the mass spectrometry (MS)-based analytical methods recently reported for the quantification of circulating levels of NO(2)Tyr and NO(2)TyrProt are discussed. These methodologies differ in sensitivity, selectivity, specificity and accessibility to interferences with the latter mainly arising from artifactual formation of NO(2)Tyr and NO(2)TyrProt during sample treatment such as acidification and chemical derivatization. Application of these methodologies to healthy normal humans revealed basal circulating levels for NO(2)Tyr which range between 0.7 and 64 nM, i.e. by two orders of magnitude. Application of gas chromatography-tandem mass spectrometry (GC-tandem MS) methods by two independent research groups by using two different protocols to avoid artifactual nitration of L-tyrosine revealed almost identical mean plasma levels of the order of 1.0 nM in healthy humans. The lower limits of quantitation (LOQ) of these methods were 0.125 and 0.3n M, respectively. This order of magnitude for basal NO(2)Tyr is supported by two liquid chromatography-tandem mass spectrometry (LC-tandem MS) methods with LOQ values of 4.4 and 1.4 nM. On the basis of the data provided by GC-tandem MS and LC-tandem MS the use of a range of 0.5-3 nM for NO(2)Tyr and of 0.6 pmol/mg plasma protein or a molar ratio of 3-nitrotyrosine to tyrosine in plasma proteins of the order of 1:10(6) for NO(2)TyrProt in plasma of healthy humans as reference values appear reasonably justified. Recently reported clinical studies involving 3-nitrotyrosine as a biomarker of oxidative stress are discussed in particular from the analytical point of view.     

ReviewMethods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources

Shikimic acid properties and its available analytical techniques are discussed. Plants having the highest content of shikimic acid are shown. The existing isolation methods are analyzed and the most optimal approaches to extracting this acid from natural sources (plants and microorganisms) are considered.     

Radical reactions of nitric oxide synthases

NOSs (nitric oxide synthases) are flavohaem enzymes that function broadly in human health and disease. We are combining mutagenesis, crystallographic and rapid kinetic methods to understand their mechanism and regulation. The NOSs create a transient tetrahydrobiopterin radical within the enzyme to generate their free radical product (NO). Recent work is revealing how critically important this process is at all levels of catalysis. This article will synthesize four seemingly disparate but related aspects of NOS tetrahydrobiopterin radical formation: (i) how it enables productive O2 activation by providing an electron to the enzyme haem, (ii) what structural features help to regulate this electron transfer, (iii) how it enables NOS to synthesize NO from its diamagnetic substrate and (iv) how it allows NOS to release NO after each catalytic cycle instead of other nitorgen oxide-containing products.     

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide-co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide-co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor (N-diazeniumdiolated dibutylhexanediamine, DBHD/N?O?) can release NO at a physiologically relevant rate > 1 × 10?1?mol min?1 cm?2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.     

Negative NO3- difference in human coronary circulation with severe atherosclerotic stenosis

To examine whether or not the levels of NOx (nitrite; NO2- and nitrate; NO3-) in coronary circulating blood reflect endothelial dysfunction due to coronary atherosclerosis, NOx levels in plasma obtained from ostium of left coronary artery and coronary sinus of patients who complained of chest pain were evaluated in relation to their coronary angiographic findings. Prior to the study, a HPLC-Griess system for NOx measurement was critically evaluated. This system has a detection limit of 0.1 microM of NO2- and NO3- by 10 microl of loading and was able to distinguish a difference of 0.1-0.2 microM of these substances. Heparin (1 U/10 microl) did not affect the detective and discriminative abilities. NO3- difference, calculated from sino-arterial difference of NO3-, was almost zero (-0.2 +/- 0.2 microM) in patients with either normal coronary arteries or mild organic coronary stenosis (< or = 20% narrowing), while a significant negative value (-5.9 +/- 1.7 microM) was obtained from patients with significant stenosis (> or = 70% narrowing) in the left coronary arteries. These results demonstrate reliable ability on the HPLC-Griess system in evaluating NO2- and NO3- in biological samples, and that the negative NO3- difference through coronary circulation may reflect endothelial dysfunction in the patients with coronary atherosclerosis with severe organic stenosis.     

Comparing Artificial Neural Networks,General Linear Models and Support Vector Machines in Building Predictive Models for Small Interfering RNAs

Background

Exogenous short interfering RNAs (siRNAs) induce a gene knockdown effect in cells by interacting with naturally occurring RNA processing machinery. However not all siRNAs induce this effect equally. Several heterogeneous kinds of machine learning techniques and feature sets have been applied to modeling siRNAs and their abilities to induce knockdown. There is some growing agreement to which techniques produce maximally predictive models and yet there is little consensus for methods to compare among predictive models. Also, there are few comparative studies that address what the effect of choosing learning technique, feature set or cross validation approach has on finding and discriminating among predictive models.

Principal Findings

Three learning techniques were used to develop predictive models for effective siRNA sequences including Artificial Neural Networks (ANNs), General Linear Models (GLMs) and Support Vector Machines (SVMs). Five feature mapping methods were also used to generate models of siRNA activities. The 2 factors of learning technique and feature mapping were evaluated by complete 3×5 factorial ANOVA. Overall, both learning techniques and feature mapping contributed significantly to the observed variance in predictive models, but to differing degrees for precision and accuracy as well as across different kinds and levels of model cross-validation.

Conclusions

The methods presented here provide a robust statistical framework to compare among models developed under distinct learning techniques and feature sets for siRNAs. Further comparisons among current or future modeling approaches should apply these or other suitable statistically equivalent methods to critically evaluate the performance of proposed models. ANN and GLM techniques tend to be more sensitive to the inclusion of noisy features, but the SVM technique is more robust under large numbers of features for measures of model precision and accuracy. Features found to result in maximally predictive models are not consistent across learning techniques, suggesting care should be taken in the interpretation of feature relevance. In the models developed here, there are statistically differentiable combinations of learning techniques and feature mapping methods where the SVM technique under a specific combination of features significantly outperforms all the best combinations of features within the ANN and GLM techniques.     

Process analytical technology case study,part III: Calibration monitoring and transfer

This is the third of a series of articles detailing the development of near-infrared spectroscopy methods for solid dosage form analysis. Experiments were conducted at the Duquesne University Center for Pharmaceutical Technology to develop a system for continuous calibration monitoring and formulate an appropriate strategy for calibration transfer. Indcators of high-flux noise (noise factor level) and wave-length uncertainty were developed. These measurements, in combination with Hotelling’s T² and Q residual, are used to continuously monitor instrument performance and model relevance. Four calibration transfer techniques were compared. Three established techniques, finite impulse response filtering, generalized least squares weighting, and piecewise direct standardization were evaluated. A fourth technique, baseline subtraction, was the most effective for calibration transfer. Using as few as 15 transfer samples, predictive capability of the analytical method was maintained across multiple instruments and major instrument maintenance.     

The radioreceptor assay: a simple, sensitive and rapid analytical procedure

Radioreceptor assays (RRAs) are analogous in concept to radioimmuno assays. Characteristic to both methods is the saturable, specific, competitive and reversible ligand/receptor interaction. The RRA is simple, sensitive and reproducible and provides a degree of precision comparable to more sophisticated analytical techniques. Since RRAs require little specialized equipment, they can be used routinely by any laboratory engaged in biochemical research or, as an inexpensive exercise to teach the fundamentals of ligand binding and analytical pharmacology.     

Endogenous presynaptic nitric oxide supports an anterograde signaling in the central nervous system

The source size and density determine the extent of nitric oxide (NO) diffusion which critically influences NO signaling. In the brain, NO released from postsynaptic somas following NMDA-mediated activation of neuronal nitric oxide synthase (nNOS) retrogradely affects smaller presynaptic targets. By contrast, in guinea pig trigeminal motor nucleus (TMN), NO is produced presynaptically by tiny and disperse nNOS-containing terminals that innervate large nNOS-negative motoneurons expressing the soluble guanylyl-cyclase (sGC); consequently, it is uncertain whether endogenous NO supports an anterograde signaling between pre-motor terminals and postsynaptic trigeminal motoneurons. In retrogradely labeled motoneurons, we indirectly monitored NO using triazolofluorescein (DAF-2T) fluorescence, and evaluated sGC activity by confocal cGMP immunofluorescence. Multiple fibers stimulation enhanced NO content and cGMP immunofluorescence into numerous nNOS-negative motoneurons; NOS inhibitors prevented depolarization-induced effects, whereas NO donors mimicked them. Enhance of cGMP immunofluorescence required extracellular Ca(2+), a nNOS-physiological activator, and was prevented by inhibiting sGC, silencing neuronal activity or impeding NO diffusion. In conclusion, NO released presynaptically from multiple cooperative tiny fibers attains concentrations sufficient to activate sGC in many motoneurons despite of the low source/target size ratio and source dispersion; thus, endogenous NO is an effective anterograde neuromodulator. By adjusting nNOS activation, presynaptic Ca(2+) might modulate the NO diffusion field in the TMN.     

Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length,chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.     

Early Diagnosis of the Effects of Long-Term Neurotoxic Action of Lead, Based on the Analysis of Heart Rate

A method for subclinical diagnosis of the consequences of exposure to lead with an example of heart rate analysis is substantiated, and methods for the study of neurotoxic effects on the autonomic nervous system are critically evaluated.     

A simulation-based evaluation of methods for inferring linear barriers to gene flow

Different analytical techniques used on the same data set may lead to different conclusions about the existence and strength of genetic structure. Therefore, reliable interpretation of the results from different methods depends on the efficacy and reliability of different statistical methods. In this paper, we evaluated the performance of multiple analytical methods to detect the presence of a linear barrier dividing populations. We were specifically interested in determining if simulation conditions, such as dispersal ability and genetic equilibrium, affect the power of different analytical methods for detecting barriers. We evaluated two boundary detection methods (Monmonier's algorithm and WOMBLING), two spatial Bayesian clustering methods (TESS and GENELAND), an aspatial clustering approach (STRUCTURE), and two recently developed, non-Bayesian clustering methods [PSMIX and discriminant analysis of principal components (DAPC)]. We found that clustering methods had higher success rates than boundary detection methods and also detected the barrier more quickly. All methods detected the barrier more quickly when dispersal was long distance in comparison to short-distance dispersal scenarios. Bayesian clustering methods performed best overall, both in terms of highest success rates and lowest time to barrier detection, with GENELAND showing the highest power. None of the methods suggested a continuous linear barrier when the data were generated under an isolation-by-distance (IBD) model. However, the clustering methods had higher potential for leading to incorrect barrier inferences under IBD unless strict criteria for successful barrier detection were implemented. Based on our findings and those of previous simulation studies, we discuss the utility of different methods for detecting linear barriers to gene flow.