Discriminative EPR detection of NO and HNO by encapsulated nitronyl nitroxides

Abstract

Nitric oxide, ^?NO, is one of the most important molecules in the biochemistry of living organisms. By contrast, nitroxyl, NO^?, one-electron reduced analog of ^?NO which exists at physiological conditions in its protonated form, HNO, has been relatively overlooked. Recent data show that HNO might be produced endogenously and display unique biological effects. However, there is a lack of specific and quantitative methods of detection of endogenous HNO production. Here we present a new method for discriminative ^?NO and HNO detection by nitronyl nitroxides (NNs) using electron paramagnetic resonance (EPR). It was found that NNs react with ^?NO and HNO with similar rate constants of about 10⁴ M^{? 1}s^{? 1} but yield different products: imino nitroxides and the hydroxylamine of imino nitroxides, correspondingly. An EPR approach for discriminative ^?NO and HNO detection using liposome-encapsulated NNs was developed. The membrane barrier of liposomes protects NNs against reduction in biological systems while is permeable to both analytes, ^?NO and HNO. The sensitivity of this approach for the detection of the rates of ^?NO/HNO generation is about 1 nM/s. The application of encapsulated NNs for real-time discriminative ^?NO/HNO detection might become a valuable tool in nitric oxide-related studies.     

NO detection in biological samples: Differentiation of NO and NO using infrared laser spectroscopy

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between ¹⁴NO and ¹⁵NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples.With the cavity leak-out spectroscopy setup (CALOS) a differentiation between ¹⁴NO and ¹⁵NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of ¹⁴NO/N₂ and ¹⁵NO/N₂ were used to calibrate the system. and of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation.The correlation received for both methods for the detection of NO in the gas phase was r = 0.999, p < 0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for 0.3 pmol and 0.5 pmol; correlation ¹⁴NO: p < 0.0001, r = 0.975, ¹⁵NO: p < 0.0001, r = 0.969).The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.     

In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.     

Reactions of PTIO and carboxy-PTIO with *NO, *NO2, and O2-*

Nitronyl nitroxides, such as derivatives of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIOs), react with *NO to form the corresponding imino nitroxides (PTIs) and *NO2. PTIOs are considered as monitors of *NO, stoichiometric sources of *NO2, biochemical and physiological effectors, specific tools for the elimination of *NO, and potential therapeutic agents. However, a better understanding of the chemical properties of PTIOs, especially following their reaction with *NO, is necessary to resolve many of the reported discrepancies surrounding the effects of PTIOs and to better characterize their potential therapeutic activity. We have generated electrochemically the oxidized and reduced forms of PTIO and carboxy-PTIO (C-PTIO), characterized their absorption spectra, and determined the reduction potentials for the oxoammonium/nitroxide and nitroxide/hydroxylamine couples. The rate constants for the reaction of *NO2 with PTIO and C-PTIO to form the corresponding oxoammonium cations (PTIO+s) and nitrite were determined to be (1.5 - 2) x 10(7) m-1 s-1. We have also shown that the reactions of PTIO+s with *NO form PTIOs and NO2-. The rate constants for these reactions are approximately 30-fold higher than those for the reactions of PTIOs with *NO or O2-*. The present results show that (i) the reaction of PTIOs with *NO forms solely PTIs and NO2- where [NO2-]/[PTI] varies between 1 and 2 depending on the steady-state concentrations of *NO. Consequently, quantitation of *NO is valid only at sufficiently low fluxes of *NO; (ii) the reaction of PTIOs with *NO can be used as a valid source of *NO2 only when the latter is effectively scavenged by an appropriate reductant; and (iii) the formation of peroxynitrite cannot be efficiently inhibited by PTIOs even under relatively low fluxes of *NO and O2-* and millimolar levels of PTIOs.     

Bacterium-based NO2- biosensor for environmental applications

A sensitive NO2- biosensor that is based on bacterial reduction of NO2- to N2O and subsequent detection of the N2O by a built-in electrochemical N2O sensor was developed. Four different denitrifying organisms lacking NO3- reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO2- tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO3- and N2O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N2O reductase. The macroscale biosensors constructed exhibited a linear NO2- response at concentrations up to 1 to 2 mM. The detection limit was around 1 microM NO2-, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO2-, and interference was observed only with NH2OH, NO, N2O, and H2S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO2- biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO2- biosensor were constructed and used to measure NO2- profiles in marine sediment.     

Selective liberation of NO from S-nitrosocysteine with potassium thiocyanate, as monitored by an amperometric sensor

S-Nitrosocysteine (CysNO) releases either NO (in the presence of divalent cations) or NO+ (in the presence of chelating agents). NO+ is then transferred to peptides or protein SH groups to form high-mass nitrosothiols. The aim of this work was the development of a specific reaction between thiocyanate (SCN-) and CysNO. This reaction selectively liberates NO from CysNO in the presence of high-mass nitrosothiols. Free NO is measured with an amperometric sensor. We examine with this system the transnitrosylation reaction between CysNO and BSA at low molecular ratios and could assay nitrites, SNO-BSA, and CysNO in the incubation mixture without any preliminary purification steps.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N(2)O-Producing Denitrifier to Convert NO(3) or NO(3) to N(2)O

A more sensitive analytical method for NO(3) was developed based on the conversion of NO(3) to N(2)O by a denitrifier that could not reduce N(2)O further. The improved detectability resulted from the high sensitivity of the Ni electron capture gas chromatographic detector for N(2)O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO(3) to N(2)O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 mug/liter) of NO(3) N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO(3) concentrations of <2 ppb of NO(3) N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of N in NO(3). This method avoids the incomplete reduction and contamination of the NO(3) -N by the NH(4) and N(2) pools which can occur by the conventional method of NO(3) analysis. N(2)O-producing denitrifier strains were also used to measure the apparent K(m) values for NO(3) use by these organisms. Analysis of N(2)O production by use of a progress curve yielded K(m) values of 1.7 and 1.8 muM NO(3) for the two denitrifier strains studied.     

Reevaluation of the Griess method for determining NO/NO2- in aqueous and protein-containing samples.

Nitric oxide (NO) is an important intracellular and extracellular signal substance. Nitrite is one product of the oxidative metabolism of NO. The purpose of this study was to establish a simple method of determining nitrite (NO2-) to provide a means of estimating the endogenous formation of NO or NO2-. A flow injection analysis (FIA) based on the Griess reaction was developed for this purpose. Using a standard additive method, it is possible to eliminate matrix effects such as those that can occur in samples containing protein. This measuring method is suitable for measurements in effluates or protein-rich cellular supernatants. The sensitivity of the method is 2 nmol/L for samples in aqueous phases and 8 nmol/L for protein-containing phases. The two-point discrimination is 2 nmol/L. A linear correlation between nitrite and signal level can be demonstrated over a range of 0.002-5 micromol/L. Reproducibility, including sample preparation and analysis, can be specified with a coefficient of variation (C.V.) of 6.7%. Day-to-day variability for identical samples 0.8% (C.V.). This study presents examples of the application of this method (measurements in blood samples and in isolated perfused hearts) and compares them to established methods of measuring NO and NO2. We found the FIA method to be equally sensitive as NO measurement by means of oxyhemoglobin assay. The FIA method is seven times more sensitive than HPLC methods, and its design is significantly simpler. Compared to the traditional Griess method, its sensitivity is higher by a factor of 500. With its high sensitivity, high reproducibility, and its unsurpassed low susceptibility to interference, this method of analysis provides a means of reliably determining nitrite concentration as a marker of NO formation in various matrices. Therefore, it can be a valuable instrument in experimental and clinical studies to determine the physiologic and pathophysiologic relevance of NO.     

开放式空气CO2浓度增高试验中的NO和NO2地-气交换观测研究

介绍了农田FACE(free airCO2 enrichment)试验中的NO和NO2 地 气交换观测方法 ,即静态暗箱采样—NO和NO2 化学发光分析法 ,并对观测结果进行了分析讨论 .此观测方法简单、易于操作 ,并可获得可靠的NO和NO2 净交换通量观测结果 .在稻麦轮作农田的旱地阶段 ,无论FACE还是对照处理 ,NO主要表现为地面净排放 ,NO2 主要表现为地面净吸收 .逐日的NO净排放不依赖于土壤温度 ,但却与土壤含水量呈线性负相关 (R2 =0 .82 ,P <0 .0 0 1) .NO2 净吸收具有明显的季节变化特征 ,逐日的净吸收通量随土壤温度和土壤含水量的变化可分别用抛物线方程拟合 (温度 :R2 =0 .74 ,P <0 .0 0 1;含水量 :R2 =0 .6 9,P <0 .0 0 1) .大气CO2 浓度升高 2 0 0± 4 0 μmol·mol-1使NO净排放减弱 19% (t 检验P =0 .0 96 ) ,NO2 净吸收减弱 10 % (t 检验P =0 .2 6 ) ,这主要是植物生长受到促进的缘故 .     

Quantitative measurements of NO reaction kinetics with a Clark-type electrode.

Nitric oxide (NO) plays important physiological roles in the body. Knowledge regarding the kinetics of NO catabolism is important for understanding the biological functions of NO. Clark-type NO electrodes have been frequently employed in measuring the kinetics of NO reactions; however, the slow response time of these electrodes can cause measurement errors and limit the application of the electrode in measurements of fast NO reactions. In this study, a simplified diffusion model is given for describing the response process of the NO electrode to the change of NO concentration. The least-square method is used in fitting the currents calculated from the diffusion equation to the experimental curves for determining the diffusion parameters and rate constants. The calculated currents are in excellent accordance with the experimental curves for different NO reaction kinetics. It has been demonstrated that when using an NO electrode with a response time of approximately 6 s to measure fast NO reactions with a half-life of approximately 1s, the response currents of the electrode have large differences compared to the curve of actual NO concentration in the solution; however, the rate constant of NO decay can still be accurately determined by computer simulations with the simplified diffusion model. Theoretical analysis shows that an NO electrode with a response time of 6 s (D/L2=0.06 s-1) and the lowest detection limit of 1 nM NO can be used in measuring kinetics of extremely rapid NO reactions with a half-life below 10 ms.     

Clinical and biological investigation of NO

Furchgott et al. demonstrated in 1980 that relaxation of arterial smooth muscle cells in response to acetylcholine is dependent on the integrity of endothelium. They named the factor responsible of this intercellular relationship EDRF (Endothelium Derived Relaxing Factor), which was identified 7 years latter as nitric oxide (NO), a free radical gas. In vessels, NO is generated locally by the endothelial NO synthase and its effect is mainly paracrine (relaxation of the underlying smooth muscle cells, and inhibition of platelet aggregation). The in vivo half-life of NO is short, and the assessment of its production is thus difficult. Invasive and non invasive techniques are now available to explore the variations of arterial diameter or flow. Furchgott's pioneering work anticipated the whole pathophysiology of endothelial-dependent relaxation. Indeed, numerous diseases, in particular atherosclerosis, are accompanied by abnormalities of endothelial-dependent vasodilation ("endothelial dysfunction"). Whereas acetylcholine (or serotonin) infused in a normal artery elicits a vasodilation, in contrast, it promotes a vasoconstriction in an atheromatous artery, as a consequence of a decrease in NO bioavailability. This defect in NO favors arterial spasm, interaction between platelets and arterial wall and thrombosis, and thus probably cardiovascular events. NO cannot be measured directly in humans, except in exhaled NO. In vivo, NO is rapidly oxidized in nitrite (NO2-) and in nitrate (NO3-), the summation being NOx. We shall detail the limitations of this measurement as a biochemical index of NO production from "endothelial" origin.     

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.     

Determination of N Abundance in Nanogram Pools of NO(3) and NO(2) by Denitrification Bioassay and Mass Spectrometry

Suspensions of two strains of Pseudomonas aeruginosa (ON12 and ON12-1) were used to reduce NO(3) and NO(2), respectively, to N(2)O. The evolved N(2)O was quantified by gas chromatography with electron capture detection, and the N abundance was determined by mass spectrometry with a special inlet system and triple-collector detection. Sample gas containing unknown N(2)O pools as small as 0.5 ng of N was analyzed by use of a spike technique, in which a reference gas of N(2)O of natural N abundance was added to obtain enough total N for the mass spectrometer. In NO(3) or NO(2) pools, the N abundance could be determined in samples as small as approximately 3.5 ng of N. No cross-contamination took place between the NO(3) and NO(2) pools. The excellent separation of NO(3) and NO(2) pools, small sample size required, and low contamination risk during N(2)O analysis offer great advantages in isotope studies of inorganic N transformations by, e.g., nitrifying or denitrifying bacteria in the environment.     

Involvement of phospholipid, biomembrane integrity, and NO peroxidase activity in the NO catabolism by cytochrome c oxidase

The physiological regulation of mitochondrial respiration by NO has been reported to result from the reversible binding of NO to the two-electron reduced binuclear center (Fe(2+)(a3)-Cu(1+)(B)) of cytochrome c oxidase (CcO). Although the role of CcO and its derived catalytic intermediates in the catabolism of NO has been documented, little has been established for the enzyme in its fully oxidized state (Fe(3+)(a3)-Cu(2+)(B)). We report: (1) CcO, in its fully oxidized state, represents the major component of the mitochondrial electron transport chain for NO consumption as controlled by the binding of NO to its binuclear center. Phospholipid enhances NO consumption by fully oxidized CcO, whereas the consumption of NO is slowed down by membrane structure and membrane potential when CcO is embedded in the phospholipid bilayer. (2) In the presence of H(2)O(2), CcO was shown to serve as a mitochondria-derived NO peroxidase. A CcO-derived protein radical intermediate was induced and involved in the modulation of NO catabolism.     

Pulmonary NO uptake in interstitial lung disease

Because lung nitric oxide (NO) diffusing capacity (DL) represents alveolar-capillary gas diffusion, we queried as to whether disturbances of pulmonary gas exchange in interstitial lung disease (ILD) are appropriately reflected by using NO. In this pilot study, we applied the (15)N-labeled stable isotope (15)NO (relative abundance 0.37% of total NO) in order to ignore the endogenous NO production. In 10 ILD-outpatients, we measured DL (15)NO by performing the single-breath method. Lung function parameters as well as arterial oxygen partial pressure (PaO(2)) were also tested. Values of DL (15)NO ranged within 50-151 ml (15)NO/(mmHg min). Ratios of DL (15)NO/reference were between 43 and 108% of predicted data as taken from our previous work on healthy volunteers [Eur. J. Physiol. 446 (2003) 256]. We found a significant reduction of DL (15)NO/reference in five patients. Additionally, values of PaO(2) were significantly correlated to ratios of DL (15)NO/reference (adjusted R2 +/-SEE=0.407+/-8.051). In conclusion, (15)NO represents an appropriate indicator gas for reflecting an ILD-induced impairment of alveolar-capillary gas exchange.     

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ

At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.     

Use of NO donors in biological systems

Owing to the increased interest in the biological roles of nitric oxide (NO) the use of NO donors is a desired method of delivering NO to the tissues of interest. This article gives an overview of the most commonly used classes of NO donors and their biotranstformation to release NO. A major consideration when choosing an NO donor is the preparation and handling of the compounds. A method has been outlined for the preparation of S-nitrosothiols which eliminates the problem of the overall instability of these compounds both as a solid and in solution. The main aim of this article is to outline the methods used in assessing the ability of NO donors to elicit a biological response in vitro in particular relaxation of vascular smooth muscle and inhibition of platelet aggregation. In addition a method is described for assessing the toxicological potential of NO donors in vitro.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.     

Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging

The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.