Maximin principle, emotional aversion, and integrative judgment in the NIMBY context, including social dilemma and moral dilemma: The roles of the amygdala, angular gyrus, and ventromedial prefrontal cortex.

Public facilities that have NIMBY (not in my backyard) structure involve both a social dilemma, in which individuals' decisions to prevent the worst outcomes for themselves undermine the public interest, and a moral dilemma focused on the majority versus the minority. This study examined the cognitive-neural processes in judging whether to prioritize the site residents or the citizenry as a whole within the context of NIMBY. Our ROIs were the right angular gyrus being related to concern about the worst possible outcomes for others and oneself, the amygdala associating with emotional aversion to prioritizing the majority, and the vmPFC, which integrates the aversion into "all things considered" judgments. As a result of comparing ingroup conditions for which a NIMBY facility may make participants worst-off position and outgroup conditions for which this possibility is denied, the right angular gyrus was activated in both conditions. The amygdala was activated only in the ingroup, and the vmPFC exhibited a stronger tendency in the ingroup. We concluded that the cognitive-neural processes in judgments on NIMBY facilities are common to both decision-making to avoid the worst-off position for others and for oneself and moral judgments between the majority and the minority.

Dynamics and stabilization mechanism of mitochondrial cristae morphofunction associated with turgor-driven cardiolipin biosynthesis under salt stress conditions.

Maintaining energy production efficiency is of vital importance to plants growing under changing environments. Cardiolipin localized in the inner mitochondrial membrane plays various important roles in mitochondrial function and its activity, although the regulation of mitochondrial morphology to various stress conditions remains obscure, particularly in the context of changes in cellular water relations and metabolisms. By combining single-cell metabolomics with transmission electron microscopy, we have investigated the adaptation mechanism in tomato trichome stalk cells at moderate salt stress to determine the kinetics of cellular parameters and metabolisms. We have found that turgor loss occurred just after the stress conditions, followed by the contrasting volumetric changes in mitochondria and cells, the accumulation of TCA cycle-related metabolites at osmotic adjustment, and a temporal increase in cardiolipin concentration, resulting in a reversible topological modification in the tubulo-vesicular cristae. Because all of these cellular events were dynamically observed in the same single-cells without causing any disturbance for redox states and cytoplasmic streaming, we conclude that turgor pressure might play a regulatory role in the mitochondrial morphological switch throughout the temporal activation of cardiolipin biosynthesis, which sustains mitochondrial respiration and energy conversion even under the salt stress conditions.

Direct evidence for dynamics of cell heterogeneity in watercored apples: turgor-associated metabolic modifications and within-fruit water potential gradient unveiled by single-cell analyses.

Watercore is a physiological disorder in apple (Malus × domestica Borkh.) fruits that appears as water-soaked tissues adjacent to the vascular core, although there is little information on what exactly occurs at cell level in the watercored apples, particularly from the viewpoint of cell water relations. By combining picolitre pressure-probe electrospray-ionization mass spectrometry (picoPPESI-MS) with freezing point osmometry and vapor pressure osmometry, changes in cell water status and metabolisms were spatially assayed in the same fruit. In the watercored fruit, total soluble solid was lower in the watercore region than the normal outer parenchyma region, but there was no spatial difference in the osmotic potentials determined with freezing point osmometry. Importantly, a disagreement between the osmotic potentials determined with two methods has been observed in the watercore region, indicating the presence of significant volatile compounds in the cellular fluids collected. In the watercored fruit, cell turgor varied across flesh, and a steeper water potential gradient has been established from the normal outer parenchyma region to the watercore region, retaining the potential to transport water to the watercore region. Site-specific analysis using picoPPESI-MS revealed that together with a reduction in turgor, remarkable metabolic modifications through fermentation have occurred at the border, inducing greater production of watercore-related volatile compounds, such as alcohols and esters, compared with other regions. Because alcohols including ethanol have low reflection coefficients, it is very likely that these molecules would have rapidly penetrated membranes to accumulate in apoplast to fill. In addition to the water potential gradient detected here, this would physically contribute to the appearance with high tissue transparency and changes in colour differences. Therefore, it is concluded that these spatial changes in cell water relations are closely associated with watercore symptoms as well as with metabolic alterations.

Endosperm cell size reduction caused by osmotic adjustment during nighttime warming in rice.

High night temperature (HNT) often reduces yield in field crops. In rice, HNT during the ripening stage diminishes endosperm cell size, resulting in a considerable reduction in final kernel weight; however, little is known about the underlying mechanisms at cell level. In this study, we performed picolitre pressure-probe-electrospray-ionization mass spectrometry to directly determine metabolites in growing inner endosperm cells of intact seeds produced under HNT conditions, combining with 13C feeding and water status measurements including in situ turgor assay. Microscopic observation in the inner zone suggested that approximately 24.2% of decrease in cell expansion rate occurred under HNT at early ripening stage, leading to a reduction in cell volume. It has been shown that HNT-treated plants were subjected to mild shoot water deficit at night and endosperm cell turgor was sustained by a decline in osmotic potential. Cell metabolomics also suggests that active solute accumulation was caused by a partial inhibition of wall and starch biosynthesis under HNT conditions. Because metabolites were detected in the single cells, it is concluded that a partial arrest of cell expansion observed in the inner endosperms was caused by osmotic adjustment at mild water deficit during HNT conditions.

Probe Electrospray Ionization (PESI) and Its Modified Versions: Dipping PESI (dPESI), Sheath-Flow PESI (sfPESI) and Adjustable sfPESI (ad-sfPESI).

In 2007, probe electrospray ionization/mass spectrometry (PESI/MS) was developed. In this technique, the needle is moved down along a vertical axis and the tip of the needle touched to the sample. After capturing the sample at the needle tip, the needle is then moved up and a high voltage is applied to the needle at the highest position to generate electrospray. Due to the discontinuous sampling followed by the generation of spontaneous electrospray, sequential and exhaustive electrospray takes place depending on the surface activity of the analytes. As modified versions of PESI, dipping PESI (dPESI), sheath-flow PESI (sfPESI) and adjustable sfPESI (ad-sfPESI) have been developed. These methods are complementary to each other and they can be applicable to surface and bulk analysis of various biological samples. In this article, the characteristics of these methods and their applications to real samples will be reviewed.

Robotic sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS): development of a touch sensor for samples in a multiwell plastic plate.

In the previous work, sheath-flow probe electrospray ionization (sfPESI) equipped with a touch sensor was developed for conducting samples. In this work, a capacitiance-sensitive touch sensor that can be applicable to samples prepared in a nonconducting plastic multiwell plate was developed. The radiofrequency with 5 kHz and 4.5 Vpp was applied to the metal substrate on which the plastic plate was placed. The probe tip stopped at the position where it touched the surface of the liquid solution prepared in the plastic multiwell plate by detecting the displacement current flowing through the capacitance of the circuit. By coupling a nondisposable sfPESI probe with a table-top 3-axis robot, consecutive analysis of peptides, proteins, drugs, and real samples was performed. The carry-over by the consecutive analyses was suppressed to minimal by cleansing the probe tip using the solvent of water/methanol/acetonitrile (1/1/1).

On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice.

Although a loss of healthy pollen grains induced by metabolic heat responses has been indicated to be a major cause of heat-induced spikelet sterility under global climate change, to date detailed information at pollen level has been lacking due to the technical limitations. In this study, we used picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS) to directly determine the metabolites in heat-treated single mature pollen grains in two cultivars, heat-tolerant cultivar, N22 and heat-sensitive cultivar, Koshihikari. Heat-induced spikelet fertility in N22 and Koshihikari was 90.0% and 46.8%, respectively. While no treatment difference in in vitro pollen viability was observed in each cultivar, contrasting varietal differences in phosphatidylinositol (PI)(34:3) have been detected in mature pollen, together with other 106 metabolites. Greater PI content was detected in N22 pollen regardless of the treatment, but not for Koshihikari pollen. In contrast, there was little detection for phosphoinositide in the single mature pollen grains in both cultivars. Our findings indicate that picoPPESI-MS analysis can efficiently identify the metabolites in intact single pollen. Since PI is a precursor of phosphoinositide that induces multiple signaling for pollen germination and tube growth, the active synthesis of PI(34:3) prior to germination may be closely associated with sustaining spikelet fertility even at high temperatures.

Point Analysis of Foods by Sheath-Flow Probe Electrospray Ionization/Mass Spectrometry (sfPESI/MS) Coupled with a Touch Sensor.

For quick, noninvasive, and high-sensitivity surface analysis of foods and agricultural products, a touch sensor was developed and applied to sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS). Upon making contact with the sample, the probe stopped by detecting the current flowing through the circuit and analytes on the sample surface were extracted in the solvent preloaded in the plastic capillary. By lifting up the probe to the default position, an electrospray ionization mass spectrum of the sample was obtained. By scanning the sample stage using a programming tool, a point analysis of targeted positions of biological samples with a spot diameter of ≤0.3 mm was achieved. It took less than 10 s for one sample spot. This method was applied to various plants and animal tissues.

Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy.

BACKGROUND: Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins. RESULTS: Here, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins. CONCLUSIONS: We present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes.

Component Profiling in Agricultural Applications Using an Adjustable Acupuncture Needle for Sheath-Flow Probe Electrospray Ionization/Mass Spectrometry.

In previous work, probe electrospray ionization/mass spectrometry (PESI/MS) and sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS) were reported for the rapid and minimally invasive analysis of food. In this work, a modified version of sfPESI will be reported. The sample surface was pricked with an acupuncture needle inserted in the sfPESI probe that protruded from the terminus of the tip by 5 mm. The invasion depth of the needle into the sample was ∼1 mm. After sampling, the needle was retracted into the solvent-preloaded capillary with a protrusion length of 0.1-0.2 mm from the tip. A mass spectrum of the sample captured on the needle was obtained by applying a high voltage to the needle. This method could be applicable to profiling analyses of plants with the epicuticular wax covering on the surfaces that are difficult to analyze by sf-PESI. The on-site mass spectrometric analysis for a growing apricot in the field was performed to monitor the developing stage of the fruit.

Multiple strategies for heat adaptation to prevent chalkiness in the rice endosperm.

Heat-induced chalkiness of rice grains is a major concern for rice production, particularly with respect to climate change. Although the formation of chalkiness in the endosperm is suppressed by nitrogen, little is known about the cell-specific dynamics of this process. Here, using picolitre pressure-probe electrospray-ionization mass spectrometry together with transmission electron microscopy and turgor measurements, we examine heat-induced chalkiness in single endosperm cells of intact rice seeds produced under controlled environmental conditions. Exposure to heat stress decreased turgor pressure and increased the cytosolic accumulation of sugars, glutathione, and amino acids, particularly cysteine. Heat stress also led to a significant enlargement of the protein storage vacuoles but with little accumulation of storage proteins. Crucially, this heat-induced partial arrest of amyloplast development led to formation of chalkiness. Whilst increased nitrogen availability also resulted in increased accumulation of amino acids, there was no decrease in turgor pressure. The heat-induced accumulation of cysteine and glutathione was much less marked in the presence of nitrogen, and storage proteins were produced without chalkiness. These data provide important information on the cell dynamics of heat acclimation that underpin the formation of chalkiness in the rice endosperm. We conclude that rice seeds employ multiple strategies to mitigate the adverse effects of heat stress in a manner that is dependent on nitrogen availability, and that the regulation of protein synthesis may play a crucial role in optimizing organelle compartmentation during heat adaption.

Electrospray Generated from the Tip-Sealed Fine Glass Capillary Inserted with an Acupuncture Needle Electrode.

In electrospray, excess charges are supplied to a sample solution by the occurrence of electrochemical reactions. Recently, different versions of electrospray, e.g., dielectric barrier electrospray ionization, inductive desorption electrospray ionization, and electrostatic-ionization driven by dielectric polarization, have been reported in which the sample solution was not in direct contact with the metal electrode but separated by dielectric materials. The objective of the current work is to elucidate the mechanism of dielectric barrier electrospray. A sealed borosilicate glass capillary inserted with a fine acupuncture needle was used as a probe. A sample solution (~ 400 nL) was captured on the glass capillary tip and a positive high voltage (HV) pulse (+ 4.5 kV) was applied to the internal metal electrode. Mass spectra were measured as a function of the HV pulse width from µs to 10 s. Ions started to be detected with the pulse width of ~ 5 ms. The ion intensities increased slowly with time and reached a plateau in a few seconds. The charge distribution of cytochrome c [M + nH]n+ shifted to higher n values from a few ms to seconds. In addition to cone-jet mode normal electrospray that lasted until all the liquid sample was depleted from the glass tip, the polarization-induced electrospray ionization was observed at the early stage of the HV application. Graphical Abstract ᅟ.

Evidence for preservation of vacuolar compartments during foehn-induced chalky ring formation of Oryza sativa L.

MAIN CONCLUSION: Vacuolar compartments being sustained among the amyloplasts inadequately accumulated in rice endosperm cells are the main cause of chalky ring formation under dry wind conditions. Foehn-induced dry wind during the grain-filling stage induces shoot water deficit in rice (Oryza sativa L.) plants, which form a ring-shaped chalkiness in their endosperm that degrades milling quality and rice appearance. Air spaces formed in several inner cells cause significant transparency loss due to irregular light reflection. Although starch synthesis was suggested to be retarded by osmotic adjustment at foehn-induced moderately low water potential, the source of these air spaces remains unknown. We hypothesised that the preservation of vacuoles accompanied by a temporary reduction in starch biosynthesis in the inner cells leads to the chalky ring formation. Panicle water status measurement, light and transmission electron microscopic (TEM) observations, and an absolute qPCR analysis were conducted. Most starch synthesis-related genes exhibited temporarily reduced expression in the inner zone in accordance with the decrease in panicle water status. TEM observations provided evidence that vacuolar compartments remained among the loosely packed starch granules in the inner endosperm cells, where a chalky ring appeared after kernel dehydration. Taken together, we propose that vacuolar compartments sustained among the amyloplasts inadequately accumulated in rice endosperm cells and caused air space formation that leads to ring-shaped chalkiness under dry wind conditions.

Dipping probe electrospray ionization/mass spectrometry for direct on-site and low-invasive food analysis.

Rapid, direct, on-site and noninvasive food analysis is strongly needed for quality control of food. To satisfy this demand, the technique of dipping probe electrospray ionization/mass spectrometry (dPESI/MS) was developed. The sample surface was pricked with a fine acupuncture needle and a sample of ∼200 pL was captured at the needle tip. After drying the sample, the needle tip was dipped into the solvent for ∼50 ms and was moved upward. A high-voltage was applied to the needle to generate electrospray when the needle reached the highest position, and mass spectra were measured with a time-of-flight mass spectrometer. For evaluation of the method, the technique was used to analyze foods such as vegetables, salmon flesh, cow's milk, yogurt, and soy-bean milk. The detected major ions for cow's milk and yogurt were [(Lac)n + Ca]2+ with n = 1-6 (where (Lac) is lactose), indicating that Ca2+ is tightly bound by Lac molecules.

Remote sampling mass spectrometry for dry samples: Sheath-flow probe electrospray ionization (PESI) using a gel-loading tip inserted with an acupuncture needle.

RATIONALE: Probe electrospray ionization (PESI) is only applicable to liquid or wet samples. In this study, a sheath-flow PESI method for remote sampling mass spectrometry that can be applied to dry samples was developed. METHODS: An acupuncture needle (0.12 mm outer diameter, 700 nm tip diameter) was inserted into a gel-loading tip with a 0.1 mm protrusion out of the tip. Analytes were extracted by filling the latter tip with solvent and softly touching the sample surface for a short time (<1 s). A high voltage was applied to the acupuncture needle, and mass spectra of analytes were obtained by self-aspirating electrospray. RESULTS: Dry samples, such as lines of ballpoint pen ink on paper, pharmaceutical tablets, instant coffee, brown rice, and narcotics, gave strong ion signals. The sample carryover was negligible. The sequential electrospray was observed to be similar to conventional PESI. The limits of detection (LODs) for morphine and rhodamine B were found to be of the order of picograms. CONCLUSIONS: Because of its simplicity and versatility, sheath-flow PESI is a promising technique for on-site and nondestructive profile analysis of dry samples with bulky and complicated shapes, with a spatial resolution of ~0.3 mm.

Turgor-responsive starch phosphorylation in Oryza sativa stems: A primary event of starch degradation associated with grain-filling ability.

Grain filling ability is mainly affected by the translocation of carbohydrates generated from temporarily stored stem starch in most field crops including rice (Oryza sativa L.). The partitioning of non-structural stem carbohydrates has been recognized as an important trait for raising the yield ceiling, yet we still do not fully understand how carbohydrate partitioning occurs in the stems. In this study, two rice subspecies that exhibit different patterns of non-structural stem carbohydrates partitioning, a japonica-dominant cultivar, Momiroman, and an indica-dominant cultivar, Hokuriku 193, were used as the model system to study the relationship between turgor pressure and metabolic regulation of non-structural stem carbohydrates, by combining the water status measurement with gene expression analysis and a dynamic prefixed 13C tracer analysis using a mass spectrometer. Here, we report a clear varietal difference in turgor-associated starch phosphorylation occurred at the initiation of non-structural carbohydrate partitioning. The data indicated that starch degradation in Hokuriku 193 stems occurred at full-heading, 5 days earlier than in Momiroman, contributing to greater sink filling. Gene expression analysis revealed that expression pattern of the gene encoding α-glucan, water dikinase (GWD1) was similar between two varieties, and the maximum expression level in Hokuriku 193, reached at full heading (4 DAH), was greater than in Momiroman, leading to an earlier increase in a series of amylase-related gene expression in Hokuriku 193. In both varieties, peaks in turgor pressure preceded the increases in GWD1 expression, and changes in GWD1 expression was correlated with turgor pressure. Additionally, a threshold is likely to exist for GWD1 expression to facilitate starch degradation. Taken together, these results raise the possibility that turgor-associated starch phosphorylation in cells is responsible for the metabolism that leads to starch degradation. Because the two cultivars exhibited remarkable varietal differences in the pattern of non-structural carbohydrate partitioning, our findings propose that the observed difference in grain-filling ability originated from turgor-associated regulation of starch phosphorylation in stem parenchyma cells. Further understanding of the molecular mechanism of turgor-regulation may provide a new selection criterion for breaking the yield barriers in crop production.

Non-proximate mass spectrometry using a heated 1-m long PTFE tube and an air-tight APCI ion source.

Direct and rapid trace-level gas analysis is highly needed in various fields such as safety and security, quality control, food analysis, and forensic medicine. In many cases, the real samples are bulky and are not accessible to the space-limited ion source of the mass spectrometer. In order to circumvent this problem, we developed an airtight atmospheric-pressure chemical ionization (APCI) ion source equipped with a flexible 1-m-long, 2-mm-i.d. PTFE sniffing tube. The ambient air bearing sample gas was sucked into the heated PTFE tube (130 °C) and was transported to the air-tight ion source without using any extra pumping system or a Venturi device. Analytes were ionized by an ac corona discharge located at 1.5 mm from the inlet of the mass spectrometer. By using the airtight ion source, all the ionized gas in the ion source was introduced into the vacuum of the mass spectrometer via only the evacuation of the mass spectrometer (1.6 l min-1). Sub-pg limits of detection were obtained for carbaryl and trinitrotoluene. Owing to its flexibility and high sensitivity, the sniffing tube coupled with a mass spectrometer can be used as the stethoscope for the high-sensitive gas analysis. The experimental results obtained for drugs, hydrogen peroxide and small alkanes were discussed by DFT calculations.

Desorption in Mass Spectrometry.

In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed.

Mass spectrometric monitoring of oxidation of aliphatic C6-C8 hydrocarbons and ethanol in low pressure oxygen and air plasmas.

Experimental and theoretical studies on the oxidation of saturated hydrocarbons (n-hexane, cyclohexane, n-heptane, n-octane and isooctane) and ethanol in 28 Torr O2 or air plasma generated by a hollow cathode discharge ion source were made. Ions corresponding to [M + 15]+ and [M + 13]+ in addition to [M - H]+ and [M - 3H]+ were detected as major ions where M is the sample molecule. The ions [M + 15]+ and [M + 13]+ were assigned as oxidation products, [M - H + O]+ and [M - 3H + O]+ , respectively. By the tandem mass spectrometry analysis of [M - H + O]+ and [M - 3H + O]+ , H2 O, olefins (and/or cycloalkanes) and oxygen-containing compounds were eliminated from these ions. Ozone as one of the terminal products in the O2 plasma was postulated as the oxidizing reagent. As an example, the reactions of C6 H14+• with O2 and of C6 H13+ (CH3 CH2 CH+ CH2 CH2 CH3 ) with ozone were examined by density functional theory calculations. Nucleophilic interaction of ozone with C6 H13+ leads to the formation of protonated ketone, CH3 CH2 C(=OH+ )CH2 CH2 CH3 . In air plasma, [M - H + O]+ became predominant over carbocations, [M - H]+ and [M - 3H]+ . For ethanol, the protonated acetic acid CH3 C(OH)2+ (m/z 61.03) was formed as the oxidation product. The peaks at m/z 75.04 and 75.08 are assigned as protonated ethyl formate and protonated diethyl ether, respectively, and that at m/z 89.06 as protonated ethyl acetate. Copyright © 2016 John Wiley & Sons, Ltd.

Nitrogen incorporation in saturated aliphatic C6-C8 hydrocarbons and ethanol in low-pressure nitrogen plasma generated by a hollow cathode discharge ion source.

Ion/molecule reactions of saturated hydrocarbons (n-hexane, cyclohexane, n-heptane, n-octane and isooctane) in 28-Torr N2 plasma generated by a hollow cathode discharge ion source were investigated using an Orbitrap mass spectrometer. It was found that the ions with [M+14](+) were observed as the major ions (M: sample molecule). The exact mass analysis revealed that the ions are nitrogenated molecules, [M+N](+) formed by the reactions of N3 (+) with M. The reaction, N3 (+) + M → [M+N](+) + N2 , were examined by the density functional theory calculations. It was found that N3 (+) abstracts the H atom from hydrocarbon molecules leading to the formation of protonated imines in the forms of R'R″CNH2 (+) (i.e. C-H bond nitrogenation). This result is in accord with the fact that elimination of NH3 is the major channel for MS/MS of [M+N](+) . That is, nitrogen is incorporated in the C-H bonds of saturated hydrocarbons. No nitrogenation was observed for benzene and acetone, which was ascribed to the formation of stable charge-transfer complexes benzene⋅⋅⋅⋅N3 (+) and acetone⋅⋅⋅⋅N3 (+) revealed by density functional theory calculations. Copyright © 2016 John Wiley & Sons, Ltd.