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Nitrate ( NO 3 − ) has properties that can improve muscle function, leading to improvements in metabolic cost of exercise as well as enhance force production. Gymnastics is a whole-body sport, involving events that demand a high level of strength and fatigue resistance. However, the effect of NO 3 − supplementation on both upper- and lower-body function in gymnasts is unknown. This study examined the effect of acute beetroot juice (BRJ) supplementation on isokinetic strength and endurance of the upper- and lower-body in highly trained international-level male gymnasts. In a double-blind, randomized crossover design, 10 international-level male gymnasts completed two acute supplementation periods, consuming either 2 × 70 ml NO 3 − -rich (∼12.8 mmol/L of NO 3 − ) or NO 3 − -depleted (PLA) BRJ. Maximal strength of the upper-leg and upper-arm at 60°/s, 120°/s, 180°/s, and 300°/s, and muscular endurance (50 repeated isokinetic contractions at 180°/s) were assessed. Plasma NO 3 − (BRJ: 663 ± 164 μM, PLA: 89 ± 48 μM) and nitrite ( NO 2 − ) concentrations (BRJ: 410 ± 137 nmol/L, PLA: 125 ± 36 nmol/L) were elevated following BRJ compared to PLA (both p <.001). Maximal strength of knee and elbow extensors and flexors did not differ between supplements (p >.05 for all velocities). Similarly, fatigue index of knee and elbow extension and flexion was not different between supplements (all p >.05). Acute BRJ supplementation, containing ∼12.8 mmol/L of NO 3 − , increased plasma NO 3 − and NO 2 − concentrations, but did not enhance isokinetic strength or fatigue resistance of either upper or lower extremities in international-level male gymnasts. [ABSTRACT FROM AUTHOR]

We investigated collision induced transitions in the (0, 0) band of the A2Σ+–X2Π electronic transition of nitric oxide (NO) using two-color polarization spectroscopy (TCPS). Two sets of TCPS spectra for 1% NO, diluted in different buffer gases at 295 K and 1 atm, were obtained with the pump beam tuned to the R11(11.5) and OP12(1.5) transitions. The buffer gases were He, Ar, and N2. The probe was scanned while the pump beam was tuned to the line center. Theoretical TCPS spectra, calculated by solving the density matrix formulation of the time-dependent Schrödinger wave equation, were compared with the experimental spectra. A collision model based on the modified exponential-gap law was used to model the rotational level-to-rotational level collision dynamics. A model for collisional transfer from an initial to a final Zeeman state was developed based on the difference in cosine of the rotational quantum number J projection angle with the z-axis for the two Zeeman states. Rotational energy transfer rates and Zeeman state collisional dynamics were varied to obtain good agreement between theory and experiment for the two different TCPS pump transitions and for the three different buffer gases. One key finding, in agreement with quasi-classical trajectory calculations, is that the spin-rotation changing transition rate in the A2Σ+ level of NO is almost zero for rotational quantum numbers ≥8. It was necessary to set this rate to near zero to obtain agreement with the TCPS spectra. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) molecules in pulsed supersonic beams have been excited to long-lived Rydberg-Stark states in series converging to the lowest vibrational level in the ground electronic state of NO+ with rotational quantum numbers N+ = 2, 4, and 6. The molecules in these excited states were then guided, or decelerated and trapped in a chip-based Rydberg-Stark decelerator, and detected in situ by pulsed electric field ionization. Time constants, reflecting the decay of molecules in N+ = 2 Rydberg-Stark states, with principal quantum numbers n between 38 and 44, from the electrostatic traps were measured to be ∼ 300 μ s. Molecules in Rydberg-Stark states with N+ = 4 and 6, and the same range of values of n were too short-lived to be trapped, but their decay time constants could be determined from complementary sets of delayed pulsed electric field ionization measurements to be ∼100 and ∼25 μs, respectively. [ABSTRACT FROM AUTHOR]

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

We developed a high-content image-based screen that utilizes the pro-inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti-inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS-induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS-stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen-activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti-inflammatory drug leads.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

Statin therapy is a cornerstone in the treatment of systemic vascular diseases. However, statins have failed to translate as therapeutics for pulmonary vascular disease. Early pulmonary vascular disease in the setting of congenital heart disease (CHD) is characterized by endothelial dysfunction, which precedes the more advanced stages of vascular remodeling. These features make CHD an ideal cohort in which to re-evaluate the potential pulmonary vascular benefits of statins, with a focus on endothelial biology. However, it is critical that the full gamut of the pleiotropic effects of statins in the endothelium are uncovered. The purpose of this investigation was to evaluate the therapeutic potential of simvastatin for children with CHD and pulmonary over-circulation, and examine mechanisms of simvastatin action on the endothelium. Our data demonstrate that daily simvastatin treatment preserves endothelial function in our shunt lamb model of pulmonary over-circulation. Further, using pulmonary arterial endothelial cells (PAECs) isolated from Shunt and control lambs, we identified a new mechanism of statin action mediated by increased expression of the endogenous Akt1 inhibitor, C-terminal modifying protein (CTMP). Increases in CTMP were able to decrease the Akt1-mediated mitochondrial redistribution of endothelial nitric oxide synthase (eNOS) which correlated with increased enzymatic coupling, identified by increases in NO generation and decreases in NOS-derived superoxide. Together our data identify a new mechanism by which simvastatin enhances NO signaling in the pulmonary endothelium and identify CTMP as a potential therapeutic target to prevent the endothelial dysfunction that occurs in children born with CHD resulting in pulmonary over-circulation.

Nitric oxide (●NO) participates in many biological activities, including enhancing DNA radiosensitivity in ionizing radiation-based radiotherapy. To help understand the radiosensitization of ●NO, we report reaction dynamics between ●NO and the radical cations of guanine (a 9HG●+ conformer) and 9-methylguanine (9MG●+). On the basis of the formation of 9HG●+ and 9MG●+ in the gas phase and the collisions of the radical cations with ●NO in a guided-ion beam mass spectrometer, the charge transfer reactions of 9HG●+ and 9MG●+ with ●NO were examined. For both reactions, the kinetic energy-dependent product ion cross sections revealed a threshold energy that is 0.24 (or 0.37) eV above the 0 K product 9HG (or 9MG) + NO+ asymptote. To interrogate this abnormal threshold behavior, the reaction potential energy surface for [9MG + NO]+ was mapped out at closed-shell singlet, open-shell singlet, and triplet states using density functional and coupled cluster theories. The results showed that the charge transfer reaction requires the interaction of a triplet-state surface originating from a reactant-like precursor complex 3[9MG●+(↑)⋅(↑)●NO] with a closed-shell singlet-state surface evolving from a charge-transferred complex 1[9MG⋅NO+]. During the reaction, an electron is transferred from π∗(NO) to perpendicular π∗(9MG), which introduces a change in orbital angular momentum. The latter offsets the change in electron spin angular momentum and facilitates intersystem crossing. The reaction threshold in excess of the 0 K thermochemistry and the low charge-transfer efficiency are rationalized by the vibrational excitation in the product ion NO+ and the kinetic shift arising from a long-lived triplet intermediate. [ABSTRACT FROM AUTHOR]

[Display omitted] The challenge of mitigating pollution stemming from industrial exhaust emissions is a pressing issue in both academia and industry. This study presents the successful synthesis of nitrogen-defect-enriched graphite carbon nitride (g-C 3 N 4) using a two-step calcination technique. Furthermore, a g-C 3 N 4 -Au heterostructure was fabricated through the photo-deposited Au quantum dots (QDs). When subjected to visible light irradiation, this heterostructure exhibited robust nitric oxide (NO) photooxidation activity and stability. With its fluffy, porous structure and large surface area, the nitrogen-defect-enriched g-C 3 N 4 provides more active sites for photooxidation processes. The ability of g-C 3 N 4 to absorb visible light is enhanced by the local surface plasmon resonance (LSPR) effect of Au QDs. Additionally, the lifetime of photogenerated charge carriers is extended by the presence of N defects and Au, which effectively prevent photogenerated electron-hole pairs from recombining during the photooxidation process. Moreover, the oxidation pathway of NO was analyzed through In-situ Fourier transform infrared (FT-IR) spectroscopy and Density Functional Theory (DFT) calculation. Computational findings revealed that the introduction of Au QDs decreases the activation energy of the oxidation reaction, thereby facilitating its occurrence while diminishing the formation of intermediate products. As a result, NO is predominantly converted to nitrate (NO 3 −). This work unveils a novel approach to constructing semiconductor-cocatalyst heterostructures and elucidates their role in NO photooxidation. [ABSTRACT FROM AUTHOR]

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Background: Random-pattern skin flaps are commonly used to repair skin tissue defects in surgical tissue reconstruction. However, flap necrosis in the distal area due to ischemia injury is still challenging for its applications in plastic surgery. The complications of diabetes will further increase the risk of infection and necrosis. Methods: This study induced type 2 diabetes mellitus (T2DM) rats with a high-fat diet and STZ. The survival rate of the skin flap was observed by adding inorganic sodium nitrate to drinking water. Histology and immunohistochemistry were used to detect the damage to the skin flap. The nitrate content was measured by total nitric oxide and nitrate/nitrite parameter assay. Dihydroethidium and malondialdehyde (MDA) assays were used to value oxidative stress. Rat colon feces were collected for 16s rRNA gene sequence. Results: Our studies showed that nitrate administration leads to anti-obesity and anti-diabetic effects. Nitrate directly increased the survival area of skin flaps in diabetic rats and mean blood vessel density by enhancing angiogenesis, inhibiting apoptosis, and reducing oxidative stress. The 16s rRNA sequence revealed that nitrate may regulate the homeostasis of the gut microbiota and re-store energy metabolism. Conclusion: Dietary nitrate has been shown to maintain the homeostasis of oxidative stress and gut microbiota to promote flap survival in rats with T2DM. [ABSTRACT FROM AUTHOR]

Key points Sodium thiosulfate (STS) is gaining increasing attention in research for its potential therapeutic applications across a spectrum of disease processes beyond its current uses. However, the precise mechanisms of action remain incompletely understood. We investigated the efficacy of STS in treating hyperglycaemia‐induced pronephros damage in zebrafish to gain further insight into the underlying mechanisms. Hyperglycaemia was induced in zebrafish by suppressing the pdx1 transcription factor, which plays a crucial role in maintaining physiological pancreatic function. STS was administered by introducing it into the medium of zebrafish larvae. The pronephros structure was analysed at 48 h post‐fertilization. Metabolomic profiling and RNA sequencing were conducted on groups exposed to various experimental conditions. Our findings reveal a downregulation of nitric oxide (NO) signalling in zebrafish with a knocked‐down pdx1 gene, both metabolomically and transcriptionally. Notably, treatment with STS led to a compensatory upregulation of the NO signalling, ultimately resulting in the rescue of the pronephros structure. Our study provides compelling evidence that targeting NO metabolism by the administration of STS offers a promising strategy for addressing hyperglycaemia‐induced organ damage. These findings underscore the potential of STS as a promising therapeutic agent for diabetic complications and warrant further investigation of its clinical applications. Sodium thiosulfate (STS) is increasingly drawing attention in research for its potential therapeutic applications across a spectrum of disease processes. Here, we demonstrate that STS treatment rescues hyperglycaemia‐induced pronephros damage in zebrafish. We identified upregulation of nitric oxide signalling as the major driver behind STS‐mediated rescue. Our data suggest that STS offers a promising strategy for addressing hyperglycaemia‐induced organ damage, including diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca2+ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca2+ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) utilizes reactive oxygen species (ROS) for eradication of cancer cells. Its effectiveness is governed by the oxygen content, which is scarce in the hypoxic tumor microenvironment. We report herein two zinc(II) phthalocyanines substituted with two or four nitric oxide (NO)‐releasing moieties, namely ZnPc‐2NO and ZnPc‐4NO, which can suppress the mitochondrial respiration, thereby sparing more intracellular oxygen for PDT. Using HT29 human colorectal adenocarcinoma cells and A549 human lung carcinoma cells, we have demonstrated that both conjugates release NO upon interaction with the intracellular glutathione, which can reduce the cellular oxygen consumption rate and adenosine triphosphate generation and alter the mitochondrial membrane potential. They can also relieve the hypoxic status of cancer cells and decrease the expression of hypoxia‐inducible factor protein HIF‐1α. Upon light irradiation, both conjugates can generate ROS and induce cytotoxicity even under a hypoxic condition, overcoming the oxygen‐dependent nature of PDT. Interestingly, the photodynamic action of ZnPc‐2NO elicits the release of damage‐associated molecular patterns, inducing the maturation of dendritic cells and triggering an antitumor immune response. The immunogenic cell death caused by this oxygen‐economized PDT has been demonstrated through a series of in vitro and in vivo experiments. [ABSTRACT FROM AUTHOR]

C-Nitroso compounds (RNO, R = alkyl and aryl) are byproducts of drug metabolism and bind to heme proteins, and their heme-RNO adducts are isoelectronic to ferrous nitroxyl (NO−/HNO) complexes. Importantly, heme-HNO compounds are key intermediates in the reduction of NO to N2O and nitrite to ammonium in the nitrogen cycle. Ferrous heme-RNO complexes act as stable analogs of these species, potentially allowing for the investigation of the vibrational and electronic properties of unstable heme-HNO intermediates. In this paper, a series of six-coordinate ferrous heme-RNO complexes (where R = iPr and Ph) were prepared using the TPP2− and 3,5-Me-BAFP2− co-ligands, and tetrahydrofuran, pyridine, and 1-methylimidazole as the axial ligands (bound trans to RNO). These complexes were characterized using different spectroscopic methods and X-ray crystallography. The complex [Fe(TPP)(THF)(iPrNO)] was further utilized for nuclear resonance vibrational spectroscopy (NRVS), allowing for the detailed assignment of the Fe–N(R)O vibrations of a heme-RNO complex for the first time. The vibrational properties of these species were then correlated with those of their HNO analogs, using DFT calculations. Our studies support previous findings that RNO ligands in ferrous heme complexes do not elicit a significant trans effect. In addition, the complexes are air-stable, and do not show any reactivity of their RNO ligands towards NO. So although ferrous heme-RNO complexes are suitable structural and electronic models for their HNO analogs, they are unsuitable to model the reactivity of heme-HNO complexes. We further investigated the reaction of our heme-RNO complexes with different Lewis acids. Here, [Fe(TPP)(THF)(iPrNO)] was found to be unreactive towards Lewis acids. In contrast, [Fe(3,5-Me-BAFP)(iPrNO)2] is reactive towards all of the Lewis acids investigated here, but in most cases the iron center is simply oxidized, resulting in the loss of the iPrNO ligand. In the case of the Lewis acid B2(pin)2, the reduced product [Fe(3,5-Me-BAFP)(iPrNH2)(iPrNO)] was identified by X-ray crystallography. [ABSTRACT FROM AUTHOR]

Ginseng, from the roots of Panax ginseng C. A. Meyer, is a widely used herbal medicine in Asian countries, known for its excellent therapeutic properties. The growth of P. ginseng is depend on specific and strict environments, with a preference for wetness but intolerance for flooding. Under excessive soil moisture, some irregular rust-like substances are deposited on the root epidermis, causing ginseng rusty symptoms (GRS). This condition leads to a significant reduce in yield and quality, resulting in substantial economic loses. However, there is less knowledge on the cause of GRS and there are no effective treatments available for its treatment once it occurs. Unsuitable environments lead to the generation of large amounts of reactive oxygen species (ROS). We investigated the key indicators associated with the stress response during different physiological stages of GRS development. We observed a significant change in ROS level, MDA contents, antioxidant enzymes activities, and non-enzymatic antioxidants contents prior to the GRS. Through the analysis of soil features with an abundance of moisture, we further determined the source of ROS. The levels of nitrate reductase (NR) and nitric oxide synthase (NOS) activities in the inter-root soil of ginseng with GRS were significantly elevated compared to those of healthy ginseng. These enzymes boost nitric oxide (NO) levels, which in turn showed a favorable correlation with the GRS. The activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase first rose and then decreased as GRS developed. Excess soil moisture causes a decrease in oxygen levels. This activated NR and NOS in the soil, resulting in a production of excess NO. The NO then diffused into the ginseng root and triggered a burst of ROS through NADPH located on the cell membrane. Additionally, Fe2+ in soil was oxidized to red Fe3+, and finally led to GRS. This conclusion was also verified by the Sodium Nitroprusside (SNP), a precursor compound producing NO. The presence of NO from NR and NOS in water-saturated soil is responsible for the generation of ROS. Among these, NO is the main component that contribute to the occurrence of GRS. [ABSTRACT FROM AUTHOR]