Nitric Oxide Releasing Nanomaterials for Cardiovascular Applications.

A central paradigm of cardiovascular homeostasis is that impaired nitric oxide (NO) bioavailability results in a wide array of cardiovascular dysfunction including incompetent endothelium-dependent vasodilatation, thrombosis, vascular inflammation, and proliferation of the intima. Over the course of more than a century, NO donating formulations such as organic nitrates and nitrites have remained a cornerstone of treatment for patients with cardiovascular diseases. These donors primarily produce NO in the circulation and are not targeted to specific (sub)cellular sites of action. However, safe, and therapeutic levels of NO require delivery of the right amount to a precise location at the right time. To achieve these aims, several recent strategies aimed at therapeutically generating or releasing NO in living systems have shown that polymeric and inorganic (silica, gold) nanoparticles and nanoscale metal-organic frameworks could either generate NO endogenously by the catalytic decomposition of endogenous NO substrates or can store and release therapeutically relevant amounts of NO gas. NO-releasing nanomaterials have been developed for vascular implants (such as stents and grafts) to target atherosclerosis, hypertension, myocardial ischemia-reperfusion injury, and cardiac tissue engineering. In this review, we discuss the advances in design and development of novel NO-releasing nanomaterials for cardiovascular therapeutics and critically examine the therapeutic potential of these nanoplatforms to modulate cellular metabolism, to regulate vascular tone, inhibit platelet aggregation, and limit proliferation of vascular smooth muscle with minimal toxic effects.

The multi-factorial modes of action of urease in the pathogenesis of incontinence associated dermatitis.

Background: Incontinence Associated Dermatitis (IAD) is a type of skin inflammation caused by chronic exposure to urine and/or faeces. Current treatment strategies involve creating a barrier between the skin and urine/faeces rather than targeting specific irritants. Urease expressing pathogens catalyse the conversion of urea, present in urine, into ammonia. The accumulation of ammonia causes an elevation in skin pH which is believed to activate faecal enzymes which damage skin, and opportunistic pathogens, which lead to secondary infections. Objectives: To develop a better, multi-factorial model of IAD pathogenesis, including the effect of urease-expressing bacteria on skin, mechanism of damage of urease and urease-triggered activity of faecal enzymes and secondary pathogens. To study the effect of urease inhibition on preventing IAD skin damage. Methods: Five separate studies were made using ex vivo porcine skin and in vivo human skin models. Measurements of the change in skin barrier function were made using skin impedance, trans-epidermal water loss (TEWL), stratum corneum moisture and pH. Skin was exposed to artificial urine, inoculated with various microbes, enzymes and chemicals to examine the influence of: 1) urease-positive Proteus mirabilis 2) ammonia, 3) combination of P. mirabilis and a faecal enzyme, trypsin, 4) combination of P. mirabilis and opportunistic pathogens, Candida albicans and Staphylococcus aureus, 5) inhibition of urease using acetohydroxamic acid (AHA) on barrier function. Results: The urease-mediated production of ammonia had two principal effects: it elevated skin pH and caused inflammation, leading to significant breakdown in skin (stratum corneum) barrier function. Urease was found to further increase the activity of faecal enzymes and opportunistic pathogens, due to elevated skin pH. The urease inhibitor, AHA, was shown to have significantly reduced damage to skin barrier function, measured as its electrical resistance. Conclusions: Targeted therapeutic strategies should be developed to prevent the manifestation of IAD, rather than creating a generic barrier between skin and urine/faeces. Urease has been identified as a crucial component in the manifestation of IAD, due to its role in the production of ammonia. Urease inhibition provides a promising therapeutic target to halt the progression of IAD.

S-nitrosocysteamine-functionalised porous graphene oxide nanosheets as nitric oxide delivery vehicles for cardiovascular applications.

Nitric oxide (NO) is a key signalling molecule released by vascular endothelial cells that is essential for vascular health. Low NO bioactivity is associated with cardiovascular diseases, such as hypertension, atherosclerosis, and heart failure and NO donors are a mainstay of drug treatment. However, many NO donors are associated with the development of tolerance and adverse effects, so new formulations for controlled and targeted release of NO would be advantageous. Herein, we describe the design and characterisation of a novel NO delivery system via the reaction of acidified sodium nitrite with thiol groups that had been introduced by cysteamine conjugation to porous graphene oxide nanosheets, thereby generating S-nitrosated nanosheets. An NO electrode, ozone-based chemiluminescence and electron paramagnetic resonance spectroscopy were used to measure NO released from various graphene formulations, which was sustained at >5 × 10-10 mol cm-2 min-1 for at least 3 h, compared with healthy endothelium (cf. 0.5-4 × 10-10 mol cm-2 min-1). Single cell Raman micro-spectroscopy showed that vascular endothelial and smooth muscle cells (SMCs) took up graphene nanostructures, with intracellular NO release detected via a fluorescent NO-specific probe. Functionalised graphene had a dose-dependent effect to promote proliferation in endothelial cells and to inhibit growth in SMCs, which was associated with cGMP release indicating intracellular activation of canonical NO signalling. Chemiluminescence detected negligible production of toxic N-nitrosamines. Our findings demonstrate the utility of porous graphene oxide as a NO delivery vehicle to release physiologically relevant amounts of NO in vitro, thereby highlighting the potential of these formulations as a strategy for the treatment of cardiovascular diseases.

Cellular Pre-Adaptation to the High O2 Concentration Used in Standard Cell Culture Confers Resistance to Subsequent H2O2-Induced Cell Death.

The addition of hydrogen peroxide (H2O2) to cultured cells is widely used as a method to modulate redox-regulated cellular pathways, including the induction of programmed cell death in cell culture experiments and the testing of pro- and antioxidant compounds. Here, we assessed the effect on the cellular response to H2O2 of pre-adapting squamous cell carcinoma cells (A431) to the standard cell culture oxygenation of 18.6% O2, compared to cells pre-adapted to a physiological skin O2 concentration (3.0% O2). We showed that cells pre-adapted to 18.6% O2 resisted H2O2-induced cell death compared to cells pre-adapted to 3.0% O2 for 96 h prior to treatment with H2O2. Moreover, the enzymatic activities of catalase and glutathione reductase, as well as the protein expression levels of catalase, were higher in cells pre-adapted to 18.6% O2 compared to cells pre-adapted to 3.0% O2. H2O2-resistant cells, pre-adapted to 18.6% O2, exhibited increased nuclear Nrf-2 levels. It is concluded that A431 cells pre-adapted to standard cell culture oxygenation conditions resist H2O2-induced cell death. This effect may be related to their heightened activation of Nrf-2.

Naturally Derived Phenethyl Isothiocyanate Modulates Induction of Oxidative Stress via Its N-Acetylated Cysteine Conjugated form in Malignant Melanoma.

Phenethyl isothiocyanate (PEITC) is a secondary metabolic product yielded upon the hydrolysis of gluconasturtiin and it is highly accumulated in the flowers of watercress. The aim of the current study was to assess the role of a naturally derived PEITC-enriched extract in the induction of oxidative stress and to evaluate its anti-melanoma potency through the regulation of its metabolism with the concurrent production of the N-acetyl cysteine conjugated by-product. For this purpose, an in vitro melanoma model was utilized consisting of human primary (A375) cells as well as metastatic (COLO-679) malignant melanoma cells together with non-tumorigenic immortalized keratinocytes (HaCaT). Cytotoxicity was assessed via the Alamar Blue assay whereas the antioxidant/prooxidant activity of PEITC was determined via spectrophotometric assays. Finally, kinetic characterization of the end-product of PEITC metabolism was monitored via UPLC coupled to a tandem mass spectrometry (MS/MS). Our results indicate that although PhEF showed very minor antioxidant activity in a cell-free system, in a cell-based system, it can modulate the activity of key enzyme(s) involved in cellular antioxidant defense mechanism(s). In addition, we have shown that PhEF induces lipid and protein oxidation in a concentration-dependent manner, while its cytotoxicity is not only dependent on PEITC itself but also on its N-acetylated cysteine conjugated form.

Localisation of nitrate-reducing and highly abundant microbial communities in the oral cavity.

The nitrate (NO3-) reducing bacteria resident in the oral cavity have been implicated as key mediators of nitric oxide (NO) homeostasis and human health. NO3--reducing oral bacteria reduce inorganic dietary NO3- to nitrite (NO2-) via the NO3--NO2--NO pathway. Studies of oral NO3--reducing bacteria have typically sampled from either the tongue surface or saliva. The aim of this study was to assess whether other areas in the mouth could contain a physiologically relevant abundance of NO3- reducing bacteria, which may be important for sampling in clinical studies. The bacterial composition of seven oral sample types from 300 individuals were compared using a meta-analysis of the Human Microbiome Project data. This analysis revealed significant differences in the proportions of 20 well-established oral bacteria and highly abundant NO3--reducing bacteria across each oral site. The genera included Actinomyces, Brevibacillus, Campylobacter, Capnocytophaga, Corynebacterium, Eikenella, Fusobacterium, Granulicatella, Haemophilus, Leptotrichia, Microbacterium, Neisseria, Porphyromonas, Prevotella, Propionibacterium, Rothia, Selenomonas, Staphylococcus, Streptococcus and Veillonella. The highest proportion of NO3--reducing bacteria was observed in saliva, where eight of the bacterial genera were found in higher proportion than on the tongue dorsum, whilst the lowest proportions were found in the hard oral surfaces. Saliva also demonstrated higher intra-individual variability and bacterial diversity. This study provides new information on where samples should be taken in the oral cavity to assess the abundance of NO3--reducing bacteria. Taking saliva samples may benefit physiological studies, as saliva contained the highest abundance of NO3- reducing bacteria and is less invasive than other sampling methods. These results inform future studies coupling oral NO3--reducing bacteria research with physiological outcomes affecting human health.

A Naturally Derived Watercress Flower-Based Phenethyl Isothiocyanate-Enriched Extract Induces the Activation of Intrinsic Apoptosis via Subcellular Ultrastructural and Ca2+ Efflux Alterations in an In Vitro Model of Human Malignant Melanoma.

The aim of the current study was to (i) extract isolated fractions of watercress flowers enriched in polyphenols, phenethyl isothiocyanate and glucosinolates and (ii) characterize the anticancer mode of action of non-lethal, sub-lethal and lethal concentrations of the most potent extract fraction in primary (A375) and metastatic (COLO-679) melanoma cells as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. Cytotoxicity was assessed via the Alamar Blue assay, whereas ultrastructural alterations in mitochondria and the endoplasmic reticulum were determined via transmission electron microscopy. Mitochondrial membrane depolarization was determined using Mito-MP dye, whereas apoptosis was evaluated through the activation of caspases-3, -8 and -9. Among all extract fractions, the phenethyl isothiocyanate-enriched one (PhEF) possessed significant cytotoxicity against A375 and COLO-679 cells, while HaCaT cells remained relatively resistant at sub-lethal and lethal concentrations. Additionally, ultrastructural subcellular alterations associated with apoptosis were observed by means of increased mitochondrial area and perimeter, decreased cristae density and a shorter distance of the endoplasmic reticulum to the mitochondria, all taking place during "early" time points (2-4 h) of exposure. Moreover, PhEF induced mitochondrial membrane depolarization associated with "late" time points (24 h) of exposure, thereby leading to the activation of intrinsic apoptosis. Finally, the inhibition of cytosolic Ca2+ efflux reduced levels of caspases-9 and -3 activity, suggesting the involvement of Ca2+ efflux in modulating the activation of intrinsic apoptosis. To conclude, our data demonstrate an association of "early" ultrastructural alterations in mitochondria and the endoplasmic reticulum with the "late" induction of intrinsic apoptosis via the modulation of Ca2+ efflux.

Reduction in blood pressure following acute dietary nitrate ingestion is correlated with increased red blood cell S-nitrosothiol concentrations.

Dietary nitrate (NO3-) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2-]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3-. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3- ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3-, ∼11 mg NO3-/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3-, NO2-, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2-] and reduced BP, but increased RBC [NO2-] was correlated with decreased systolic BP (rs = -0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = -0.68, P = 0.001), diastolic (rs = -0.59, P = 0.008) and mean arterial pressure (rs = -0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2-] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3- supplementation.

Detecting and monitoring incontinence associated dermatitis: Does impedance spectroscopy have a part to play?

In this review, current understanding of the prevention and treatment of Incontinence Associated Dermatitis (IAD) is discussed. The need for preventative measures which target specific faecal/urinary irritants is highlighted, including the role of urease inhibitors. There is no existing internationally and clinically accepted method to diagnose and categorise the severity of IAD. Diagnosis currently relies on visual inspection; non-invasive techniques to assess skin barrier function could remove subjectiveness, particularly in darker skin tones. Impedance spectroscopy is a non-invasive technique which can be used to monitor skin barrier function, supporting visual assessments. Six studies (2003-2021) which used impedance to assess dermatitis were reviewed; inflamed skin was distinguishable from healthy skin in each case. This suggests that impedance spectroscopy could be useful in diagnosis early-stage IAD, potentially enabling earlier intervention. Finally, the authors present their initial findings on the role of urease in skin breakdown in an in vivo IAD model, using impedance spectroscopy.

Oral Temperature and pH Influence Dietary Nitrate Metabolism in Healthy Adults.

This study tested the hypothesis that the increases in salivary and plasma [NO2-] after dietary NO3- supplementation would be greater when oral temperature and pH were independently elevated, and increased further when oral temperature and pH were elevated concurrently. Seven healthy males (mean ± SD, age 23 ± 4 years) ingested 70 mL of beetroot juice concentrate (BR, which provided ~6.2 mmol NO3-) during six separate laboratory visits. In a randomised crossover experimental design, salivary and plasma [NO3-] and [NO2-] were assessed at a neutral oral pH with a low (TLo-pHNorm), intermediate (TMid-pHNorm), and high (THi-pHNorm) oral temperature, and when the oral pH was increased at a low (TLo-pHHi), intermediate (TMid-pHHi), and high (THi-pHHi) oral temperature. Compared with the TMid-pHNorm condition (976 ± 388 µM), the mean salivary [NO2-] 1-3 h post BR ingestion was higher in the TMid-pHHi (1855 ± 423 µM), THi-pHNorm (1371 ± 653 µM), THi-pHHi (1792 ± 741 µM), TLo-pHNorm (1495 ± 502 µM), and TLo-pHHi (2013 ± 662 µM) conditions, with salivary [NO2-] also higher at a given oral temperature when the oral pH was increased (p < 0.05). Plasma [NO2-] was higher 3 h post BR ingestion in the TMid-pHHi, THi-pHHi, and TLo-pHHi conditions, but not the TLo-pHNorm and THi-pHNorm conditions, compared with TMid-pHNorm (p < 0.05). Therefore, despite ingesting the same NO3- dose, the increases in salivary [NO2-] varied depending on the temperature and pH of the oral cavity, while the plasma [NO2-] increased independently of oral temperature, but to a greater extent at a higher oral pH.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes.

AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.

Polyphenolics, glucosinolates and isothiocyanates profiling of aerial parts of Nasturtium officinale (Watercress).

Watercress (Nasturtium officinale) is a rich source of secondary metabolites with disease-preventing and/or health-promoting properties. Herein, we have utilized extraction procedures to isolate fractions of polyphenols, glucosinolates and isothiocyanates to determine their identification, and quantification. In doing so, we have utilized reproducible analytical methodologies based on liquid chromatography with tandem mass spectrometry by either positive or negative ion mode. Due to the instability and volatility of isothiocyanates, we followed an ammonia derivatization protocol which converts them into respective ionizable thiourea derivatives. The analytes' content distribution map was created on watercress flowers, leaves and stems. We have demonstrated that watercress contains significantly higher levels of gluconasturtiin, phenethyl isothiocyanate, quercetin-3-O-rutinoside and isorhamnetin, among others, with their content decreasing from flowers (82.11 ± 0.63, 273.89 ± 0.88, 1459.30 ± 12.95 and 289.40 ± 1.37 ng/g of dry extract respectively) to leaves (32.25 ± 0.74, 125.02 ± 0.52, 1197.86 ± 4.24 and 196.47 ± 3.65 ng/g of det extract respectively) to stems (9.20 ± 0.11, 64.7 ± 0.9, 41.02 ± 0.18, 65.67 ± 0.84 ng/g of dry extract respectivbely). Pearson's correlation analysis has shown that the content of isothiocyanates doesn't depend only on the bioconversion of individual glucosinolates but also on other glucosinolates of the same group. Overall, we have provided comprehensive analytical data of the major watercress metabolites thereby providing an opportunity to exploit different parts of watercress for potential therapeutic applications.

Evaluation of Bioactive Properties of Lipophilic Fractions of Edible and Non-Edible Parts of Nasturtium officinale (Watercress) in a Model of Human Malignant Melanoma Cells.

Watercress is an enriched source of phenethyl isothiocyanate (PEITC), among other phytochemicals, with an antioxidant capacity. The aim of this study was to (i) chemically characterize and (ii) biologically evaluate the profile of the main health-promoting compounds contained in edible (i.e., mixture of leaves and lateral buds) and non-edible (i.e., stems) parts of watercress in an in vitro model of malignant melanoma consisting of human malignant melanoma (A375), non-melanoma (A431) and keratinocyte (HaCaT) cells. The extraction of the main constituents of watercress was performed by subjecting the freeze-dried edible and non-edible samples through different extraction protocols, whereas their concentration was obtained utilizing analytical methodologies. In addition, cell viability was evaluated by the Alamar Blue assay, whereas levels of oxidative stress and apoptosis were determined by commercially available kits. The edible watercress sample contained a higher amount of various nutrients and phytochemicals in the hexane fraction compared to the non-edible one, as evidenced by the presence of PEITC, phenolics, flavonoids, pigments, ascorbic acid, etc. The cytotoxicity potential of the edible watercress sample in the hexane fraction was considerably higher than the non-edible one in A375 cells, whereas A431 and HaCaT cells appeared to be either more resistant or minimally affected, respectively. Finally, levels of oxidative stress and apoptotic induction were increased in both watercress samples, but the magnitude of the induction was much higher in the edible than the non-edible watercress samples. Herein, we provide further evidence documenting the potential development of watercress extracts (including watercress waste by-products) as promising anti-cancer agent(s) against malignant melanoma cells.

S-nitrosothiols, and other products of nitrate metabolism, are increased in multiple human blood compartments following ingestion of beetroot juice.

Ingested inorganic nitrate (NO3⁻) has multiple effects in the human body including vasodilation, inhibition of platelet aggregation, and improved skeletal muscle function. The functional effects of oral NO3⁻ involve the in vivo reduction of NO3⁻ to nitrite (NO2⁻) and thence to nitric oxide (NO). However, the potential involvement of S-nitrosothiol (RSNO) formation is unclear. We hypothesised that the RSNO concentration ([RSNO]) in red blood cells (RBCs) and plasma is increased by NO3⁻-rich beetroot juice ingestion. In healthy human volunteers, we tested the effect of dietary supplementation with NO3⁻-rich beetroot juice (BR) or NO3⁻-depleted beetroot juice (placebo; PL) on [RSNO], [NO3⁻] and [NO2⁻] in RBCs, whole blood and plasma, as measured by ozone-based chemiluminescence. The median basal [RSNO] in plasma samples (n = 22) was 10 (5-13) nM (interquartile range in brackets). In comparison, the median values for basal [RSNO] in the corresponding RBC preparations (n = 19) and whole blood samples (n = 19) were higher (p < 0.001) than in plasma, being 40 (30-60) nM and 35 (25-80) nM, respectively. The median RBC [RSNO] in a separate cohort of healthy subjects (n = 5) was increased to 110 (93-125) nM after ingesting BR (12.8 mmol NO3⁻) compared to a corresponding baseline value of 25 (21-31) nM (Mann-Whitney test, p < 0.01). The median plasma [RSNO] in another cohort of healthy subjects (n = 14) was increased almost ten-fold to 104 (58-151) nM after BR supplementation (7 × 6.4 mmol of NO3⁻ over two days, p < 0.01) compared to PL. In conclusion, RBC and plasma [RSNO] are increased by BR ingestion. In addition to NO2⁻, RSNO may be involved in dietary NO3⁻ metabolism/actions.

Network analysis of nitrate-sensitive oral microbiome reveals interactions with cognitive function and cardiovascular health across dietary interventions.

Many oral bacteria reduce inorganic nitrate, a natural part of a vegetable-rich diet, into nitrite that acts as a precursor to nitric oxide, a regulator of vascular tone and neurotransmission. Aging is hallmarked by reduced nitric oxide production with associated detriments to cardiovascular and cognitive function. This study applied a systems-level bacterial co-occurrence network analysis across 10-day dietary nitrate and placebo interventions to test the stability of relationships between physiological and cognitive traits and clusters of co-occurring oral bacteria in older people. Relative abundances of Proteobacteria increased, while Bacteroidetes, Firmicutes and Fusobacteria decreased after nitrate supplementation. Two distinct microbiome modules of co-occurring bacteria, that were sensitive to nitrate supplementation, showed stable relationships with cardiovascular (Rothia-Streptococcus) and cognitive (Neisseria-Haemophilus) indices of health across both dietary conditions. A microbiome module (Prevotella-Veillonella) that has been associated with pro-inflammatory metabolism was diminished after nitrate supplementation, including a decrease in relative abundance of pathogenic Clostridium difficile. These nitrate-sensitive oral microbiome modules are proposed as potential pre- and probiotic targets to ameliorate age-induced impairments in cardiovascular and cognitive health.

Graphene Oxide-Based Targeting of Extracellular Cathepsin D and Cathepsin L As A Novel Anti-Metastatic Enzyme Cancer Therapy.

Overexpression and secretion of the enzymes cathepsin D (CathD) and cathepsin L (CathL) is associated with metastasis in several human cancers. As a superfamily, extracellularly, these proteins may act within the tumor microenvironment to drive cancer progression, proliferation, invasion and metastasis. Therefore, it is important to discover novel therapeutic treatment strategies to target CathD and CathL and potentially impede metastasis. Graphene oxide (GO) could form the basis of such a strategy by acting as an adsorbent for pro-metastatic enzymes. Here, we have conducted research into the potential of targeted anti-metastatic therapy using GO to adsorb these pro-tumorigenic enzymes. Binding of CathD/L to GO revealed that CathD/L were adsorbed onto the surface of GO through its cationic and hydrophilic residues. This work could provide a roadmap for the rational integration of CathD/L-targeting agents into clinical settings.

Biocompatibility and toxicity of graphene quantum dots for potential application in photodynamic therapy.

AIM: Achieving reliably high production of reactive oxygen species (ROS) in photodynamic therapy (PDT) remains challenging. Graphene quantum dots (GQDs) hold great promise for PDT. However, the photochemical processes leading to GQD-derived ROS generation have not yet been fully elucidated. MATERIALS & METHODS: Physicochemical characteristics of GQDs were comprehensively investigated, including electron paramagnetic resonance analysis of singlet oxygen production. Dark toxicity was assessed in vitro and in vivo. RESULTS: GQDs demonstrated excellent photoluminescent features, corrosion resistance, high water solubility, high photo/pH-stability, in vitro and in vivo biocompatibility and very efficient singlet oxygen/ROS generation. CONCLUSION: The enhanced ROS generation, combined with good biocompatibility and minimal toxicity in vitro and in vivo support the potential of GQDs for future PDT application.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Oxidative stress in autoimmune rheumatic diseases.

The management of patients with autoimmune rheumatic diseases such as rheumatoid arthritis (RA) remains a significant challenge. Often the rheumatologist is restricted to treating and relieving the symptoms and consequences and not the underlying cause of the disease. Oxidative stress occurs in many autoimmune diseases, along with the excess production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The sources of such reactive species include NADPH oxidases (NOXs), the mitochondrial electron transport chain, nitric oxide synthases, nitrite reductases, and the hydrogen sulfide producing enzymes cystathionine-ß synthase and cystathionine-γ lyase. Superoxide undergoes a dismutation reaction to generate hydrogen peroxide which, in the presence of transition metal ions (e.g. ferrous ions), forms the hydroxyl radical. The enzyme myeloperoxidase, present in inflammatory cells, produces hypochlorous acid, and in healthy individuals ROS and RNS production by phagocytic cells is important in microbial killing. Both low molecular weight antioxidant molecules and antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and peroxiredoxin remove ROS. However, when ROS production exceeds the antioxidant protection, oxidative stress occurs. Oxidative post-translational modifications of proteins then occur. Sometimes protein modifications may give rise to neoepitopes that are recognized by the immune system as 'non-self' and result in the formation of autoantibodies. The detection of autoantibodies against specific antigens, might improve both early diagnosis and monitoring of disease activity. Promising diagnostic autoantibodies include anti-carbamylated proteins and anti-oxidized type II collagen antibodies. Some of the most promising future strategies for redox-based therapeutic compounds are the activation of endogenous cellular antioxidant systems (e.g. Nrf2-dependent pathways), inhibition of disease-relevant sources of ROS/RNS (e.g. isoform-specific NOX inhibitors), or perhaps specifically scavenging disease-related ROS/RNS via site-specific antioxidants.

Nitrate-responsive oral microbiome modulates nitric oxide homeostasis and blood pressure in humans.

Imbalances in the oral microbial community have been associated with reduced cardiovascular and metabolic health. A possible mechanism linking the oral microbiota to health is the nitrate (NO3-)-nitrite (NO2-)-nitric oxide (NO) pathway, which relies on oral bacteria to reduce NO3- to NO2-. NO (generated from both NO2- and L-arginine) regulates vascular endothelial function and therefore blood pressure (BP). By sequencing bacterial 16S rRNA genes we examined the relationships between the oral microbiome and physiological indices of NO bioavailability and possible changes in these variables following 10 days of NO3- (12 mmol/d) and placebo supplementation in young (18-22 yrs) and old (70-79 yrs) normotensive humans (n = 18). NO3- supplementation altered the salivary microbiome compared to placebo by increasing the relative abundance of Proteobacteria (+225%) and decreasing the relative abundance of Bacteroidetes (-46%; P < 0.05). After NO3-supplementation the relative abundances of Rothia (+127%) and Neisseria (+351%) were greater, and Prevotella (-60%) and Veillonella (-65%) were lower than in the placebo condition (all P < 0.05). NO3- supplementation increased plasma concentration of NO2- and reduced systemic blood pressure in old (70-79 yrs), but not young (18-22 yrs), participants. High abundances of Rothia and Neisseria and low abundances of Prevotella and Veillonella were correlated with greater increases in plasma [NO2-] in response to NO3- supplementation. The current findings indicate that the oral microbiome is malleable to change with increased dietary intake of inorganic NO3-, and that diet-induced changes in the oral microbial community are related to indices of NO homeostasis and vascular health in vivo.