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Background:Brain cholesterol levels are tightly regulated but increasing evidence indicates that cholesterol metabolism may drive Alzheimer’s disease (AD)-associated pathological changes. Recent advances in understanding of mitochondrial dysfunction in AD brain have presented a vital role played by mitochondria in oxysterol biosynthesis and their involvement in pathophysiology. Oxysterol accumulation in brain is controlled by various enzymatic pathways including sulfation. While research into oxysterol is under the areas of active investigation, there is less evidence for oxysterol sulfate levels in human brain.Objective:This study investigates the hypothesis that AD brain oxysterol detoxification via sulfation is impaired in later stages of disease resulting in oxysterol accumulation.Methods:Lipids were extracted from postmortem frozen brain tissue and cerebrospinal (CSF) from late- (Braak stage III-IV) and early- (Braak stage I-II) stage AD patients. Samples were spiked with internal standards prior to lipid extraction. Oxysterols were enriched with a two-step solid phase extraction using a polymeric SPE column and further separation was achieved by LC-MS/MS.Results:Oxysterols, 26-hydroxycholesterol (26-OHC), 25-hydroxycholesterol (25-OHC), and 7-oxycholesterol levels were higher in brain tissue and mitochondria extracted from late-stage AD brain tissue except for 24S-hydroxycholesterol, which was decreased in late AD. However, oxysterol sulfates are significantly lower in the AD frontal cortex. Oxysterols, 25-OHC, and 7-oxocholesterol was higher is CSF but 26-OHC and oxysterol sulfate levels were not changed.Conclusion:Our results show oxysterol metabolism is altered in AD brain mitochondria, favoring synthesis of 26-OHC, 25-OHC, and 7-oxocholesterol, and this may influence brain mitochondrial function and acceleration of the disease.

Aims Patients with chronic kidney disease (CKD) are also susceptible to periodontitis. The causal link between periodontitis and CKD may be mediated via systemic inflammation/oxidative stress. Using structural equation modelling (SEM), this cross‐sectional study aimed to explore the causal relationship between periodontal inflammation (PI) and renal function. Materials and methods Baseline data on 770 patients with stage 3–5 (pre‐dialysis) CKD from an ongoing cohort study were used. Detailed, bioclinical data on PI and renal function, as well as potential confounders and mediators of the relationship between the two, were collected. SEMs of increasing complexity were created to test the causal assumption that PI affects renal function and vice versa. Results Structural equation modelling confirmed the assumption that PI and renal function are causally linked, mediated by systemic oxidative stress. The magnitude of this effect was such that a 10% increase in PI resulted in a 3.0% decrease in renal function and a 10% decrease in renal function resulted in a 25% increase in PI. Conclusions Periodontal inflammation represents an occult source of oxidative stress in patients with CKD. Further clinical studies are needed to confirm whether periodontal therapy, as a non‐pharmacological approach to reducing systemic inflammatory/oxidative stress burden, can improve outcomes in CKD.

Cholesterol and oxysterol sulfates are important regulators of lipid metabolism, inflammation, cell apoptosis, and cell survival. Among the sulfate-based lipids, cholesterol sulfate (CS) is the most studied lipid both quantitatively and functionally. Despite the importance, very few studies have analysed and linked the actions of oxysterol sulfates to their physiological and pathophysiological roles. Overexpression of sulfotransferases confirmed the formation of a range of oxysterol sulfates and their antagonistic effects on liver X receptors (LXRs) prompting further investigations how are the changes to oxysterol/oxysterol sulfate homeostasis can contribute to LXR activity in the physiological milieu. Here, we aim to bring together for novel roles of oxysterol sulfates, the available techniques and the challenges associated with their analysis. Understanding the oxysterol/oxysterol sulfate levels and their pathophysiological mechanisms could lead to new therapeutic targets for metabolic diseases. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Background: Abnormal cholesterol metabolism changes the neuronal membrane and may promote amyloidogenesis. Oxysterols in cerebrospinal fluid (CSF) are related to Alzheimer's disease (AD) biomarkers in mild cognitive impairment and dementia. Cholesterol turnover is important for axonal and white matter (WM) microstructure maintenance. Objective: We aim to demonstrate that the association of oxysterols, AD biomarkers, and WM microstructure occurs early in asymptomatic individuals. Methods: We studied the association of inter-individual variability of CSF 24-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), 7-ketocholesterol (7-KC), 7 beta-hydroxycholesterol (7 beta-OHC), amyloid-beta(42) (A beta(42)), total-tau (t-tau), phosphorylated-tau (p-tau), neurofilament (NfL), and WM microstructure using diffusion tensor imaging, generalized linear models and moderation/mediation analyses in 153 healthy adults. Results: Higher 7-KC levels were related to lower A beta(42), indicative of greater AD pathology (p = 0.041). Higher 7-KC levels were related to lower fractional anisotropy (FA) and higher mean (MD), axial (AxD), and radial (RD) diffusivity. 7-KC modulated the association between AxD and NfL in the corpus callosum splenium (B = 39.39, p = 0.017), genu (B = 68.64, p = 0.000), and fornix (B = 10.97, p = 0.000). Lower A beta(42) levels were associated to lower FA and higher MD, AxD, and RD in the fornix, corpus callosum, inferior longitudinal fasciculus, and hippocampus. The association between AxD and A beta(42) was moderated by 7K-C (p = 0.048). Conclusion: This study adds clinical evidence to support the role of 7K-C on axonal integrity and the involvement of cholesterol metabolism in the A beta(42) generation process.

The aim of the present paper is to assess the relationship between oxysterol levels and retinal microvascular function in individuals of various age groups, free of clinically evident diseases. Forty-two apparently healthy individuals were included in the present study (group 1: 19–30 years, group 2: 31–50 years, and group 3: 51–70 years). Retinal microvascular function was assessed using the dynamic retinal vessel analyzer (DVA, IMEDOS GmbH, Jena, Germany). Fasting plasma was obtained from all subjects and quantification of monohydroxy and dihydroxy oxysterols assessment was performed using LC-MS/MS following reverse phase chromatography. A Griess assay was used to evaluate the Nitric Oxide (NO) concentration in all individuals. The glutathione redox ratio was also analyzed by means of whole blood glutathione recycling assay. In all participants, the levels of 7-Ketocholesterol, 25-hydroxycholesterol and 7β-hydroxycholesterol correlated significantly and positively with the time to maximum arteriolar dilation. In addition, 25-hydroxycholesterol and 7β-hydroxycholesterol negatively correlated to the percentage of maximum arteriolar dilation. A negative correlation was observed for 27-hydroxycholesterol and 7β-hydroxycholesterol with microvascular arteriolar constriction. These results suggest that, with age, abnormal oxysterol levels correlate with early changes in microvascular bed function. This relationship could signal early risk for cardiovascular diseases (CVDs) in an ageing population.

Neuroinflammation has been implicated in Alzheimer’s disease onset and progression. Chronic neuroinflammation is initiated by amyloid-β-activated microglial cells that secrete immuno-modulatory molecules within the brain and into the vasculature. Inflammation is normally self-limiting and actively resolves by “switching off” the generation of pro-inflammatory mediators and by non-phlogistic clearance of spent cells and their debris to restore tissue homeostasis. Deficits in these anti-inflammatory/pro-resolution pathways may predispose to the development of chronic inflammation. The synthesis of endogenous lipid mediators from arachidonic acid, lipoxins via cyclooxygenase 2 and lipoxygenases, and conversion of exogenous polyunsaturated fatty acids, namely docosahexaenoic acid and eicosapentaenoic acid, to resolvins contributes to effective, timely resolution of acute inflammation. Work by Xiuzhe et al., 2020 in the Journal of Alzheimer’s Disease reported that plasma level of LXA4 is related to cognitive status in ischemic stroke patients suggesting that decreased LXA4 may be a potential risk factor for post post-stroke cognitive impairment. As evident by recent clinical trials and development of drug analogues, there is recent drive to search for lipoxin analogues as therapeutics for inflammatory diseases. Understanding how bioactive lipid signaling is involved in resolution will increase our understanding of controlling inflammation and may facilitate the discovery of new classes of therapeutic pro-resolution agents for evaluation in AD prevention studies.

The raw datasets of oxysterol quantifications from whole cell and mitochondrial fractions of THP-1 monocytes and macrophages, neuronal-like SH-SH5Y cells and human peripheral blood mononuclear cells are presented. Oxysterols were quantified using a new liquid chromatography-mass spectrometry (LC-MS) and multiple reaction monitoring analysis published in the article "A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism" in Redox Biology [1]. This method showed improved extraction efficiency and recovery of mono and dihydroxycholesterols from cellular matrix. The datasets derived from the three cell lines are included in the appendix. These datasets provide new information about the oxysterol distribution in THP-1 monocytes and macrophages, SH-SY5Y cells and peripheral blood mononuclear cells. These datasets can be used as a guide for oxysterol distribution in the three cell lines for future studies, and can used for future method optimization, and for comparison of oxysterol recovery with other analytical techniques.

Endothelial dysfunction is a hallmark of preeclampsia, a life-threatening complication of pregnancy characterised by hypertension and elevated soluble Fms-Like Tyrosine Kinase-1 (sFlt-1). Dysregulation of hydrogen sulfide (H2S) by inhibition of cystathionine γ-lyase (CSE) increases sFlt-1 and soluble endoglin (sEng) release. We explored whether compromise in CSE/H2S pathway is linked to dysregulation of the mitochondrial bioenergetics and oxidative status. We investigated whether these effects were linked to CSE-induced sFlt-1 and sEng production in endothelial cells. Here, we demonstrate that CSE/H2S pathway sustain endothelial mitochondrial bioenergetics and loss of CSE increases the production of mitochondrial-specific superoxide. As a compensatory effect, low CSE environment enhances the reliance on glycolysis. The mitochondrial-targeted H2S donor, AP39, suppressed the antiangiogenic response and restored the mitochondrial bioenergetics in endothelial cells. AP39 revealed that upregulation of sFlt-1, but not sEng, is independent of the mitochondrial H2S metabolising enzyme, SQR. These data provide new insights into the molecular mechanisms for antiangiogenic upregulation in a mitochondrial-driven environment. Targeting H2S to the mitochondria may be of therapeutic benefit in the prevention of endothelial dysfunction associated with preeclampsia.

Preeclampsia affects one in twelve of the 130 million pregnancies a year. The lack of an effective therapeutic to prevent or treat it is responsible for an annual global cost burden of 100 billion US dollars. Preeclampsia also affects these women later in life as it is a recognised risk factor for cardiovascular disease, stroke and vascular dementia. Our laboratory demonstrated that preeclampsia is associated with high soluble fms-like tyrosine kinase 1 (sFlt-1) and low heme oxygenase-1 (HO1/Hmox1) expression. Here we sought to determine the therapeutic value of a novel H2S-releasing aspirin (MZe786) in HO-1 haploid deficient (Hmox1+/−) pregnant mice in a high sFlt-1 environment. Pregnant Hmox1+/− mice were injected with adenovirus encoding sFlt-1 or control virus at gestation day E11.5. Subsequently, Hmox1+/− dams were treated daily with a number of treatment regimens until E17.5, when maternal and fetal outcomes were assessed. Here we show that HO-1 compromised mice in a high sFlt-1 environment during pregnancy exhibit severe preeclampsia signs and a reduction in antioxidant genes. MZe786 ameliorates preeclampsia by reducing hypertension and renal damage possibly by stimulating antioxidant genes. MZe786 also improved fetal outcome in comparison with aspirin alone and appears to be a better therapeutic agent at preventing preeclampsia than aspirin alone., Graphical abstract Image 1, Highlights • Partial loss of heme oxygenase-1 under high soluble Flt-1 causes severe preeclampsia compared to high sFlt-1 alone. • MZe786, hydrogen sulfide releasing aspirin prevents preeclampsia by suppressing maternal hypertension and kidney injury. • MZe786 is able to rescue pregnancy and improves fetal outcome despite the persistent high levels of sFlt-1. • MZe786 is a superior therapeutic candidate than aspirin in preventing preeclampsia.

Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples., Highlights • New method for extraction and quantification of mono and dihydroxysterols from mitochondria. • Method offers high sensitivity and selectivity allowing quantification of oxysterols at ≥5pg.μl−1. • Successful application to tissue, primary cells and cell lines. • Common oxysterol metabolites are present in blood and brain cell mitochondria.

The current study aims to investigate retinal vascular function and its relationship with systemic anti-oxidative defence capacity in normal individuals versus those with early hypertensive changes according to the current ESC/ESH guidelines. Retinal microvascular function was assessed in 201 participants by means of dynamic retinal vessel analysis. Blood pressure, lipid panel, oxidized (GSH) & reduced glutathione (GSSG) were also evaluated for each participant. Individuals classed as grade 1 hypertension demonstrated higher retinal arterial baseline diameter fluctuation (p = 0.0012), maximum dilation percentage (p = 0.0007), time to maximum constriction (p = 0.0003) and lower arterial constriction slope (p = 0.0131). Individuals classed as high normal and grade 1 hypertension also demonstrated higher time to maximum dilation than individuals classed as optimal or normal. GSH levels correlated negatively with SBP, DBP and MBP values in all participants (p = 0.0010; p = 0.0350 and p = 0.0050) as well as with MBP values in high normal and grade 1 hypertension (p = 0.0290). The levels of GSSG correlated positively with SBP, DBP and MBP values in all participants (p = 0.0410; p = 0.0330 and, p = 0.0220). Our results point to the fact that microvascular alterations can be identifiable at BP values still considered within normal values and go in parallel with the changes observed in the level of oxidative stress.

Significance: Inflammation increases during the aging process. It is linked to mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Mitochondrial macromolecules are critical targets of oxidative damage; they contribute to respiratory uncoupling with increased ROS production, redox stress, and a cycle of senescence, cytokine production, and impaired oxidative phosphorylation. Targeting the formation or accumulation of oxidized biomolecules, particularly oxidized lipids, in immune cells and mitochondria could be beneficial for age-related inflammation and comorbidities. Recent Advances: Inflammation is central to age-related decline in health and exhibits a complex relationship with mitochondrial redox state and metabolic function. Improvements in mass spectrometric methods have led to the identification of families of oxidized phospholipids (OxPLs), cholesterols, and fatty acids that increase during inflammation and which modulate nuclear factor erythroid 2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor gamma (PPARγ), activator protein 1 (AP1), and NF-κB redox-sensitive transcription factor activity. Critical Issues: The kinetic and spatial resolution of the modified lipidome has profound and sometimes opposing effects on inflammation, promoting initiation at high concentration and resolution at low concentration of OxPLs. Future Directions: There is an emerging opportunity to prevent or delay age-related inflammation and vascular comorbidity through a resolving (oxy)lipidome that is dependent on improving mitochondrial quality control and restoring redox homeostasis.

Mitochondria are important (patho)physiological sources of reactive oxygen species (ROS) that mediate mitochondrial dysfunction and phospholipid oxidation; an increase in mitochondrial content of oxidized phospholipid (OxPL) associates with cell death. Previously we showed that the circulating OxPL 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) increases in patients with Alzheimer's disease (AD), and associates with lower plasma antioxidant oxocarotenoids, zeaxanthin, and lutein. Since oxocarotenoids are metabolized in mitochondria, we propose that during AD, lower concentrations of mitochondrial zeaxanthin and lutein may result in greater phospholipid oxidation and predispose to neurodegeneration. Here, we have investigated whether non-toxic POVPC concentrations impair mitochondrial metabolism in differentiated (d)SH-SY5Y neuronal cells and whether there is any protective role for oxocarotenoids against mitochondrial dysfunction. After 24 hours, glutathione (GSH) concentration was lower in neuronal cells exposed to POVPC (1-20 μM) compared with vehicle control without loss of viability compared to control. However, mitochondrial ROS production (determined by MitoSOX oxidation) was increased by 50% only after 20 μM POVPC. Following delivery of lutein (0.1-1 μM) and zeaxanthin (0.5-5 μM) over 24 hours in vitro, oxocarotenoid recovery from dSH-SY5Y cells was 50%. Co-incubation with oxocarotenoids prevented loss of GSH after 1 μM but not 20 μM POVPC, whereas the increase in ROS production induced by 20 μM POVPC was prevented by lutein and zeaxanthin. Mitochondrial uncoupling increases and ATP production is inhibited by 20 μM but not 1 μM POVPC; carotenoids protected against uncoupling although did not restore ATP production. In summary, 20 μM POVPC induced loss of GSH and a mitochondrial bioenergetic deficit in neuronal cells that was not mitigated by oxocarotenoids.

Oxysterols play important roles in development and diseases, but can be highly challenging to analyze. To ensure satisfactory measurements, oxysterols must typically be separated with chromatography prior to detection. Here, we will devote attention to the chromatography of oxysterols, focusing on gas chromatography and liquid chromatography. We will present the role of stationary phases, mobile phases, and dimensions and geometries of particles/columns. We discuss how these parameters may affect the chromatography, regarding factors such as speed and resolution. Finally, we present some less explored avenues for separation of oxysterols.

The health benefits of playing football and the importance of exercise and social contact for healthy ageing are well established, but few older adults in the UK take enough exercise. Football is popular, flexible in format and draws players into engrossing, effortful and social exercise, but the physical demands of play at full speed may make it unsustainable for some older adults. Restricted to walking pace, will play still be engaging? Will health benefits be retained? Will physical demands remain manageable? This pilot study aims to investigate: (1) the experience of older adults playing walking football every week, is it sustainable and rewarding, (2) the intensity and locomotor pattern of walking football, (3) the scale and nature of walking football health benefits and (4) possible cognitive benefits of playing walking football through measures of processing speed, selective and divided attention and updating and inhibition components of executive function. 'Walking football' and 'waiting list' groups were compared before and after 12 weeks of one-hour per week football. Walking football was found to be engaging, sustainable for older adults and moderately intensive; however, selective health and cognitive benefits were not found from this brief intervention. Highlights Walking football is a lower impact but authentic form of football that enables older players to extend their active participation. Walking football is enjoyable and moderately demanding and may be a sustainable form of exercise for older adults. Health and cognitive benefits to playing walking football were not found.

Individuals with chronic kidney disease (CKD) and periodontitis as a co-morbidity have a higher mortality rate than individuals with CKD and no periodontitis. The inflammatory burden associated with both diseases contributes to an increased risk of cardiovascular and all-cause mortality. We previously demonstrated that periodontitis is associated with increasing circulating markers of inflammation and oxidative stress. We propose that inflammatory oxidised phosphocholines may contribute to the increased risk of cardiovascular disease in patients with CKD. However, the analysis of oxidised phospholipids has been limited by a lack of authentic standards for absolute quantification. Here, we have developed a comprehensive quantification liquid chromatography-mass spectrometry-based multiple reaction monitoring method for oxidised phospholipids (including some without available authentic species) that enables us to simultaneously measure twelve oxidised phosphatidylcholine species with high levels of sensitivity and specificity. The standard curves for commercial standards 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidylcholine (PGPC); 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphatidylcholine (PONPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphatidylcholine (PAzPC) and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphatidylcholine (POVPC), were linear with a correlation coefficient greater than 0.99 for all analytes. The method is reproducible, with intra- and inter-day precision

So far, the investigation in cancer cell lines of the modulation of cancer growth and progression by oxysterols, in particular 27-hydroxycholesterol (27HC), has yielded controversial results. The primary aim of this study was the quantitative evaluation of possible changes in 27HC levels during the different steps of colorectal cancer (CRC) progression in humans. A consistent increase in this oxysterol in CRC mass compared to the tumor-adjacent tissue was indeed observed, but only in advanced stages of progression (TNM stage III), a phase in which cancer has spread to nearby sites. To investigate possible pro-tumor properties of 27HC, its effects were studied in vitro in differentiated CaCo-2 cells. Relatively high concentrations of this oxysterol markedly increased the release of pro-inflammatory interleukins 6 and 8, monocyte chemoattractant protein-1, vascular endothelial growth factor, as well as matrix metalloproteinases 2 and 9. The up-regulation of all these molecules, which are potentially able to favor cancer progression, appeared to be dependent upon a net stimulation of Akt signaling exerted by supra-physiological amounts of 27HC.

Sulfate-based lipids (SL) have been proposed as players in inflammation, immunity and infection. In spite of the many biochemical processes linked to SL, analysis on this class of lipids has only focused on specific SL sub-classes in individual fluids or cells leaving a range of additional SL in other biological samples unaccounted for. This study describes the mass spectrometry screening of SL in lipid extracts of human fluids (saliva, plasma, urine, seminal fluid) and primary human cells (RBC, neutrophils, fibroblasts and skin epidermal) using targeted precursor ion scanning (PIS) approach. The PIS 97 mass spectra reveal a wide diversity of SL including steroid sulfates, sulfoglycolipids and other unidentified SL, as well as metabolites such as taurines, sulfated polyphenols and hypurate conjugates. Semi-quantification of SL revealed that plasma exhibited the highest content of SL whereas seminal fluid and epithelial cells contained the highest sulphur to phosphorous (S/P) ratio. The complexity of biofluids and cells sulfateome presented in this study highlight the importance of expanding the panel of synthetic sulfate-based lipid standards. Also, the heterogenous distribution of SL provides evidence for the interplay of sulfotransferases/sulfatases, opening new avenues for biomarker discovery in oral health, cardiovascular, fertility and dermatology research areas. published

The association between poor paternal diet, perturbed embryonic development, and adult offspring ill health represents a new focus for the Developmental Origins of Health and Disease hypothesis. However, our understanding of the underlying mechanisms remains ill-defined. We have developed a mouse paternal low-protein diet (LPD) model to determine its impact on semen quality, maternal uterine physiology, and adult offspring health. We observed that sperm from LPD-fed male mice displayed global hypomethylation associated with reduced testicular expression of DNA methylation and folate-cycle regulators compared with normal protein diet (NPD) fed males. Furthermore, females mated with LPD males display blunted preimplantation uterine immunological, cell signaling, and vascular remodeling responses compared to controls. These data indicate paternal diet impacts on offspring health through both sperm genomic (epigenetic) and seminal plasma (maternal uterine environment) mechanisms. Extending our model, we defined sperm- and seminal plasma-specific effects on offspring health by combining artificial insemination with vasectomized male mating of dietary-manipulated males. All offspring derived from LPD sperm and/or seminal plasma became heavier with increased adiposity, glucose intolerance, perturbed hepatic gene expression symptomatic of nonalcoholic fatty liver disease, and altered gut bacterial profiles. These data provide insight into programming mechanisms linking poor paternal diet with semen quality and offspring health.

Alzheimer’s disease (AD) etiology is complex; gene and environmental risk factors may interact to predispose to disease. From single nucleotide polymorphism analyses and genome-wide association studies, a number of candidate risk genes for the onset of AD have been identified and cluster around lipid metabolism and inflammation. We hypothesized that endothelial cells which line the blood-brain barrier are likely to be critical mediators of systemic metabolism within the brain. Therefore, we have studied the effect of 27 hydroxycholesterol (27-OHC) on microvascular endothelial cell (HMVEC) redox state, inflammatory cytokine secretion, and microRNA (miR) expression. Using a transwell method, we have studied directional secretion profiles for the proinflammatory cytokines TNFα and IL-6 and confirmed that 27-OHC induces discrete and directional inflammatory molecular signatures from HMVEC. The lipids caused depletion of cellular glutathione and cytokine secretion is HMVEC-redox state-dependent. Discovery miR expression changes in HMVEC with and without 27-OHC treatment was undertaken. We selected three genes for further analysis by qPCR; miR-144 and 146 expression, which are anti-inflammatory and redox regulating modulators, were not affected significantly by 27-OHC. However, increased expression of a putative neurotrophic regulatory factor miR933 in HMVEC with 27-OHC was confirmed by qPCR. In plasma from patients with dementia, all three miR were found at significantly elevated levels compared to healthy older adults. These data highlight that 27-OHC has an important regulatory effect on endothelial microvascular cells to increase expression of a miR (–933) and secretion of inflammatory cytokines that are elevated in plasma from dementia patients.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 µM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells., Graphical abstract, Highlights • Oxidized LDL lipids but not proteins oxidize SHSY-5Y cell glutathione, triggering lipid rafts. • Oxidized lipid-induced rafts promote β-secretase activity and β-amyloid secretion. • These effects are mimicked by 27-hydroxycholesterol present in oxidized LDL.

Oxysterols are oxidized derivatives of cholesterol that are formed enzymatically or via reactive oxygen species or both. Cholesterol or oxysterols ingested as food are absorbed and packed into lipoproteins that are taken up by hepatic cells. Within hepatic cells, excess cholesterol is metabolised to form bile acids. The endoplasmic reticulum acts as the main organelle in the bile acid synthesis pathway. Metabolised sterols originating from this pathway are distributed within other organelles and in the cell membrane. The alterations to membrane oxysterol:sterol ratio affects the integrity of the cell membrane. The presence of oxysterols changes membrane fluidity and receptor orientation. It is well documented that hydroxylase enzymes located in mitochondria facilitate oxysterol production via an acidic pathway. More recently, the presence of oxysterols was also reported in lysosomes. Peroxisomal deficiencies favour intracellular oxysterols accumulation. Despite the low abundance of oxysterols compared to cholesterol, the biological actions of oxysterols are numerous and important. Oxysterol levels are implicated in the pathogenesis of multiple diseases ranging from chronic inflammatory diseases (atherosclerosis, Alzheimer's disease and bowel disease), cancer and numerous neurodegenerative diseases. In this article, we review the distribution of oxysterols in sub-cellular organelles and in biological fluids.

Oxidation and S-nitrosylation of cysteinyl thiols (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H), sulfonic acids (Cys-SO3H), disulphides and S-nitrosothiols are suggested as important post-translational modifications that can activate or deactivate the function of many proteins. Non-enzymatic post-translational modifications to cysteinyl thiols have been implicated in a wide variety of physiological and pathophysiological states but have been difficult to monitor in a physiological setting because of a lack of experimental tools. The purpose of this review is to bring together the approaches that have been developed for stably trapping cysteine either in its reduced or oxidised forms for enrichment and or subsequent mass spectrometric analysis. These tools are providing insight into potential targets for post-translational modifications to cysteine modification in vivo. This article is part of a Special Issue entitled: Special Issue: Posttranslational Protein modifications in biology and Medicine. © 2013.

Aim: To determine the effect of periodontitis patients' plasma on the neutrophil oxidative burst and the role of albumin, immunoglobulins (Igs) and cytokines. Materials and Methods: Plasma was collected from chronic periodontitis patients (n=11) and periodontally healthy controls (n=11) and used with/without depletion of albumin and Ig or antibody neutralization of IL-8, GM-CSF or IFN-a to prime/stimulate peripheral blood neutrophils, isolated from healthy volunteers. The respiratory burst was measured by lucigenin-dependent chemiluminescence. Plasma cytokine levels were determined by ELISA. Results: Plasmas from patients were significantly more effective in both directly stimulating neutrophil superoxide production and priming for subsequent formyl-met-leu-phe (fMLP)-stimulated superoxide production than plasmas from healthy controls (p

T cell receptor signalling requires a reduced cell surface protein environment and increased reactive oxygen species (ROS) inhibit activation. The main objective of this study was to identify sulphenated proteins on the membrane of T cells at rest, during activation and under oxidising conditions. Naive CD4+ T cells were activated using anti-CD3/CD28 antibodies in the presence or absence H2O2 (20 µM) for 24 h. Activated CD4+ T cells in the presence of H2O2 show a significant decrease in CD25 expression by twofold and IL-2 secretion (0.67±0.14ng/ml versus 1.16±0.09ng/ml). NAC (10 mM) did not reverse the effect of H2O2 on IL-2 or CD25 after 24 h. CD4+ T cell-membrane proteins were isolated using MEM-PR kit after T cell activation with or without H2O2 and sulphenated proteins were captured by a dimedone based technique. Captured proteins were identified by LC-MS/MS and the table below shows the number of sulphenated proteins; Activation alone did not affect the number of sulphenated membrane proteins. Activation in the presence of H2O2, which associated with lower intracellular calcium flux, show a reduction in number of sulphenated proteins possibly due to disulphide bond formation.

Oxidized lipids have been recently recognized as important mediators that are involved in a wide range of cellular functions and stress responses. These lipid species are formed transiently and present in low concentrations in biological systems which creates challenges to detection techniques. Therefore, high quality of methods must be employed to improve understanding of the roles oxidised lipids. The recent development of high sensitivity mass spectrometry tools has enabled the field of lipidomics to reach a level that was impossible years ago. Due to a lack of bioinformatics tools to support high-throughput identification of previously unconsidered, oxidized lipids, the detection of oxidized lipids is mostly performed by targeted approaches. This approach can significantly improve the sensitivity for detection in limited biological sample volumes. In our laboratory, we have established mass spectrometry based targeted methods to analyse oxidised cholesterol (oxysterols), oxidised phospholipids and isoprostane species. Using these techniques, we have demonstrated discrete oxidised lipids fingerprints in plasma during health and chronic inflammatory diseases such as chronic kidney disease, Alzheimer's disease and hypercholesterolaemia (Dias et. al., Free Rad Biol 2018).

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-872aa]) and nonimmune (IL-877aa) cells. IL-877aahas low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-877aasignificantly enhance burst activation by fMLP and chemotactic activity (P< 0.05) but decrease burst activation and chemotactic activity of IL-872aatoward neutrophil-like HL60 cells and primary neutrophils (P< 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-877aaand the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.

WOS: 000367338700040, PubMed ID: 26381917, Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathics., (C) 2015 The Authors. Published by Elsevier B.V., European Cooperation in Science and Technology (COST Action)European Cooperation in Science and Technology (COST) [BM1203/EU-ROS]; Kidney Research UKKidney Research UK (KRUK) [PDF3/2014], Several authors of this review were supported by the European Cooperation in Science and Technology (COST Action BM1203/EU-ROS). HKID is funded by Kidney Research UK (PDF3/2014).

Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal tissues, alongside a reduced antimicrobial capacity, create a locally destructive chronic inflammatory cycle in Papillon-Lefévre syndrome.

Elevated low-density lipoprotein (LDL) concentration in mid-life increases the risk of developing Alzheimer's disease (AD) in later life. Increased oxidized LDL (oxLDL) modification and nitration is observed during dementia and hypercholesterolaemia. We investigated the hypothesis that statin intervention in mid-life mitigates the inflammatory effects of oxLDL on the microvasculature. Human microvascular endothelial cells (HMVECs) were maintained in transwells to mimic the microvasculature and exposed to patient and control LDL. Blood was obtained from statin-naive, normo- and hyper-lipidaemic subjects, AD with vascular dementia (AD-plus) and AD subjects (n=10/group) at baseline. Only hyperlipidaemic subjects with normal cognitive function received 40 mg of simvastatin intervention/day for 3 months. Blood was re-analysed from normo- and hyper-lipidaemic subjects after 3 months. LDL isolated from statin-naive hyperlipidaemic, AD and AD-plus subjects was more oxidized (agarose gel electrophoretic mobility, protein carbonyl content and 8-isoprostane F2α) compared with control subjects. Statin intervention decreased protein carbonyls (2.5±0.4 compared with 3.95±0.2 nmol/mg; P

Alzheimer's disease (AD) aetiology is complex with gene and environmental risk factor interaction. Our recent studies have confirmed that oxLDL is higher in vascular dementia patients and correlated with cognitive function. We hypothesised that endothelial cells of the blood-brain barrier are critical mediators of systemic nutrient effects within the brain. Therefore we have studied the effect of 27 hydroxycholesterol and F2alpha isoprostane on microvascular endothelial cell redox state, inflammatory cytokines and regulatory microRNA (miR) profile. We showed that lipids from patients with dementia or hypercholesterolaemia release directional inflammatory molecular signatures via an endothelial redox state-dependent mechanism. miR expression in endothelial cells with and without oxidised lipid treatment was undertaken using an Agilent DNA microarray scanner and microarray data was analysed using GeneSpring GX software. Upregulated miR showed an increase in oxidative stress and inflammatory pathways. Downregulated pathways included growth factor signalling. Using qPCR, we determined that miR-144 and 146 which are anti-inflammatory and redox regulating modulators were decreased by oxidised lipids. A neurotrophic factor-targeting miR was increased in expression. These data highlight that oxidised lipids have important regulatory effects on endothelial microvascular cell function.

Blood cholesterol levels are not consistently elevated in subjectswith age-related cognitive decline, although epidemiological studies suggest that Alzheimer's disease and cardiovascular diseases share common risk factors. These include the presence of an unusual genetic variant, the APOE4 (apolipoprotein E4) allele, which modulates LDL (low-density lipoproteins) metabolism, increases free radical formation and reduces plasma antioxidant concentrations. Together, these risk factors support a mechanism for increased LDL circulation time and free radical modification of LDL. Plasma oxycholesterols, hydroxylated metabolites of cholesterol, are carried by oxidized LDL, and elevated lipids in mid-life are associated with increased longterm risk of dementia. Although brain cholesterol metabolism is segregated from the systemic circulation, during oxidative stress, plasma oxycholesterols could have damaging effects on BBB (blood-brain barrier) function and consequently on neuronal cells. Cholesterol-lowering drugs such as statins may prevent the modifications to LDL in mid-life and might show beneficial effects in later life. © The Authors Journal compilation © 2014 Biochemical Society.

Elevated serum cholesterol concentrations in mid-life increase risk for Alzheimer's disease (AD) in later life. However, lower concentrations of cholesterol-carrying high density lipoprotein (HDL) and its principal apolipoprotein A1 (ApoA1) correlate with increased risk for AD. As HDL transports oxocarotenoids, which are scavengers of peroxynitrite, we have investigated the hypothesis that lower HDL and oxocarotenoid concentrations during AD may render HDL susceptible to nitration and oxidation and in turn reduce the efficiency of reverse cholesterol transport (RCT) from lipid-laden cells. Fasting blood samples were obtained from subjects with (1) AD without cardiovascular comorbidities and risk factors (AD); (2) AD with cardiovascular comorbidities and risk factors (AD Plus); (3) normal cognitive function; for carotenoid determination by HPLC, analysis of HDL nitration and oxidation by ELISA, and 3H-cholesterol export to isolated HDL. HDL concentration in the plasma from AD Plus patients was significantly lower compared to AD or control subject HDL levels. Similarly, lutein, lycopene, and zeaxanthin concentrations were significantly lower in AD Plus patients compared to those in control subjects or AD patients, and oxocarotenoid concentrations correlated with Mini-Mental State Examination scores. At equivalent concentrations of ApoA1, HDL isolated from all subjects irrespective of diagnosis was equally effective at mediating RCT. HDL concentration is lower in AD Plus patients' plasma and thus capacity for RCT is compromised. In contrast, HDL from patients with AD-only was not different in concentration, modifications, or function from HDL of healthy age-matched donors. The relative importance of elevating HDL alone compared with elevating carotenoids alone or elevating both to reduce risk for dementia should be investigated in patients with early signs of dementia.

Individuals with both Type 1 and Type 2 diabetes show vascular complications that may remain undetected for many years until the disease is at an advanced stage. Clinical trials to date have been unsuccessful in identifying a therapeutic approach that addresses both the underlying problem of poor glycaemic control in diabetes and the high incidence of microvascular disease. Oxidative stress, an imbalance of oxidants and antioxidants in the favour of oxidants, has been thought to play a central part in the complications of diabetes arising from protein glycation. Neutralization of oxidants by increased antioxidant availability has been considered as one possible way to mitigate oxidative stress. Indeed, several human intervention studies have been undertaken to determine whether dietary antioxidants can exert beneficial effects for patients with Type 2 diabetes. Chronically elevated concentrations of glucose increase the frequency of non-enzymatic glucose adduction with proteins, lipids and DNA to form advanced glycation end products (AGEs). One of the major plasma proteins that can undergo secondarymodification by glucose or oxidised lipids is the cholesterolcarrying low density lipoprotein (LDL). In Type 2 diabetes, LDL is more glycated and more susceptible to oxidation, increasing its clearance bymacrophages in a nonregulated manner and increasing foam cell formation. Complications such as albuminuria are associated with renal protein oxidation, which is often preceded by LDL fatty acid oxidation. The initiator is H2O2 produced from protein glycation, auto-oxidation of homocysteine and increased metabolism of arachidonic acid towards its pro-inflammatory eicosanoids. The backdrop of increased oxidative stress promotes a state of diffuse vasculopathy. In both Type 1 and Type 2 diabetes, there is an increased risk of cognitive impairment in later life. In the glucose control intervention study, cognitive function was an independent predictor of glycaemic control, but the risk of cognitive decline or dementia was not decreased by improving glycaemic control. AGEs, carbonylation of LDL, which is indicative of oxidative stress, and glycation are shared common risk factors for both diabetes and cognitive impairment.

The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites., Highlights • Loss of redox homeostasis may affect the secretome content and protein concentration, transmitting redox signals to distant cells through extracellular vesicles. • Damaged glycoforms may arise from oxidants or aberrant biosynthetic regulation. • Reactive species generation by NOX and NOS is controlled through redox regulation. • Cell surface and plasma thiol-oxidised proteins can be reduced and their activity modulated by thioredoxin, protein disulphide isomerase and reductases.

Oxidative stress has been implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease. The transcription factor, Nrf2 (nuclear factor E2-related factor 2) that binds to the antioxidant responsive element (ARE) activates a battery of genes encoding enzymes and factors essential for neuronal survival. We have investigated the hypothesis that a downstream product of cyclooxygenase(COX-2), 15-deoxy-delta (12, 14)-prostagland in J2 (15d-PGJ2) has protective effects by activating the Nrf2 pathway during oxidative stress.Human neuroblastoma cells (SHSY5Y) were differentiated intoneuronal-like cells as described previously (Gimenez-Cassina et al.,2006). SHSY5Y cells were co-treated with 10 mM buthionine sulfoximine (BSO) 7 10 mM 15d-PGJ2. Cell viability was measured by MTT assay and cellular glutathione (GSH) levels were measured after treating cells for0.5-24 hours by GSH recycling assay. Cellular Nrf2 levels were determined by immunoblotting. IL-6 levels were measured by ELISA.15d-PGJ2 alone lowered GSH levels 30min after the treatment(12.870.64 nmol/mg protein) and returned to untreated control levels at 16hours (28.173.6 nmol/mg protein; Po0.01). Compared to intracellular GSH levels in untreated cells (27.871.8 nmol/mg protein) BSO treatment alone significantly decreased GSH (9.672.1 nmol/mg protein;Po0.001) but co-incubation with 15d-PGJ2 for 24 hours prevented the depletion elicited by BSO(21.372.7 nmol/mg protein). Compared to untreated cells BSO treatment decrease dIL-6 secretion (from 0.941.6ng/ml to 0.6971.3ng/ml) and total Nrf2 protein levels (by21%). Co-incubation with15d-PGJ2 for 24 hours with BSO did not change IL-6(0.6771.4ng/ml) or total Nrf2 level at any time point. This study suggests that neuronal toxicity resulting from glutathione depletion canbere stored by the induction of Nrf2-ARE pathway and the role of the Nrf2 signalling merits further investigation in neurodegenerative diseases.

The ageing process results from a complex interplay between genes and the environment that can precipitate an uncontrolled inflammation. Epigenetic changes are believed to provide a link between the environment and nutrition to gene expression by altering the activity of some histone-modifying protein. Epigenetic modifications of DNA and histone proteins have been proposed as important contributory mechanisms to the retention of metabolic memory over time. A thorough understanding of the posttranscriptional and epigenetic factors involved in both normal ageing and age-related disease may inform new strategies and approaches to diagnose, treat, or suppress many aspects of age-dependent frailty.

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al.

Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2−• scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2−•.To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4 h hypoxia with and without 2 h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage., Graphical abstract ROS and RNS in cell survival and death during hypoxia and reoxygenation; the role of Nrf2. There is evidence of superoxide anion radical and nitric oxide production in both hypoxia and H/R. While MnTBap inhibited the toxicity of both stressors, L-NAME only protected against the toxicity of hypoxia implying that nitric oxide was required for survival during H/R. Both superoxide anion radical and nitric oxide elicited Nrf2 activation during H/R and to a lesser extent during hypoxia. Nrf2 knockdown prevented GSH induction and also reduced survival during H/R by 50% but only by 30% during hypoxia. RNS are essential for Nrf2 activation but may also exert cytoprotective effects during H/R by other means., Highlights • Cardiomyoblast toxicity during hypoxia is dependent on O2−• and NO•. • Nrf2 activation is important for cardiomyoblast survival during hypoxia or hypoxia/reoxygenation, but, restoration of GSH is not required. • NOS activity is essential for the adaptation of cardiomyoblasts to hypoxia/reoxygenation but survival may be independent of Nrf2.

During ageing an altered redox balance has been observed in both intracellular and extracellular compartments, primarily due to glutathione depletion and metabolic stress. Maintaining redox homeostasis is important for controlling proliferation and apoptosis in response to specific stimuli for a variety of cells. For T cells, the ability to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols. Here we describe the effects of depleting intracellular glutathione concentration for T cell exofacial expression of thioredoxin 1 and IL-2 production, and have determined the distribution of Trx1 with ageing. Using buthionine sulfoximine to deplete intracellular glutathione in Jurkat T cells we show using Western blotting that cell surface thioredoxin-1 is lowered and that the response to the lectin phytohaemagglutinin measured by ELISA as IL-2 production is also decreased. Using flow cytometry we show that the distribution of Trx1 on primary CD4+ T cells is age-dependent, with lower surface Trx1 expression and greater variability of surface expression observed with age. Together these data suggest that a relationship exists between the intracellular redox compartment and exofacial surface. Redox imbalance may be important for impaired T cell function during ageing.

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.