Unfolded stress response (UPR) is a conserved cellular pathway involved in protein quality control to maintain homeostasis under different conditions and disease states characterized by cell stress. Although three general schemes of and genes induced by UPR are rather well-established, open questions remain including the precise role of UPR in human diseases and the interactions between different sensor systems during cell stress signaling. Particularly, the issue how the normally adaptive and pro-survival UPR pathway turns into a deleterious process causing sustained endoplasmic reticulum (ER) stress and cell death requires more studies. UPR is also named a friend with multiple personalities that we need to understand better to fully recognize its role in normal physiology and in disease pathology. UPR interacts with other organelles including mitochondria, and with cell stress signals and degradation pathways such as autophagy and the ubiquitin proteasome system. Here we review current concepts and mechanisms of UPR as studied in different cells and model systems and highlight the relevance of UPR and related stress signals in various human diseases. Progress made in recent years has greatly increased our understanding about mechanisms of protein homeostasis at the organelle level such as the endoplasmic reticulum (ER) and mitochondria observed during cell stress and in disease. In the ER, the unfolded protein response (UPR) exerts an adaptive function in protein quality control to ensure the proper synthesis, secretion, and correct folding of proteins  caused by misfolded/mutant proteins, or by alterations in signaling networks or in the ionic balance such as calcium in the cell. Three major signaling pathways characterize the UPR; the inositol-requiring enzyme 1α (IRE1α), the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and the activating transcription factor 6  is a protein kinase that phosphorylates downstream targets such as the eukaryotic translation initiation factor-2α (eIF2α). This leads to a reduction in general protein synthesis with a decrease in the load of misfolded/mutant proteins in the ER. However, the translational block is not complete and a subset of mRNAs including that encoding the transcription factor ATF4 are still being translated leading to increased expression of among others the growth arrest and DNA damage-inducible 34 (GADD34), the transcription factor C/EBP homologous protein (CHOP) as well as pro-apoptotic BCL-2 family proteins. GADD34, also called PPP1R15A, is a phosphatase for p-IF2α with the ability to restore protein synthesis. The degree of eIF2α phosphorylation is dynamically regulated in the cell and this can be interfered with using specific compounds to control ER stress in various diseases (see below).The timing of induction of the different UPR pathways and their mutual interactions are important issues and ultimately determine the cell response to stress. Thus, an early and transient increase in IRE1α mediates cell protection, whilst an early-onset increase in PERK-ATF4 causes cell degeneration . ER stress is part of the normal development of the brain and of notochord in medaka, caused by the preponderate synthesis of extracellular matrix (EM) proteins required for organogenesis  protein family are transcription factors that are latent in the ER and activated in a process resembling that of ATF6  is a transcription factor residing latent in the ER membrane involved in the regulation of expression of antioxidants and ERAD components. NRF1 shares many of its activities with the related factor NRF2, but NRF1 can specifically increase proteins associated with the proteasomes to promote protein degradation. NRF1 can be activated by different factors and events including ER stress  is a transmembrane ER resident protein encoded by the WFS1 gene that in a mutant form causes Wolfram syndrome 1 (WS1), an autosomal recessive degenerative disease characterized by insulin-dependent diabetes mellitus, optic atrophy, deafness and various neurological symptoms  initiates a cascade of reactions to promote nuclear transcription of mitochondrial chaperones and protective factors for organelle repair and homeostasis. Recently it was shown using myoblasts that the catalytic ATP-dependent Clp protease ATP-binding subunit (ClpX) in the protease complex, ClpXP, degrades misfolded/unfolded or oxidized proteins in the mitochondria during mammalian mitochondrial stress   is a key factor in the control of UPR(mt) mediating a crosstalk between mitochondria and nucleus. The protein is normally imported into the mitochondria but during cell stress the mitochondrial import machinery is impaired increasing ATFS-1 in the nucleus inducing expression of UPR(mt)-associated genes , PD, amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD) have all distinct clinical signs band are characterized by the accumulation of misfolded and aggregated proteins  in brain cortex undergo cell degeneration by mechanisms only partly understood. Transgenic mice expressing fluorescent labeled ubiquitin C-terminal hydrolase-L1 (UCHL1) in the CSMNs showed that these neurons are particularly sensitive to ER stress and to the lack of UCHL1 the ALS-associated gene, In vivo sephin1 was neuroprotective in the mutant SOD1-ALS mouse model, and improved behavior with no obvious adverse effects  and increasing cell survival  peptides and with intracellular neurofibrillary tangles of hyperphosphorylated tau. The mechanisms for cell toxicity in AD are complex and related to changes in synaptic transmission, an altered calcium homeostasis, increased ER stress and a chronic state of neuroinflammation  and in animals  caused by the infectious prion protein  in the brain. Glutamate acts via different types of glutamate receptors and thereby influences cell signaling cascades and elevates intracellular calcium in neurons. Increased ER stress is linked to brain ischemia . The pathophysiology of BD is complex and the disease is associated with other mental as well as metabolic disorders  or cones (responsible for day-light and color vision) are often caused by mutations in specific genes involved in phototransduction or in protein quality control  is a genetically heterogeneous disorder that primarily affects the rods leading to blindness. Mutations in rhodopsin is a common cause of RF and induce an increase in UPR particularly in IRE1α/XBP1 signaling attempting to reduce the amount of mutant rhodopsin in the ER  that can occur at any age. It is though that this can be the result of an autoimmune process or caused by reactive oxidative species and subsequent ER stress mediated cell degeneration  and TLR4 in macrophages can activate IRE1 and induced XBP1 splicing increasing the production of proinflammatory cytokines contributing the inflammation  in peripheral tissues  was found to mitigate ER stress and reduce the amount of atherosclerotic lesions in vivo  with the responses in other organs. In many metabolic diseases ER stress and UPR are activated in hepatocytes that eventually can lead to liver damage. UPR in hepatocytes is important to balance fluctuations in blood glucose levels and under diabetic conditions sustained UPR leads to ER stress in the liver  is characterized by hyperglycemia, increased proteinuria and UPR activation causing podocyte malfunctions and increased cell stress, with advanced glycation end products and free fatty acids present in the kidney (Zhuang and Forbes, C. elegans the XBP1 pathway regulates resistance to ER stress and lifespan in conjunction with the DAF-16/FOXO signaling (Ben-Zvi et al., C. elegans (Taylor and Dillin, Caloric restriction and altered metabolic signaling including insulin/insulin-like growth factor, and mTOR pathways have been linked to longevity in different organisms (Henis-Korenblit et al., Saccharomyces cerevisiae have shown that that damaged and aggregates proteins are segregated between mother and daughter cells so that potentially harmful proteins are eliminated from the progeny. Vacuole fusion in the yeast is important for this process that is controlled by asymmetry-generating genes including the myosin-dependent adaptor protein Vac17 (Hill et al., Studies of aging and protein quality control in the yeast, Rapidly growing tumor cells face different types of environmental stressors in form of hypoxia, glucose deprivation, inadequate vascularization, and metabolic challenges. In addition to the external stressors, internal activation of oncogenes, accumulation of somatic mutations, genome instability, changes in chromosome number lead to the additional demands for ER pathways. Tumor cells also adapt to the relative lack of glucose by an increased rate of aerobic glycolysis known as the Warburg effect. To overcome restrains for growth, cancer cells mount an increase in UPR to cope with cell stress and to counteract cell death (Vandewynckel et al., As ER stress has obvious role in the survival of cancer cells, IRE1α, and PERK are attractive candidates for drug development (Maly and Papa, Potent and selective inhibitors of PERK's kinase domain have also been developed, and one such compound, GSK2656157 revealed promising anti-tumor effects in preclinical models of pancreatic adenocarcinoma and multiple myeloma (Atkins et al., UPR as part of the cell protein quality control system is an important process to optimize cell functions and correct aberrations caused by cell stress. Recent developments in the field have shown that UPR plays a role not only in different diseases but also in physiological processes including the regulation of cell metabolism, inflammation, aging and neuronal plasticity, and cognition. Figure All authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genome-wide association studies have confirmed the polygenic nature of schizophrenia and suggest that there are hundreds or thousands of alleles associated with increased liability for the disorder. However, the generalizability of any one allelic marker of liability is remarkably low and has bred the notion that schizophrenia may be better conceptualized as a pathway(s) disorder. Here, we empirically tested this notion by conducting a pathway-wide association study (PWAS) encompassing 255 experimentally validated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among 5033 individuals diagnosed with schizophrenia and 5332 unrelated healthy controls across three distinct ethnic populations; European-American (EA), African-American (AA) and Han Chinese (CH). We identified 103, 74 and 87 pathways associated with schizophrenia liability in the EA, CH and AA populations, respectively. About half of these pathways were uniquely associated with schizophrenia liability in each of the three populations. Five pathways  were shared across all three populations and the single-nucleotide polymorphism sets representing these five pathways were enriched for single-nucleotide polymorphisms with regulatory function. Our findings provide empirical support for schizophrenia as a pathway disorder and suggest schizophrenia is not only a polygenic but likely also a poly-pathway disorder characterized by both genetic and pathway heterogeneity. Thus, identifying schizophrenia-associated SNPs that are generalizable to diverse populations of people with the disorder is unlikely and suggests alternative approaches at identifying genetic markers of schizophrenia liability are needed.Schizophrenia is a severe psychiatric disorder characterized by significant genetic heterogeneity and is commonly referred to as a polygenic disorder.3 whereby one or a number of SNPs within a pathway could result in an increased liability to schizophrenia by altering sensitivity to environmental insults and/or disruption of brain development. In the context of schizophrenia, this pathway approach has been applied in a variety of forms ranging from pathway clustering analysis,4 where SNPs in key genes within a single pathway are examined, to post-hoc pathway enrichment analyses of candidate SNP-sets using the SNP ratio test5 or bioinformatics resources .8 These approaches undoubtedly have a pathway focus but provide an incomplete examination of the compendium of known human biological pathways. Our primary aim was to conduct a comprehensive pathway-wide association study (PWAS) of schizophrenia. Here, we report results of that analysis in which we tested 255 biological pathway-based SNP-sets for their association and potential function in schizophrenia in three ancestral distinct populations.One such approach is to examine SNPs within the biological pathway(s) in which they reside. Underlying this approach is the notion that schizophrenia is a pathway(s) disorder,n=5033) and healthy controls (n=5332) across three distinct ethnic populations; European-American (EA) , Han Chinese (CH)  and African-American (AA)  were obtained . CH GWAS data were collected from multiple collaborating hospitals included in the Chinese Schizophrenia Collaboration Group  and the other a validation dataset .GWAS data from individuals with schizophrenia . A mega KEGG pathway  that encompasses several other pathways was excluded, leaving 300 pathways available for further analysis.SNPs surviving quality control were mapped to gene loci using the annotation provided by the National Center for Biotechnology Information (see 14 For each pathway the discovery dataset for the EA and CH cohorts as well as the single dataset available for the AA cohort were randomly split (maintaining the case:control ratio of the full dataset) 100 times into two subsets, a SNP  selection set (80% of the participants) and a test set (20% of participants). Within each SNP selection set, 80% of participants were randomly selected 10 times (maintaining the case:control ratio of the full dataset) and the resulting subsets were subjected to the maximum relevance minimum redundancy (mRmR) feature selection procedure  using 80% of the discovery dataset followed by testing of the classifiers in the remaining 20% of the discovery dataset. To address inherent imbalances in the case:control ratios of our datasets, under-sampling of the majority class (cases or controls) of each dataset was employed before running the random forest algorithm, as this strategy has previously been shown to be useful for classification in the presence of imbalanced classes.P-values for each of the 255 pathways were generated by comparing the mean OR (based on 100 iterations) from the independent  dataset with the corresponding mean OR from the permutation dataset using a t-test. The Benjamini–Hochberg (BH) procedure was used to adjust for multiple comparisons.18 Furthermore, using the independent validation dataset for the EA and CH cohorts, we calculated the Nagelkerke R2  and odds ratio (OR)) were calculated for each of the pathways using the independent validation dataset for EA and CH cohorts, and the 20% test set for the AA cohort. To further evaluate our pathway analysis pipeline, we selected 129 previously identified gene ontology (GO) pathways associated with schizophrenia and applied our pipeline to each of the 129 pathways in all three populations.19 as well as the functional annotation information obtained from the RegulomeDB, a database that annotates SNPs with known and predicted regulatory elements.20 We hypothesized that the selected features with greater appearance rates within significant schizophrenia liability pathways would be enriched for functional SNPs compared with SNP sets derived from non-significant pathways.To assess whether the mRmR feature selection approach was capable of selecting informative features and to evaluate the potential functional relevance of selected features, we utilized the brain expression quantitative trait loci (eQTLs) dataset obtained from the genotype-tissue expression (GTEx) portal v6.0,cis-eQTLs using genotype and gene expression data derived from human post-mortem frontal cortex (Brodmann area 9) of 92 donors included in the GTEx portal.19 For each of the three cohorts, SNPs within each of our candidate pathways was assigned an appearance rate based on the number of times (out of 100 iterations) the SNP represented the candidate pathway during our feature selection procedure  described above. SNPs were then grouped into quartiles based on their appearance rate  and the proportion of SNPs within each quartile associated  with expression of its corresponding gene was calculated. For comparison, the same analysis was conducted on 152, 181 and 168 non-significant pathways in the EA, CH and AA population, respectively. A one-sample t-test was used to determine if the proportion of eQTLs observed in our overlapping pathway SNP sets differed from the SNP sets derived from non-significant pathways.SNP sets representing pathways associated with schizophrenia in all three cohorts were further assessed as potential http://www.regulomedb.org).20 Similar to the eQTL analysis, SNPs were grouped into quartiles based on their appearance rate and a weighted Regulome score was computed for each quartile group as well as SNP sets derived from non-significant pathways. A one-sample t-test was used to determine if the weighted Regulome score in our candidate SNP sets differed from SNP sets derived from non-significant pathways  and DNA–RNA interaction , we utilized the RegulomeDB (R2=0.03–0.57%) of the variance in the liability to schizophrenia (R2=0.31–0.57%), although when features from all significant pathways were assessed the variance explained ranged from 0.66% in the EA cohort to 2.46% in the CH cohort. Furthermore, among the 129 previously identified schizophrenia-associated GO pathways our analysis pipeline replicated 45, 20 and 56 of these pathways in the EA, CH and AA populations  of the pathways we tested were uniquely associated with schizophrenia liability in only one of the three populations we examined—raising the possibility that schizophrenia is not only a polygenic but also a poly-pathway disorder. In fact, all pathways had an OR<1.35, suggesting multiple pathways of small effect collectively contribute to schizophrenia liability.Pathway heterogeneity has only recently been discussed in the context of schizophreniaFurthermore, our results suggest disruption of certain pathways may be necessary (but perhaps not sufficient) for the development of schizophrenia across populations. About one-fourth (27%) of the pathways we tested were associated with liability to schizophrenia in two or more of the populations, among which five pathways were associated with schizophrenia liability in all three cohorts. These pathways included the serotonergic synapse, ubiquitin mediated proteolysis, hedgehog signaling, renin secretion and adipocytokine signaling, all of which have been implicated in schizophrenia and/or related phenotypes.24 Thus, identification of the serotonergic synapse pathway in the current study is perhaps not surprising. In fact, the largest schizophrenia GWAS to date found SNPs in three genes  within the serotonergic synapse pathway reached GWAS significance (P<5 × 10−8) and SNPs in another 17 genes within this pathway were nominally significant (P<1 × 10−5).2 Furthermore, a recent gene-set enrichment analysis of the SZGene database25 identified 24 pathways significantly enriched for schizophrenia candidate genes among which the serotonin receptor signaling pathway was ranked second.7A number of post-mortem, functional neuroimaging and peripheral biomarker studies have implicated the serotonergic system in the pathophysiology of schizophrenia , a critical system for the removal of damaged/toxic proteins in the cell, has been shown to be dysregulated at the transcript37 dopaminergic neuron development,38 and promotes brain expression of disc1, a candidate gene in schizophrenia.39 The pathway's most well characterized ligand, sonic hedgehog, regulates the generation of functional synaptic contacts,41 and is abundant in the adult human central nervous system.43 Furthermore, hedgehog signaling interacts with the UPP45The hedgehog signaling pathway is a key regulator of oligodendrocyte production,46 and in general exhibit dysregulation of body temperature.47 Rodent studies have demonstrated renin is also synthesized in the brain48 and has considerable effects on anxiety-related behaviors and cognition .49 In the brain, renin is proposed to enzymatically process angiotensinogen to angiotensin, which is then further processed by angiotensin-converting enzyme (ACE).50 ACE activity has been shown to modulate dopamine turnover51 and abnormal levels of ACE in cerebrospinal fluid have been reported in individuals with schizophrenia,53 albeit potential neurotropic and length of illness effects have been noted.55 The interaction between angiotensin II (AT II), a neuropeptide substrate for ACE, and central dopamine has also been associated with schizophrenia.57 Moreover, numerous genetic studies suggested polymorphisms in ACE are associated with susceptibility to schizophrenia and major depression.62The renin secretion pathway is typically associated with regulation of arterial blood pressure, thirst and thermoregulation via the kidney secreted enzyme renin and its interaction with the renin–angiotensin–aldosterone system. Epidemiological studies have reported up to 25% of schizophrenia have polydipsia (excessive thirst)63 hundreds of adipocytokines have been found, such as adiponentin, tumor necrosis factor-alpha and members of the interleukin family. Increased expression of tumor necrosis factor-alpha and a number of interleukins have recently been proposed as markers of schizophrenia in brain64 and blood.65 Furthermore, adipocytokines are recognized not only as regulators of energy metabolism, but also as factors that may be associated with mental disorders. Decreased serum levels of adiponectin have been identified in major depressive disorder and schizophrenia,68 and serum levels of leptin correlate with less severe positive symptoms in schizophrenia patients69 and may regulate the mesolimbic dopamine system.70The adipocytokine signaling pathway is a collective destination of cytokines secreted by the adipose tissue. Since the first adipocytokine leptin was discovered in 1994,71 Third, the dopamine hypothesis of schizophrenia is an enduring, widely accepted, idea but among the three populations we studied the dopaminergic synapse pathway was only a significant liability pathway in the EA  and CH  populations. Although, we failed to detect this pathway in the AA population, the expected trend was evident  and is likely a result of the smaller sample size available for this population. Fourth, our analysis did not look at potential clinical subtypes of schizophrenia based on symptom profiles, despite recognition that schizophrenia is a broader spectrum disorder including a range of symptoms. A PWAS of clinical subtypes may lead to stronger associations by reducing the noise associated with the broad schizophrenia phenotype. However, the clinical symptom data available for this study was inconsistent or minimal across the three cohorts inhibiting such an analysis. Fifth, SNPs eligible for inclusion in our analysis were limited to those that were within a gene using a narrow ‘5 and 3' intergenic window . Intergenic SNPs are known to play functional regulatory roles on genes72 and as such exclusion of more distal intergenic SNPs may have biased our results. In the most recent schizophrenia GWAS,2 45% (57) of the 128 SNPs identified were intergenic but 52% (30) of these intergenic SNPs are in linkage disequilibrium (R2⩾0.50) with one or more SNPs within a gene according to our intergenic window. Thus, our intergenic window was capable of capturing a majority  of the 128 SNPs, suggesting the bias conferred by our SNP inclusion criteria are likely modest. Finally, our feature selection procedure (mRmR) resulted in the loss of many pathways containing smaller SNP pools. In total, 45 pathways cataloged within KEGG were not included in our pathway association study as the number of SNPs selected to represent these pathways was fewer than the required number of SNPs  to run our random forest algorithm.Despite the novelty and many strengths of our study, our findings should be interpreted in the context of several limitations. First, the detection of population differences in schizophrenia liability at the pathway level may, in part, be a result of sampling, allelic frequency and/or linkage disequilibrium differences across the populations studied. These potential confounding factors may also explain why we only identified five overlapping pathways rather than the expected 10 overlaps given the number of significant pathways identified in each population. Although we attempted to reduce these confounding influences by selecting features independently in each population using an mRmR approach, complete restraint of these confounds is not possible and as such our results should be interpreted with caution. Second, an independent validation dataset was not available for the AA population and as such all estimates were based on the 20% holdout dataset derived from the discovery dataset. This may have resulted in over-estimation of associations within this population, although the random forest algorithm we employed internally mitigates this potential bias via the out-of-bag error estimate mechanism.In conclusion, our results empirically support the notion that schizophrenia is a pathway disorder and further suggest that there is a considerable amount of pathway heterogeneity within and across different ethnic populations. We also identified five pathways that may serve as harbors of genotypic markers for schizophrenia across populations. However, future application of our pathway-wide association approach in larger cohorts as well as among ethnic groups not examined here are required before firm conclusions can be drawn.


Markers of oxidative stress are increased in chronic obstructive pulmonary disease (COPD) and reactive oxygen species (ROS) are able to alter biological molecules, signaling pathways and antioxidant molecule function, many of which have been implicated in the pathogenesis of COPD. However, the involvement of ROS in the development and progression of COPD is not proven. Here, we discuss the sources of ROS, and the defences that have evolved to protect against their harmful effects. We address the role that ROS may have in the development and progression of COPD, as well as current therapeutic attempts at limiting the damage they cause. Evidence has indicated that the function of several key cells appears altered in COPD patients, and expression levels of important oxidant and antioxidant molecules may be abnormal. Therapeutic trials attempting to restore equilibrium to these molecules have not impacted upon all facets of disease and whilst the theory behind ROS influence in COPD appears sound, current models testing relevant pathways to tissue damage are limited. The heterogeneity seen in COPD patients presents a challenge to our understanding, and further research is essential to identify potential targets and stratified COPD patient populations where ROS therapies may be maximally efficacious. Chronic obstructive pulmonary disease (COPD) is a chronic respiratory lung condition with significant systemic manifestations and associated co-morbidities that deleteriously impacts on quality of life . It is cSmoking and air pollution have been identified as significant initiating and risk factors for COPD and cell damage and death seen in COPD airways has been attributed to oxidative and carbonyl stress . Only 152·−) which contain unpaired electrons, the unstable nature of these ROS permit transfer of electrons to other molecules via oxidation, resulting in damage, inactivation or creation of further ROS. Potential targets for damage by ROS include proteins, lipids or DNA. Lungs are particularly vulnerable to oxidative stress due to the relatively high oxygen environment, increased blood supply and exposure to environmental pathogens and toxins. Cigarette and biomass smoke add significantly to this burden, a single puff of cigarette smoke is estimated to have in excess of 1 × 1015 oxidant molecules within [Oxidative stress occurs when exposure to free radicals is sufficient to overwhelm antioxidant defences. Such free radicals, termed reactive oxygen species (ROS), are ubiquitous, arising during mitochondrial respiration, signaling and when contributing to the damage and destruction of pathogens. Common ROS include hydroxyl radical (·OH) and superoxide anion , whilst subunits p40ph°x, p47ph°x, p67ph°x exist as a complex in the cytosol. Activation of a phagocyte via pattern recognition receptors (PRRs) on the cell surface, such as toll-like receptors, results in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling and subsequent phosphorylation of p47ph°x, initiating translocation of the cytosolic complex to the membrane. Activation of NOX2 is completed by the co-localisation of two final proteins, Rac2 and Rap1A. Upon activation, cytosolic Rac2 binds guanosine triphosphate and translocates to the membrane with p40ph°x, p47ph°x and p67ph°x, whereupon it associates with cyt b558 and Rap1A. As the process of activation of PRRs also initiates phagocytic envelopment of the microbes, and fusion with a lysosome, the membrane localisation of the assembled NOX2 protein becomes the membrane of the phagolysosome. Active NOX2 transfers an electron from NADPH to O2 resulting in the formation of superoxide anion (O2·−) which is released into the phagolysosome [Antimicrobial ROS production in phagocytic cells relies upon nicotinamide adenine dinucleotide phosphate(NADPH)-oxidase (NOX2). NOX2 consists of multiple subunits, spatially separated and inactivated in a resting phagocyte in order to prevent ROS generation in the quiescent state. Subunits gp91lysosome . This pr2O2), catalysed by the enzyme superoxide dismutase (SOD) in the following Reaction (2):The superoxide anion is unstable due to the absence of a hydrogen ion and resolving this state is energetically favourable, resulting in a willingness of superoxide to bind to other substances in order to share electrons. Direct oxidation via superoxide is considered to contribute little to oxidative stress, in particular because of the rapid dismutation of superoxide into hydrogen peroxide . Peroxynitrite is an extremely damaging molecule thanks to its enhanced stability, allowing it to diffuse throughout tissues where it is capable of oxidising multiple targets, including protein thiols [A further species of ROS common in phagocytes is nitric oxide (·NO), this is readily produced via inducible nitric oxide synthase following the stimulation of phagocytes by pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and tumour necrosis factor (TNF)α . It is an thiols , and tyrn thiols . 2O2, producing highly oxidising hypochlorous acid (HOCl). Furthermore, iron and other metal ions can catalyse the production of hydroxyl radicals from H2O2 via Haber–Weiss and Fenton reactions. This chain of oxidation events leads to the formation of reactive nitrogen and carbonyl species (RNS and RCS) alongside ROS, with similar damaging potential. This pathway of events and chief ROS outcomes are illustrated in Phagocyte lysosomes also contain the enzyme myeloperoxidase  which caEnvironmental derived ROS are common at the lung epithelium, found not only within cigarette smoke and combustion of organic matter , but alsReactive species and by-products are capable of altering thiols  and aminProduction of ROS is not limited to pathogen defence. ROS are key in mitochondrial respiration as part of the electron transport chain, during which electrons are passed from donors to acceptors in the process powering the transfer of protons across membranes. Under certain conditions (reviewed elsewhere ), ROS ca2O2, which is reversible with glutathione and other thiols [ROS are also involved in cell signalling. Typical intracellular signalling occurs on the macromolecular level, proteins interacting with proteins, ligands with receptors, with shape and surface charge being key to specificity. By their nature, ROS react indiscriminately with vulnerable atomic sequences, contrasting with the traditional view of specificity required in signalling. However, this indiscriminate, atomic level signalling allows for influence in multiple pathways simultaneously, permitting a more nuanced response from effector proteins, responsive to intracellular conditions and other pathways not directly linked via macromolecules. For example, protein tyrosine phosphatases (PTPs) can be inactivated in the presence of Hr thiols . Via thir thiols  plays a 2O and the much less damaging vitamin E radical. Ascorbate acts by donating electrons to lipid peroxyls, or to vitamin E radical, returning them to safe states, whilst itself becoming the very stable ascorbate radical [ROS can be inactivated by both enzymatic and non-enzymatic means, and these are present in abundance in health. Non-enzymatic antioxidant defence consists of antioxidant molecules (ascorbic acid), metal binding proteins, sacrificial proteins and unsaturated lipids, which act as electron donors or recipients. Two such antioxidant molecules found in lung epithelial lining fluid are vitamin C (ascorbate) and vitamin E (tocopherol). Tocopherol acts as an antioxidant by donating an electron from its aromatic ring to ·OH to produce H radical .Antioxidant sacrificial proteins, such as albumin and mucin in epithelial fluid, and glutathione within cells, also function as antioxidants by the nature of their surface exposed methionine and cysteine residues. These residues are readily oxidised by a number of ROS ,37, scav2− converting it to H2O2. It functions as an antioxidant by removing superoxide anions before they have the time to cause damage, or the opportunity to interact with other radicals to produce extremely damaging peroxyl radicals. However, in this process, H2O2 is produced, itself less damaging than O2−, but H2O2 can take part in Haber–Weiss and Fenton reactions to produce ·OH hydroxyl radicals which very readily react with substrates to become hydroxyl groups  acts as a hydrogen ion donor, becoming glutathione disulphide (GSSG), the donated hydrogen ion can then inactivate the ROS molecule.Enzymatic antioxidants include superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). Superoxide dismutase in its three forms  has very high affinity for Ooups see . The enzAntioxidant molecules are maintained at very high concentrations in vivo in order to rapidly swamp ROS that are released extracellularly. Albumin is the most abundant protein in serum, whilst glutathione levels in alveolar lining fluid is approximately 140 times higher than in blood in healthy individuals  and thesA variety of methods have been employed to assess the presence of oxidative stress in the lungs of COPD patients, and there is clear evidence of an increased oxidative burden in COPD compared with non-smoking controls.2O2 is greatly increased in the exhaled breath condensate of COPD subjects compared to healthy controls [2O2 increased even further. Arachidonic acid, an abundant polyunsaturated fatty acid in cell membranes, can be peroxidised by free radicals in vivo, to form isoprostanes [1) [Exhaled breath condensate (EBC) is an effective method used to identify oxidative stress products found in the lungs . Multiplcontrols ,44 with rostanes , which crostanes . This strostanes ,48. A fuanes [1) , suggestSerum levels of MDA and GPx (determined by activity) correlate with COPD severity ,51, with1 [Using immunohistological staining, it is possible to visualise some products of oxidative stress within distinct cellular components of the lung, such as 4-hydroxy-2-nonenal (4HNE), an end product of lipid peroxidation, which readily forms adducts with several protein residues. The markers of nitrogen derived oxidative stress, nitrotyrosine and inducible nitric oxide synthase (iNOS) have been found to be increased in COPD  correlat1 . Neutrophils are thought to be key effector cells in COPD. Their numbers in COPD lungs are significantly increased , correlaThese altered functions provide a possible mechanism for the damage seen in COPD; as neutrophils migrate through lung tissues they are known to release proteinases and ROS in sequence as they move through complex tissues. Inaccurate migration may result in moving across a larger surface area, whilst undergoing increased degranulation. Products of degranulation include pro-inflammatory cytokines such as interleukin 8 (IL8), ROS and proteases such as neutrophil elastase  increasOf note, migration by neutrophils is known to be affected by ROS, which interact with the enzyme PTEN at the leading edge, reducing its activity , allowinThere are also important associations between neutrophil proteinases and ROS. Neutrophil elastase, released from primary granules during neutrophil activation, can digest many of the proteins in the extra cellular matrix , contribIn health, macrophages are the most common immune cell found in the peripheral lung , where tOnce recruited, differentiation of monocytes is influenced by environmental conditions and stimuli, typical pro-inflammatory cytokines such as bacterial fragments and interferon-γ encourage differentiation into M1 phenotype, often referred to as “classical” or pro-inflammatory, as opposed to anti-inflammatory M2 phenotype. In COPD, there is evidence of altered monocyte and monocyte derived macrophage function, including reduced phagocytosis  and an iAn element of M2 macrophage anti-inflammatory phenotype is the secretion of the cytokine interleukin(IL)-10. This has an anti-inflammatory effect on other monocytes by inducing the intracellular protein histone deacetylase(HDAC)2 to inhibit the transcription of the pro-inflammatory cytokine IL-8. Interestingly, Ito et al.  have sho2O2 and physiological in vivo antioxidants were absent. This has been linked to oxidation of an essential part of the caspase apoptosis pathway [Epithelial cells within the lung are likely to have an impact upon COPD progression, whilst there is little evidence of abnormal ROS production from the cells directly, lung epithelial cells are capable of contributing to the pro-inflammatory environment that is key to ROS production. Exposure to oxidative stress results in the release of pro-inflammatory cytokines ,84, whil pathway , caspase pathway ,88, prov pathway , and subThe functions of ROS in health can be linked logically to many features seen in COPD, as described in One common model is chronic exposure of mice to ozone, which results in the display of COPD-like pathological changes such as lung inflammation, airway hyper-responsiveness, and neutrophil and macrophage infiltration of the lung. Using these models, research has shown that various anti-inflammatory or antioxidant molecules ,92,93 ha2O2 induced mitochondrial dysfunction in healthy ASM but did not worsen COPD dysfunction. Mitochondrial targeted antioxidant treatment inhibited mitochondrial dysfunction in healthy patients, and reduced excessive proliferation and cytokine production of ASM cells isolated from COPD patients with moderate to severe disease. Furthermore, Belchamber et al. [Abnormal mitochondrial function related to ROS has recently been described in a number of studies supporting the hypothetical link between mitochondrial dysfunction and COPD. Wiegman et al.  studied r et al.  found evr et al. ,97 suggeMarkers of oxidative stress appear increased in COPD patients and it has been hypothesised that there may be an imbalance between oxidants and antioxidants in COPD that would contribute to disease pathogenesis and progression.Some studies report reduced GSH in induced sputum of stable COPD subjects when compared to age matched healthy smokers and non-smokers, this level was even lower in COPD subjects who were exacerbating . However1 decline in COPD. In vitro studies suggest that the 213 Gly SNP infers a resistance to post translational cleavage by furin proteases, which permits an extended half-life to SOD3, increasing tissue levels nine-fold or more [1 and FVC, markers of disease severity, independent of the SOD3 213Gly variant. These polymorphisms occurred in exon 1 and intron 1, occurring with complete linkage, but were not deleterious, as tissue levels of SOD3 remain similar as in health.Some studies have suggested that mutations in SOD genetics may have an impact upon COPD incidence or pathogenesis. Siedlinski et al.  found a  or more ,106 and  or more . Dahl et or more  describeCatalase activity has been shown to be significantly reduced in COPD sufferers compared to smokers and non-COPD subjects ,99. Bets1 [Some studies have described reduced levels of glutathione peroxidase in plasma of COPD patients , and lev1 . However1 , and the1 ,101 suppStudies of peripheral lung expression of inducible nitric oxide synthase (iNOS) (an enzyme responsible for creating ·NO) have reported no difference in iNOS expression in alveolar macrophages, however, type 2 pneumocytes expressing iNOS were more numerous in severe COPD , suggestCurrently, there are no clinically available treatments that prevent COPD progression. Studies of ROS related damage in COPD have been observational or described using cell and animal models, and therefore cannot prove causality. To further investigate the role of ROS in COPD pathogenesis, researchers have instigated clinical trials of antioxidants to determine efficacy on characteristic disease features. NAC in vivo is rapidly metabolised to cysteine, where the thiol group is capable of reducing oxidant activity, reducing disulphide bonds and becoming a substrate for the formation of new GSH molecules . NAC is 1 decline and exacerbation frequency was not improved by NAC treatment, however in a subset of patients not using inhaled corticosteroid, risk of exacerbation was lowered. Secondary analysis found that functional residual capacity was significantly reduced in the NAC group but the authors concluded that NAC was ineffective at this dose in COPD. The PANTHEON trial [The BRONCUS trial  treated ON trial  assessedA recent meta-analysis  of NAC tGlutathione is an abundant antioxidant molecule but older clinical studies have shown mixed results using this therapeutically. Borok et al.  providedGSH was not tolerated in a trial of eight asthmatic patients who experienced cough/breathlessness and a reduction in FEV1 of 19%  potentiaNuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcriptional factor in the production of antioxidant molecules ,117,118 A trial in COPD patients of the Nrf2 activating molecule sulforaphane improved the reduced phagocytosis of bacteria witnessed in alveolar macrophages from COPD patients . Nrf2 ac1 . Therapiewed in  supplemeiewed in  found noiewed in .Overall, studies of anti-oxidants in COPD have been disappointing. This might reflect an inappropriately chosen medicant, or its dose or route; a lack of power for clinically based endpoints, or the use of the treatment in the wrong patient subset . Further studies are warranted, but enhanced mechanistic insights are needed in order to focus such studies appropriately, particularly in light of new studies highlighting the role of mitochondrial dysfunction in ROS related tissue damage.15 oxidants/free radicals contained in cigarette smoke and that many of these oxidants are relatively long-lived . HoweveMany products of oxidative stress have been shown to be increased in COPD compared with healthy smoking and non-smoking controls, whilst corresponding levels of enzymes responsible for removing ROS appear reduced in some studies. Cell studies suggest increased ROS release from key mediators of the inflammatory response in COPD including neutrophils, airway macrophages and monocytes. Although there is no animal model of COPD that replicates all clinical facets of disease, murine models have described increased oxidative burden following cigarette smoke exposure and subsequent tissue damage including the development of emphysema, which can be partially attenuated by targeting oxidation pathways. It is unclear whether the loss of balance between oxidants and anti-oxidants are a product of chronic inflammation seen in COPD or an initiating event. Specific oxidant/antioxidant genetic mutations have been associated with disease in humans, but these are relatively rare and could only account for a small proportion of cases.The great heterogeneity in COPD, including clinical presentation and course as well as the variation in proteins, enzymes, molecules and cells involved in COPD are a significant challenge when looking for potential therapies. It is currently unclear whether over- or under-expression of oxidant/anti-oxidants is uniform amongst COPD sufferers, or whether sub populations exist. Understanding this is vital in order to stratify which groups may benefit most from anti-oxidant therapies. Clearly, further basic and translational research is needed to identify which patients become susceptible to ROS related damage and to clarify whether ROS is an effective target for change in COPD.


Inflammatory reactions occurring in the central nervous system (CNS), known as neuroinflammation, are key components of the pathogenic mechanisms underlying several neurological diseases. The chemokine system plays a crucial role in the recruitment and activation of immune and non-immune cells in the brain, as well as in the regulation of microglia phenotype and function. Chemokines belong to a heterogeneous family of chemotactic agonists that signal through the interaction with G protein-coupled receptors (GPCRs). Recently, a small subset of chemokine receptors, now identified as “atypical chemokine receptors” (ACKRs), has been described. These receptors lack classic GPCR signaling and chemotactic activity and are believed to limit inflammation through their ability to scavenge chemokines at the inflammatory sites. Recent studies have highlighted a role for ACKRs in neuroinflammation. However, in the CNS, the role of ACKRs seems to be more complex than the simple control of inflammation. For instance, CXCR7/ACKR3 was shown to control T cell trafficking through the regulation of CXCL12 internalization at CNS endothelial barriers. Furthermore, D6/ACKR2 KO mice were protected in a model of experimental autoimmune encephalomyelitis (EAE). D6/ACKR2 KO showed an abnormal accumulation of dendritic cells at the immunization and a subsequent impairment in T cell priming. Finally, CCRL2, an ACKR-related protein, was shown to play a role in the control of the resolution phase of EAE. Indeed, CCRL2 KO mice showed exacerbated, non-resolving disease with protracted inflammation and increased demyelination. This phenotype was associated with increased microglia and macrophage activation markers and imbalanced M1 vs. M2 polarization. This review will summarize the current knowledge on the role of the ACKRs in neuroinflammation with a particular attention to their role in microglial polarization and function. The central nervous system (CNS) degeneration, which characterizes several chronic neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS), is closely linked to immune activation occurring in the CNS called neuroinflammation  in microglial activation and polarization.in vivo activation. Transcriptomic and proteomic analysis will help to define the heterogeneity of microglial phenotype associated to different pathological contexts , which results in the receptor activation and the triggering of intracellular signaling cascade that regulates several cell activities such as migration, adhesion, phagocytosis, cytokine secretion, proliferation and apoptosis (Bachelerie et al., In CNS, chemokines play a role in physiological conditions being involved in neuronal migration, cell proliferation and synaptic activities. During neuroinflammation, in addition to their primary role in leukocytes recruitment, they can also have direct effects on neuronal cells and mediate the cross talk between neurons and inflammatory cells (Cartier et al., In active MS patients, the pathogenic Th1 axis, involving CXCL10, CCL3, CCL4, CCL5 and the receptors CXCR3 and CCR5, is activated in blood and cerebrospinal fluid (Uzawa et al., The dysregulation of the CX3CL1-CX3CR1 communications between neurons and microglia has been investigated in AD patients showing opposite effects. In one study CX3CR1 deficiency was reported to reduce beta-amyloid deposition in AD experimental model (Lee et al., Overall, neuroinflammation is a complex process involved in several CNS diseases ranging from autoimmune to degenerative pathologies and chemokine system plays key role in immune surveillance, in microglial activation and functions, in neurocommunications among several CNS cell types and can orchestrate both neuroprotective and detrimental effects.i signaling, they are unable to activate canonical G protein-dependent receptor signaling and chemotactic functions (Bachelerie et al., ACKRs represent a small subset of proteins expressing high degree of homology with chemokine receptors. Since ACKRs lack structural domains required for proper Gαin vivo (Pruenster et al., ACKR1 binds, with high affinity over 20 inflammatory chemokines belonging to the CC and CXC families (Novitzky-Basso and Rot, ACKR2 binds a broad panel of inflammatory CC chemokines and is expressed by lymphatic endothelial cells, trophoblasts, keratinocytes and some leukocyte subsets, mostly innate-like B cells (Lee et al., ACKR3 binds two chemokines, namely CXCL12 and CXCL11. ACKR3 is expressed by endothelial cells, some hematopoietic cells, mesenchymal cells, neurons and astrocytes. ACKR3 is able to internalize CXCL12 and modulate CXCR4 expression and signaling by forming heterodimers with CXCR4 (Levoye et al., ACKR4 binds the homeostatic CC chemokines CCL19, CCL21, CCL25 and with lower affinity CXCL13. ACKR4 is expressed by thymic epithelial cells, keratinocytes and lymphatic endothelial cells. After chemokine binding ACKR4 internalizes and drives its ligand to degradation (Comerford et al., in vitro and in vivo studies showed CCRL2 expression in mouse microglia and astrocytes and upregulation in macrophages in the EAE model (Zuurman et al., CCRL2 is a seven transmembrane protein that shares some structural and functional aspects with ACKRs, such as the lack of conventional GPCR signaling and the inability to induce cell migration (Zabel et al., aa35–55 immunization and paralleled the kinetics of the clinical symptoms, reaching the peak induction at the acme of the disease and declining thereafter (Mazzon et al., CCRL2 expression was found upregulated following MOGNeuroinflammation is a key component of neurodegenerative diseases. This process involves the effector components of both innate and adaptive immunity. In the CNS, microglia represent the resident immune component involved in tissue homeostasis. Being an immune sensor of altered homeostasis, microglia accumulate in response to neuron injury and after the entry of foreign substances in the CNS. The way microglia become activated represents a crucial element in the regulation of neuroinflammation and may be associated with either beneficial or detrimental effects resulting in neuroprotection or neurotoxicity. The chemokine system plays multiple roles in the modulation of this multifaceted process. Our growing understanding on the functions of the ACKRs increases the level of complexity of this scenario and provides new potential targets to be exploited in the control of neuroinflammation.VS and FS wrote and edited the manuscript and prepared the Table and Figure. SS and ADP contributed in writing and supervised the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The efficacy of these lead compounds for decreasing AVP release and increasing both renal blood flow and diuresis, make them promising candidates for the treatment of water retention and/or hyponatremic disorders.Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling body fluid homeostasis and cardiovascular functions. In animal models, experimental data demonstrate that intracerebroventricular injection of apelin into lactating rats inhibits the phasic electrical activity of arginine vasopressin (AVP) neurons, reduces plasma AVP levels, and increases aqueous diuresis. In the kidney, apelin increases diuresis by increasing the renal microcirculation and by counteracting the antidiuretic effect of AVP at the tubular level. Moreover, after water deprivation or salt loading, in humans and in rodents, AVP and apelin are conversely regulated to facilitate systemic AVP release and to avoid additional water loss from the kidney. Furthermore, apelin and vasopressin secretion are significantly altered in various water metabolism disorders including hyponatremia and polyuria-polydipsia syndrome. Since the  They used a Cytosensor microphysiometer to detect the metabolic activation of cells expressing the human APJ receptor through measurement of the extracellular acidification rate . This 36In vivo, various molecular forms of apelin are present, differing only in length     may cleave proapelin (amino acids 23–77 of preproapelin sequence) directly into apelin 13 without generating longer isoforms . More recently, it has been shown isoforms . For apeisoforms . In rat isoforms , 10.The gene encoding for the ApelinR is intronless in human and rodents and it is located on chromosome 11q12 in human , 6. The 8-Leu9 of K17F and Arg4-Leu5 of pE13F leading to two truncated inactive peptides , and pE13F have been shown to have a similar potency (in the subnanomolar range) to inhibit FSK-induced cAMP production in CHO cells expressing the rat ApelinR and human embryonic kidney (HEK) cells expressing the human ApelinR . Hosoya  protein , 22.2+]i mobilization in Ntera 2 human teratocarcinoma (NT2N) cells, which differentiate into postmitotic neurons following retinoic acid stimulation and also in cells derived from basophils (RBL-2H3) or HEK 293 cells stably expressing human ApelinR (2+]i mobilization in glomerular arterioles in a nitric oxide (NO)-dependent manner. Interestingly, several studies have shown that the stimulation of ApelinR by apelin induces vasodilation and modulates vascular tone through NO production -dependent, and Ras-independent manner /Akt pathway and the ERK1/2 pathway  model of the ApelinR using the validated cholecystokinin receptor-1 3D model as a template revealed the presence at the bottom of the binding site of a hydrophobic cavity in which the C-terminal Phe of pE13F was embedded by Trp in TMVI   when compared with the corresponding natural peptides pE13F and K17F . On the other hand, an original strategy for improving the protection of endogenous peptides against enzymatic degradation on the basis of the introduction of a fluorocarbon chain (FC) directly into the N-terminal part of K17F was used to generate LIT01-196. The presence of the FC on the apelin peptide had no impact on the affinity of LIT01-196 for ApelinR (Ki = 0.08 nM), or on its solubility in water (>10 mM).Given these findings, metabolically stable K17F analogs were recently developed , 33. ForAltogether these chemical modifications allowed to an extend the plasma half-lives of P92 by a factor of 6–11 and that of LIT01-196 by a factor of >100 relative to that of K17F. LIT01-196 displayed remarkable resistance to degradation by plasma enzymes, as >90% of the peptide remained unchanged after 24 h of incubation at 37°C. FC acylation of an endogenous peptide therefore seems to be an efficient way to extend its half-life in plasma.P92 and LIT01-196 displayed full agonist activity for cAMP production, ERK1/2 phosphorylation (nanomolar range), induction of ApelinR internalization (subnanomolar range), and β-arrestin recruitment .in situ hybridization . This results in the insertion of AQP2 at the apical membrane of the principal cells of the CD , 76, leaThus, the aquaretic effect of apelin is not only due to a central effect by inhibiting AVP release in the blood circulation but also to a direct action of apelin at the kidney level by increasing renal blood flow and counteracting the antidiuretic effect of AVP occurring via V2R in CD.These results also show that apelin and AVP have opposite effects on the CD and contribute to control plasma osmolality by regulating the reabsorption of water by the kidney Figure .−/−) were unable to concentrate their urine to the same extent , chronic heart failure (CHF), chronic liver disease, and the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Copeptin corresponds to the C-terminal part of the AVP precursor and is composed of 77 amino acids. Copeptin is released mole to mole with AVP from the posterior pituitary in the blood circulation and plasma copeptin levels are used as a surrogate marker for AVP release in humans , 96. CopIn SIADH, abnormal AVP osmoregulation is the primary defect . A recenThis study showed that apelin levels and apelin to copeptin ratios were inappropriate to natremia in SIADH patients, suggesting that the increase in plasma apelin secretion cannot compensate for the higher levels of vasopressin release and may contribute to the corresponding water metabolism defect.In CHF, the decrease in cardiac output and in effective circulating volume results in activation of baroreceptors. These activate the sympathetic nervous system, the renin–angiotensin–aldosterone system, and the release of AVP in the bloodstream. The increase in plasma AVP concentration leads to water reabsorption by the kidney . Mild toIn response to an increase in plasma osmolality, i.e. in case of dehydration or of hypertonic saline infusion, it has been shown both in humans and in animals that AVP secretion is increased and apelin secretion is decreased, allowing the reabsorption of water by the kidney. Thirst is also stimulated by the increase in plasma osmolality. In human pathology, an increase in both fluid intake (polydipsia) and diuresis (hypotonic urine output) is characteristic of the polyuria-polydipsia syndrome (PPS). Three entities are comprised in the PPS: central , diabetes insipidus (DI), nephrogenic  DI, and PP. Central DI (CDI) is secondary to complete or partial brain AVP deficiency mirrored by low plasma copeptin levels. In most cases, thirst perception is intact, resulting in polyuria, polydipsia. Severe hyperosmolality only occurs in case of fluid deprivation. Nephrogenic DI (NDI) is secondary to renal insensitivity to AVP and can be acquired or hereditary, due to mutations of the V2R or of AQP2. PP is not due to a deficiency or resistance of AVP but rather results from an excessive fluid intake over an extended period of time, in the absence of plasma osmolality regulation disorder. PP results from an abnormal thirst mechanism or a psychiatric disorder . Plasma In CDI, plasma copeptin levels were decreased by 52% compared to healthy volunteers whereas plasma apelin levels were only decreased by 18%. However, the plasma apelin to copeptin ratio was higher in CDI patients than in healthy volunteers. Possibly, the decrease in plasma apelin levels of patients with CDI is insufficient as compared to the decrease of AVP/copeptin to reestablish a balance between the antidiuretic effect of AVP and the diuretic effect of apelin, resulting in polyuria.Patients with PP had normal plasma copeptin and slightly but significantly lower plasma apelin levels compared to healthy volunteers. However, the apelin to copeptin ratio was similar to that measured in healthy volunteers. This suggests that the normal apelin to copeptin ratio attests for balanced water homeostasis, whereas plasma apelin to copeptin ratio in CDI or NDI is increased or decreased compared to healthy volunteers, reflecting a disturbed water homeostasis.Another interesting explanation is that apelin itself might also be involved directly in the pathogenesis of PP. Indeed, the decrease in plasma apelin levels in PP patients could account for altered drinking behavior (leading to an inadequate fluid intake in these patients) since i.c.v. injection of apelin into water deprived rats decreases drinking behavior . FurtherThe discovery of apelin as the endogenous ligand of the ApelinR is an important step in basic research and has clinical implications. In animal models, experimental data demonstrate that central injection of apelin into lactating rats inhibits the phasic electrical activity of AVP neurons, reduces plasma AVP levels, and increases aqueous diuresis. In the kidney, apelin increases diuresis by increasing renal blood flow and by counteracting the antidiuretic effect of AVP at the tubular level. Moreover, following water deprivation or salt loading, in humans and in rodents, AVP and apelin are conversely regulated to facilitate systemic AVP release and to avoid additional water loss at the kidney level. These data show that, together with AVP, apelin play a crucial role in the maintenance of body fluid homeostasis. In addition, in SIADH or CHF patients with hyponatremia, the apelin to copeptin balance is altered contributing to the water metabolism defect. The administration of apelin agonists in these patients could induce aqueous diuresis and therefore increase water excretion, reversing hyponatremia.i signaling pathway like the endogenous apelin fragment K16P (biased agonist), represent promising candidates for the treatment of water retention/hyponatremic disorders.To date, there are no published preclinical or clinical studies utilizing ApelinR agonists/analogs in pathological animal models or patients with water metabolism disorders. The development of metabolically stable apelin analogs that exert longer-lasting and more potent effects than the endogenous peptide on aqueous diuresis and which additionally could only target the GAll appropriate permissions have been obtained from the copyright holders of Figures All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite numerous correlative data, a causative role for oxidative stress in mammalian longevity has remained elusive. However, there is strong evidence that increased oxidative stress is associated with exacerbation of many diseases and pathologies that are also strongly related to advanced age. Obesity, or increased fat accumulation, is one of the most common chronic conditions worldwide and is associated with not only metabolic dysfunction but also increased levels of oxidative stress in vivo. Moreover, obesity is also associated with significantly increased risks of cardiovascular disease, neurological decline and cancer among many other diseases as well as a significantly increased risk of mortality. In this review, we investigate the possible interpretation that the increased incidence of these diseases in obesity may be due to chronic oxidative stress mediating segmental acceleration of the aging process. Understanding how obesity can alter cellular physiology beyond that directly related to metabolic function could open new therapeutic areas of approach to extend the period of healthy aging among people of all body composition. A large number of diseases, pathologies and other maladies become much more prevalent with advanced age. There is substantial evidence that the molecular regulators of most of these age-related diseases overlap with many of those that are thought to regulate the aging process in general. Now more than ever, understanding how to delay the constant acceleration of aging could profoundly impact health and healthy aging in a growing aged population.The oxidative stress theory of aging (OSTA) has been exceedingly well-studied as a primary mechanistic theory to describe how the aging process is regulated. Evolution of life in an aerobic environment has afforded species many benefits. However, this lifestyle also increases the likelihood of generating cellular oxidative stress. At the most basic description, cellular oxidative stress is an imbalance between the production of pro-oxidants (including reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, etc.) and the activity of the cellular antioxidant defense (including enzymatic and non-enzymatic antioxidants). More than 60 years ago, Denham Harman developed an original theory suggesting that the oxidative stress associated with aerobic life plays an essential role in the regulation of the aging process . The salAt its most basic, the mechanistic framework of the OSTA predicts (1) that older organisms should exhibit higher levels of oxidative stress and/or damage than younger organisms and (2) that altering levels of oxidative stress should significantly impact the aging process. Thorough literature searches published elsewhere show for the most part that aging is associated with accumulation of oxidative damage to cellular macromolecules, reduced antioxidant defenses and increased susceptibility to oxidative stress in vivo ,5. MoreoCaenorhabditis elegans and the fruit fly Drosophila melanogaster) have supported this prediction [As to the prediction that altering levels of oxidative stress will impact aging, the data from experimental approaches designed to test this prediction are still ambiguous. Studies using genetic manipulation of invertebrate models . ediction ,15. The Sod1−/− mice have a shorter lifespan (~30%) than control mice, thereby strongly supporting the OSTA [Sod1−/− mice may be directly attributed to an increase in cancer rate, particularly of a rare (for C57BL/6 mice) cancer, hepatocellular carcinoma [Sod1−/− mice potentially hint at an acceleration of the aging process as a whole in these mice. These mice have been reported to exhibit acceleration in the rate of development (or increase in the incidence of) multiple pathological changes associated with age-related diseases including sarcopenia and neuromuscular junction degeneration [Sod1−/− mice, more so than all other antioxidant mutant mice tested, show a dramatic increase in oxidative damage to cellular macromolecules [Sod1−/− than in mice lacking glutathione peroxide 1 (Gpx1) or with reductions in other antioxidants such as Sod2 or Gpx4 [Sod1−/− mice reach a “theoretical threshold limit of oxidative damage” required to accelerate the aging process as a whole, or perhaps to drive the increase in cancer rate, that is unmet in other antioxidant mutant mouse models. Interestingly, one antioxidant mutant mouse model that has shown repeatable, robust effects on longevity is a mouse generated with complete ablation of superoxide dismutase 1 (Sod1). Several groups have reported that the OSTA ,18,19. Iarcinoma ,18,19. Hneration ,21,22, aneration , eye [24neration , pancreaneration  and neurneration . Why theolecules ,29,30. F or Gpx4 . Similar or Gpx4 ,30. ThusDigging deeper into other antioxidant mutant mouse models more generally, there is sufficient evidence to suggest that altering the accumulation of oxidative stress/damage does indeed often correlate with changes in health associated with age if not longevity itself ,15. To gMsrA−/−) in mammalian longevity. Moskovitz et al. first reported that MsrA−/− mice were short-lived; however, even control mice in this study were relatively short-lived for the strain of mice used (a mixed background of C57BL/6 and 129) [MsrA−/− mice under conditions designed to maximize control mouse lifespan . Under these conditions, we found no effect on longevity in mice lacking MsrA, providing support for the hypothesis described above [Because natural animal aging does not take place in a vacuum, it is important to also consider the role that external inputs play on the development of age-related disease, if not aging itself. Life on Earth is constantly exposed to numerous generators of ROS both natural  and man-made  in addition to the oxidative stress generated by mitochondrial oxidative phosphorylation. Aging experiments in the lab have rightfully focused on elimination of hard-to-control external variables, but it would be remiss to completely dismiss the role these play in healthy aging. In particular, external sources of stress might significantly alter the outcome of longevity experiments through oxidative-stress mediated mechanisms. A potential example of this is in the study of the role of the protein oxidation repair enzyme methionine sulfoxide reductase A (and 129) . One posed above . IdentifIt is becoming increasingly clear that obesity has become one of the 21st century’s most significant public health concerns. The proportion of those characterized as obese has risen dramatically over the last few decades world-wide, with Western society currently suffering to the greatest degree ,46. MuchUnderstanding how obesity alters cellular homeostasis in ways that drive the etiology of these physiological defects could profoundly affect how diseases are prevented and could have significant impact on public health. In this context, there are myriad potential molecular pathways that are affected by obesity in both time- and tissue-specific patterns. One approach to begin to simplify this complexity may be to focus on those common pathways that are (a) affected by obesity and (b) associated with many, or all, the common pathologies associated with obesity. In this regard, we and others have noted that oxidative stress may be a likely target on which to focus. Obesity is known to promote oxidative stress ,52,53,54Oxidative stress associated with obesity originates from many sources; here we briefly discuss the roles of mitochondrial dysfunction and increased inflammation. Mitochondrial respiration, through the oxidation of glucose and/or fatty acids, can generate oxidative stress through the byproduct of generation of ATP in an oxygenated environment ,56. ThisTaken together, it seems clear that persistence of obesity is associated with chronically increased levels of oxidative stress. Due to the growing numbers of juveniles now classified as clinically obese, there is a growing portion of the population that will remain obese nearly their entire lives and who are thus likely to be show increased risk of developing what we generally term age-related diseases at earlier points in life and reduced health span if not lifespan. It then seems entirely possible that this oxidative stress may be the molecular tipping point in the progression of poor health associated with obesity. Importantly, clarification of oxidative stress at this key role then identifies a potential therapeutic target to slow or prevent the associated health problems in this population group.The most well-known physiological defect associated with obesity is the development of metabolic disorders, including and primarily type 2 diabetes mellitus (T2DM). The primary etiology in T2DM is the development of insulin resistance, or the pathological decline in insulin response in peripheral tissues . A deficBased on observational as well as experimental findings, there is now reasonable evidence that oxidative stress likely plays a key role in the development of insulin resistance and metabolic dysfunction. Perhaps most well-publicized are results from the Framingham Heart Study; several reports from this longitudinal study have identified increased oxidative stress as a significant correlate of metabolic disease ,39,40,77Together, these studies paint a picture in which obesity causes high levels of oxidative stress, though do not necessarily define oxidative stress/damage as the root cause of associated metabolic dysfunction. However, several mouse studies using either genetic or pharmacological interventions have fairly clearly defined the modulation of oxidative stress as a key step between high fat feeding/obesity and the development of metabolic dysfunction. As an example, transgenic mice with an increase in cytosolic (Sod1) or mitochondrial (Sod2) superoxide dismutase, those with an increase in catalase targeted to the mitochondria (mCAT), an increase in peroxiredoxin (Prdx3), those with an increase in cytosolic thioredoxin (Trx1), and those with an increase in mitochondria-targeted methionine sulfoxide reductase A (TgMito MsrA) have all been shown to be protected against the development of insulin resistance when fed high fat diets ,90,91,92MsrA−/− mice became more insulin resistant than expected based on their diet-matched controls [The precise mechanisms by which oxidative stress gives rise to insulin resistance or metabolic dysfunction, though there are likely roles for the common agents of action including induction of stress signaling pathways such as JNK or stimulation of pro-inflammation ,96,97. Hcontrols . Furthercontrols . TogetheIn addition to diabetes and metabolic dysfunction, there is now ample evidence that obesity can accelerate the progression of other age-related diseases and pathologies in both mouse models of disease as well as normally aging mice ,105,106.These findings regarding obesity and disease raise an important, and rather unexplored, question of whether obesity may actually be accelerating the aging process. Paraphrasing the definition of aging established by Miller, aging is a process that converts healthy, young adults into frail, older adults with diminished capacity to resist and increased vulnerability to most diseases and death . As desc2-isoprostanes) and protein oxidation  and suggestion of elevated levels of circulating pro-inflammatory cytokines. These high levels of oxidative stress were associated with a significant increase in glucose intolerance and increased plasma insulin concentrations, both indicative of metabolic dysfunction consistent with insulin resistance. Most relevant to this review, we also found a significant lifespan shortening of approximately 25% in mice fed the high fat diet. This lifespan effect was in similar size and direction to previous studies comparing lifespan of rodents fed various high fat diets compared to normal diet-fed controls [With a relative dearth of data to address this conflict, we recently published a study designed to assess the effects of high fat diet-induced obesity on the health span of mice using a broad approach . In thiscontrols ,121. During the course of this study, we assessed the performance of these mice in tests of several physiological parameters in a longitudinal fashion, i.e., after 4 months on high fat diet (or 12 months of age) and again after 10 months on high fat diet . We selected a serious of physiological tests that were, prior to this study, not thought to be largely related to the increased fat accumulation such as the relationship between insulin resistance and obesity. Thus, we designed this study to address the effect of aging, diet and their interaction to whether obesity, and the associated chronic oxidative stress and inflammation, altered the age-related trajectories of physiological performance. For example, after 10 months of feeding, high fat-fed mice showed a significant decline in activity whereas control-fed mice activity was unchanged. Similarly, the swing-time of both front and rear limbs measured during gait analysis was significantly reduced by high fat feeding over this time period and unchanged in control fed mice. In addition, mice fed a high fat diet for 10 months showed a significantly reduced ability to stay on a rotating rod, a marker of balance and coordination. We found the significance of these differences to exist even after accounting for differences in body weight and fat mass. Further, these markers have been reported to decline with normal aging in mice; thus, obesity caused by a high fat diet accelerated this decline in function and contributed to a decline in health span. The exact nature of these declines are unclear, i.e., are they due to dysfunctional muscles, or motor neurons, or other, and this is one of the significant challenges in future studies. In addition, ongoing studies are addressing whether these declines in function can be attributed directly to the increased levels of oxidative stress/damage. The larger question remains though whether this increased incidence of disease is evidence for acceleration of the aging process, if not wholly then at least segmentally? While still unclear, the prevalence of data certainly suggests this and, moreover, implicates oxidative stress as a key player in this process. Ultimately, the goal is to improve the period of healthy aging in all people which will include both the lean and metabolically normal as well as the obese and metabolically compromised. The data above point to the idea that perhaps addressing oxidative stress, either by preventing it through dietary interventions or reducing it through pharmaceutical antioxidants, may be a potential therapeutic approach in this process. On the other hand, mouse studies have largely shown no effect of altering oxidative stress on longevity in lean, healthy rodents and antioxidant treatment has largely failed as a human intervention against obesity and metabolic dysfunction ,123. How


A number of crucial technical considerations for designing experiments and analyzing data with stable isotope labelling were discussed which included replication, instrumentation, methods of labelling, tracer dilution and data analysis. This report reflects the current viewpoint on the use of stable isotope labelling in metabolomics experiments, identifying it as a great tool with the potential to improve biological interpretation of metabolomics data in a number of ways.This is a perspective from the peer session on stable isotope labelling and fluxomics at the Australian & New Zealand Metabolomics Conference (ANZMET) held from 30 March to 1 April 2016 at La Trobe University, Melbourne, Australia. This report summarizes the key points raised in the peer session which focused on the advantages of using stable isotopes in modern metabolomics and the challenges in conducting flux analyses. The session highlighted the utility of stable isotope labelling in generating reference standards for metabolite identification, absolute quantification, and in the measurement of the dynamic activity of metabolic pathways. The advantages and disadvantages of different approaches of fluxomics analyses including flux balance analysis, metabolic flux analysis and kinetic flux profiling were also discussed along with the use of stable isotope labelling in  In the postgenomic era, metabolomics has emerged as a powerful approach for enabling the study of the intricate biochemistry of cells, organisms, and systems in response to different conditions such as stress, disease, or nutrition. This detailed analysis of metabolite profiles can provide functionally relevant biochemical information about biological processes. It forms one of the pillars of systems biology, which advocates the need for a holistic view for understanding biological systems. Metabolomics relies on identification and measurement of small molecule metabolites in a biological system, and is usually driven by global data acquisition which leads to hypothesis generation for further research. This approach is typically built on readouts of steady-state metabolite levels/concentrations and thus only presents a snapshot of biological activity. While useful in some circumstances, for example, in detecting in-born errors of metabolism, measurement of static metabolite levels is unable to directly determine the activity of metabolic pathways, which are intrinsically dynamic in nature and often experience minimal metabolite (reactant and product) concentration changes ,2. In coin vitro or in vivo [Combining the use of stable isotope labelled metabolic precursors (tracers), and modern analytical techniques (such as high resolution chromatography and mass spectrometry) for separation and detection of chemical species permits characterization of the metabolism of a broad range of biological systems. This can include direct and accurate measurement of turnover of a specific metabolite or even determining entire pathway fluxes  in vivo ,5,7. A t in vivo ,9, paras in vivo ,11, dise in vivo  and plan in vivo  amongst  in vivo ,15,16.Stable isotope labelling is becoming increasingly popular in the field of metabolomics for a number of reasons. Being non-hazardous, stable isotopes clearly have a marked advantage over traditionally-used radioisotopes and have almost completely replaced the use of radionuclides in mass spectrometry and NMR based modern metabolomics. The discussion during the peer session encompassed the following broad areas.Stable isotope labelling can be used for generating reference standards, which can aid in metabolite identification and absolute quantification by stable isotope dilution strategies. Confident identification and accurate quantification are major bottlenecks in global discovery-based experiments owing to problematic matrix effects which contaminate spectra and suppress signal responses. Stable isotope dilution is the gold standard for accurate quantification and stable isotope reference standards are becoming increasingly available. However, for metabolome-wide approaches the use of individual stable isotope standards is neither practical nor economical. An attractive strategy is to apply labelled standards in a metabolome-wide approach by mixing the experimental biological sample extract with a known biological extract grown in the presence of an exclusively labelled single carbon source and analyzing the resulting isotope labelling patterns, thus permitting precise, relative quantification (corrected for matrix effects and response factors) of many hundreds of metabolites simultaneously ,18,19,20The major interest in stable isotope labelling for metabolomics stems from its ability to aid in the measurement of dynamic activity of metabolic pathways, in order to provide mechanistic explanations for the perturbations in steady-state metabolite levels observed in classical metabolomics studies. Stable isotope labelled precursors with uniform or positional labelling of constituent atoms have been used to generate biological samples that allow the study of their metabolic fate throughout the network in an untargeted fashion ,15,16. SFluxomics, in theory refers to the measurement of all the metabolic reaction rates in a biological system, including the directionality of metabolic conversions and import/efflux of metabolites. Like metabolomics, fluxomics requires identification and analysis of metabolites, and in practice, is often limited to known central metabolic pathways involving a subset of metabolites due to an inability to actually measure the entire biological “metabolome” using any single analytical platform. A number of approaches for fluxomics analyses were discussed in the peer session.Owing to the highly dynamic nature of any biological system at a given time, it is difficult to directly measure the metabolic flux. An indirect measurement however can be obtained using the concept of metabolite balancing or flux balance analysis . This trin vivo studies in animals or humans, owing to a much higher degree of complexity which results from an interconnection of different cell and organ systems that simultaneously produce, transfer, and consume multiple substrates.Therefore, for successful application of this approach, a thorough understanding of the biological system being studied is critical in order to accurately reconstruct the metabolic network . This me2H, 13C, 15N, and 34S) and performing labelling experiments over a time-course to observe the dynamic nature of metabolism in a variety of biological systems including unicellular pathogens, plants, and mammalian cells [13C-glucose and 13C-glutamine, as they are metabolized in a cascade of enzymatic reactions can be exploited to undertake isotopomer analysis in order to pinpoint specific adaptations in central carbon metabolism [Flux balance analysis can be improved by implementing a method called metabolic flux analysis and using stable isotope labelling to fill in the gaps in data that arise due to compartmentalization or cycling of metabolites. This can be achieved by using a number of stable isotopes of atoms which occur naturally in biomolecules  has been used to analyze mass isotopomer distributions of metabolites based on their atomic rearrangements in autotrophs. These organisms are sampled before achieving isotopic steady state following abolites ,42. Thisabolites ,44.Kinetic flux profiling is an approach which can provide the most dynamic information in a metabolomics experiment, but requires extensive modelling and specialized experimental methodology. It uses dynamic isotopomer distribution data in order to estimate absolute flux along metabolic pathways. Typically, this approach assumes that the magnitude of measured metabolic flux is proportional to the rate of transmission of labelled isotopes from the precursor to subsequent products. It considers steady state dynamics for influx and efflux of a given metabolite and the sample preparation involves switching from an unlabelled precursor to an isotopically labelled precursor at a given time. These assumed steady state dynamics are true for cells undergoing rapid proliferation, provided nutrient resources are not limiting. Under these conditions, when a precursor is switched from an unlabelled to labelled form, the immediate product of the precursor is replaced by its labelled form. Measurement of concentrations of the labelled forms over time—as the label gradually infiltrates the metabolite pool—can be used to estimate the influx and efflux . Kineticin vivo animal and human experimentation. Due to the unpredictable nature of a multi-organ system that is shared by the one continually recirculating blood supply, it is difficult, perhaps even impossible, to model (“constrain”) the entire metabolism of an organ (or organs) in an intact animal or human. For example, when an isotopic tracer is administered  in animals or humans, the tracer can be utilized by one organ where it is metabolized and recycled to another form and released into the circulation, ready to be taken up and recycled again by another organ. This can occur many times, thus resulting in the formation of a range of secondary tracers (metabolites) with “scrambled” labelling patterns when compared to the originally administered tracer [in vivo to provide both qualitative and quantitative information on the fate of substrates across multiple metabolic pathways in different organs. By administering stable isotope labelled metabolic precursors, then rapidly harvesting animal organs, or collecting surgical biopsy/resection specimens from humans, targeted metabolomics analyses have been used to determine the contribution that specific substrates make to pathways of central carbon metabolism in both healthy and diseased states [While metabolic flux analysis has been extensively used in microbial, plant, and—more recently—mammalian cell culture systems ,38, its d tracer . Howeverd states ,55,56,57In this session, peers also discussed the importance of a number of technical issues which should be addressed early to ensure reproducibility and acquisition of high quality metabolomics data for stable isotope labelling experiments. Some of these are discussed below.Any good experimental design incorporates a plan for replication of the experimental conditions and the power of the study is heavily dependent on the reproducibility of the data obtained, irrespective of the approach. Replicates are essential as they provide multiple measurement points for any given metabolite in a sample, and averaging across replicates improves accuracy and data resolution. Furthermore, replication provides a means of identifying anomalies in the experimental procedure as outliers and, most importantly, they provide measurements of the variability in experimental data that are used for determination of significant differences between study factors. As metabolomics experiments involve multiple steps between sample preparation and analysis, the probability of user-induced variation increases with every step. Usually, with validated procedures and good laboratory practice, technical variability can be kept to the minimum and the balance of the resources  should be tipped towards obtaining biological replicates. However, due to the nature of analytical methods used in metabolomics, it may be desirable to capture technical variability either by obtaining technical replicates, or by the addition of batch-wise quality control samples .If internal standardization is achieved in an identical manner for all variables under testing  in stable isotope labelled metabolomics experiments, the technical variance is negligible compared to label-free comparative metabolomics studies as the isotopologue peaks are detected in the same sample as the unlabelled (or other isotopologue) peaks. For this reason, fewer technical replicates can be justified in order to offset the increased costs associated with stable isotopes. Nevertheless, intrinsic biological variance remains in even the most tightly controlled experiments, and some degree of biological replication remains essential in order to obtain statistical significance.2H) labelling but is still apparent for 13C and therefore this shift must be taken into account in data processing. Retention time shifts due to 13C incorporation are insignificant for small molecules, for example TCA cycle intermediates, however a small shift may be observed for larger molecules, such as fatty acids, when using a highly selective GC method and therefore the data processing method must ensure peak areas for each isotopologue are measured accurately [Only MS instruments with resolving powers above 100,000 are able to resolve most co-eluting isobaric metabolite mass signals . Howevercurately .The linear dynamic range of a method is an important consideration while developing quantitative analyses, however, it is often overlooked when adapting methods to different instruments. Increasing sensitivity of MS instrumentation is not necessarily associated with an increase in linear dynamic range. Most modern MS instruments report a within-scan linear dynamic range of 4–5 orders of magnitude, which does not cover the full concentration range of metabolites and isotopologues likely to be found in a complex metabolomics sample. Therefore, sample loading and/or analytical methods must be adapted to ensure that the instrument can measure isotope profiles within its linear range. Without this consideration, large errors in the measurement of isotopologue ratios can occur and the inclusion of standard curves is therefore necessary to validate the linear dynamic range of a method.The procedures for addition of stable isotope precursors must be considered in the context of the biological system under study and the aim of the experiment. In many cases, for metabolic flux analysis and generation of reference standards, an isotopic steady state is desired. The time taken to reach isotopic steady state is dependent on the metabolic flux within the pathways of interest under the specific experimental conditions. Often a pilot study is necessary to determine an appropriate experiment duration.In the case of kinetic flux profiling experiments, a series of samples are generally obtained shortly after addition of the labelled precursor. Meaningful interpretation of these studies require the system to be at metabolic steady state throughout the experiment, which necessitates careful consideration of the method used to introduce the stable isotope labelled precursor. Media exchange techniques that rely on centrifugation or filtration, or washing protocols that utilize nutrient-free media, are likely to significantly perturb metabolism and influence the resulting flux measurements.13C labelled central carbon source such as glucose (or CO2 in plants if a time course study is desired) can be highly informative. However, it is common to perform labelling studies subsequent to the initial hypothesis-generating metabolomics study, and labelled nutrients can be selected based on the published or predicted precursor(s) for the metabolic pathway(s) of interest [The choice of stable isotope labelled nutrient is a critical first step in these studies, and is highly dependent on the research questions ,61,62. Finterest .A critical point that was raised in the peer session discussion was the importance of accounting for tracer dilution from unlabelled precursors. It was noted that even when care is taken to account for any pre-existing unlabelled precursor at the start of such studies, such forms may further originate from recycling of other metabolic intermediates or environmental fixation and may dilute the labelled tracer along the path of synthesis of downstream products. To overcome this issue, determination of precursor dilution by unlabelled metabolites is necessary, and this can often be achieved by quantitative mass isotopomer distribution analysis ,64,65.13C-glucose allows differentiation of the flux in each of the two branches (oxidative vs. non-oxidative) of the pentose phosphate pathway (PPP) in a highly glycolytic cell, which would not be possible from a 100% U-13C-glucose precursor  [13C-glucose for differentiating glycolytic and PPP flux.In some cases, it is informative to study the metabolism of partly-labelled tracer mixtures, due to the complex architecture of metabolic pathways. For example, a 50% labelled/unlabelled mixture of U-y state) ,16. Non-2H and 13C) cannot be differentiated from one another. This isobaric background signal can be minimized by using ultra-high resolution mass spectrometers for data acquisition and post-processing computation which incorporates background correction [Due to the presence of naturally occurring isotopes of metabolites, background subtraction is required prior to isotopologue data interpretation ,66,67,68rrection . Backgrorrection . In some13C glucose, amino acids, acetate, etc.) and observing their metabolic incorporation and/or dilution in biological systems, it is also possible to use other labelling approaches combined with mass spectrometric and/or NMR analysis to understand cellular metabolism and growth characteristics. For example, the use of deuterated “heavy” water (2H2O) has proven to be a particularly powerful approach for measuring the synthesis rates of proteins [de novo lipogenesis, esterification, and chain elongation) [in vivo and in vitro. Furthermore, 2H2O labelling can be used to quantify the pathways used to produce glucose and glycogen, thus providing a way to measure gluconeogenic and glycogenolytic/glycogenic pathway activity [2H2O labelling lies in the fact that many different biomolecules incorporate hydrogen atoms (protons) from body/cellular water via enzyme catalyzed reactions [2H2O into a living system, deuterium (2H) atoms become stably incorporated into C-H bonds of newly synthesized biomolecules, essentially “tagging” them. The degree to which molecules (particularly macromolecules) become enriched with 2H atoms depends on both their rates of synthesis and the pathways used to synthesize them. 2H2O can be administered to animals or humans in drinking water, or directly into the cell culture media in vitro, which results in the enrichment of the body/cellular water pool. A major advantage of using 2H2O is that it can be used to probe multiple metabolic processes simultaneously and can be easily and safely administered for long periods of time in vivo and in vitro, permitting flux analysis in free living conditions or long term culture experiments [2H2O enrichment can have toxic effects on cellular systems due to the slightly stronger bonds that deuterium forms with other atoms when compared to hydrogen, thus creating kinetic isotope effects on chemical reactions [2H2O enrichments of body water/cell culture media to <10%, although the kinetic isotope effect should be considered when modelling and interpreting data from 2H2O labelling [2H2O has also recently been combined with other “omics” technologies including proteomics [in vivo, and imaging mass spectrometry [2H2O labelling can provide complementary information on the synthetic rates of numerous cellular components and their biosynthetic pathways .In addition to administration of isotopically labelled precursor molecules  ,76,77,78ngation) ,80, and ngation) , thus practivity ,85,86,87eactions ,89,90. Teriments ,91. Howeeactions ,93. The abelling ,93. 2H2Ooteomics ,95,96, ptrometry , permittWhether being used as a hypothesis-generating holistic approach, or for hypothesis-driven analysis of specific pathways, stable isotope labelling is a powerful tool that can enhance the biological interpretation of metabolomics data. Stable isotope labelling can help alleviate a number of issues associated with metabolomics data analysis and provide confidence in assigning correct identification to metabolite features. Quantification of metabolites in real time using stable isotope labelling allows accurate measurements for fluxomics studies. For most biological experiments investigating mechanisms of metabolic perturbation, targeted tracer studies or metabolic flux analysis are the preferred methods, due to ease of experimental design and data analysis. These approaches often reveal sufficient information about the metabolic pathways of interest to interpret observed biological phenomena without the complexity associated with detailed kinetic flux analysis.Thanks to stable isotope labelling, we are increasingly advancing from discovery-based global metabolomics approaches and becoming capable of teasing out mechanisms of interplay of metabolic pathways in intricate networks. We hope that the use of stable isotope labelling will become routine in metabolomics experiments in the future and will continue to play a major role in biochemical elucidation of existing pathways along with identification of novel metabolic networks.