A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).

Targeted metabolite assays that measure tens or hundreds of pre-selected metabolites, typically using liquid chromatography-mass spectrometry, are increasingly being developed and applied to metabolic phenotyping studies. These are used both as standalone phenotyping methods and for the validation of putative metabolic biomarkers obtained from untargeted metabolomics studies. However, there are no widely accepted standards in the scientific community for ensuring reliability of the development and validation of targeted metabolite assays (referred to here as 'targeted metabolomics'). Most current practices attempt to adopt, with modifications, the strict guidance provided by drug regulatory authorities for analytical methods designed largely for measuring drugs and other xenobiotic analytes. Here, the regulatory guidance provided by the European Medicines Agency, US Food and Drug Administration and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use are summarized. In this Perspective, we have adapted these guidelines and propose a less onerous 'tiered' approach to evaluate the reliability of a wide range of metabolomics analyses, addressing the need for community-accepted, harmonized guidelines for tiers other than full validation. This 'fit-for-purpose' tiered approach comprises four levels-discovery, screening, qualification and validation-and is discussed in the context of a range of targeted and untargeted metabolomics assays. Issues arising with targeted multiplexed metabolomics assays, and how these might be addressed, are considered. Furthermore, guidance is provided to assist the community with selecting the appropriate degree of reliability for a series of well-defined applications of metabolomics.

Association of gut-related metabolites with respiratory symptoms in COVID-19: A proof-of-concept study.

Gut-related metabolites have been linked with respiratory disease. The crosstalk between the gut and lungs suggests that gut health may be compromised in COVID-19. The aims of the present study were to analyze a panel of gut-related metabolites (acetyl-L-carnitine, betaine, choline, L-carnitine, trimethylamine, and trimethylamine N-oxide) in patients with COVID-19, matched with healthy individuals and patients with non-COVID-19 respiratory symptoms. As results, metabolites from this panel were impaired in patients with COVID-19 and were associated with the symptoms of breathlessness and temperature, and it was possible to differentiate between COVID-19 and asthma. Preliminary results showed that lower levels of betaine appeared to be associated with poor outcomes in patients with COVID-19, suggesting betaine as a marker of gut microbiome health.

Sex specific inflammatory profiles of cerebellar mitochondria are attenuated in Parkinson's disease.

Response to inflammation is a key determinant in many diseases and their outcomes. Diseases that commonly affect older people are frequently associated with altered inflammatory processes. Neuroinflammation has been described in Parkinson's disease (PD) brain. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and at the sub-cellular level, mitochondrial dysfunction is a key feature. However, there is evidence that a different region of the brain, the cerebellum, is involved in the pathophysiology of PD. We report relative levels of 40 pro- and anti-inflammatory cytokines measured in PD and control cerebellar mitochondria. These data were obtained by screening cytokine antibody arrays. In parallel, we present concentrations of 29 oxylipins and 4 endocannabinoids measured in mitochondrial fractions isolated from post-mortem PD cerebellum with age and sex matched controls. Our oxylipin and endocannabinoid data were acquired via quantitation by LC-ESI-MS/MS. The separate sample sets both show there are clearly different inflammatory profiles between the sexes in control samples. Sex specific profiles were not maintained in cerebellar mitochondria isolated from PD brains.

A novel nucleoside rescue metabolic pathway may be responsible for therapeutic effect of orally administered cordycepin.

Although adenosine and its analogues have been assessed in the past as potential drug candidates due to the important role of adenosine in physiology, only little is known about their absorption following oral administration. In this work, we have studied the oral absorption and disposition pathways of cordycepin, an adenosine analogue. In vitro biopharmaceutical properties and in vivo oral absorption and disposition of cordycepin were assessed in rats. Despite the fact that numerous studies showed efficacy following oral dosing of cordycepin, we found that intact cordycepin was not absorbed following oral administration to rats. However, 3'-deoxyinosine, a metabolite of cordycepin previously considered to be inactive, was absorbed into the systemic blood circulation. Further investigation was performed to study the conversion of 3'-deoxyinosine to cordycepin 5'-triphosphate in vitro using macrophage-like RAW264.7 cells. It demonstrated that cordycepin 5'-triphosphate, the active metabolite of cordycepin, can be formed not only from cordycepin, but also from 3'-deoxyinosine. The novel nucleoside rescue metabolic pathway proposed in this study could be responsible for therapeutic effects of adenosine and other analogues of adenosine following oral administration. These findings may have importance in understanding the physiology and pathophysiology associated with adenosine, as well as drug discovery and development utilising adenosine analogues.

Cysteines and Disulfide-Bridged Macrocyclic Mimics of Teixobactin Analogues and Their Antibacterial Activity Evaluation against Methicillin-Resistant Staphylococcus Aureus (MRSA).

Teixobactin is a highly potent cyclic depsipeptide which kills a broad range of multi-drug resistant, Gram-positive bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA) without detectable resistance. In this work, we describe the design and rapid synthesis of novel teixobactin analogues containing two cysteine moieties, and the corresponding disulfide-bridged cyclic analogues. These analogues differ from previously reported analogues, such as an Arg10-teixobactin, in terms of their macrocyclic ring size, and feature a disulfide bridge instead of an ester linkage. The new teixobactin analogues were screened against Methicillin-resistant Staphylococcus aureus and Methicillin-sensitive Staphylococcus aureus. Interestingly, one teixobactin analogue containing all l-amino acid building blocks showed antibacterial activity against MRSA for the first time. Our data indicates that macrocyclisation of teixobactin analogues with disulfide bridging is important for improved antibacterial activity. In our work, we have demonstrated the unprecedented use of a disulfide bridge in constructing the macrocyclic ring of teixobactin analogues.

Age-dependent plasticity in endocannabinoid modulation of pain processing through postnatal development.

Significant age- and experience-dependent remodelling of spinal and supraspinal neural networks occur, resulting in altered pain responses in early life. In adults, endogenous opioid peptide and endocannabinoid (ECs) pain control systems exist which modify pain responses, but the role they play in acute responses to pain and postnatal neurodevelopment is unknown. Here, we have studied the changing role of the ECs in the brainstem nuclei essential for the control of nociception from birth to adulthood in both rats and humans. Using in vivo electrophysiology, we show that substantial functional changes occur in the effect of microinjection of ECs receptor agonists and antagonists in the periaqueductal grey (PAG) and rostroventral medulla (RVM), both of which play central roles in the supraspinal control of pain and the maintenance of chronic pain states in adulthood. We show that in immature PAG and RVM, the orphan receptor, GPR55, is able to mediate profound analgesia which is absent in adults. We show that tissue levels of endocannabinoid neurotransmitters, anandamide and 2-arachidonoylglycerol, within the PAG and RVM are developmentally regulated (using mass spectrometry). The expression patterns and levels of ECs enzymes and receptors were assessed using quantitative PCR and immunohistochemistry. In human brainstem, we show age-related alterations in the expression of key enzymes and receptors involved in ECs function using PCR and in situ hybridisation. These data reveal that significant changes on ECs that to this point have been unknown and which shed new light into the complex neurochemical changes that permit normal, mature responses to pain.

The role of CB1 in intestinal permeability and inflammation.

The endocannabinoid system has previously been shown to play a role in the permeability and inflammatory response of the human gut. The goal of our study was to determine the effects of endogenous anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) on the permeability and inflammatory response of intestinal epithelium under normal, inflammatory, and hypoxic conditions. Human intestinal mucosa was modeled using Caco-2 cells. Human tissue was collected from planned colorectal resections. Accumulation of AEA and 2-AG was achieved by inhibiting their metabolizing enzymes URB597 (a fatty acid amide hydrolase inhibitor) and JZL184 (a monoacylglycerol lipase inhibitor). Inflammation and ischemia were simulated with TNF-α and IFN-γ and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB1 receptor was explored using CB1-knockdown (CB1Kd) intestinal epithelial cells. Endocannabinoid levels were measured using liquid chromatography-mass spectrometry. Cytokine secretion was measured using multiplex and ELISA. URB597 and JZL184 caused a concentration-dependent increase in permeability via CB1 (P < 0.0001) and decreased cytokine production. Basolateral application of JZL184 decreased permeability via CB1 (P < 0.0001). URB597 and JZL184 increased the enhanced (worsened) permeability caused by inflammation and hypoxia (P < 0.0001 and P < 0.05). CB1Kd cells showed reduced permeability response to inflammation (P < 0.01) but not hypoxia. 2-AG levels were increased in response to inflammation and hypoxia in Caco-2 cells. In human mucosal tissue, inflammation increased the secretion of granulocyte macrophage-colony stimulating factor, IL-12, -13, and -15, which was prevented with ex vivo treatment with URB597 and JZL184, and was inhibited by a CB1 antagonist. The results of this study show that endogenous AEA and 2-AG production and CB1 activation play a key modulatory roles in normal intestinal mucosa permeability and in inflammatory and hypoxic conditions.-Karwad, M. A., Couch, D. G., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. The role of CB1 in intestinal permeability and inflammation.

RECAST (Remote Ischemic Conditioning After Stroke Trial): A Pilot Randomized Placebo Controlled Phase II Trial in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Repeated episodes of limb ischemia and reperfusion (remote ischemic conditioning [RIC]) may improve outcome after acute stroke. METHODS: We performed a pilot blinded placebo-controlled trial in patients with acute ischemic stroke, randomized 1:1 to receive 4 cycles of RIC within 24 hours of ictus. The primary outcome was tolerability and feasibility. Secondary outcomes included safety, clinical efficacy (day 90), putative biomarkers (pre- and post-intervention, day 4), and exploratory hemodynamic measures. RESULTS: Twenty-six patients (13 RIC and 13 sham) were recruited 15.8 hours (SD 6.2) post-onset, age 76.2 years (SD 10.5), blood pressure 159/83 mm Hg (SD 25/11), and National Institutes of Health Stroke Scale (NIHSS) score 5 (interquartile range, 3.75-9.25). RIC was well tolerated with 49 out of 52 cycles completed in full. Three patients experienced vascular events in the sham group: 2 ischemic strokes and 2 myocardial infarcts versus none in the RIC group (P=0.076, log-rank test). Compared with sham, there was a significant decrease in day 90 NIHSS score in the RIC group, median NIHSS score 1 (interquartile range, 0.5-5) versus 3 (interquartile range, 2-9.5; P=0.04); RIC augmented plasma HSP27 (heat shock protein 27; P<0.05, repeated 2-way ANOVA) and phosphorylated HSP27 (P<0.001) but not plasma S100-ß, matrix metalloproteinase-9, endocannabinoids, or arterial compliance. CONCLUSIONS: RIC after acute stroke is well tolerated and appears safe and feasible. RIC may improve neurological outcome, and protective mechanisms may be mediated through HSP27. A larger trial is warranted. CLINICAL TRIAL REGISTRATION: URL: http://www.isrctn.com. Unique identifier: ISRCTN86672015.

Production of N-acyl homoserine lactones by the sponge-associated marine actinobacteria Salinispora arenicola and Salinispora pacifica.

The structures of acyl homoserine lactone (AHL) compounds and their quantification were accomplished using an integrated liquid chromatography-mass spectrometry approach. The precursor and product ions, along with retention times of peaks, were searched against an in-house database of AHLs and structures confirmed by accurate mass and by comparison with authentic AHL standards. The two compounds, N-(3-oxodecanoyl)-L-homoserine lactone and N-(3-oxododecanoyl)-L-homoserine lactone, were characterised and quantified in Salinispora sp. cultures.

Oleoylethanolamine and palmitoylethanolamine modulate intestinal permeability in vitro via TRPV1 and PPARα.

Cannabinoids modulate intestinal permeability through cannabinoid receptor 1 (CB1). The endocannabinoid-like compounds oleoylethanolamine (OEA) and palmitoylethanolamine (PEA) play an important role in digestive regulation, and we hypothesized they would also modulate intestinal permeability. Transepithelial electrical resistance (TEER) was measured in human Caco-2 cells to assess permeability after application of OEA and PEA and relevant antagonists. Cells treated with OEA and PEA were stained for cytoskeletal F-actin changes and lysed for immunoassay. OEA and PEA were measured by liquid chromatography-tandem mass spectrometry. OEA (applied apically, logEC50 -5.4) and PEA (basolaterally, logEC50 -4.9; apically logEC50 -5.3) increased Caco-2 resistance by 20-30% via transient receptor potential vanilloid (TRPV)-1 and peroxisome proliferator-activated receptor (PPAR)-α. Preventing their degradation (by inhibiting fatty acid amide hydrolase) enhanced the effects of OEA and PEA. OEA and PEA induced cytoskeletal changes and activated focal adhesion kinase and ERKs 1/2, and decreased Src kinases and aquaporins 3 and 4. In Caco-2 cells treated with IFNγ and TNFα, OEA (via TRPV1) and PEA (via PPARα) prevented or reversed the cytokine-induced increased permeability compared to vehicle (0.1% ethanol). PEA (basolateral) also reversed increased permeability when added 48 or 72 h after cytokines (P < 0.001, via PPARα). Cellular and secreted levels of OEA and PEA (P < 0.001-0.001) were increased in response to inflammatory mediators. OEA and PEA have endogenous roles and potential therapeutic applications in conditions of intestinal hyperpermeability and inflammation.-Karwad, M. A., Macpherson, T., Wang, B., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. Oleoylethanolamine and palmitoylethanolamine modulate intestinal permeability in vitro via TRPV1 and PPARα.

Robust anti-nociceptive effects of monoacylglycerol lipase inhibition in a model of osteoarthritis pain.

BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. EXPERIMENTAL APPROACH: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight-bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. KEY RESULTS: Acute MJN110 (5 mg·kg-1 ) significantly reversed MIA-induced weight-bearing asymmetry (MIA/vehicle: 68 ± 6 g; MIA/MJN110: 35 ± 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 ± 0.8 g; MIA/MJN110: 11 ± 0.6 g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg-1 ) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg-1 ) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane-associated PGE synthase-1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle-treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-arachidonoylglycerol. CONCLUSIONS AND IMPLICATIONS: Our data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain.

Changes in plasma levels of N-arachidonoyl ethanolamine and N-palmitoylethanolamine following bariatric surgery in morbidly obese females with impaired glucose homeostasis.

AIM: We examined endocannabinoids (ECs) in relation to bariatric surgery and the association between plasma ECs and markers of insulin resistance. METHODS: A study of 20 participants undergoing bariatric surgery. Fasting and 2-hour plasma glucose, lipids, insulin, and C-peptide were recorded preoperatively and 6 months postoperatively with plasma ECs (AEA, 2-AG) and endocannabinoid-related lipids (PEA, OEA). RESULTS: Gender-specific analysis showed differences in AEA, OEA, and PEA preoperatively with reductions in AEA and PEA in females postoperatively. Preoperatively, AEA was correlated with 2-hour glucose (r = 0.55, P = 0.01), HOMA-IR (r = 0.61, P = 0.009), and HOMA %S (r = -0.71, P = 0.002). OEA was correlated with weight (r = 0.49, P = 0.03), waist circumference (r = 0.52, P = 0.02), fasting insulin (r = 0.49, P = 0.04), and HOMA-IR (r = 0.48, P = 0.05). PEA was correlated with fasting insulin (r = 0.49, P = 0.04). 2-AG had a negative correlation with fasting glucose (r = -0.59, P = 0.04). CONCLUSION: Gender differences exist in circulating ECs in obese subjects. Females show changes in AEA and PEA after bariatric surgery. Specific correlations exist between different ECs and markers of obesity and insulin and glucose homeostasis.

Depolarizing and calcium-mobilizing stimuli fail to enhance synthesis and release of endocannabinoids from rat brain cerebral cortex slices.

The concentrations of the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (anandamide) were examined in rat brain cerebral cortex slices and surrounding medium. Basal concentrations of endocannabinoids were similar to those identified previously in rat brain, with anandamide content being much lower (19 pmol/g) than that of 2-AG (7300 pmol/g). In contrast, basal concentrations in the surrounding medium were proportionally much lower for 2-arachidonoylglycerol (16 pmol/mL) compared to anandamide (0.6 pmol/mL). Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue concentrations of endocannabinoids, while endocannabinoids in the medium were unaltered by elevated KCl. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue concentrations of anandamide (and related N-acylethanolamines), without altering 2-AG, while evoking proportional elevations of anandamide in the medium. Removal of extracellular calcium ions failed to alter tissue concentrations of anandamide, but significantly reduced 2-AG in the tissue by 90% and levels in the medium to below the detection limit. Supplementation of the medium with 50 µM N-oleoylethanolamine only raised tissue concentrations of N-oleoylethanolamine in the presence of cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester and failed to alter either tissue or medium anandamide or 2-AG concentrations. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-AG concentrations in this tissue.

Repeated novel cage exposure-induced improvement of early Alzheimer's-like cognitive and amyloid changes in TASTPM mice is unrelated to changes in brain endocannabinoids levels.

Environmental factors (e.g. stress, exercise, enrichment) are thought to play a role in the development of Alzheimer's disease later in life. We investigated the influence of repeated novel cage exposure on the development of early Alzheimer's-like pathology in adult (4 months old) double transgenic mice over-expressing the amyloid precursor protein and presenilin-1 genes (TASTPM mouse line). The procedure involves the repeated placement of the animal into a novel clean cage, a manipulation which induces a stress response and exploratory activity and, as such, can also be seen as a mild form of enrichment. Before and after exposure to the novel cage procedure, separate groups of mice were evaluated for locomotor performance and short-term contextual memory in the fear-conditioning test. Repeated novel cage exposure prevented the onset of a short-term memory deficit that was apparent in 5.5- but not 4-month-old TASTPM mice, without reversing the deficit in extinction already evident at 4 months of age. Brain regional levels of soluble and insoluble amyloid and of endocannabinoids were quantified. Novel cage exposure attenuated soluble and insoluble amyloid accumulation in the hippocampus and frontal cortex, without affecting the age-related increases in regional brain endocannabinoids levels. These beneficial effects are likely to be the consequence of the increase in physical and exploratory activity induced by novel cage exposure and suggest that the impact of environmental factors on Alzheimer's-like changes may be dependent on the degree of activation of stress pathways.