Four Weeks of Probiotic Supplementation Alters the Metabolic Perturbations Induced by Marathon Running: Insight from Metabolomics

Few data are available that describe how probiotics influence systemic metabolism during endurance exercise. Metabolomic profiling of endurance athletes will elucidate mechanisms by which probiotics may confer benefits to the athlete. In this study, twenty-four runners (20 male, 4 female) were block randomised into two groups using a double-blind matched-pairs design according to their most recent Marathon performance. Runners were assigned to 28-days of supplementation with a multi-strain probiotic (PRO) or a placebo (PLB). Following 28-days of supplementation, runners performed a competitive track Marathon race. Venous blood samples and muscle biopsies (vastus lateralis) were collected on the morning of the race and immediately post-race. Samples were subsequently analysed by untargeted 1H-NMR metabolomics. Principal component analysis (PCA) identified a greater difference in the post-Marathon serum metabolome in the PLB group vs. PRO. Univariate tests identified 17 non-overlapped metabolites in PLB, whereas only seven were identified in PRO. By building a PLS-DA model of two components, we revealed combinations of metabolites able to discriminate between PLB and PRO post-Marathon. PCA of muscle biopsies demonstrated no discernible difference post-Marathon between treatment groups. In conclusion, 28-days of probiotic supplementation alters the metabolic perturbations induced by a Marathon. Such findings may be related to maintaining the integrity of the gut during endurance exercise

Synovial Fluid Metabolites Differentiate between Septic and Nonseptic Joint Pathologies

Osteoarthritis (OA), osteochondrosis (OC), and synovial sepsis in horses cause loss of function and pain. Reliable biomarkers are required to achieve accurate and rapid diagnosis, with synovial fluid (SF) holding a unique source of biochemical information. Nuclear magnetic resonance (NMR) spectroscopy allows global metabolite analysis of a small volume of SF, with minimal sample preprocessing using a noninvasive and nondestructive method. Equine SF metabolic profiles from both nonseptic joints (OA and OC) and septic joints were analyzed using 1D 1H NMR spectroscopy. Univariate and multivariate statistical analyses were used to identify differential metabolite abundance between groups. Metabolites were annotated via 1H NMR using 1D NMR identification software Chenomx, with identities confirmed using 1D 1H and 2D 1H 13C NMR. Multivariate analysis identified separation between septic and nonseptic groups. Acetate, alanine, citrate, creatine phosphate, creatinine, glucose, glutamate, glutamine, glycine, phenylalanine, pyruvate, and valine were higher in the nonseptic group, while glycylproline was higher in sepsis. Multivariate separation was primarily driven by glucose; however, partial-least-squares discriminant analysis plots with glucose excluded demonstrated the remaining metabolites were still able to discriminate the groups. This study demonstrates that a panel of synovial metabolites can distinguish between septic and nonseptic equine SF, with glucose the principal discriminator

Ex-Vivo Equine Cartilage Explant Osteoarthritis Model - A Metabolomics and Proteomics Study.

Osteoarthritis is an age-related degenerative musculoskeletal disease characterised by loss of articular cartilage, synovitis and subchondral bone sclerosis. Osteoarthritis pathogenesis is yet to be fully elucidated with no osteoarthritis specific biomarkers in clinical use. Ex-vivo equine cartilage explants (n=5) were incubated in TNF-α/IL-1β supplemented culture media for 8 days, with media removed and replaced at 2, 5 and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent 1D 1H nuclear magnetic resonance metabolomic analysis with media samples also undergoing mass spectrometry proteomic analysis. Within the cartilage, glucose and lysine were elevated following TNF-α/IL-1β treatment whilst adenosine, alanine, betaine, creatine, myo-inositol and uridine decreased. Within the culture media, four, four and six differentially abundant metabolites and 154, 138 and 72 differentially abundant proteins were identified at 1-2 days, 3-5 days and 6-8 days respectively, including reduced alanine and increased isoleucine, enolase 1, vimentin and lamin A/C following treatment. Nine potential novel osteoarthritis neopeptides were elevated in treated media. Implicated pathways were dominated by those involved in cellular movement. Our innovative study has provided insightful information on early osteoarthritis pathogenesis, enabling potential translation for clinical markers and possible new therapeutic targets

"Fuel for the Damage Induced": Untargeted Metabolomics in Elite Rugby Union Match Play

The metabolic perturbations caused by competitive rugby are not well characterized. Our aim is to utilize untargeted metabolomics to develop appropriate interventions, based on the metabolic fluctuations that occur in response to this collision-based team sport. Seven members of an English Premiership rugby squad consented to provide blood, urine, and saliva samples daily, over a competitive week including gameday (GD), with physical demands and dietary intake also recorded. Sample collection, processing and statistical analysis were performed in accordance with best practice set out by the metabolomics standards initiative employing 700 MHz NMR spectroscopy. Univariate and multivariate statistical analysis were employed to reveal the acute energy needs of this high intensity sport are met via glycolysis, the TCA cycle and gluconeogenesis. The recovery period after cessation of match play and prior to training recommencing sees a re-entry to gluconeogenesis, coupled with markers of oxidative stress, structural protein degradation, and reduced fatty acid metabolism. This novel insight leads us to propose that effective recovery from muscle damaging collisions is dependent upon the availability of glucose. An adjustment in the periodisation of carbohydrate to increase GD+1 provision may prevent the oxidation of amino acids which may also be crucial to allay markers of structural tissue degradation. Should we expand the 'Fuel for the work required' paradigm in collision-based team sports to include 'Fuel for the damage induced'

Metabolic profiling of maternal serum of women a high-risk of spontaneous preterm birth using NMR and MGWAS approach

Preterm birth (PTB) is a leading global cause of infant mortality. Risk factors include genetics, lifestyle choices and infection. Understanding the mechanism of PTB could aid the development of novel approaches to prevent PTB. This study aimed to investigate the metabolic biomarkers of PTB in early pregnancy and the association of significant metabolites with participant genotypes. Maternal sera collected at 16 and 20 weeks of gestation, from women who previously experienced PTB (high-risk) and women who did not (low-risk controls), were analysed using (1)H nuclear magnetic resonance (NMR) metabolomics and genome-wide screening microarray. ANOVA and probabilistic neural network (PNN) modelling were performed on the spectral bins. Metabolomics genome-wide association (MGWAS) of the spectral bins and genotype data from the same participants was applied to determine potential metabolite-gene pathways. Phenylalanine, acetate and lactate metabolite differences between PTB cases and controls were obtained by ANOVA and PNN showed strong prediction at week 20 (AUC = 0.89). MGWAS identified several metabolite bins with strong genetic associations. Cis-eQTL analysis highlighted TRAF1 (involved in the inflammatory pathway) local to a non-coding SNP associated with lactate at week 20 of gestation. MGWAS of a well-defined cohort of participants highlighted a lactate-TRAF1 relationship that could potentially contribute to PTB

Serum ¹H nuclear magnetic resonance-based metabolomics of sole lesion development in Holstein cows.

Sole hemorrhage and sole ulcers, referred to as sole lesions, are important causes of lameness in dairy cattle. We aimed to compare the serum metabolome of dairy cows that developed sole lesions in early lactation with that of cows that remained unaffected. We prospectively enrolled a cohort of 1,169 Holstein dairy cows from a single dairy herd and assessed animals at 4 time points: before calving, immediately after calving, early lactation, and late lactation. Sole lesions were recorded by veterinary surgeons at each time point, and serum samples were collected at the first 3 time points. Cases were defined by the presence of sole lesions in early lactation and further subdivided by whether sole lesions had been previously recorded; unaffected controls were randomly selected to match cases. Serum samples from a case-control subset of 228 animals were analyzed with proton nuclear magnetic resonance spectroscopy. Spectral signals, corresponding to 34 provisionally annotated metabolites and 51 unlabeled metabolites, were analyzed in subsets relating to time point, parity cohort, and sole lesion outcome. We used 3 analytic methods (partial least squares discriminant analysis, least absolute shrinkage and selection operator regression, and random forest) to determine the predictive capacity of the serum metabolome and identify informative metabolites. We applied bootstrapped selection stability, triangulation, and permutation to support the inference of variable selection. The average balanced accuracy of class prediction ranged from 50 to 62% depending on the subset. Across all 17 subsets, 20 variables had a high probability of being informative; those with the strongest evidence of being associated with sole lesions corresponded to phenylalanine and 4 unlabeled metabolites. We conclude that the serum metabolome, as characterized by proton nuclear magnetic resonance spectroscopy, does not appear able to predict sole lesion presence or future development of lesions. A small number of metabolites may be associated with sole lesions although, given the poor prediction accuracies, these metabolites are likely to explain only a small proportion of the differences between affected and unaffected animals. Future metabolomic studies may reveal underlying metabolic mechanisms of sole lesion etiopathogenesis in dairy cows; however, the experimental design and analysis need to effectively control for interanimal and extraneous sources of spectral variation

α-Methyl-α-phenylsuccinimide ameliorates neurodegeneration in a C. elegans model of TDP-43 proteinopathy

The antiepileptic drug ethosuximide has recently been shown to be neuroprotective in various Caenorhabditis elegans and rodent neurodegeneration models. It is therefore a promising repurposing candidate for the treatment of multiple neurodegenerative diseases. However, high concentrations of the drug are required for its protective effects in animal models, which may impact on its translational potential and impede the identification of its molecular mechanism of action. Therefore, we set out to develop more potent neuroprotective lead compounds based on ethosuximide as a starting scaffold. Chemoinformatic approaches were used to identify compounds with structural similarity to ethosuximide and to prioritise these based on good predicated blood-brain barrier permeability and C. elegans bioaccumulation properties. Selected compounds were initially screened for anti-convulsant activity in a C. elegans pentylenetetrazol-induced seizure assay, as a rapid primary readout of bioactivity; and then assessed for neuroprotective properties in a C. elegans TDP-43 proteinopathy model based on pan-neuronal expression of human A315T mutant TDP-43. The most potent compound screened, α-methyl-α-phenylsuccinimide (MPS), ameliorated the locomotion defects and extended the shortened lifespan of TDP-43 mutant worms. MPS also directly protected against neurodegeneration by reducing the number of neuronal breaks and cell body losses in GFP-labelled GABAergic motor neurons. Importantly, optimal neuroprotection was exhibited by external application of 50 μM MPS, compared to 8 mM for ethosuximide. This greater potency of MPS was not due to bioaccumulation to higher internal levels within the worm, based on 1H-nuclear magnetic resonance analysis. Like ethosuximide, the activity of MPS was abolished by mutation of the evolutionarily conserved FOXO transcription factor, daf-16, suggesting that both compounds act via the same neuroprotective pathway(s). In conclusion, we have revealed a novel neuroprotective activity of MPS that is >100-fold more potent than ethosuximide. This increased potency will facilitate future biochemical studies to identify the direct molecular target(s) of both compounds, as we have shown here that they share a common downstream DAF-16-dependent mechanism of action. Furthermore, MPS is the active metabolite of another approved antiepileptic drug, methsuximide. Therefore, methsuximide may have repurposing potential for treatment of TDP-43 proteinopathies and possibly other human neurodegenerative diseases

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations