Exercise and ageing impact the kynurenine/tryptophan pathway and acylcarnitine metabolite pools in skeletal muscle of older adults.

Exercise-induced perturbation of skeletal muscle metabolites is a probable mediator of long-term health benefits in older adults. Although specific metabolites have been identified to be impacted by age, physical activity and exercise, the depth of coverage of the muscle metabolome is still limited. Here, we investigated resting and exercise-induced metabolite distribution in muscle from well-phenotyped older adults who were active or sedentary, and a group of active young adults. Percutaneous biopsies of the vastus lateralis were obtained before, immediately after and 3 h following a bout of endurance cycling. Metabolite profile in muscle biopsies was determined by tandem mass spectrometry. Mitochondrial energetics in permeabilized fibre bundles was assessed by high resolution respirometry and fibre type proportion was assessed by immunohistology. We found that metabolites of the kynurenine/tryptophan pathway were impacted by age and activity. Specifically, kynurenine was elevated in muscle from older adults, whereas downstream metabolites of kynurenine (kynurenic acid and NAD+ ) were elevated in muscle from active adults and associated with cardiorespiratory fitness and muscle oxidative capacity. Acylcarnitines, a potential marker of impaired metabolic health, were elevated in muscle from physically active participants. Surprisingly, despite baseline group difference, acute exercise-induced alterations in whole-body substrate utilization, as well as muscle acylcarnitines and ketone bodies, were remarkably similar between groups. Our data identified novel muscle metabolite signatures that associate with the healthy ageing phenotype provoked by physical activity and reveal that the metabolic responsiveness of muscle to acute endurance exercise is retained [NB]:AUTHOR: Please ensure that the appropriate material has been provide for Table S2, as well as for Figures S1 to S7, as also cited in the text with age regardless of activity levels. KEY POINTS: Kynurenine/tryptophan pathway metabolites were impacted by age and physical activity in human muscle, with kynurenine elevated in older muscle, whereas downstream products kynurenic acid and NAD+ were elevated in exercise-trained muscle regardless of age. Acylcarnitines, a marker of impaired metabolic health when heightened in circulation, were elevated in exercise-trained muscle of young and older adults, suggesting that muscle act as a metabolic sink to reduce the circulating acylcarnitines observed with unhealthy ageing. Despite the phenotypic differences, the exercise-induced response of various muscle metabolite pools, including acylcarnitine and ketone bodies, was similar amongst the groups, suggesting that older adults can achieve the metabolic benefits of exercise seen in young counterparts.

A Novel Endocrine Role for the BAT-Released Lipokine 12,13-diHOME to Mediate Cardiac Function.

BACKGROUND: Brown adipose tissue (BAT) is an important tissue for thermogenesis, making it a potential target to decrease the risks of obesity, type 2 diabetes, and cardiovascular disease, and recent studies have also identified BAT as an endocrine organ. Although BAT has been implicated to be protective in cardiovascular disease, to this point there are no studies that identify a direct role for BAT to mediate cardiac function. METHODS: To determine the role of BAT on cardiac function, we utilized a model of BAT transplantation. We then performed lipidomics and identified an increase in the lipokine 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME). We utilized a mouse model with sustained overexpression of 12,13-diHOME and investigated the role of 12,13-diHOME in a nitric oxide synthase type 1 deficient (NOS1-/-) mouse and in isolated cardiomyocytes to determine effects on function and respiration. We also investigated 12,13-diHOME in a cohort of human patients with heart disease. RESULTS: Here, we determined that transplantation of BAT (+BAT) improves cardiac function via the release of the lipokine 12,13-diHOME. Sustained overexpression of 12,13-diHOME using tissue nanotransfection negated the deleterious effects of a high-fat diet on cardiac function and remodeling, and acute injection of 12,13-diHOME increased cardiac hemodynamics via direct effects on the cardiomyocyte. Furthermore, incubation of cardiomyocytes with 12,13-diHOME increased mitochondrial respiration. The effects of 12,13-diHOME were absent in NOS1-/- mice and cardiomyocytes. We also provide the first evidence that 12,13-diHOME is decreased in human patients with heart disease. CONCLUSIONS: Our results identify an endocrine role for BAT to enhance cardiac function that is mediated by regulation of calcium cycling via 12,13-diHOME and NOS1.

Multiplexed LC-MS/MS analysis of methylsuccinic acid, ethylmalonic acid, and glutaric acid in plasma and urine.

Low molecular-mass aliphatic carboxylic acids are critically important for intermediate metabolism and may serve as important biomarkers for metabolic homeostasis. Here in, we focused on multiplexed method development of aliphatic carboxylic analytes, including methylsuccinic acid (MSA), ethylmalonic acid (EMA), and glutaric acid (GA). Also assessed was their utility in a population's health as well as metabolic disease screening in both plasma and urine matrices. MSA, EMA, and GA are constitutional isomers of dicarboxylic acid with high polarity and poor ionization efficiency, resulting in such challenges as poor signal intensity and retention, particularly in reversed-phase liquid chromatography with electrospray mass spectrometry (RP-LC-ESI-MS/MS). Derivatization using n-butanol was performed in the sample preparation to enhance the signal intensity accompanied with a positive charge from ionization in complicated biomatrices as well as to improve the separation of these isomers with optimal retention. Fit-for-purpose method validation results demonstrated quantitative ranges for MSA/EMA/GA from 5/10/20 ng/mL to 400 ng/mL in plasma analysis, and 100/200/100 ng/mL to 5000/10000/5000 ng/mL in urine analysis. This validated method demonstrates future utility when exploring population health analysis and biomarker development in metabolic diseases.

Transplantation of Brown Adipose Tissue with the Ability of Converting Omega-6 to Omega-3 Polyunsaturated Fatty Acids Counteracts High-Fat-Induced Metabolic Abnormalities in Mice.

A balanced omega (ω)-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio has been linked to metabolic health and the prevention of chronic diseases. Brown adipose tissue (BAT) specializes in energy expenditure and secretes signaling molecules that regulate metabolism via inter-organ crosstalk. Recent studies have uncovered that BAT produces different PUFA species and circulating oxylipin levels are correlated with BAT-mediated energy expenditure in mice and humans. However, the impact of BAT ω-6/ω-3 PUFAs on metabolic phenotype has not been fully elucidated. The Fat-1 transgenic mice can convert ω-6 to ω-3 PUFAs. Here, we demonstrated that mice receiving Fat-1 BAT transplants displayed better glucose tolerance and higher energy expenditure. Expression of genes involved in thermogenesis and nutrient utilization was increased in the endogenous BAT of mice receiving Fat-1 BAT, suggesting that the transplants may activate recipients' BAT. Using targeted lipidomic analysis, we found that the levels of several ω-6 oxylipins were significantly reduced in the circulation of mice receiving Fat-1 BAT transplants than in mice with wild-type BAT transplants. The major altered oxylipins between the WT and Fat-1 BAT transplantation were ω-6 arachidonic acid-derived oxylipins via the lipoxygenase pathway. Taken together, these findings suggest an important role of BAT-derived oxylipins in combating obesity-related metabolic disorders.

The Next Frontier: Translational Development of Ubiquitination, SUMOylation, and NEDDylation in Cancer.

Post-translational modifications of proteins ensure optimized cellular processes, including proteostasis, regulated signaling, cell survival, and stress adaptation to maintain a balanced homeostatic state. Abnormal post-translational modifications are associated with cellular dysfunction and the occurrence of life-threatening diseases, such as cancer and neurodegenerative diseases. Therefore, some of the frequently seen protein modifications have been used as disease markers, while others are targeted for developing specific therapies. The ubiquitin and ubiquitin-like post-translational modifiers, namely, small ubiquitin-like modifier (SUMO) and neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8), share several features, such as protein structures, enzymatic cascades mediating the conjugation process, and targeted amino acid residues. Alterations in the regulatory mechanisms lead to aberrations in biological processes during tumorigenesis, including the regulation of tumor metabolism, immunological modulation of the tumor microenvironment, and cancer stem cell stemness, besides many more. Novel insights into ubiquitin and ubiquitin-like pathways involved in cancer biology reveal a potential interplay between ubiquitination, SUMOylation, and NEDDylation. This review outlines the current understandings of the regulatory mechanisms and assay capabilities of ubiquitination, SUMOylation, and NEDDylation. It will further highlight the role of ubiquitination, SUMOylation, and NEDDylation in tumorigenesis.

The influence of low-carbohydrate diets on the metabolic response to androgen-deprivation therapy in prostate cancer.

BACKGROUND: Prostate cancer (PC) is the second most lethal cancer for men. For metastatic PC, standard first-line treatment is androgen deprivation therapy (ADT). While effective, ADT has many metabolic side effects. Previously, we found in serum metabolome analysis that ADT reduced androsterone sulfate, 3-hydroxybutyric acid, acyl-carnitines but increased serum glucose. Since ADT reduced ketogenesis, we speculate that low-carbohydrate diets (LCD) may reverse many ADT-induced metabolic abnormalities in animals and humans. METHODS: In a multicenter trial of patients with PC initiating ADT randomized to no diet change (control) or LCD, we previously showed that LCD intervention led to significant weight loss, reduced fat mass, improved insulin resistance, and lipid profiles. To determine whether and how LCD affects ADT-induced metabolic changes, we analyzed serum metabolites after 3-, and 6-months of ADT on LCD versus control. RESULTS: We found androsterone sulfate was most consistently reduced by ADT and was slightly further reduced in the LCD arm. Contrastingly, LCD intervention increased 3-hydroxybutyric acid and various acyl-carnitines, counteracting their reduction during ADT. LCD also reversed the ADT-reduced lactic acid, alanine, and S-adenosyl methionine (SAM), elevating glycolysis metabolites and alanine. While the degree of androsterone reduction by ADT was strongly correlated with glucose and indole-3-carboxaldehyde, LCD disrupted such correlations. CONCLUSIONS: Together, LCD intervention significantly reversed many ADT-induced metabolic changes while slightly enhancing androgen reduction. Future research is needed to confirm these findings and determine whether LCD can mitigate ADT-linked comorbidities and possibly delaying disease progression by further lowering androgens.

A phase I safety study of topical calcitriol (BPM31543) for the prevention of chemotherapy-induced alopecia.

PURPOSE: Chemotherapy-induced alopecia (CIA) negatively affects psychosocial health and quality of life (QoL). Currently, there are no approved pharmacologic agents to prevent CIA. Here, we evaluated the safety, tolerability, and potential signal of efficacy of topical calcitriol (BPM31543) on CIA prevention. MATERIALS AND METHODS: This Phase 1 trial included 23 female patients with breast cancer, gynecologic cancer, or sarcomas receiving a taxane-based chemotherapy. Patients received a 3 + 3 dose-escalation regimen at 5, 10, 20, 40, 60, and 80 µg/mL, with 3-6 patients per group. Patients applied topical BPM31543 to the scalp twice a day for 2 weeks prior to chemotherapy and continued until chemotherapy treatment was completed. The maximum tolerated dose (MTD) during first 28 day application was determined. Adverse event (AE) monitoring, pharmacokinetics, blinded photographic assessments, and patient self-assessment were evaluated. RESULTS: Out of 23 patients treated with BPM31543, 8 patients experienced at least 1 treatment-related adverse event (AE). The majority of AEs were mild to moderate in severity. Only 1 patient experienced SAEs (vomiting, nausea, fever, and flank pain) considered treatment related. Alopecia < 50% from baseline was observed in 8 patients at Week 7, and, of which 2 patients had < 50% alopecia maintained at Week 15. There were no detectable effects of topical BPM31543 on serum levels of calcitriol. CONCLUSIONS: BPM31543 applied topically twice daily to the scalp is safe and well tolerated in patients receiving taxane-based chemotherapy. No DLT was observed at up to 80 µg/mL, and MTD was not reached. Based on the data from this trial, BPM31543 represents a promising therapy and warrants further investigation in Phase 2/3 trials.

Multi-omic serum biomarkers for prognosis of disease progression in prostate cancer.

BACKGROUND: Predicting the clinical course of prostate cancer is challenging due to the wide biological spectrum of the disease. The objective of our study was to identify prostate cancer prognostic markers in patients 'sera using a multi-omics discovery platform. METHODS: Pre-surgical serum samples collected from a longitudinal, racially diverse, prostate cancer patient cohort (N = 382) were examined. Linear Regression and Bayesian computational approaches integrated with multi-omics, were used to select markers to predict biochemical recurrence (BCR). BCR-free survival was modeled using unadjusted Kaplan-Meier estimation curves and multivariable Cox proportional hazards analysis, adjusted for key pathologic variables. Receiver operating characteristic (ROC) curve statistics were used to examine the predictive value of markers in discriminating BCR events from non-events. The findings were further validated by creating a training set (N = 267) and testing set (N = 115) from the cohort. RESULTS: Among 382 patients, 72 (19%) experienced a BCR event in a median follow-up time of 6.9 years. Two proteins-Tenascin C (TNC) and Apolipoprotein A1V (Apo-AIV), one metabolite-1-Methyladenosine (1-MA) and one phospholipid molecular species phosphatidic acid (PA) 18:0-22:0 showed a cumulative predictive performance of AUC = 0.78 [OR (95% CI) = 6.56 (2.98-14.40), P < 0.05], in differentiating patients with and without BCR event. In the validation set all four metabolites consistently reproduced an equivalent performance with high negative predictive value (NPV; > 80%) for BCR. The combination of pTstage and Gleason score with the analytes, further increased the sensitivity [AUC = 0.89, 95% (CI) = 4.45-32.05, P < 0.05], with an increased NPV (0.96) and OR (12.4) for BCR. The panel of markers combined with the pathological parameters demonstrated a more accurate prediction of BCR than the pathological parameters alone in prostate cancer. CONCLUSIONS: In this study, a panel of serum analytes were identified that complemented pathologic patient features in predicting prostate cancer progression. This panel offers a new opportunity to complement current prognostic markers and to monitor the potential impact of primary treatment versus surveillance on patient oncological outcome.

Comparative analysis of differentially abundant proteins quantified by LC-MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples.

BACKGROUND: Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry (MS). However, FF specimens are comprised of multiple cell types, making it difficult to ascertain the proteomic profiles of specific cells. Conversely, OCT-embedded (Optimal Cutting Temperature compound) specimens can undergo laser microdissection (LMD) to capture and study specific cell types separately from the cell mixture. In the current study, we compared proteomic data obtained from FF and OCT samples to determine if samples that are stored and processed differently produce comparable results. METHODS: Proteins were extracted from FF and OCT-embedded invasive breast tumors from 5 female patients. FF specimens were lysed via homogenization (FF/HOM) while OCT-embedded specimens underwent LMD to collect only tumor cells (OCT/LMD-T) or both tumor and stromal cells (OCT/LMD-TS) followed by incubation at 37 °C. Proteins were extracted using the illustra triplePrep kit and then trypsin-digested, TMT-labeled, and processed by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Proteins were identified and quantified with Proteome Discoverer v1.4 and comparative analyses performed to identify proteins that were significantly differentially expressed amongst the different processing methods. RESULTS: Among the 4,950 proteins consistently quantified across all samples, 216 and 171 proteins were significantly differentially expressed (adjusted p-value < 0.05; |log2 FC|> 1) between FF/HOM vs. OCT/LMD-T and FF/HOM vs. OCT/LMD-TS, respectively, with most proteins being more highly abundant in the FF/HOM samples. PCA and unsupervised hierarchical clustering analysis with these 216 and 171 proteins were able to distinguish FF/HOM from OCT/LMD-T and OCT/LMD-TS samples, respectively. Similar analyses using significantly differentially enriched GO terms also discriminated FF/HOM from OCT/LMD samples. No significantly differentially expressed proteins were detected between the OCT/LMD-T and OCT/LMD-TS samples but trended differences were detected. CONCLUSIONS: The proteomic profiles of the OCT/LMD-TS samples were more similar to those from OCT/LMD-T samples than FF/HOM samples, suggesting a strong influence from the sample processing methods. These results indicate that in LC-MS/MS proteomic studies, FF/HOM samples exhibit different protein expression profiles from OCT/LMD samples and thus, results from these two different methods cannot be directly compared.

Mycoplasma infection and hypoxia initiate succinate accumulation and release in the VM-M3 cancer cells.

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1ß production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.

The total and mitochondrial lipidome of Artemia franciscana encysted embryos.

Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the 'late embryogenesis abundant' proteins, which are known to interact with lipid membranes.

A next generation sequencing based approach to identify extracellular vesicle mediated mRNA transfers between cells.

BACKGROUND: Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. RESULTS: In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the "sequence varying mRNAs" transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. CONCLUSIONS: Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.

CoQ10 selective miscibility and penetration into lipid monolayers with lower lateral packing density.

CoQ10 is ubiquitously present in eukaryotic cells. It acts as electron carrier in the electron transport chain of the inner membrane of the mitochondria to facilitate aerobic cellular respiration. A highly stable lipid nanodispersion formulation containing CoQ10 (BPM31510) is currently in clinical investigation for treatment of cancer. This study was designed to determine whether biophysical interactions between CoQ10 and lipid, in part, explain the observed stability and cellular accumulation of CoQ10 in cells and tissues. A lipid monolayer at the air-water interface was used as an experimental membrane model to measure CoQ10 penetration and solubility. Lipid monolayers with varying proportions of CoQ10 were laterally compressed to measure CoQ10 miscibility and lateral organization. Additionally, lipid monolayers with varying lateral packing densities were spread at the air-water interface and CoQ10 was injected in proximity to measure its rate of penetration. Our results demonstrate that CoQ10 selectively penetrates into lipid monolayers with a lower lateral packing density, and is excluded by monolayers of higher packing densities. Data also indicates that CoQ10-lipid mixing is non-ideal. CoQ10 presence in lipid monolayers is biphasic, with one phase occupying the interstitial space between the DMPC lipids, and the other phase is present as pure CoQ10 domains. This work provides further insight into mechanism of action of CoQ10 based formulations that can significantly increase intracellular CoQ10 concentration to show pleotropic effects on cellular functions.

Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses.

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1ß, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity.

Filamin A Is a Prognostic Serum Biomarker for Differentiating Benign Prostatic Hyperplasia from Prostate Cancer in Caucasian and African American Men.

Prostate cancer represents a significant health risk to aging men, in which diagnostic challenges to the identification of aggressive cancers remain unmet. Prostate cancer screening is driven by the prostate-specific antigen (PSA); however, in men with benign prostatic hyperplasia (BPH) due to an enlarged prostate and elevated PSA, PSA's screening utility is diminished, resulting in many unnecessary biopsies. To address this issue, we previously identified a cleaved fragment of Filamin A (FLNA) protein (as measured with IP-MRM mass spectrometry assessment as a prognostic biomarker for stratifying BPH from prostate cancer and subsequently evaluated its expanded utility in Caucasian (CA) and African American (AA) men. All men had a negative digital rectal examination (DRE) and PSA between 4 and 10 ng/mL and underwent prostate biopsy. In AA men, FLNA serum levels exhibited diagnostic utility for stratifying BPH from patients with aggressive prostate cancer (0.71 AUC and 12.2 OR in 48 men with BPH and 60 men with PCa) and outperformed PSA (0.50 AUC, 2.2 OR). In CA men, FLNA serum levels also exhibited diagnostic utility for stratifying BPH from patients with aggressive prostate cancer (0.74 AUC and 19.4 OR in 191 men with BPH and 109 men with PCa) and outperformed PSA (0.46 AUC, 0.32 OR). Herein, we established FLNA alone as a serum biomarker for stratifying men with BPH vs. those with high Gleason (7-10) prostate cancers compared to the current diagnostic paradigm of using PSA. This approach demonstrates clinical actionability of FLNA alone without the requirement of prostate volume measurement as a test with utility in AA and CA men and represents a significant opportunity to decrease the number of unnecessary biopsies in aggressive prostate cancer diagnoses.

Identification and validation of N-acetylputrescine in combination with non-canonical clinical features as a Parkinson's disease biomarker panel.

Parkinson's disease is a progressive neurodegenerative disorder in which loss of dopaminergic neurons in the substantia nigra results in a clinically heterogeneous group with variable motor and non-motor symptoms with a degree of misdiagnosis. Only 3-25% of sporadic Parkinson's patients present with genetic abnormalities that could represent a risk factor, thus environmental, metabolic, and other unknown causes contribute to the pathogenesis of Parkinson's disease, which highlights the critical need for biomarkers. In the present study, we prospectively collected and analyzed plasma samples from 194 Parkinson's disease patients and 197 age-matched non-diseased controls. N-acetyl putrescine (NAP) in combination with sense of smell (B-SIT), depression/anxiety (HADS), and acting out dreams (RBD1Q) clinical measurements demonstrated combined diagnostic utility. NAP was increased by 28% in Parkinsons disease patients and exhibited an AUC of 0.72 as well as an OR of 4.79. The clinical and NAP panel demonstrated an area under the curve, AUC = 0.9 and an OR of 20.4. The assessed diagnostic panel demonstrates combinatorial utility in diagnosing Parkinson's disease, allowing for an integrated interpretation of disease pathophysiology and highlighting the use of multi-tiered panels in neurological disease diagnosis.

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia.

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

Chronic exercise improves hepatic acylcarnitine handling.

Exercise mediates tissue metabolic function through direct and indirect adaptations to acylcarnitine (AC) metabolism, but the exact mechanisms are unclear. We found that circulating medium-chain acylcarnitines (AC) (C12-C16) are lower in active/endurance trained human subjects compared to sedentary controls, and this is correlated with elevated cardiorespiratory fitness and reduced adiposity. In mice, exercise reduced serum AC and increased liver AC, and this was accompanied by a marked increase in expression of genes involved in hepatic AC metabolism and mitochondrial ß-oxidation. Primary hepatocytes from high-fat fed, exercise trained mice had increased basal respiration compared to hepatocytes from high-fat fed sedentary mice, which may be attributed to increased Ca2+ cycling and lipid uptake into mitochondria. The addition of specific medium- and long-chain AC to sedentary hepatocytes increased mitochondrial respiration, mirroring the exercise phenotype. These data indicate that AC redistribution is an exercise-induced mechanism to improve hepatic function and metabolism.

Multiomic foundations of human prefrontal cortex tissue function.

The prefrontal cortex (PFC) is a region of the brain that in humans is involved in the production of higher-order functions such as cognition, emotion, perception, and behavior. Neurotransmission in the PFC produces higher-order functions by integrating information from other areas of the brain. At the foundation of neurotransmission, and by extension at the foundation of higher-order brain functions, are an untold number of coordinated molecular processes involving the DNA sequence variants in the genome, RNA transcripts in the transcriptome, and proteins in the proteome. These "multiomic" foundations are poorly understood in humans, perhaps in part because most modern studies that characterize the molecular state of the human PFC use tissue obtained when neurotransmission and higher-order brain functions have ceased (i.e., the postmortem state). Here, analyses are presented on data generated for the Living Brain Project (LBP) to investigate whether PFC tissue from individuals with intact higher-order brain function has characteristic multiomic foundations. Two complementary strategies were employed towards this end. The first strategy was to identify in PFC samples obtained from living study participants a signature of RNA transcript expression associated with neurotransmission measured intracranially at the time of PFC sampling, in some cases while participants performed a task engaging higher-order brain functions. The second strategy was to perform multiomic comparisons between PFC samples obtained from individuals with intact higher-order brain function at the time of sampling (i.e., living study participants) and PFC samples obtained in the postmortem state. RNA transcript expression within multiple PFC cell types was associated with fluctuations of dopaminergic, serotonergic, and/or noradrenergic neurotransmission in the substantia nigra measured while participants played a computer game that engaged higher-order brain functions. A subset of these associations - termed the "transcriptional program associated with neurotransmission" (TPAWN) - were reproduced in analyses of brain RNA transcript expression and intracranial neurotransmission data obtained from a second LBP cohort and from a cohort in an independent study. RNA transcripts involved in TPAWN were found to be (1) enriched for RNA transcripts associated with measures of neurotransmission in rodent and cell models, (2) enriched for RNA transcripts encoded by evolutionarily constrained genes, (3) depleted of RNA transcripts regulated by common DNA sequence variants, and (4) enriched for RNA transcripts implicated in higher-order brain functions by human population genetic studies. In PFC excitatory neurons of living study participants, higher expression of the genes in TPAWN tracked with higher expression of RNA transcripts that in rodent PFC samples are markers of a class of excitatory neurons that connect the PFC to deep brain structures. TPAWN was further reproduced by RNA transcript expression patterns differentiating living PFC samples from postmortem PFC samples, and significant differences between living and postmortem PFC samples were additionally observed with respect to (1) the expression of most primary RNA transcripts, mature RNA transcripts, and proteins, (2) the splicing of most primary RNA transcripts into mature RNA transcripts, (3) the patterns of co-expression between RNA transcripts and proteins, and (4) the effects of some DNA sequence variants on RNA transcript and protein expression. Taken together, this report highlights that studies of brain tissue obtained in a safe and ethical manner from large cohorts of living individuals can help advance understanding of the multiomic foundations of brain function.

Development and characterization of phospholipid-stabilized submicron aqueous dispersions of coenzyme Q10 presenting continuous vibrating-mesh nebulization performance.

Coenzyme Q10 (CoQ10) is a poorly-water soluble compound that is being investigated for the treatment of carcinomas. The aim of this research was to develop a suitable formulation for pulmonary delivery of this anticancer agent. An appropriate selection of excipients (phospholipids) and a suitable device (Aeroneb Pro® vibrating-mesh nebulizer) were selected initially after reviewing the literature. After characterization of the bulk drug, a feasible manufacturing process was selected to obtain small particle size dispersions of CoQ10. Following selection of an appropriate process, the parameters affecting drug particle size were studied. Using LD and gravimetrical analysis, nebulization was evaluated to assess the performance of the inhalation system triad: drug-excipients-device. CoQ10 powder studied was crystalline with a melting point approximately at 51 °C and with a particle size of 30 µm. Microfluidization was found to be a suitable method to prepare submicron drug particles in aqueous dispersions. Increasing microfluidization processing to more than 50 passes did not provide further particle downsizing for both soya phosphatidylcholine (lecithin) and dipalmitoyl phosphatidylcholine (DPPC) dispersions of CoQ10, presenting Z-average values of approximately 130 and 70 nm, respectively. Nebulization performance of lecithin-stabilized CoQ10 dispersions varied according to number of passes in the microfluidizer. Formulations processed with 10 passes presented steadier nebulization over time and different rheological behavior compared to those processed with 30 or 50 passes. In conclusion, aqueous dispersions of CoQ10 were adequately produced using a microfluidizer with characteristics that were suitable for pulmonary delivery with an Aeroneb Pro® nebulizer. Furthermore, the rheology of these dispersions appeared to play a significant role in the aerosol generation from the active vibrating-mesh nebulizer used.