Novel odd-chain cyclopropane fatty acids: detection in a mammalian lipidome and uptake by hepatosplanchnic tissues.

Microbe-produced molecules (xenometabolites) found in foods or produced by gut microbiota are increasingly implicated in microbe-microbe and microbe-host communication. Xenolipids, in particular, are a class of metabolites for which the full catalog remains to be elaborated in mammalian systems. We and others have observed that cis-3,4-methylene-heptanoylcarnitine is a lipid derivative that is one of the most abundant medium-chain acylcarnitines in human blood, hypothesized to be a product of incomplete ß-oxidation of one or more "odd-chain" long-chain cyclopropane fatty acids (CpFAs). We deduced two possible candidates, cis-11,12-methylene-pentadecanoic acid (cis-11,12-MPD) and cis-13,14-methylene-heptadecanoic acid (cis-13,14-MHD). Authentic standards were synthesized: cis-11-pentadecenoic acid and cis-13-heptadecenoic acid were generated (using Jones reagent) from cis-11-pentadecene-1-ol and cis-13-heptadecene-1-ol, respectively, and these were converted to CpFAs via a reaction involving diiodomethane. Using these standards in mass spectrometry analyses, we determined the presence/absence of cis-11,12-MPD and cis-13,14-MHD in archived piglet biospecimens. Both CpFAs were detected in rectal contents of sow and soy-fed piglets. Archived mass spectra were analyzed post hoc from a second independent study that used tissue-specific catheterization to monitor net metabolite flux in growing pigs. This confirmed the presence of both CpFAs in plasma and revealed a significant net uptake of the odd-chain CpFAs across the splanchnic tissue bed and liver. The results confirm that the novel xenolipids cis-11,12-MPD and cis-13,14-MHD can be components of the mammalian lipidome and are viable candidate precursors of cis-3,4-methylene-heptanoylcarnitine produced from partial ß-oxidation in liver or other tissues.

Elamipretide treatment during pregnancy ameliorates the progression of polycystic kidney disease in maternal and neonatal mice with PKD1 mutations.

Pregnancy is proposed to aggravate cyst progression in autosomal dominant polycystic kidney disease (ADPKD) but Tolvaptan, the only FDA-approved drug for adult ADPKD, is not recommended for pregnant ADPKD patients because of potential fetal harm. Since pregnancy itself may increase the risk for ADPKD progression, we investigated the safety and efficacy of Elamipretide, a mitochondrial-protective tetrapeptide. Elamipretide was found to ameliorate the progression of kidney disease in pregnant Pkd1RC/RC mice, in parallel with attenuation of ERK1/2 phosphorylation and improvement of mitochondrial supercomplex formation. Furthermore, Elamipretide was found to pass through the placenta and breast milk and ameliorate aggressive infantile polycystic kidney disease without any observed teratogenic or harmful effect. Elamipretide has an excellent safety profile and is currently tested in multiple phase II and phase III clinical trials. These preclinical studies support a potential clinical trial of Elamipretide for the treatment of ADPKD, particularly for patients that cannot take Tolvaptan.

Gut Microbiome and Metabolome Modulation by Maternal High-Fat Diet and Thermogenic Challenge.

The gut microbiota plays a critical role in energy homeostasis and its dysbiosis is associated with obesity. Maternal high-fat diet (HFD) and ß-adrenergic stimuli alter the gut microbiota independently; however, their collective regulation is not clear. To investigate the combined effect of these factors on offspring microbiota, 20-week-old offspring from control diet (17% fat)- or HFD (45% fat)-fed dams received an injection of either vehicle or ß3-adrenergic agonist CL316,243 (CL) for 7 days and then cecal contents were collected for bacterial community profiling. In a follow-up study, a separate group of mice were exposed to either 8 °C or 30 °C temperature for 7 days and blood serum and cecal contents were used for metabolome profiling. Both maternal diet and CL modulated the gut bacterial community structure and predicted functional profiles. Particularly, maternal HFD and CL increased the Firmicutes/Bacteroidetes ratio. In mice exposed to different temperatures, the metabolome profiles clustered by treatment in both the cecum and serum. Identified metabolites were enriched in sphingolipid and amino acid metabolism in the cecum and in lipid and energy metabolism in the serum. In summary, maternal HFD altered offspring's response to CL and altered microbial composition and function. An independent experiment supported the effect of thermogenic challenge on the bacterial function through metabolome change.

Metabolomic profiles of current cigarette smokers.

Smoking-related biomarkers for lung cancer and other diseases are needed to enhance early detection strategies and to provide a science base for tobacco product regulation. An untargeted metabolomics approach by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF MS) totaling 957 assays was used in a novel experimental design where 105 current smokers smoked two cigarettes 1 h apart. Blood was collected immediately before and after each cigarette allowing for within-subject replication. Dynamic changes of the metabolomic profiles from smokers' four blood samples were observed and biomarkers affected by cigarette smoking were identified. Thirty-one metabolites were definitively shown to be affected by acute effect of cigarette smoking, uniquely including menthol-glucuronide, the reduction of glutamate, oleamide, and 13 glycerophospholipids. This first time identification of a menthol metabolite in smokers' blood serves as proof-of-principle for using metabolomics to identify new tobacco-exposure biomarkers, and also provides new opportunities in studying menthol-containing tobacco products in humans. Gender and race differences also were observed. Network analysis revealed 12 molecules involved in cancer, notably inhibition of cAMP. These novel tobacco-related biomarkers provide new insights to the effects of smoking which may be important in carcinogenesis but not previously linked with tobacco-related diseases. © 2016 Wiley Periodicals, Inc.

Optimization of Mass Spectrometric Parameters in Data Dependent Acquisition for Untargeted Metabolomics on the Basis of Putative Assignments.

Optimization of mass spectrometric parameters for a data dependent acquisition (DDA) experiment is essential to increase the MS/MS coverage and hence increase metabolite identifications in untargeted metabolomics. We explored the influence of mass spectrometric parameters including mass resolution, radio frequency (RF) level, signal intensity threshold, number of MS/MS events, cycle time, collision energy, maximum ion injection time (MIT), dynamic exclusion, and automatic gain control (AGC) target value on metabolite annotations on an Exploris 480-Orbitrap mass spectrometer. Optimal annotation results were obtained by performing ten data dependent MS/MS scans with a mass isolation window of 2.0 m/z and a minimum signal intensity threshold of 1 × 104 at a mass resolution of 180,000 for MS and 30,000 for MS/MS, while maintaining the RF level at 70%. Furthermore, combining an AGC target value of 5 × 106 and MIT of 100 ms for MS and an AGC target value of 1 × 105 and an MIT of 50 ms for MS/MS scans provided an improved number of annotated metabolites. A 10 s exclusion duration and a two stepped collision energy were optimal for higher spectral quality. These findings confirm that MS parameters do influence metabolomics results, and propose strategies for increasing metabolite coverage in untargeted metabolomics. A limitation of this work is that our parameters were only optimized for one RPLC method on single matrix and may be different for other protocols. Additionally, no metabolites were identified at level 1 confidence. The results presented here are based on metabolite annotations and need to be validated with authentic standards.

Circulating long-chain acylcarnitine concentrations are not affected by exercise training in pregnant women with obesity.

The purpose of this study was to determine the effect of exercise during pregnancy in sedentary women with obesity on longitudinal changes in long-chain acylcarnitine (LC-AC) concentrations. We hypothesized that exercise training would significantly decrease circulating LC-ACs throughout gestation compared with a nonexercise control group. Pregnant women with obesity considered otherwise healthy [n = 80, means ± SD; body mass index (BMI): 36.9 ± 5.7 kg/m2] were randomized into an exercise (n = 40, aerobic/resistance 3 times/wk, ∼13th gestation week until birth) or a nonexercise control (n = 40) group. At gestation week 12.2 ± 0.5 and 36.0 ± 0.4, a submaximal exercise test was conducted, and indirect calorimetry was used to measure relative resting energy expenditure (REE), as well as respiratory exchange ratio (RER) at rest. Fasting blood samples were collected and analyzed for LC-AC concentrations. Fitness improved with prenatal exercise training; however, exercise training did not affect circulating LC-AC. When groups were collapsed, LC-ACs decreased during gestation (combined groups, P < 0.001), whereas REE (kcal/kg/day, P = 0.008) increased. However, average REE relative to fat-free mass (FFM) (kcal/kg FFM/day) and RER did not change. There was an inverse relationship between the change in RER and all LC-ACs (except C18:2) throughout gestation (C14: r = -0.26, P = 0.04; C16: r = -0.27, P = 0.03; C18:1: r = -0.28, P = 0.02). In summary, a moderate-intensity exercise intervention during pregnancy in women with obesity did not alter LC-ACs concentrations versus control, indicating that the balance between long-chain fatty acid availability and oxidation neither improved nor worsened with an exercise intervention.NEW & NOTEWORTHY This research showed that a moderate-intensity prenatal exercise program, consisting of aerobic and resistance training, did not negatively impact normal alterations in substrate supply and demand for the mother and the offspring throughout gestation. Findings provide support for metabolic safety of exercise during pregnancy.

Associations between maternal obesity and offspring gut microbiome in the first year of life.

BACKGROUND: Maternal obesity is an important determinant of offspring obesity risk, which may be mediated via changes in the infant microbiome. OBJECTIVES: We examined infant faecal microbiome, short-chain fatty acids (SCFA), and maternal human milk oligosaccharides (HMO) in mothers with overweight/obese body mass index (BMI) (OW) compared with normal weight (NW) (Clinicaltrials.gov NCT01131117). METHODS: Infant stool samples at 1, 6, and 12 months were analysed by 16S rRNA sequencing. Maternal (BODPOD) and infant (quantitative nuclear magnetic resonance [QMR]) adiposity were measured. HMOs at 2 months postpartum and faecal SCFAs at 1 month were also assessed. Statistical analyses included multivariable and mixed linear models for assessment of microbiome diversity, composition, and associations of taxonomic abundance with metabolic and anthropometric variables. RESULTS: At 1 month, offspring of women with obesity had lower abundance of SCFA-producing bacteria (including Ruminococcus and Turicibacter) and lower faecal butyric acid levels. Lachnospiraceae abundance was lower in OW group at 6 months, and infant fat mass was negatively associated with the levels of Sutterella. Gradient boosting machine models indicated that higher α-diversity and specific microbial taxa at 1 month predicted elevated adiposity at 12 months with overall accuracy of 76.5%. Associations between maternal HMO concentrations and infant bacterial taxa differed between NW and OW groups. CONCLUSIONS: Elevated maternal BMI is associated with relative depletion of butyrate-producing microbes and faecal butyrate in the early infant faecal microbiome. Overall microbial richness may aid in prediction of elevated adiposity in later infancy.

Phosphoproteomics Provides Novel Insights into the Response of Primary Acute Lymphoblastic Leukemia Cells to Microtubule Depolymerization in G1 Phase of the Cell Cycle.

Microtubule targeting agents (MTAs) have been used for the treatment of cancer for many decades and are among the most successful chemotherapeutic agents. However, their application and effectiveness are limited because of toxicity and resistance as well as a lack of knowledge of molecular mechanisms downstream of microtubule inhibition. Insights into key pathways that link microtubule disruption to cell death is critical for optimal use of these drugs, for defining biomarkers useful in patient stratification, and for informed design of drug combinations. Although MTAs characteristically induce death in mitosis, microtubule destabilizing agents such as vincristine also induce death directly in G1 phase in primary acute lymphoblastic leukemia (ALL) cells. Because many signaling pathways regulating cell survival and death involve changes in protein expression and phosphorylation, we undertook a comprehensive quantitative proteomic study of G1 phase ALL cells treated with vincristine. The results revealed distinct alterations associated with c-Jun N-terminal kinase signaling, anti-proliferative signaling, the DNA damage response, and cytoskeletal remodeling. Signals specifically associated with cell death were identified by pre-treatment with the CDK4/6 inhibitor palbociclib, which caused G1 arrest and precluded death induction. These results provide insights into signaling mechanisms regulating cellular responses to microtubule inhibition and provide a foundation for a better understanding of the clinical mechanisms of MTAs and for the design of novel drug combinations. The mass spectrometry proteomics data have been deposited to the PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD027190 and 10.6019/PXD027190.

Glutathione peroxidase-1 overexpression reduces oxidative stress, and improves pathology and proteome remodeling in the kidneys of old mice.

This study investigated the direct roles of hydrogen peroxide (H2 O2 ) in kidney aging using transgenic mice overexpressing glutathione peroxidase-1 (GPX1 TG). We demonstrated that kidneys in old mice recapitulated kidneys in elderly humans and were characterized by glomerulosclerosis, tubular atrophy, interstitial fibrosis, and loss of cortical mass. Scavenging H2 O2 by GPX1 TG significantly reduced mitochondrial and total cellular reactive oxygen species (ROS) and mitigated oxidative damage, thus improving these pathologies. The potential mechanisms by which ROS are increased in the aged kidney include a decreased abundance of an anti-aging hormone, Klotho, in kidney tissue, and decreased expression of nuclear respiratory factor 2 (Nrf2), a master regulator of the stress response. Decreased Klotho or Nrf2 was not improved in the kidneys of old GPX1 TG mice, even though mitochondrial morphology was better preserved. Using laser capture microdissection followed by label-free shotgun proteomics analysis, we show that the glomerular proteome in old mice was characterized by decreased abundance of cytoskeletal proteins (critical for maintaining normal glomerular function) and heat shock proteins, leading to increased accumulation of apolipoprotein E and inflammatory molecules. Targeted proteomic analysis of kidney tubules from old mice showed decreased abundance of fatty acid oxidation enzymes and antioxidant proteins, as well as increased abundance of glycolytic enzymes and molecular chaperones. GPX1 TG partially attenuated the remodeling of glomerular and tubule proteomes in aged kidneys. In summary, mitochondria from GPX1 TG mice are protected and kidney aging is ameliorated via its antioxidant activities, independent and downstream of Nrf2 or Klotho signaling.

ProteoViz: a tool for the analysis and interactive visualization of phosphoproteomics data.

Quantitative proteomics generates large datasets with increasing depth and quantitative information. With the advance of mass spectrometry and increasingly larger data sets, streamlined methodologies and tools for analysis and visualization of phosphoproteomics are needed both at the protein and modified peptide levels. To assist in addressing this need, we developed ProteoViz, which includes a set of R scripts that perform normalization and differential expression analysis of both the proteins and enriched phosphorylated peptides, and identify sequence motifs, kinases, and gene set enrichment pathways. The tool generates interactive visualization plots that allow users to interact with the phosphoproteomics results and quickly identify proteins and phosphorylated peptides of interest for their biological study. The tool also links significant phosphosites with sequence motifs and pathways that will help explain the experimental conditions and guide future experiments. Here, we present the workflow and demonstrate its functionality by analyzing a phosphoproteomic data set from two lymphoma cell lines treated with kinase inhibitors. The scripts and data are freely available at and via the ProteomeXchange with identifier PXD015606.

Granisetron Alleviates Alzheimer's Disease Pathology in TgSwDI Mice Through Calmodulin-Dependent Protein Kinase II/cAMP-Response Element Binding Protein Pathway.

Alzheimer's disease (AD) is characterized by a compromised blood-brain barrier (BBB) and disrupted intracellular calcium homeostasis in the brain. Therefore, rectifying the BBB integrity and restoring calcium homeostasis could provide an effective strategy to treat AD. Recently, we developed a high throughput-screening assay to screen for compounds that enhance a cell-based BBB model integrity, which identified multiple hits among which is granisetron, a Food and Drug Administration approved drug. Here, we evaluated the therapeutic potential of granisetron against AD. Granisetron was tested in C57Bl/6J young and aged wild-type mice, and in a transgenic mouse model of AD namely TgSwDI for its effect on BBB intactness and amyloid-ß (Aß)-related pathology. Our study findings showed that granisetron enhanced BBB integrity in both aged and TgSwDI mice. This effect was associated with an overall reduction in Aß load and neuroinflammation in TgSwDI mice brains. In addition, and supported by proteomics analysis, granisetron significantly reduced Aß induced calcium influx in vitro, and rectified calcium dyshomeostasis in TgSwDI mice brains by restoring calmodulin-dependent protein kinase II/cAMP-response element binding protein pathway, which was associated with cognitive improvement. These results support granisetron repurposing as a potential drug to hold, slow, and/or treat AD.

Local and Relayed Effects of Deep Brain Stimulation of the Pedunculopontine Nucleus.

Our discovery of low-threshold stimulation-induced locomotion in the pedunculopontine nucleus (PPN) led to the clinical use of deep brain stimulation (DBS) for the treatment of disorders such as Parkinson's disease (PD) that manifest gait and postural disorders. Three additional major discoveries on the properties of PPN neurons have opened new areas of research for the treatment of motor and arousal disorders. The description of (a) electrical coupling, (b) intrinsic gamma oscillations, and (c) gene regulation in the PPN has identified a number of novel therapeutic targets and methods for the treatment of a number of neurological and psychiatric disorders. We first delve into the circuit, cellular, intracellular, and molecular organization of the PPN, and then consider the clinical results to date on PPN DBS. This comprehensive review will provide valuable information to explain the network effects of PPN DBS, point to new directions for treatment, and highlight a number of issues related to PPN DBS.

Menthol Smokers: Metabolomic Profiling and Smoking Behavior.

BACKGROUND: The use of menthol in cigarettes and marketing is under consideration for regulation by the FDA. However, the effects of menthol on smoking behavior and carcinogen exposure have been inconclusive. We previously reported metabolomic profiling for cigarette smokers, and novelly identified a menthol-glucuronide (MG) as the most significant metabolite directly related to smoking. Here, MG is studied in relation to smoking behavior and metabolomic profiles. METHODS: This is a cross-sectional study of 105 smokers who smoked two cigarettes in the laboratory one hour apart. Blood nicotine, MG, and exhaled carbon monoxide (CO) boosts were determined (the difference before and after smoking). Spearman correlation, χ2, and ANCOVA adjusted for gender, race, and cotinine levels for menthol smokers assessed the relationship of MG boost, smoking behavior, and metabolic profiles. Multivariate metabolite characterization using supervised partial least squares-discriminant analysis (PLS-DA) was carried out for the classification of metabolomics profiles. RESULTS: MG boost was positively correlated with CO boost, nicotine boost, average puff volume, puff duration, and total smoke exposure. Classification using PLS-DA, MG was the top metabolite discriminating metabolome of menthol versus nonmenthol smokers. Among menthol smokers, 42 metabolites were significantly correlated with MG boost, which linked to cellular functions, such as of cell death, survival, and movement. CONCLUSIONS: Plasma MG boost is a new smoking behavior biomarker that may provide novel information over self-reported use of menthol cigarettes by integrating different smoking measures for understanding smoking behavior and harm of menthol cigarettes. IMPACT: These results provide insight into the biological effect of menthol smoking. Cancer Epidemiol Biomarkers Prev; 26(1); 51-60. ©2016 AACR.