Matching Items (6)

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Epigallocatechin-3-gallate (EGCG) Helps Maintain Mitochondrial Function in MPP+-induced PC12 Cells

Description

Impairments to mitochondrial function and metabolism can make neurons vulnerable to stress and degeneration. Several studies have shown that aberrations in the electron transport chain (ETC) and the Krebs cycle

Impairments to mitochondrial function and metabolism can make neurons vulnerable to stress and degeneration. Several studies have shown that aberrations in the electron transport chain (ETC) and the Krebs cycle are involved in the pathogenesis of Parkinson’s disease (PD). Therefore, targeting these pathways is becoming increasingly important in the discovery of new treatment for neurodegenerative diseases like PD. (−)-epigallocatechin-3-gallate (EGCG), the most common polyphenol found in Green tea, has been shown to exert neuroprotective effects and lower the risk of developing PD. However, the mechanism by which it accomplishes this remains to be elucidated. The purpose of this study was to shed light on these mechanisms by exploring the effects of EGCG against MPP+-induced mitochondrial dysfunction with PC12 cells being used as a PD pathological cell model. The cell viability differences between cells treated with varying combinations of MPP+ and EGCG were measured using a CCK-8 assay. The morphology changes induced by the different treatments were then identified with fluorescence microscopy. Next, a Seahorse assay was carried out to investigate mitochondrial function followed by GC-MS and LC-MS analysis to evaluate mitochondrial metabolism. 13C metabolic flux analysis was then used to trace the metabolic flux of the Krebs cycle. The results of the CCK-8 assay and fluorescence microscopy showed that EGCG helps attenuate the decreased viability of PC12 cells as well as the morphology changes induced by MPP+. The Seahorse and GC-MS assay found that the it also helps prevent impaired mitochondrial respiration caused by MPP+. The impaired mitochondrial respiration manifested as alterations to the Krebs cycle and glycolysis. In addition, 13C metabolic flux analysis revealed significant increases in Krebs cycle activity in MPP+-induced PC12 cells if treated with EGCG beforehand. Moreover, LC-MS showed a distinct metabolite profile for each group and identified 26 potent biomarkers. In conclusion, this study demonstrated that EGCG exerts a neuroprotective effect on PC12 cells and helps maintain mitochondrial metabolic balance in the presence of MPP+.

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Date Created
  • 2020-05

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Ovarian Cancer Detection Using Targeted Plasma Metabolic Profiling

Description

Ovarian cancer (OC) is the second most common form of gynecologic cancer and is the most fatal among all forms of gynecologic malignancies. Despite the pivotal role of metabolic processes

Ovarian cancer (OC) is the second most common form of gynecologic cancer and is the most fatal among all forms of gynecologic malignancies. Despite the pivotal role of metabolic processes in the molecular pathogenesis of OC, robust metabolic markers to enable effective screening, rapid diagnosis, accurate surveillance, and therapeutic monitoring of OC are still lacking. In this study, we present a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolic profiling approach for the identification of metabolite biomarker candidates that could enable expedited, highly sensitive and specific OC detection. Using this targeted approach, 90 plasma metabolites from many metabolic pathways of potential biological significance were reliably detected and monitored in 218 plasma samples taken from three groups of subjects (78 OC patients, 50 benign samples, and 90 healthy controls). Univariate significance testing and receiver operating characteristic (ROC) analysis revealed 7 metabolites with high predictive accuracy [area under curve (AUC) > 0.90] for distinguishing healthy controls from OC patients. The results of our multivariate model development informed the construction of a 5-metabolite panel of potential plasma biomarkers for enhanced discrimination of OC samples from benign specimens, exhibiting roughly 75% predictive accuracy using a 50% random-split training set. ROC analysis that was generated based on a logistic regression classifier showed enhanced classification performance relative to individual metabolites, with more than 75% accuracy using a testing data set for external validation. Pathway analysis revealed significant disturbances in glycine, serine, and threonine metabolism; glyoxylate and dioxylate metabolism; the pentose phosphate pathway; and histidine metabolism. The results expand basic knowledge of the metabolome related to OC pathogenesis relative to healthy controls and benign samples, revealing potential pathways or markers that can be targeted therapeutically. This study also provides a promising basis for the development of larger multi-site projects to validate our findings across population groups and further advance the development of improved clinical care for OC patients.

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Date Created
  • 2020-05

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LC-MS/MS Analysis of Renal Cell Carcinoma Treated with Sulforaphane

Description

Sulforaphane(SFN)isanisothiocyanate(ITC)derivedfromcruciferousvegetables,suchas
broccoli,thatisgrowinginpopularityforitsantioxidantandanti-inflammatorycapabilities.
Furthermore,SFNhasbeendemonstratedtoimproverenalcancercarcinoma(RCC)treatment
outcomesinconjunctionwithmultipleotherformsoftherapy,whichisespeciallyimportant
consideringRCC’spoortherapeuticoutcomeswithchemotherapy.Theaimofthisstudywasto
determinetheeffectsofSFNonRCC ​invitro utilizingcellviabilityanalysisandLC/MS-MS
targetedmetabolicprofilingtorevealpathwaysresponsibleforSFN’spossibleenhancementof
chemotherapytreatmentinRCC.CCK-8resultsshowthat15 ​μ​MofSFNcausedasignificant(p
<0.05)increaseinRCCproliferation.Kruskal-Wallistestsrevealed16metabolitesinourcell,
and28inthemediumtobesignificant(p<0.05).Anorthogonalpartialleastsquares-discriminant
analysis,OPLS-DA,ofsignificantmetaboliteswasusedtocomparedtreatedandnon-treated
samplesforbothdatasetsandshoweda100%predictiveaccuracy(AUC=1).Enrichment
analysisdeterminedthatatotalof7metabolicpathwaysweresignificantlyenriched(VLCFA
β-oxidation,glutamatemetabolism,theureacycle,ammoniarecycling,glycine/serine,alanine,
andglucose-alaninecycle).Pathwayanalysisshowedhistidinemetabolismtobetheonly
significantlyaffectedpathwaybetweenbothdatasets.SFN-inducedmetaboliccharacteristics
foundinRCCwereconsistentwithknownantioxidantandanti-inflammatorypathways.Ourdata
suggeststhatthetherapeuticmechanismsofSFNarelikelyduetointeractionswithTandNKT
cellsthatprotectthemfromoxidativestress.Futureexperimentsregardingantioxidantresearch
incancershouldbecompletely ​invivo​,asopposedto ​invitro, ​inordertomaintainthenatural
physiology of cancer cells in the presence of host immune cells.

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Agent

Created

Date Created
  • 2019-05

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Targeted Metabolomics Reveals the Effect of Nitrate Supplementation on Vascular Function

Description

In the United States, two-thirds of adults are considered hypertensive orprehypertensive. In addition, chronic illness, such as hypertension, cardiovascular disease, and type II diabetes, results in $3.5 trillion in annual

In the United States, two-thirds of adults are considered hypertensive orprehypertensive. In addition, chronic illness, such as hypertension, cardiovascular disease, and type II diabetes, results in $3.5 trillion in annual healthcare cost and is the primary cause of disability and death. As a result, many individuals seek cheaper and simpler alternatives to combat their conditions. In this exploratory analysis, a study assessing nitrate intake and its effects on vascular function in 39 young adult males was investigated for underlying metabolic variations through a liquid chromatography – mass spectrometry-based large-scale targeted metabolomics approach. A two-way repeated measures ANOVA was used, and 18 significant metabolites were discovered across the time, treatment, and time & treatment groups, including prostaglandin E2 (p<0.001), stearic acid (p=0.002), caprylic acid (p=0.016), pentadecanoic acid (p=0.027), and heptadecanoic acid (p=0.005). In addition, log-transformed principal component analysis and orthogonal partial least squares – discriminant analysis models demonstrated distinct separation among the treatment, control, and time variables. Moreover, pathway and enrichment analyses validated the effect of nitrate intake on the metabolite sets and its possible function in fatty acid oxidation. This better understanding of altered metabolic pathways may help explicate the benefits of nitrate on vascular function and reveal any unknown mechanisms of its supplementation.

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Agent

Created

Date Created
  • 2020

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Microbiome after bariatric surgery and microbial insights into surgical weight loss

Description

Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is

Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery.

Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB.

Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity.

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Agent

Created

Date Created
  • 2016

Physiological and Genetic Mechanisms Underlying Variation in Anoxia Tolerance in Drosophila Melanogaster

Description

The ability to tolerate bouts of oxygen deprivation varies tremendously across the animal kingdom. Adult humans from different regions show large variation in tolerance to hypoxia; additionally, it is widely

The ability to tolerate bouts of oxygen deprivation varies tremendously across the animal kingdom. Adult humans from different regions show large variation in tolerance to hypoxia; additionally, it is widely known that neonatal mammals are much more tolerant to anoxia than their adult counterparts, including in humans. Drosophila melanogaster are very anoxia-tolerant relative to mammals, with adults able to survive 12 h of anoxia, and represent a well-suited model for studying anoxia tolerance. Drosophila live in rotting, fermenting media and a result are more likely to experience environmental hypoxia; therefore, they could be expected to be more tolerant of anoxia than adults. However, adults have the capacity to survive anoxic exposure times ~8 times longer than larvae. This dissertation focuses on understanding the mechanisms responsible for variation in survival from anoxic exposure in the genetic model organism, Drosophila melanogaster, focused in particular on effects of developmental stage (larval vs. adults) and within-population variation among individuals.

Vertebrate studies suggest that surviving anoxia requires the maintenance of ATP despite the loss of aerobic metabolism in a manner that prevents a disruption of ionic homeostasis. Instead, the abilities to maintain a hypometabolic state with low ATP and tolerate large disturbances in ionic status appear to contribute to the higher anoxia tolerance of adults. Furthermore, metabolomics experiments support this notion by showing that larvae had higher metabolic rates during the initial 30 min of anoxia and that protective metabolites were upregulated in adults but not larvae. Lastly, I investigated the genetic variation in anoxia tolerance using a genome wide association study (GWAS) to identify target genes associated with anoxia tolerance. Results from the GWAS also suggest mechanisms related to protection from ionic and oxidative stress, in addition to a protective role for immune function.

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Agent

Created

Date Created
  • 2018