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Article ; Online: Microbial modulation of host body composition and plasma metabolic profile.

Nazmul Huda, M / Winnike, Jason H / Crowell, Jocelyn M / O'Connor, Annalouise / Bennett, Brian J

2020 Volume 10, Issue 1, Page(s) 6545

Abstract: The gut microbiota is a critical mediator of nutrition and disease risk. Like most complex traits, the microbiome is under genetic regulation and differs between inbred strains of mice. We tested the effect of fecal microbiota transplantation (FMT) on ... ...

Abstract	The gut microbiota is a critical mediator of nutrition and disease risk. Like most complex traits, the microbiome is under genetic regulation and differs between inbred strains of mice. We tested the effect of fecal microbiota transplantation (FMT) on obesity, and plasma glucose. For this study, we collected microbiota from 2 inbred strains of mice which differ in adiposity and glucose tolerance, C57BL/6J and WSB/EiJ. C57BL/6J female mice (n = 18) were first treated with antibiotics for 4 weeks to ablate the microbiota. Following ablation, the mice were transplanted with microbiota from a C57BL/6J or a WSB/EiJ mouse and clinical traits and plasma metabolomic profiles were interrogated at 2- and 4-weeks post-transplantation. Unexpectedly, the mice receiving WSB/EiJ microbiota increased adiposity but decreased plasma glucose. Metabolomic and 16S microbiota profiling indicated broad metabolic changes occurred during and after FMT. Detailed analysis of these interactions demonstrated specific microbiota-host metabolite interactions which may alter disease susceptibility.
MeSH term(s)	Adiposity ; Animals ; Anti-Bacterial Agents/pharmacology ; Biodiversity ; Biomarkers/blood ; Blood Glucose/metabolism ; Body Composition/drug effects ; Cholesterol/blood ; Fecal Microbiota Transplantation ; Feces/microbiology ; Female ; Gastrointestinal Microbiome/drug effects ; Metabolomics ; Mice, Inbred C57BL ; Multivariate Analysis ; Phenotype ; Phylogeny ; Principal Component Analysis
Chemical Substances	Anti-Bacterial Agents ; Biomarkers ; Blood Glucose ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2020-04-16
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-63214-1
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Article: Stable Isotope-Resolved Metabolomic Differences between Hormone-Responsive and Triple-Negative Breast Cancer Cell Lines.

Winnike, Jason H / Stewart, Delisha A / Pathmasiri, Wimal W / McRitchie, Susan L / Sumner, Susan J

International journal of breast cancer

2018 Volume 2018, Page(s) 2063540

Abstract: Purpose: To conduct an exploratory study to identify mechanisms that differentiate Luminal A (BT474 and MCF-7) and triple-negative (MDA-MB-231 and MDA-MB-468) breast cancer (BCa) cell lines to potentially provide novel therapeutic targets based on ... ...

Abstract	Purpose: To conduct an exploratory study to identify mechanisms that differentiate Luminal A (BT474 and MCF-7) and triple-negative (MDA-MB-231 and MDA-MB-468) breast cancer (BCa) cell lines to potentially provide novel therapeutic targets based on differences in energy utilization. Methods: Cells were cultured in media containing either [U- Results: MCF-7 cells consumed the most glucose, producing the most lactate, demonstrating the greatest Warburg effect-associated energy utilization. BT474 cells had the highest tricarboxylic acid cycle (TCA) activity. The majority of energy utilization patterns in MCF-7 cells were more similar to MDA-MB-468 cells, while the patterns for BT474 cells were more similar to MDA-MB-231 cells. Compared to the Luminal A cell lines, TNBC cell lines consumed more glutamine and less glucose. BT474 and MDA-MB-468 cells produced high amounts of Conclusions: Stable isotopic resolved metabolomics using
Language	English
Publishing date	2018-09-30
Publishing country	Egypt
Document type	Journal Article
ZDB-ID	2603566-2
ISSN	2090-3189 ; 2090-3170
ISSN (online)	2090-3189
ISSN	2090-3170
DOI	10.1155/2018/2063540
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Article ; Online: Comparison of GC-MS and GC×GC-MS in the analysis of human serum samples for biomarker discovery.

Winnike, Jason H / Wei, Xiaoli / Knagge, Kevin J / Colman, Steven D / Gregory, Simon G / Zhang, Xiang

Journal of proteome research

2015 Volume 14, Issue 4, Page(s) 1810–1817

Abstract: We compared the performance of gas chromatography time-of-flight mass spectrometry (GC-MS) and comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) for metabolite biomarker discovery. Metabolite extracts from 109 human serum ... ...

Abstract	We compared the performance of gas chromatography time-of-flight mass spectrometry (GC-MS) and comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) for metabolite biomarker discovery. Metabolite extracts from 109 human serum samples were analyzed on both platforms with a pooled serum sample analyzed after every 9 biological samples for the purpose of quality control (QC). The experimental data derived from the pooled QC samples showed that the GC×GC-MS platform detected about three times as many peaks as the GC-MS platform at a signal-to-noise ratio SNR ≥ 50, and three times the number of metabolites were identified by mass spectrum matching with a spectral similarity score Rsim ≥ 600. Twenty-three metabolites had statistically significant abundance changes between the patient samples and the control samples in the GC-MS data set while 34 metabolites in the GC×GC-MS data set showed statistically significant differences. Among these two groups of metabolite biomarkers, nine metabolites were detected in both the GC-MS and GC×GC-MS data sets with the same direction and similar magnitude of abundance changes between the control and patient sample groups. Manual verification indicated that the difference in the number of the biomarkers discovered using these two platforms was mainly due to the limited resolution of chromatographic peaks by the GC-MS platform, which can result in severe peak overlap making subsequent spectrum deconvolution for metabolite identification and quantification difficult.
MeSH term(s)	Biomarkers/analysis ; Chromatography, Gas/methods ; Gas Chromatography-Mass Spectrometry/methods ; Humans ; Serum/chemistry ; Signal-To-Noise Ratio
Chemical Substances	Biomarkers
Language	English
Publishing date	2015-04-03
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/pr5011923
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Article: Comparison of GC-MS and GC×GC-MS in the Analysis of Human Serum Samples for Biomarker Discovery

Winnike, JasonH / Wei, Xiaoli / Knagge, Kevin J / Colman, Steven D / Gregory, Simon G / Zhang, Xiang

Journal of Proteome Research. 2015 Apr. 03, v. 14, no. 4

2015

Abstract	We compared the performance of gas chromatography time-of-flight mass spectrometry (GC-MS) and comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) for metabolite biomarker discovery. Metabolite extracts from 109 human serum samples were analyzed on both platforms with a pooled serum sample analyzed after every 9 biological samples for the purpose of quality control (QC). The experimental data derived from the pooled QC samples showed that the GC×GC-MS platform detected about three times as many peaks as the GC-MS platform at a signal-to-noise ratio SNR ≥ 50, and three times the number of metabolites were identified by mass spectrum matching with a spectral similarity score Rsim ≥ 600. Twenty-three metabolites had statistically significant abundance changes between the patient samples and the control samples in the GC-MS data set while 34 metabolites in the GC×GC-MS data set showed statistically significant differences. Among these two groups of metabolite biomarkers, nine metabolites were detected in both the GC-MS and GC×GC-MS data sets with the same direction and similar magnitude of abundance changes between the control and patient sample groups. Manual verification indicated that the difference in the number of the biomarkers discovered using these two platforms was mainly due to the limited resolution of chromatographic peaks by the GC-MS platform, which can result in severe peak overlap making subsequent spectrum deconvolution for metabolite identification and quantification difficult.
Keywords	biomarkers ; blood serum ; comprehensive two-dimensional gas chromatography ; data collection ; gas chromatography-mass spectrometry ; humans ; metabolites ; patients ; proteome ; quality control
Language	English
Dates of publication	2015-0403
Size	p. 1810-1817.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021%2Fpr5011923
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Article ; Online: Metabolomics Analysis of Hormone-Responsive and Triple-Negative Breast Cancer Cell Responses to Paclitaxel Identify Key Metabolic Differences.

Stewart, Delisha A / Winnike, Jason H / McRitchie, Susan L / Clark, Robert F / Pathmasiri, Wimal W / Sumner, Susan J

Journal of proteome research

2016 Volume 15, Issue 9, Page(s) 3225–3240

Abstract: To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor- ... ...

Abstract	To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor-negative TNBC cell lines and two hormone receptor-positive Luminal A cell lines. Studies were conducted in the presence and absence of paclitaxel (Taxol). TNBC cell lines had higher levels of amino acids, branched-chain amino acids, nucleotides, and nucleotide sugars and lower levels of proliferation-related metabolites like choline compared with Luminal A cell lines. In the presence of paclitaxel, each cell line showed unique metabolic responses, with some similarities by type. For example, in the Luminal A cell lines, levels of lactate and creatine decreased while certain choline metabolites and myo-inositol increased with paclitaxel. In the TNBC cell lines levels of glutamine, glutamate, and glutathione increased, whereas lysine, proline, and valine decreased in the presence of drug. Profiling secreted inflammatory cytokines in the conditioned media demonstrated a greater response to paclitaxel in the hormone-positive Luminal cells compared with a secretion profile that suggested greater drug resistance in the TNBC cells. The most significant differences distinguishing the cell types based on pathway enrichment analyses were related to amino acid, lipid and carbohydrate metabolism pathways, whereas several biological pathways were differentiated between the cell lines following treatment.
MeSH term(s)	Amino Acids/metabolism ; Carbohydrate Metabolism ; Cell Line, Tumor ; Female ; Hormones/pharmacology ; Humans ; Lipid Metabolism ; Metabolic Networks and Pathways ; Metabolism/drug effects ; Metabolomics/methods ; Paclitaxel/pharmacology ; Paclitaxel/therapeutic use ; Phenobarbital ; Triple Negative Breast Neoplasms/drug therapy ; Triple Negative Breast Neoplasms/metabolism
Chemical Substances	Amino Acids ; Hormones ; Paclitaxel (P88XT4IS4D) ; Phenobarbital (YQE403BP4D)
Language	English
Publishing date	2016-09-02
Publishing country	United States
Document type	Journal Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.6b00430
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Article: Effects of a prolonged standardized diet on normalizing the human metabolome

Winnike, Jason H / Busby, Marjorie G / Watkins, Paul B / O'Connell, Thomas M

American journal of clinical nutrition AJN. 2009 Dec., v. 90, no. 6

2009

Abstract: BACKGROUND: Although the effects of acute dietary interventions on the human metabolome have been studied, the extent to which the metabolome can be normalized by extended dietary standardization has not yet been examined. OBJECTIVE: We examined the ... ...

Abstract	BACKGROUND: Although the effects of acute dietary interventions on the human metabolome have been studied, the extent to which the metabolome can be normalized by extended dietary standardization has not yet been examined. OBJECTIVE: We examined the metabolic profiles of healthy human subjects after extended dietary standardization to see whether the inherent variation in the human metabolome could be decreased. DESIGN: A cohort of 10 healthy volunteers was admitted to a clinical research center for 2 wk of dietary standardization. Daily serum and urine samples and serum samples at a 2-wk follow-up visit were collected. The samples were analyzed by ¹H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analyses. RESULTS: NMR spectra were collected to globally profile the higher-concentration metabolites (>μmol/L concentrations). Metabolic changes were observed in some serum samples after day 1 or the 2-wk follow-up visit. For each subject, the samples from all other days had similar profiles. The urinary metabolome reflected no effects from dietary standardization. Pooled 24-h urine samples were studied, which indicated that any normalization that does occur would do so in <24 h. CONCLUSIONS: For both the urinary and serum metabolome, a single day of dietary standardization appears to provide all of the normalization that is achievable within the strict controls implemented in a clinical research setting. After 24 h, the subjects remain in their metabolic space; the remaining intra- and intersubject variations appear to be influenced by variables such as genetics, age, and lifestyle.
Keywords	long term effects ; diet ; metabolome ; metabolites ; metabolism ; human health ; health status ; food intake ; nutritional intervention ; humans ; cohort studies ; urine ; genetics ; age ; health effects assessments ; lifestyle ; sociodemographic characteristics ; functional foods ; diet therapy
Language	English
Dates of publication	2009-12
Size	p. 1496-1501.
Publishing place	American Society for Clinical Nutrition
Document type	Article
ZDB-ID	280048-2
ISSN	1938-3207 ; 0002-9165
ISSN (online)	1938-3207
ISSN	0002-9165
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Article ; Online: Effects of a prolonged standardized diet on normalizing the human metabolome.

Winnike, Jason H / Busby, Marjorie G / Watkins, Paul B / O'Connell, Thomas M

The American journal of clinical nutrition

2009 Volume 90, Issue 6, Page(s) 1496–1501

Abstract: Background: Although the effects of acute dietary interventions on the human metabolome have been studied, the extent to which the metabolome can be normalized by extended dietary standardization has not yet been examined.: Objective: We examined the ...

Abstract	Background: Although the effects of acute dietary interventions on the human metabolome have been studied, the extent to which the metabolome can be normalized by extended dietary standardization has not yet been examined. Objective: We examined the metabolic profiles of healthy human subjects after extended dietary standardization to see whether the inherent variation in the human metabolome could be decreased. Design: A cohort of 10 healthy volunteers was admitted to a clinical research center for 2 wk of dietary standardization. Daily serum and urine samples and serum samples at a 2-wk follow-up visit were collected. The samples were analyzed by (1)H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analyses. Results: NMR spectra were collected to globally profile the higher-concentration metabolites (>micromol/L concentrations). Metabolic changes were observed in some serum samples after day 1 or the 2-wk follow-up visit. For each subject, the samples from all other days had similar profiles. The urinary metabolome reflected no effects from dietary standardization. Pooled 24-h urine samples were studied, which indicated that any normalization that does occur would do so in <24 h. Conclusions: For both the urinary and serum metabolome, a single day of dietary standardization appears to provide all of the normalization that is achievable within the strict controls implemented in a clinical research setting. After 24 h, the subjects remain in their metabolic space; the remaining intra- and intersubject variations appear to be influenced by variables such as genetics, age, and lifestyle.
MeSH term(s)	Adolescent ; Adult ; Diet ; Female ; Humans ; Magnetic Resonance Spectroscopy ; Male ; Metabolome ; Middle Aged ; Principal Component Analysis
Language	English
Publishing date	2009-10-28
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	280048-2
ISSN	1938-3207 ; 0002-9165
ISSN (online)	1938-3207
ISSN	0002-9165
DOI	10.3945/ajcn.2009.28234
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Article ; Online: Acetaminophen dosing of humans results in blood transcriptome and metabolome changes consistent with impaired oxidative phosphorylation.

Fannin, Rick D / Russo, Mark / O'Connell, Thomas M / Gerrish, Kevin / Winnike, Jason H / Macdonald, Jeffrey / Newton, Jack / Malik, Shahid / Sieber, Stella O / Parker, Joel / Shah, Ruchir / Zhou, Tong / Watkins, Paul B / Paules, Richard S

Hepatology (Baltimore, Md.)

2009 Volume 51, Issue 1, Page(s) 227–236

Abstract: Unlabelled: The diagnosis and management of drug-induced liver injury (DILI) is hindered by the limited utility of traditional clinical chemistries. It has recently been shown that hepatotoxicants can produce compound-specific changes in the peripheral ... ...

Abstract	Unlabelled: The diagnosis and management of drug-induced liver injury (DILI) is hindered by the limited utility of traditional clinical chemistries. It has recently been shown that hepatotoxicants can produce compound-specific changes in the peripheral blood (PB) transcriptome in rodents, suggesting that the blood transcriptome might provide new biomarkers of DILI. To investigate in humans, we used DNA microarrays as well as serum metabolomic methods to characterize changes in the transcriptome and metabolome in serial PB samples obtained from six healthy adults treated with a 4-g bolus dose of acetaminophen (APAP) and from three receiving placebo. Treatment did not cause liver injury as assessed by traditional liver chemistries. However, 48 hours after exposure, treated subjects showed marked down-regulation of genes involved in oxidative phosphorylation/mitochondrial function that was not observed in the placebos (P < 1.66E-19). The magnitude of down-regulation was positively correlated with the percent of APAP converted to the reactive metabolite N-acetyl-p-benzoquinone-imide (NAPQI) (r= 0.739;P= 0.058). In addition, unbiased analysis of the serum metabolome revealed an increase in serum lactate from 24 to 72 hours postdosing in the treated subjects alone (P< 0.005). Similar PB transcriptome changes were observed in human overdose patients and rats receiving toxic doses. Conclusion: The single 4-g APAP dose produced a transcriptome signature in PB cells characterized by down-regulation of oxidative phosphorylation genes accompanied by increased serum lactate. Similar gene expression changes were observed in rats and several patients after consuming hepatotoxic doses of APAP. The timing of the changes and the correlation with NAPQI production are consistent with mechanisms known to underlie APAP hepatoxicity. These studies support the further exploration of the blood transcriptome for biomarkers of DILI.
MeSH term(s)	Acetaminophen/adverse effects ; Acetaminophen/urine ; Adult ; Animals ; Biomarkers/blood ; Chemical and Drug Induced Liver Injury/diagnosis ; Chemical and Drug Induced Liver Injury/physiopathology ; Down-Regulation ; Gene Expression Profiling ; Humans ; Liver/metabolism ; Metabolome/drug effects ; Middle Aged ; Oxidative Phosphorylation/drug effects ; Placebos ; Rats
Chemical Substances	Biomarkers ; Placebos ; Acetaminophen (362O9ITL9D)
Language	English
Publishing date	2009-11-16
Publishing country	United States
Document type	Controlled Clinical Trial ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
ZDB-ID	604603-4
ISSN	1527-3350 ; 0270-9139
ISSN (online)	1527-3350
ISSN	0270-9139
DOI	10.1002/hep.23330
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Article: Metabolomic analysis of cancer cachexia reveals distinct lipid and glucose alterations

O'Connell, Thomas M / Ardeshirpour, Farhad / Asher, Scott A / Winnike, Jason H / Yin, Xiaoying / George, Jonathan / Guttridge, Denis C / He, Wei / Wysong, Ashley / Willis, Monte S / Couch, Marion E

Metabolomics. 2008 Sept., v. 4, no. 3

2008

Abstract: Cancer cachexia remains a challenging clinical problem with complex pathophysiology and unreliable diagnostic tools. A blood test to detect this metabolic derangement would aid in early treatment of these patients. A ¹H NMR-based metabolomics approach ... ...

Abstract	Cancer cachexia remains a challenging clinical problem with complex pathophysiology and unreliable diagnostic tools. A blood test to detect this metabolic derangement would aid in early treatment of these patients. A ¹H NMR-based metabolomics approach was used to determine the unique metabolic fingerprint of cachexia and to search for biomarkers in serum samples taken from an established murine model of cancer cachexia. Male CD2F1 mice received a subcutaneous flank injection of C26 adenocarcinoma cells to induce experimental cancer-related cachexia. Two molecular markers of muscle atrophy, upregulation of the E3 ubiquitin ligase Muscle Ring Finger 1 (MuRF1) and aberrant glycosylation of β-dystroglycan (β-DG), were used to confirm muscle wasting in the tumor-bearing mice. Serum samples were collected for metabolomic analysis during the development of the cachexia: at baseline, when the tumor was palpable, and when the mice demonstrated cachexia. The unsupervised statistical analysis demonstrated a distinct metabolic profile with the onset of cachexia. The critical metabolic changes associated with cachexia included increased levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), with decreased serum glucose levels. Regression analysis demonstrated a very high correlation of the presence of aberrant glycosylation of β-DG with the unique metabolic profile of cachexia. This study demonstrates for the first time that metabolomics has potential as a diagnostic tool in cancer cachexia, and in further elucidating simultaneous metabolic pathway alterations due to this syndrome. In addition, variations in VLDL and LDL deserve more investigation as surrogate serum biomarkers for cancer cachexia.
Language	English
Dates of publication	2008-09
Size	p. 216-225.
Publisher	Springer US
Publishing place	Boston
Document type	Article
ZDB-ID	2250617-2
ISSN	1573-3882
ISSN	1573-3882
DOI	10.1007/s11306-008-0113-7
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Article: High-throughput nuclear magnetic resonance metabolomic footprinting for tissue engineering.

Seagle, Christopher / Christie, Megan A / Winnike, Jason H / McClelland, Randall E / Ludlow, John W / O'Connell, Thomas M / Gamcsik, Michael P / MacDonald, Jeffrey M

Tissue engineering. Part C, Methods

2008 Volume 14, Issue 2, Page(s) 107–118

Abstract: We report a high-throughput (HTP) nuclear magnetic resonance (NMR) method for analysis of media components and a metabolic schematic to help easily interpret the data. Spin-lattice relaxation values and concentrations were measured for 19 components and ... ...

Abstract	We report a high-throughput (HTP) nuclear magnetic resonance (NMR) method for analysis of media components and a metabolic schematic to help easily interpret the data. Spin-lattice relaxation values and concentrations were measured for 19 components and 2 internal referencing agents in pure and 2-day conditioned, hormonally defined media from a 3-dimensional (3D) multicoaxial human bioartificial liver (BAL). The (1)H NMR spectral signal-to-noise ratio is 21 for 0.16 mM alanine in medium and is obtained in 12 min using a 400 MHz NMR spectrometer. For comparison, 2D gel cultures and 3D multicoaxial BALs were batch cultured, with medium changed every day for 15 days after inoculation with human liver cells in Matrigel-collagen type 1 gels. Glutamine consumption was higher by day 8 in the BAL than in 2D culture; lactate production was lower through the 15-day culture period. Alanine was the primary amino acid produced and tracked with lactate or urea production. Glucose and pyruvate consumption were similar in the BAL and 2D cultures. NMR analysis permits quality assurance of the bioreactor by identifying contaminants. Ethanol was observed because of a bioreactor membrane "wetting" procedure. A biochemical scheme is presented illustrating bioreactor metabolomic footprint results and demonstrating how this can be translated to modify bioreactor operational parameters or quality assurance issues.
MeSH term(s)	Alanine/metabolism ; Artificial Organs ; Bioreactors ; Cell Culture Techniques/methods ; Equipment Design ; Glutamine/metabolism ; Hepatocytes/metabolism ; Humans ; Imaging, Three-Dimensional ; Liver/pathology ; Magnetic Resonance Spectroscopy/methods ; Metabolomics ; Quality Control ; Reproducibility of Results ; Tissue Engineering/methods
Chemical Substances	Glutamine (0RH81L854J) ; Alanine (OF5P57N2ZX)
Language	English
Publishing date	2008-06-11
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2420585-0
ISSN	1937-3392 ; 1937-3384
ISSN (online)	1937-3392
ISSN	1937-3384
DOI	10.1089/ten.tec.2007.0401
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