Day :
- Metabolomics and Cancer Research
Track 2: Analytical Techniques in Metabolomics
Track 3: Applications of Separation Sciences in Metabolomics
Track 4: Frontiers of Metabolomics Research
Chair
Mukesh Verma
NIH, USA
Co-Chair
Paola Marignani
Dalhousie University, Canada
Session Introduction
Stefan Kempa
BIMSB, Germany
Title: Using stable isotope resolved metabolomics to characterize glycolytic inhibition and to decode synthetic lethality in cancer
Time : 11:10-11:30
Biography:
Stefan Kempa has completed his PhD at the University of Potsdam and performed his Postdoctoral studies at the IMBA/GMI in Vienna focusing on the crosstalk between signaling and metabolism in plants. He is leading a Research and Technology Group at the Berlin Institute for Medical Systems Biology-Max Delbruck Center in Berlin, Germany
Abstract:
Metabolic reprogramming is a key step in oncogenic transformation including the activation of energy and anabolic metabolism. The central metabolism is the ultimate source of energy and building blocks enabling growth and proliferation. Specifically, time-resolved analysis of central metabolic pathways is needed for a better understanding of metabolic dynamics and the comparison and even quantification of pathway usage. In order to quantify the usage and activity of central metabolic pathways we have developed pulsed stable isotope resolved metabolomics (pSIRM) to analyse metabolism in vitro and in vivo. The applied GC-MS based technology enables the absolute quantification of metabolites and at the same time the determination of stable isotope incorporation, thus it allows also quantifying metabolic dynamics in vivo. Since the observation that glycolysis is deregulated in cancer the central metabolism gained attention as a possible therapeutic target. We have characterized the action of glycolytic inhibitors using pSIRM in a time dependent manner to distinguish between actual metabolic inhibition and adaptive processes. We observed unexpected effects and argue that the commonly used compound 2-deoxyglucose is not a specific glycolytic inhibitor. In order to understand the metabolic vulnerabilities at a molecular level we together with our co-workers have investigated the metabolic aspect of synthetic lethal interaction around the oncogene cMyc and could uncover metabolic circuits in regulatory networks that may open new ways for combinatorial therapies including metabolic inhibition.
Mukesh Verma
NIH, USA
Title: Title: Metabolomic approaches in cancer epidemiology
Time : 11:30-11:50
Biography:
Mukesh Verma is a Program Director and Chief in the Methods and Technologies Branch (MTB), Epidemiology and Genetics Research Program (EGRP) of the Division of Cancer Control and Population Sciences (DCCPS) at the National Cancer Institute (NCI), National Institutes of Health (NIH). Before coming to the DCCPS, he was a Program Director in the Division of Cancer Prevention (DCP), NCI, providing direction in the areas of biomarkers, early detection, risk assessment and prevention of cancer, and cancers associated with infectious agents. He holds an MSc from Pantnagar University and a PhD from Banaras Hindu University. He did Postdoctoral research at George Washington University and was a faculty member at Georgetown University. He has published 136 research articles and reviews and edited three books in cancer epigenetics and epidemiology field
Abstract:
Metabolomics is the study of low molecular weight molecules or metabolites produced within cells and biological systems. It involves technologies, such as mass spectrometry (MS) and nuclear magnetic resonance spectrography (NMR), which can measure hundreds to thousands of unique chemical entities (UCE). The metabolome provides one of the most accurate reflections of cellular activity at the functional level and hence can be leveraged for discerning mechanistic information during different normal and disease states. In clinical samples metabolites are more stable than proteins or RNA. In fact, metabolomic profiling in basic, epidemiological, clinical and translational studies has revealed potential new biomarkers of disease and therapeutic outcome and led to novel mechanistic understanding of pathogenesis. These include the recent biomarkers for diabetes risk, novel metabolites associated with cancer, and the discovery of over 500 unique lipids in plasma. However, unlike genomics or even proteomics, the degree ofmetabolite complexity and heterogeneity within biological systems presents unique challenges requiring specialized skills and resources to overcome. An example of association of metabolomics predictors of body fat amount and distribution and associated risk with cancer will be discussed. Epidemiology studies with altered metabolite profiles in lung, prostate, and endometrial cancer will also be discussed.
Jian Zhi Hu
Pacific Norwest National Laboratory, USA
Title: Non-, or minimally invasive intact biological objects using slow magic angle spinning
Time : 11:50-12:10
Biography:
Jian Zhi Hu has completed his PhD at the age of 32 years from a Joint-Training Program between Wuhan Institute of Physics, the Chinese Academy of Sciences and the Department of Chemistry, University of Utah, USA, and Postdoctoral studies from University of Utah. He is a Senior Staff Scientist and Principal Investigator of the Pacific Norwest National Laboratory. He has published more than 160 papers in reputed journals. He received one US R&D 100 award and is a holder of 8 issued US patents.
Abstract:
Metabolomics studies on tissues are of significance since a disease is often associated with a specific tissue or organ malfunction. It is, therefore, expected that the changes in metabolic profile are more dramatic in the diseased tissue than body fluids. It is likely that tissue specific metabolic profiling provides a unique window of investigating the biochemistry associated with a particular disease in greater detail than possible using global body fluids. In this work, we will report a non-destructive magic angle spinning NMR metabolomics technique that is capable of high resolution and high sensitivity metabolic profiling of biological samples, in particular tissue samples, with sample volume from as small as 200 nanoliters (nL) to as large as a milliliter or more using a single probe and using only a few minutes. This has been achieved by combining a the techniques of high resolution slow-MAS 1H NMR technique and switchable inductively coupled static micro-RF coil-LC resonator and by rotating the specimen at a sample spinning rate of 40 to 200 Hz about the magic angle axis. The nanoliter capability has the potential to follow the metabolic changes through a continued investigation on a single small laboratory animal over a long period of time using minimally invasive blood and tissue biopsy samples. While the milliliter capability would allow minimally destructive studies of intact biological objects with size as large as >1 cm3. Examples of applications will be reported.
Paola Marignani
Dalhousie University, Canada
Title: Targeting cancer metabolism to overcome treatment resistance in HER2-positive breast cancer.
Time : 12:10-12:30
Biography:
Dr. Paola Marignani received her PhD from McMaster University, followed by Postdoctoral fellowships at Harvard, the Samuel Lunenfeld Research Institute and the Ontario Cancer Institute. The Marignani Discovery Research Laboratory uses animal models and high through-put screening strategies to identify novel signalling pathways involved in disease. Dr. Marignani and her team have shown that the tumour suppressor kinase LKB1 is an interacting partner and regulator of the estrogen receptor. More recently her team developed a novel spontaneous mouse model of breast cancer. In this model, proteomic and metabolic profiling of tumours confirm hyperactive mTOR and metabolic activity. By combining multiple platforms, The Marignani Discovery Research Laboratory continues to focus on identifying molecular switches that drive tumourigenesis, conduct pre-clinical trials that evaluate novel targeted therapies and develop clinically relevance animals models of human disease.
Abstract:
Metabolic adaption of tumour cells is necessary to meet biosynthesis and energy needs of a growing cancer. The energy sensing kinase AMPK is responsible for maintaining AMP/ATP ratio, serving as a metabolic checkpoint that is activated when phosphorylated by the LKB1, a tumour suppressor kinase. LKB1 is frequently found mutated in numerous cancers including 31% of HER2 breast cancer. In our LKB1-/-NIC model, loss of LKB1 expression resulted in reduced tumour latency where tumours were biochemically characterized as hyperactive mTOR, along with metabolic changes characteristic of Warburg effect, namely elevated ATP, LDH, PDH expression and enhanced lactate. Based on these finding, we conducted a pre-clinical studies to evaluate novel combinatorial therapies on tumourigenesis. We report that targeting PI3K-p70S6K pathways with competitive NVP-BEZ235 inhibitor was not as effective at reducing tumourigenesis as targeting mTOR and glycolysis with AZD8055 and 2-DG monotherapies, respectively. Interestingly, simultaneous inhibition of these pathways with AZD8055/2-DG combination was significantly more effective at reducing mitochondria function, tumour volume and burden, culminating in reduced tumourigenesis. At the molecular level, combination treatment inhibited both mTOR signalling and blocked MAPK survival signalling that is responsible for ERK-p90RSK pathway engagement. Finally, loss of LKB1 expression in cancers should be considered a marker for metabolic dysfunction given the role LKB1 plays in regulating both AMPK activity and mTOR function. The results of our pre-clinical studies suggest that combinatorial therapy that target mTORC1/mTORC2 and glycolytic pathways in cancer, is critical for inhibiting tumour growth. Importantly, our discovery showed that the drug combination inhibited the activation of feedback loops that are drivers of resistance, namely ERK and p90RSK. We believe that simultaneous targeting of these pathways will provide the best clinical outcome for the treatment of metabolically active cancers, as well as reduce the likelihood of recurrence.
Li Zhang
The University of Texas, USA
Title: Evaluating the association of heme and heme metabolites with lung cancer energetics and progression
Time : 12:30-12:50
Biography:
Li Zhangcompleted herPhD from UCLA and postdoctoral studies from MIT department of Biology.She is Cecil H. and Ida Green Distinguished Chair in Systems Biology Science at the University of Texas at Dallas. Professor Zhang has worked on studying heme signaling and function for 20 years. She has published many original research articles and a book entitled “Heme Biology: The Secret Life of Heme in Regulating Diverse Biological Processes” on this subject.
Abstract:
Emerging experimental data increasingly show that despite the enhanced glycolytic flux,many types of cancer cells exhibit intensified oxygen consumption or mitochondrial respiration. Even under hypoxia, cancer cells can maintain oxidative phosphorylation at a substantial rate. Heme is a central factor in oxygen utilization and oxidative phosphorylation. It serves as a prosthetic group in many proteins and enzymes involved in mitochondrial respiration. Notably, our recent work showed that non-small-cell lung cancer (NSCLC) cells and xenograft tumors exhibit substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins, and various types ofoxygen-utilizing hemoproteins such as cytoglobin and cytochromes. In contrast, lowering hemebiosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygenconsumption, cancer cell proliferation, migration and colony formation. Therefore, elevatedheme function and flux are likely a key feature of NSCLC cells and tumors. Based on this observation, we decided to further ascertain the relationship between heme and lung cancer. We extract heme and its metabolites from various NSCLC cancer cells and tumors. We then perform LC-mass spectrometry to quantify the amounts of heme and its metabolites. We also measure the rates of oxygen consumption in various cancer cells and compare them to the levels of heme in these cells. We expect that these experimental results will enable us to determine the extent to which heme and heme metabolites impact cancer cell bioenergetics and progression
Jayoung Kim
Cedars-Sinai Medical Center, USA
Title: Non-invasive biomarker candidates of Interstitial cystitis
Time : 12:50-13:10
Biography:
Jayoung Kim is an NIH-funded translational scientist and an Associate Professor at Cedars-Sinai Medical Center. She aims to improve the means of objectively diagnosing IC/PBS and to discover the mechanistic basis for this disorder. She was originally trained as a cancer biologist at Harvard Medical School, and is a rising leader of research on basic urological diseases including IC/PBS. One of the major focus areas in her group is the study of the metabolic signature for the diagnosis of IC/PBS, using a number of approaches, including large-scale “Omics” methods, to elucidate the signaling network in human cells that is perturbed in IC/PBS
Abstract:
Interstitial cystitis/painful bladder syndrome (IC/PBS) is a debilitating condition that presents with a constellation of symptoms including bladder pain, urinary urgency, frequency, nocturia, and small voided volumes in the absence of other identifiable etiologies. A lack of objective diagnostic criteria has affected our ability to adequately treat the disease. The goal of this proposed study is to identify/validate sensitive and non-invasive diagnostic biomarkers using urine specimens that stratify IC/PBS patients from healthy subjects. NMR spectroscopy-based metabolomics analysis was performed to search for soluble metabolites that segregate with the diagnosis of IC/PBS. Annotation of the NMR peaks was performed using MeltDB and MetaboloAnalyst software. It was able to annotate several of the discriminant peaks, including the most significant peak, which was identified as tyramine, a neuro-transmodulator related to pain. These results demonstrate our ability to assay for and provisionally identify discrete urine metabolites that are significantly associated with IC/PBS. This study is believed to provide novel insights about the etiology of IC/PBS and identify urine metabolites as biomarkers of IC/PBS that have the potential to be employed clinically.
Yongyu Zhang
Shanghai University of Traditional Chinese Medicine, China
Title: Metal Element on Non-alcoholic Fatty Liver Disease(NAFLD)
Time : 14:10-14:30
Biography:
Yongyu Zhang has completed his PhD from Meiji Pharmaceutical University in Japan, 2000. He is a Professor of Shanghai University of Traditional Chinese Medicine. He has published more than 20 papers in reputed journals and has been serving as an Editorial Board Member of 3 journals.
Abstract:
Our group has been devoted to researching the mechanism, development and treatment of the diseases under the guidance of Traditional Chinese Medicine (TCM) theory through metabolomics for many years. Results of the researches indicated that the “Tongbing Yizheng” theory, namely the same disease could be divided into different groups based on the TCM theory, has its substantial basis at the metabolic level. Meanwhile, we also found that some metal elements played a role in the classification of TCM syndrome. So we introduce the metabolomics basing on ICP-MS and HPLC-ICP-MS which is a new member of the omics family into our research on the substantial basis of dampness - heat Syndrome, one of common syndromes in TCM. Twenty-six elements were quantitatively detected in the serum samples of model rats with dampness - heat Syndrome, which was combined with sesum metabolomics to investigate the connotation of dampness-heat syndrome. Eleven differential metabolites and 4 changed elements including Zn, Fe, Se and Cu were found between normal group and dampness-heat syndrome group. After the pathway analysis it was found that the dampness-heat syndrome was related with cyanoamino acid metabolism, nitrogen metabolism, thiamine metabolism, butanoate metabolism, mineral absorption, two-component system and ABC transporters. The present results indicate that the combination of metabolomics and metabolomics could provide an approach to research the TCM subtypes of diseases.
Ih-Jen Su
National Health Research Institutes, Taiwan
Title: Dynamic pattern and terminal switch of lipid metabolomics in HBV tumorigenesis
Time : 14:30-14:50
Biography:
Dr. Su received his MD degree from National Taiwan University Medical School in 1976, and PhD degree in Pathology in 1987. His major research interest is virus and virus-associated human cancers. He was the pioneer investigator in EBV-associated T cell lymphoma, and was appointed as the member of International Lymphoma Study Group ( 1996-2008 ) for WHO lymphoma classification. During the SARS period, he served as the Director General of Taiwan CDC and successfully controlled SARS. In 2011-2013, he was appointed as the Director of National Institutes of Infectious Diseases and Vaccinology, NHRI to develop vaccines for EV71, H7N9, and BCG. In the past decade, he become interested in HBV carcinogenesis andidentified pre-S2 mutants as the new viral oncoproteins, and ground glass hepatocytes as pre-neoplastic lesions. He studied the cancer metabolomics and started to apply these biomarkers, especially mTOR and c-myc signals, for chemoprevention of high risk chronic HBV carriers. He published a total of around 300 papers, many of which in the prestigious journals like Lancet, Blood, Journal of Clinical Investigation, Hepatology, etc. His many studies have been translated into clinical application and industry development.
Abstract:
Background: The lipid metabolic disorders were frequently observed in patients of HBV and HCV-associated hepatocellular carcinoma (HCC). The underlying mechanism and significance remains to be clarified. Aim: In this study, we attempt to clarify the role of lipid metabolism in HBV tumorigenesis. Methods: The dynamic, temporal pattern of lipid metabolic profiles in serum and lipid were demonstrated in two transgenic mice models of HBx and pre-S2 deletion mutant by biochemistry and Affymetrix DNA array chip. The data were confirmed by western blot and further validated in human HCC tissues. Results: We observed an interesting biphase response pattern of lipid metabolomics in both HBx and pre-S2 mutant transgenic mice models of HBV tumorigenesis. The first peak of fatty change occurred in the early phase of 1-3 months, which subsided and then remarkably increased or terminally switched in HCC tissues. This biphasic pattern was synchronized with ATP citrate lyase (ACYL ) activation, followed by the activation of sertol regulatory element binding transcription factor 1 (SEBPF1) and fatty acid desaturase 2 (FADS2) in pre-S2 model. In HBx model, five lipid genes were specifically activated at the terminal phase including the lipoprotein lipase, fatty acid binding protein (FABP). In both models, the endoplasmic reticulum (ER) stress-induced mTOR pathway is the driving signals. Such an ER stress-dependent mTOR signal cascade is also important for cell proliferation of hepatocytes and further validated in HCC tissues. Conclusion: The mTOR signal pathway is important for the lipid metabolic disorders and the driving force for HBV tumorigenesis in animal and human models. To target on this pathway we will provide chemoprevention for HCC tumorigenesis in high risk patients of chronic HBV infection.
Chang Gong
Sun Yat-Sen University,China
Title: BRMS1L suppresses breast cancer metastasis by inducing epigenetic silence of FZD10
Time : 14:50-15:10
Biography:
Dr. Chang Gong has completed her MD in 2004 and PhD in 2010 from Sun Yat-Sen University as well as postdoctoral studies from INSERM, France. She is an associate professor of breast surgery in Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-Sen University. She focuses on the mechanisms of drug resistance and metastasis of breast cancer. She has published 15 papers in reputed journals.
Abstract:
BRMS1L (breast cancer metastasis suppressor 1 like，BRMS1-like) is a component of the SIN3A-HDAC corepressor complex that suppresses target gene transcription. However, the contribution of BRMS1L in cancer development is not well characterized. Here, we show that reduced BRMS1L in breast cancer tissues is associated with tumor metastasis and poor patient survival. Functionally, BRMS1L inhibits migration and invasion of breast cancer cells by inhibiting epithelial-mesenchymal transition (EMT). These effects are mediated by epigenetic silencing of FZD10, a receptor for Wnt signaling, by facilitating the recruitment of HDAC1 to its promoter and enhancing histone H3K9 deacetylation. Consequently, BRMS1L-induced FZD10 silencing inhibits aberrant activation of WNT3-FZD10-ï¢-catenin signaling. Furthermore, BRMS1L is a target of miR-106b and miR-106b upregulation leads to BRMS1L reduction, which is responsible for Wnt pathway activation and the ensuing EMT in breast cancer cells. RNAi-mediated silencing of BRMS1L expression promotes metastasis of breast cancer xenografts in immunocompromised mice, while ectopic BRMS1L expression inhibits metastasis. Therefore, BRMS1L provides an epigenetic regulation of Wnt signaling in breast cancer cells and acts as a breast cancer metastasis suppressor.
Houkai Li
Shanghai University of Traditional Chinese Medicine, China
Title: Combined metabolomic and transcriptomic profiling revealed the time-dependent effects of metformin on LoVo cells
Time : 15:30-15:50
Biography:
Houkai Li has completed his PhD at the age of 32 years from Shanghai Jiao Tong University and Postdoctoral studies from University of North Carolina (2011.8-2013.5) and Chinese Academy Sciences (2008.11-2011.3) respectively. He is now a Professor at Center for Traditional Chinese Medicine and Systems Biology in Shanghai University of Traditional Chinese Medicine. He has published over 22 papers in reputed journals.
Abstract:
Metformin is a commonly used anti-diabetic drug, which has been recognized of possessing potential anticancer activities in a variety of cancer models. However, the exact mechanism is poorly understood. In current study, we performed a GC-TOFMS and LC-TOFMS-based metabolomic profiling on LoVo cells, a human derived colon cancer cell line, treated by 10 mM metformin for 8, 24 and 48h respectively. Although the significant suppression on cell viability was only observed after 24h treatment, an obvious metabolic alteration was present after 8h treatment, and these metabolic changes were further amplified after 24 and 48h metformin treatment in consistence with the time-dependent suppressive effects on cell viability. Nevertheless, we observed that most differential metabolites were up-regulated at 8h, but down-regulated at 24 and 48h by metformin compared to the corresponding control cells, indicating the different modulation in cell metabolism by metformin at different time points. We found that most of the identified differential metabolites were involved in amino acids metabolism, glycolysis, TCA cycle, and nucleic acids metabolism, suggesting that the metabolic impacts of metformin on energy metabolism were prior to the phenotypic changes in cell viability. Meanwhile, we performed a transcriptomic profile of LoVo cells which were treated by 10mM metformin for 8 and 24h. The transcriptomic data showed there are over 100 and 3000 differentially expressed genes induced by 8 and 24h metformin treatment compared to the corresponding control cells. Interestingly, we found that the impacts of normal 24h culture were greater than 8h treatment of metformin on gene expression, which was different with the observed metabolic alterations. We observed that the main involved pathways of differentially expressed genes were not only related with classical cancer signaling pathways such as Wnt signaling, p53 signaling, MAPK pathway, cell cycle, apoptosis and ErbB signaling pathway, but also with energy metabolism process. Altogether, our current data indicate that metformin treatment results in a time-dependent metabolic and transcriptomic alteration on LoVo cells, and the results from these two omics approaches could be complementary and warrants a further investigation on these observed changes of genes and metabolic pathways.
Xiaoyan Wang
Shanghai Center for Systems Biomedicine, China
Title: Metabolomics approach to understanding the metabolic regulatory effects on rats under several kinds of stresses
Time : 15:50-16:10
Biography:
Xiaoyan Wang got her PhD in Pharmacology from Shanghai Jiao Tong University and now is an Associate Professor at the Shanghai Center for Systems Biomedicine. She has been engaged in stress related metabolomics and pharmacology study and has published more than 20 papers in reputed journals
Abstract:
Stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which might cause further tissue injury and could also be gradually reinstated when stress source was removed. However, such a sophisticated metabolic regulatory process has so far been poorly understood, especially from a holistic view. The series of metabolomics analysis on urine, serum and tissue samples derived from rat models of acute cold stress, forced swimming stress, Chronic Unpredictable Mild Stress (CUMS) and subacute heat stress enables us to visualize significant alterations in metabolite expression patterns as a result of stress-induced metabolic responses and post-stress compensation. The results indicate the mild and acute stress, like cold and forced swimming stress induced metabolic perturbations were reversible and nonspecific, but long-term stress, as subacute heat and chronic, unpredictable mild stress, brought sustained metabolic distributions and caused more injury to other tissues including brain (CUMS) and testicles(heat stress). The differentially expressed metabolites were involved in metabolic regulation and compensation required to restore homeostasis, especially in the epididymis, metabolites with reproductive benefits were found being up-regulated to play a self-protective function in resisting, heat stress and to maintain normal reproductive function. Meanwhile, our study provides a dynamic and systemic approach for the characterization of anti-stress and metabolic protective effects of ginsenosides from the herbal drug named Ginseng
Fuxiang Zhou
Zhongnan Hospital of Wuhan University, China
Title: Title: Mitochondrial DNA depletion in H1299 cells promotes radio-resistance and anti-apoptosis through activation of PI3K/Akt2
Time : 16:10-16:30
Biography:
Fuxiang Zhou has completed his Ph.D at the age of 40 years old from Zhongnan Hospital of Wuhan University and postdoctoral studies from CAL Cancer Center, University of Nice, France. He is the director of Hubei Key Laboratory of Tumor Biological Behaviors & Hubei Cancer Clinical Study Center. He has published more than 60 papers in reputed journals and serving as an editorial board member of repute.
Abstract:
Radiotherapy takes an important role in treatment of various cancers while the main limit for this strategy is radioresistance of cancer cells. A large number of studies have found that mitochondrial DNA (mtDNA) is not only associated with tumor development, but also affect the tumor radiosensitivity. But, the roles of mtDNA on radiosensitivity are still conflictable, and the mechanisms remain unresolved. Here, we have built a cell model with mtDNA deletion, to investigate the relationship between mtDNA and radiosensitivity and its mechanism. Human non-small cell lung cancer cells (H1299) were depleted of mtDNA (ρ0) by culturing chronically in the presence of ethidium bromide, and then verified by PCR of total DNA using primer pairs specific for mtDNA. We found that loss of mtDNA decreased proliferation rate, ATP and oxidative phosphorylation. Moreover, ρ0 cells regulate radio-sensitivity: (a)by inhibiting cell cycle progression at the G2/M transition leading to growth arrest and apoptosis; (b) by increasing the expression of G2/M checkpoint ATM/ATR-mitotic cyclinB1 and decrease the expression of apoptotic factors pro-caspases-3 and -8; and (c) accelerated the repair kinetics of DNA damage induced by irradiation. We further examined the phosphorylation of Akt2, mTOR and IKKs and found they all were significantly increased in ρ0 cells. In keeping with these findings, suppression of the PI3K/Akt2 pathway by the small molecular inhibitor MK-2206 2HCL dramatically increased the expression of apoptotic proteins in ρ0 cells. Collectively, our results indicated that mtDNA depletion resulting in downregulation of radiosensitivity, and retrograde activation the PI3K/Akt2 pathway in non-small cell lung cells.
Jayaraman G
VIT University, India
Title: Comparative metabolic profiling of halotolerant bacterial strains and identification of novel species-specific metabolites
Time : 16:30-16:50
Biography:
Jayaraman G obtained his PhD degree at the National Tsing Hua University (Taiwan) in Structural Biology in 1998 and continued as a Post-doctoral Fellow with funding from National Health Research Institute, Taiwan. Though continuing to explore the structure-function relation of snake venom proteins, he has expanded his interest in understanding the adaptative features of halotolerant organisms. Currently, he is a professor at VIT University (India) and has more than 75 research articles published in international journals.
Abstract:
The salt stress response of four representative halotolerant bacterial species (Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21 and Virgibacillus dokdonensis VITP14) isolated from a previously unexplored solar saltern in Kumta, along the Arabian Sea coast in Karnataka, India was analyzed using comparative metabolomics approach. Chemometric analysis of 1H NMR spectra revealed salt-dependent increase in the levels of metabolites, mainly from the aspartate and glutamate family, that are directed from the glycolytic pathway, pentose phosphate pathway and citric acid cycle. The composition of the metabolites was found to be different with respect to the species and the type of growth medium. Two dimensional NMR data revealed accumulation of two rare diaminoacids, Nε- acetyl-α-lysine and Nδ-acetylornithine apart from other well known compatible solutes. Metabolite profiles of species capable of synthesizing Nε-acetyl-α-lysine and Nδ-acetylornithine suggested their biosynthesis from lysine and ornithine using aspartate and glutamate as their precursors, respectively. One of the species, Planococcus maritimus VITP21 was found to accumulate an unusual sugar, (2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→2)-β-D-fructofuranose is not previously reported for its natural synthesis by any other organism. The protective effects of Nε-acetyl-α-lysine and (2-acetamido-2-deoxy-α-Dglucopyranosyl)-(1→2)-β-D-fructo furanose along with other commonly occurring bacterial osmolytes, ectoine, proline, sucrose, trehalose and glycine betaine on protein stability and activity were evaluated with a few of possible biotechnological application.
Cocktails sponsored by Journal of Metabolomics: Open Access 18:15-19:15 @ Independence Foyer
- Track 1: Metabolomics and Cancer Research
Track 4: Frontiers of Metabolomics Research
Track 5: Metabolic Syndrome
Chair
Deliang Guo,
The Ohio State University, USA
Co-Chair
Scott J Tebbutt
University of British Columbia, Canada
Session Introduction
Marvin W. Makinen
The University of Chicago, USA
Title: Preferential sequestration of a vanadyl chelate by cancer tissue facilitates 2-(fluorine-18)-2-deoxyglucose (FDG)
Time : 10:30-10:50
Biography:
Marvin W Makinen has pursued research on the structural basis of enzyme action and enzyme mechanism using electron paramagnetic resonance and electron nuclear double resonance methods. The present research directed at cancer detection is derived from earlier research on inhibition of protein tyrosine phosphatase-1B by vanadyl (VO2+) chelates.
Abstract:
PET imaging with FDG as the tracer molecule for cancer detection is biochemically based and is associated with high specificity. However, FDG PET imaging relies on tumor size and the local metabolic characteristics of the type of cancer. Some breast cancer types characterized by low glucose uptake give rise to false negatives. Bis (acetylacetonato) oxidovana-dium (IV) [VO (acac)2] exhibits high stability in solution and displays a high capacity to enhance cellular glucose uptake. We observed that enhanced FDG uptake by MDA-MB-231 cultured human breast carcinoma cells was facilitated by VO(acac)2 over the 1–8 μM range in the presence of a background 5 mM glucose concentration, equivalent to the fasting level of blood glucose in a normal adult. Generating MDA-MB-231 xenograft tumors in SCID mice, we observed maximum FDG uptake approximately 4 hours post-VO(acac)2 injection. This observation closely correlates with the time of 3.5-4 hours for maximum preferential sequestration of the chelate by tumor tissue. Longer intervals between intraperitoneal injection of VO(acac)2 and the onset of FDG PET imaging showed decreased FDG uptake. We have observed up to 6-fold enhanced FDG uptake compared to controls with a two-fold higher dose of VO(acac)2. These preliminary results indicate that use of VO(acac)2 as a pharmacologic agent to enhance FDG uptake by malignant tissue has the potential to improve detection of both small tumors and tumors of low metabolic activity by FDG PET imaging.
Scott J Tebbutt
University of British Columbia, Canada
Title: Integrated metabolite and RNA biomarker signatures of the late phase asthmatic response
Time : 10:50-11:10
Biography:
Scott J Tebbutt is Associate Professor in the Department of Medicine, University of British Columbia, and Principal Investigator at the Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, Canada. His research program is focused on molecular signatures of complex respiratory disease, including the early and late reactions in atopic asthma and allergic rhinitis. He is also Chief Scientific Officer of the Prevention of Organ Failure (PROOF) Centre of Excellence - a not-for-profit organization dedicated to moving research findings into health care, and focused on non-invasive biomarkers that can diagnose and/or predict organ failure (heart, lung and kidney). His responsibilities include the evaluation of new, high-performance technologies to improve biomarker discovery, as well as computational biology approaches to better deal with cell type heterogeneity. He holds a BA in Biochemistry from the University of Oxford and a PhD in Molecular Genetics from the University of East Anglia (Cambridge Laboratory, John Innes Centre).
Abstract:
Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). The late response is associated with inflammation and chronic disease pathobiology, and it is important to develop biomarkers that can predict which individuals might be more susceptible to late phase responses. We are using transcriptomics (RNA-Seq) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 35 study participants. We are developing analytical tools such as sparse generalized canonical correlation discriminant analysis (SGCCDA) to derive classifier signatures that combine metabolite and RNA biomarkers of the late response
Khalid Al-Qahtani
University of Oxford, UK
Title: Mass spectrometry based metabolic profiles of brain cancer
Time : 11:10-11:30
Biography:
Khalid Al-Qahtani has completed his PhD at the age of 32 years from University Oxford. He was awarded the delegate’s choice prize for best young person’s poster presentation at the BMSS annual meeting, April 2014. He has published more than 9 papers in peer reviewed journals and is focussing on building a database for cancer metabolomics linking this with changes in metabolic pathways for studying regulation in cancer cells.
Abstract:
Introduction: The author will present highly selective and sensitive LC and GC methods for quantification of intracellular metabolites involved in tricarboxylic acid cycle (TCAs) metabolism including the configuration of the enantiomers of (L/D)-2-hydroxyglutaric (2-HG). These are applied to the analysis of brain cancer cells and tissues in order to look for metabolic differences between mutant and wild type cells. Method: The author has developed two novels LC-MS methods for studying changes in TCA cycle intermediates and their concentrations in cells. The first method focuses on the quantification of metabolites involved in TCA cycle metabolism and the second uses chiral separation for enantiomeric selectivity of (L/D)-2-HG and some amino acids associated with TCA reactions. In addition, third method for untargeted metabolites by using GC-MS for studying Fold changes on all brain metabolic profiles. Results: All isomers and enantiomeric forms of the metabolites were well separated with baseline resolution. Method validation provided limits of detection (LODs) for L/D-2HG ≤3μM (±2SD, Accuracy (%) ±4, %CV ±1.5, STD <5). calibration curves showed good linearity mainly over six orders of magnitude with a correlation coefficient R2 > 0.99. Conclusions: These methods were developed and applied to the analysis of brain cancer cells and tissues to investigate changes in TCA cycle intermediates identifying selected identifying selected enantionmer concentrations and studying isocitrate dehydrogenase (IDH) metabolon. We describe the methodology used and give examples from the analysis of selected wild-type and modified cancer cell lines which show highly specific enantiomeric changes in 2-hydroxyglutarate and 2-oxoglutarate taking place in mutant cell lines. There were statistically significant differences in TCAs metabolites (D-2HG, L-HG, 2-oxoglutarate, Oxaloacetate) levels between the IDHwt and IDH1R132H and IDH2R172K cells. There were significant differences in metabolite concentrations seen with IDH inhibition (shRNA, AGI-5198) and also when adding individual TCA cycle metabolites individually into culture media.
John M. Pisciotta
West Chester University,USA
Title: Use of Electricity to direct Microbial Metabolite Production
Time : 11:30-11:50
Biography:
John M. Pisciotta received his PhD from Johns Hopkins University for research on heme and lipid metabolism in malaria. Postdoctoral stints at the University of Maryland Center for Marine Biotechnology and then Pennsylvania State University College of Engineering focused on the development of novel bioelectrochemical systems (BESs) for production of bioenergtic products from waste. Dr. Pisciotta is currently an assistant professor in the Department of Biology at West Chester University in Pennsylvania where he teaches courses on Industrial Microbiology and Microbial Physiology.
Abstract:
At the metabolic level many organisms respond adaptively to changes in external stimuli such as exposure to toxins, drugs or radiation. For instance, high light exposure can induce up regulation of photo-protective metabolites and pigments in diverse plants and algae. Here we will focus on recent findings showing how application of electrical energy, alone or in combination with other stressors, may be used to guide and direct metabolism in microorganisms. Emerging bio-electrochemical tools and strategies for the targeted metabolomic analysis of useful electrically stimulated biological products including biofuels, antioxidants and pigments will be discussed. HPLC, GC and potentiostatic methodologies are used to study microbes as pure cultures or as syntrophic associations of bacteria, archaea and phototrophs such as cyanobaceria. Global profiling approaches useful for the discovery of novel electrically induced metabolites, proteins and response pathways will also be addressed.
Biography:
Dr. Deliang Guo is Assistant Professor at Ohio State University Comprehensive Cancer Center. His major research interests are tumor metabolism, autophagy and oncogenic signaling. His research is supported by multiple grants from NIH, American Cancer Society and American Brain Tumor Association
Abstract:
Dysregulated lipid metabolism is emerging as a new hallmark in malignancies. Understanding lipid biology in cancer cells is important to identify the key player in regulating lipid reprogramming and develop an effective therapeutic strategy to treat cancer. Our studies have characterized that sterol regulatory element-binding protein (SREBP-1), an endoplasmic reticulum-bound transcription factor with central roles in lipid metabolism, is highly upregulated in glioblastoma (GBM), a most common primary brain tumor with a median survival only 12-15 months even after advanced therapies. Furthermore, we found that EGFR/PI3K/Akt signaling via SREBP-1 upregulates low-density lipoprotein receptor (LDLR) for elevated cholesterol uptake. These data demonstrate that SREBP-1 plays a central role in mediating oncogenic signaling-driven lipid metabolism reprogramming in cancer cells. Recently, we found that lipid droplets (LD) are prevalent in GBM and inversely correlated with patient overall survival. Blocking LD formation via inhibition of ACAT1 significantly suppressed GBM tumor growth via inhibition of SREBP-1-dependent de novo fatty acid synthesis. Taken together, our studies demonstrate that SREBP-1 is a central player in cancer lipid metabolism, and suggest that targeting SREBP-1 and LD formation is a promising therapeutic strategy to treat malignancies.
Ligong Chen
Tsinghua University School of Medicine, China
Title: An metabolomic approaches to identify the endogenous substrate of OCT1
Time : 12:10-12:30
Biography:
Ligong Chen has completed his PhD from University of California at Berkeley and Postdoctoral studies from UCSF School of Medicine and Pharmacy. He is the Principal Investigator in Pharmacology and Toxicology of Tsinghua University School of Medicine, a premier University in China. He has published more than 20 papers in reputed journals including Nature Genetics, PNAS, and JBC et al. He is an expert in Transporter Physiology and Pharmacology. His lab is working on various transporters’ role in human diseases, using metabolomics, genomics and proteomics as major tools.
Abstract:
Proliferating cancer cells require abundant energy, building blocks for macromolecules, as well as reducing power to counteract high oxidative stress. Glutamine is essential for the survival of cancer cells by providing carbon, nitrogen, NADPH and participating in regulatory pathways such as mTORC1 and MAPK signaling. Several transporters belonging to SLC1, 6, 7 and 38 families mediate absorption of glutamine across the plasma membrane. We focus on several members of the SLC38 family and have utilized metabolomics approach to analyze the effect of glutamine transporter on cancer cell metabolism. After overexpressing specific SLC38 transporters (SLC38A3 and SLC38A5) in pancreatic ductal adenocarcinoma cells, we analyzed alterations of metabolites with HILIC-LC/ESI-MS. We found that over expression of glutamine transporter indeed influenced TCA cycle, GSH production and nucleotide synthesis. Abundance of the intermediates of TCA cycle, glycolysis, GSH and nucleotide synthesis was changed. In addition, changes in the amount of urea cycle intermediates including citrulline and ornithine were observed, which introduces new perspective on the relationship between glutamine and ammonia metabolism in cancer cells. Intriguingly, significant rise of the amount of glutamine itself was not demonstrated in any analyzed cancer cell line, which suggests concomitant up-regulation of glutamine consumption through biosynthetic and regulatory pathways. Though more experiments are required to illuminate mechanisms contributing to the influence of glutamine transporter on relevant metabolites, metabolomics as a powerful tool strengthens our understanding of glutamine’s effect on key metabolic processes and helps to identify noncanonical metabolic pathways in glutamine metabolism.
Poster Presentations @ 13:30-14:30
- Track 10: Proteomics and Genomics
Track 11: Clinical Metabolomics and Lipidomics
Track 12: Analytical Platforms Employed in Metabolomics
Chair
Sam F. Y. Li
National University of Singapore, Singapore
Co-Chair
P K Ragunath
Sri Ramachandra University, India
Session Introduction
Lars Lofgren
Translational Science, AstraZeneca R&D, Sweden
Title: Total lipid extraction for lipidomics made easy – The BUME method
Time : 09:30-09:50
Biography:
Lars Löfgren completed his PhD in 1993 from Chalmers University of Technology, where he developed sampling devices for - and characterized human exposure to - volatile hydrocarbons in urban air. He has continued his scientific work, published in peer-reviewed journals, in the field of sample preparations for lipids and rapidly metabolized biomarkers in biological samples in his current position as an Associate Principal Scientist in Translational Science, AstraZeneca
Abstract:
An important part of the workflow of lipid analysis is the extraction procedure. While there has been an impressive development of automated and high-throughput oriented, analytical methods, the extraction procedure is still often performed manually with traditional chloroform-based methods since more than 50 years. Now is the time for a change. To overcome the drawbacks of time-consuming, manual, and chloroform-based methods we have developed an automated chloroform-free method for total lipid extraction of biofluids and tissue. The method for biofluids is 100% automated, performed using a standard pipetting 96-well pipetting robot. It is based on an initial one-phase extraction using butanol and methanol (BUME). After addition of heptane/ethyl acetate/1% acetic acid a two phase system is formed without the need for centrifugation, with the organic lipid-containing upper phase easily recovered. To validate the methods, extraction recoveries for hundreds of lipid species from 10 lipid classes were tested and compared to the Folch method. The results showed similar or better extraction yields for all investigated lipids using the BUME method. For biofluids, the method was shown to be compatible with volumes ranging from 10-100 µl. For tissue, the method was validated for 15-150mg tissue. In conclusion, we believe that the development of these two methods is a major breakthrough moving lipid extraction into the high-throughput workflows of a modern lipid laboratory.
P K Ragunath
Sri Ramachandra University, India
Title: Relevance of Machine Learning Approach in Analysing Metabolomic Data to gain insights on Comorbidity issues in COPD & Asthma
Time : 09:50-10:10
Biography:
P K Ragunath graduated in March 1983 from Vivekananda College, Chennai, affiliated to University of Madras. He obtained Post Graduate Degree from Pachaiyappa's College, Chennai and MPhil degree from Madras University. Later he completed PhD in Madras Medical College, Chennai, affiliated to The Tamil Nadu Dr. MGR University Chennai. In addition, he has completed many degree/certificate programs in Management and information Technology. He has published 15 research articles in peer-reviewed journals and has also presented around 15 papers in national and international conferences. He is holding membership in many ‘World ‘renowned organizations’ catering to the Bioinformatics & Medical Informatics.
Abstract:
Background: Comorbidity is usually defined as a disease coexisting with the disease of interest. Chronic obstructive pulmonary disease (COPD) and Asthma are the most frequent causes of respiratory ill health, covering all ages and several cases of comorbidity between the two conditions have been reported. Asthma and COPD are different diseases each with a unique natural history and pathophysiology, but differentiating the underlying cause of their symptoms is difficult and often leads to generalized treatment protocols. Metabolomics involves quantitative measurement of time-related multi-parametric metabolic response of living systems to pathophysiological stimuli or change in gene expression profile. Over the past few years a number of studies especially employing Mass Spectrometry and NMR have emerged to identify the presence of metabolite markers specific to Asthma and COPD. The study aims at identifying a consensus metabolic profile of Asthma and COPD using text mining based machine learning approach and gain understand the underlying mechanism causing comorbidity. Lacunae: There is little information available on metabolite profile specific for COPD and Asthma, such knowledge would be invaluable in gain Insights on comorbidities between the 2 conditions. Methodology: Comprehensive text mining was carried to enlist all eligible studies on metabolomic profiling studies to recognize specific metabolite signatures common for Asthma and COPD and employ Machine learning approaches to gain Insights on comorbidity issues between the 2 conditions. Results and Conclusions: Modeling using Machine Learning like ANN, SVM, GA approaches were used to get quantitative effects of exogenous compounds on pathogenesis of COPD and Asthma. Metabolic profiling through the use of pattern recognition statistics on metabolite signals has the potential to identify specific signatures for COPD and Asthma which can aid in differential diagnosis and also to gain understanding on the comorbidity between the 2 disorders. Such knowledge would help in accurate diagnostics and to devise a novel management technique for COPD and Asthma. The results will be presented and discussed.
Ningombam Sanjib Meitei
Premier Biosoft, USA
Title: SimMet: Informatics tool for automating LC-MS and MS/MS based large metabolomic data processing and analysis
Time : 10:10-10:30
Biography:
Ningombam Sanjib Meitei has completed his PhD; he is the Chief Scientific Officer at Premier Biosoft, a leading bioinformatics company that provides software solutions for mass spectrometry based proteomics, glycomics, metabolomics, lipidomics and imaging data analysis. He has been leading the development of novel informatics tools namely; SimLipid, a LC-, MALDI- MS/MS data analysis tool for lipid identification by annotating product ions corresponding to lipid head-groups, fatty acyls, Charge Remote Fragment ions etc.; SimGlycan, the only high throughput glycan/glycopeptide identification tool that can also quantitate glycans using reporter ions intensity and MALDI Vision, a comprehensive mass spectrometry imaging data visualization and analysis tool.
Abstract:
Recently, there has been rapid growth in innovations related to liquid chromatography-tandem mass spectrometry (LC-MS/MS) based metabolite profiling studies. However, the lack of high throughput software tools has been one of the bottle necks. A typical metabolomics data analysis pipeline may include multiple software tools for example, using of a data processing tool to generate peak lists, a database search tool for metabolite profiling, other tools to validate metabolites using MS/MS data pattern matching or in silico fragment matching, tools for performing statistical analysis for identifying differential metabolites and pathways analysis for the identified metabolites. We have investigated some of the challenges we commonly face while processing raw data and identifying metabolites accurately. In order to address the challenges, we have developed SimMet. We introduce data filters in the software protocol that can effectively remove significant number of peaks corresponding to noise based on shape of the LC-peaks and data from LC-MS runs of blanks, QCs, technical replicates of the biological samples. The application of these filters prior to subjecting the data into conventional statistical techniques such as ANOVA, t-test, PCA etc. may be desirable since data can be reduced without compromising the actual information. This will also enable speedy analysis of large data that are common in mass spectrometry based metabolomics work flows. The software work flow will be demonstrated based on a food metabolomics experiment with data acquired on an LC-compact Qq-TOF mass spectrometer (Bruker Daltonik) system.
Sam F. Y. Li
National University of Singapore, Singapore
Title: Application of metabolomics for toxicity assessment of solid waste re-utilization
Time : 10:30-10:50
Biography:
Professor Sam Li is a faculty member at the Department of Chemistry, National University of Singapore (NUS). He received his BSc, PhD and DSc degrees from Imperial College, UK. His research interests include environmental science and technology, metabolomics, biosensors and nanotechnology. He has authored/co-authored 325 publications in international peer review journals, more than 100 conference presentations and 10 US patents. He serves/served on editorial advisory boards of several international scientific journals, including Electrophoresis (Germany), Journal of Chromatographic Science (USA), LC-GC (Asia Pacific), and Biomedical Chromatography (UK).
Abstract:
Leachate samples from fly ash and bottom ash obtained by gasification of solid wastes were analyzed by non-targeted screening using liquid chromatography-quadrupole-time of flight-mass spectrometry (LC-QTOF-MS). The results were used to determine which organic compounds could contribute to the toxicity of the leachates of solid waste gasification. Subsequently, the effects of the leachate on mortility and immobility of Daphnia mangna were evaluated as a method for monitoring water quality, and as a screening method for toxicity assessment of solid waste re-utilization.
- Young Researchers Forum
Session Introduction
Inmaculada Martinez-Reyes
Northwestern University, USA
Title: Dissecting the distinct functions of mitochondria
Time : 11:00-11:20
Biography:
Inmaculada Martinez-Reyes received her BA in Pharmacy and completed her PhD in Molecular Biology under the guidance of Dr. Jose Manuel Cuezva at University Autonoma of Madrid, Spain. During her Doctorate studies, she focused on the role of mitochondria in cancer. She was awarded a Postdoctoral Fellowship from Ramon Areces Foundation of Spain to perform her Postdoctoral training in Dr. Navdeep Chandel’s laboratory at Northwestern University. She is currently studying fundamental biology related to mitochondrial metabolism in hopes of finding new therapies for mitochondrial-associated diseases including cancer. She has published papers in outstanding journals and participated in numerous international conferences.
Abstract:
Mitochondria are the major site of energy production in the cell. An interesting and unique feature of the Electron Transport Chain (ETC) is that it is regulated under dual genetic control. Whereas the majority of the proteins that constitute the ETC are encoded by the nuclear DNA, 13 proteins are encoded by the Mitochondrial DNA (mtDNA). It is important to note, that mutations in mtDNA lead to very different phenotypes, suggesting that mitochondrial perturbations must manifest beyond bioenergetics. Other two salient functions of mitochondria are their biosynthetic capacity and their role as signaling organelles. We hypothesize that biological outcomes derived from these functions of mitochondria are also involved in the mechanisms mediating mitochondrial-associated diseases. However, it is not fully understood the involvement of distinct mitochondrial functions in the regulation of key cellular processes such as proliferation. In this study, HEK293 cells stably expressing a dominant negative form of the mtDNA polymerase POLG were used to eliminate mtDNA from cells in culture in a doxycycline-dependent manner as a model to dissect the importance of the different mitochondrial functions. First, the metabolic changes that occur during loss of mtDNA were studied by analyzing the whole cell metabolome profile to identify unique pathways activated during mitochondrial dysfunction. Next, the TCA cycle function and the mitochondrial membrane potential in cells with mtDNA loss were independently restored to elucidate the final consequences derived from the mitochondrial ability to carry out biosynthetic processes and to overall communicate with the rest of the cell.
Christin Zasada
BIMSB, Germany
Title: Quantifying metabolic fluxes in cancer and stem cells
Time : 11:20-11:40
Biography:
Christin Zasada studied Biosystems engineering at the Otto-von-Guericke University Magdeburg, Germany. In 2011 she started her PhD thesis in the lab of Stefan Kempa (Integrative Proteomics and Metabolomics) at the Berlin Institute of Medical Systems Biology at the Max-Delbrueck-Center for Molecular Medicine in Berlin, Germany.
Abstract:
Despite their different origin, cancer and stem cells share the feature of fast growth and unlimited proliferation. It is under debate if their mode of proliferation should require likewise distinct demands of precursors for biosynthesis or energy; regardless both cell types favoring aerobic glycolysis. In our lab we combine MS-based ‘omics’-technologies (LC-MS / GC-MS) to deliver relative and absolute quantitative information about enzyme abundance and metabolite concentrations of the central carbon metabolism (CCM). We have developed pulsed stable isotope resolved metabolomics (pSIRM) to monitor how cells utilize substrates like glucose and glutamine to meet their energetic requirements. Finally, the integration of quantitative and time-resolved isotope incorporation in a mathematical framework enables the calculation of the metabolic fluxes; the only functional readout of a cell. Isotopically non-stationary (INST) metabolic flux analysis uses the collected data (absolute poolsizes, extracellular rates) in combination with a network model (material balances, carbon transitions) to jointly estimate intracellular metabolic fluxes and pool sizes in cell culture experiments. The complete workflow-from the petri dish to the metabolic flux map-has been applied to track the rerouting of carbon usage during pluripotency, reprogramming and differentiation. We applied stable isotope labeled substrates and analyzed their fate in fibroblasts and their pluripotent counter parts (iPSC), in breast cancer MDA-MB231 and in human embryonic stem cells (hESC). The analysis endorsed the switch from a glycolytic to respiratory metabolism after differentiation both in hESC and iPSC-derived fibroblasts. Specifically the comparison of the metabolic profiles of stem cells and cancer cells revealed distinct metabolic features of these cell types.
- Workshop
Session Introduction
John M Pisciotta
West Chester University, USA
Title: Metabolomics research using bioelectrochemical systems
Time : 14:30-15:30
Biography:
John M Pisciotta received his PhD from Johns Hopkins University for research on heme and lipid metabolism in malaria. Postdoctoral stints at the University of Maryland Center for Marine Biotechnology and then Pennsylvania State University College of Engineering focused on the development of novel bioelectrochemical systems (BESs) for production of bioenergtic products from waste. He is currently an Assistant Professor in the Department of Biology at West Chester University in Pennsylvania where he teaches courses on Industrial Microbiology and Microbial Physiology.
Abstract:
The metabolic profile of most species can be influenced by externally applied physicochemical factors. Electrical potentials have fairly recently been found to powerfully effect metabolite production profiles in diverse species. This workshop will explore the utility of a rapidly evolving class of bioreactor termed “Bioelectrochemical Systems” (BESs) as a research platform for conducting basic and applied metabolomic studies.
- Track 6: Metabolic Modelling
Track 7: Systems Biology and Computational Biology
Track 8: Bioinformatics and Advancements
Track 9: Data Analysis and Interpretation
Chair
John M Pisciotta
West Chester University, USA
Co-Chair
Giulia Guerriero
University of Naples Federico II, Italy
Session Introduction
Shireesh Srivastava
ICGEB, India
Title: In silico identification of gene targets for bioethanol production from lignocellulosic hydrolysates using E. coli
Time : 15:30-15:50
Biography:
Shireesh Srivastava has completed his PhD from Michigan State University and Postdoctoral studies from National Institute on Alcohol Abuse and Alcoholism, (NIAAA/NIH), Rockville, MD. He is a Team Leader in the DBT-ICGEB Center for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. He has published more than 15 papers in reputed journals and has won Fellows Award for Research Excellence at NIH as well as Sigma Xi award for excellence in graduate studies. His research interest is in metabolism and systems biology applications to solve challenging biomedical and biotechnological problems.
Abstract:
Hydrolysates of lignocellulosic biomass contain both hexoses and pentoses, with pentoses contributing to about 30% of the fermentable sugars. Thus, it is important to select an organism that can utilize both sugars effectively. E. coli has the advantage that it can utilize pentose and hexose sugars. However, in the course of its fermentation, it produces many by-products in addition to ethanol, such as acetate and formate. For efficient production of lignocellulosic biofuel, it is important to maximize product yield and minimize by-products formation. We employed in silico analyses on a recent genome-scale metabolic model of E. coli (iJO1366), to identify the knock-out targets to improve production of ethanol from glucose and xylose. Genetic Design through Local Search (GDLS) was employed to identify knock-out targets. GDLS involves reduction of the metabolic model to yield an equivalent Flux Balance Analysis model with fewer reactions and metabolites, followed by local search methodology to efficiently cover the space of genetic manipulations. Interestingly, similar knock-outs were identified for both glucose and xylose. Using these knock-outs, the ethanol was predicted to be over-90% of the theoretical yield on both glucose and xylose. The flux distribution in the mutant strain was identified through the Relative Change (RELATCH) method using available 13C-metabolic flux analysis data. The mutant strain had increased activity of Entner-Duodoroff pathway but reduced pentose-phosphate pathway and TCA cycle activity.
PatrÃciaVerardi Abdelnur
Embrapa Agroenergy, Brazil
Title: Metabolomics of xylose-fermenting yeast for increasing efficiency in the production of second generation ethanol
Time : 15:50-16:10
Biography:
Patrícia has completed his PhD at the age of 27 years from State University of Campinas, performed a part of her PhD at Mass Spectrometry Research Center (MSRC), at Vanderbilt University.She is a researcher at Brazilian Enterprise for Agricultural Research, in the National Center for Agroenergy Research. She has published more than 20 papers in reputed journals and has been serving as a reviewer in scientific journals
Abstract:
The growing demand for alternative energy sources, adding value to products and utilization of residual biomass, as well as reducing the negative impacts on the environment, have encouraged the development of processes for producing ethanol from second generation (2G), ie, ethanol produced from lignocellulosic biomass. However, a bottleneck in the production of ethanol 2G is the inability of the yeast Saccharomyces cerevisiae, used in industry worldwide microorganism to produce ethanol, to convert pentoses such as xylose to ethanol (RUDOLF et al, 2009). Xylose is the second most abundant sugar on biomass, with about 33 % of fermentable sugars in the sugarcane bagasse (SASSNER et al, 2008). Given the importance of utilizing xylose to ethanol production, research have been done to identify yeast strains able to ferment pentose, highlighting two strategies: the identification of strains naturally capable of fermenting xylose and the development of recombinant strains of S. cerevisiae (WOHLBACH et al 2011; HÄGERDAL - HAHN et al, 2007).In this work, metabolomics approaches has been used to identify and quantify targets in the metabolic pathway for ethanol production from xylose. A method has been developed using UPLC-MS (Xevo TQD, Waters). ESI(+) and ESI(-)-MS, MS/MS and MRM experiments were performedin different conditions to obtain a sensitive and seletive method of targeted metabolomics. Finally, the metabolic pathways of four strains of yeast were compared and potential targets identified.
Giulia Guerriero
University of Naples Federico II, Italy
Title: Frontiers in comparative endocrinology: Challenges and opportunities of the “Program of Relevant National Interest
Time : 16:10-16:30
Biography:
Giulia Guerriero is Associate Professor of Comparative Anatomy and Cytology, professor for the PhD School in Environmental Systems Analysis and the Advisory Board of the Center for Environmental Research (I.R.C.Env.) of the University of Naples Federico II. Her education is due to the PhD program in Evolutionary Biology and Comparative that followed the degree in Biological Sciences at the University Federico II and her research conducted in the United States of America (Thomas Jefferson University and Penn's Philadeplhia, Andrology Center of the Michael Reese Hospital in Chicago, Oregon Health Sciences University in Portland, Oregon). She’s a member of the Working Group of the Italian Society of Biological Biomarkers Marine Biology; of the Center of Research Bioactive Peptides (CIRPeB) of the University of Naples Federico II; the Young Investigator Awards Judging Annual Meeting of the Society for Free Radical Biology and Medicine, USA and Head of the Laboratory of Comparative Endocrinology (EClab) at the Department of Biology of the University of Naples Federico II. She is Associate Editor and member of the Editorial Board of international journals and member of several scientific societies. She collaborates with Italian and foreign researchers. Since 2011 she is representative of bilateral agreements between the University of Naples Federico II and Egyptians Universities and research centers of excellence. Her research papers, subject of wide dissemination and inviting as speaker at national and international conferences, have helped to define the correlation between oxidants and antioxidants in physiological defenses of embryonic and larval development of non-mammalian vertebrates and the role of steroidal neuroreceptors in the reproductive species and the importance of barcoding in the evaluation of species-specific responses. She is currently in charge of a scientific research project of national interest (PRIN) on the assessment of the health status of 'marine-coastal environment’, and an European applied research project (LIFE) for the development of protocols for the remediation of contaminated soils present in the Domitian-agro Aversa coast.
Abstract:
Comparative endocrinologists have important roles to play in many areas of the life sciences, such as the development of alternative animal model systems for discovery of novel hormones and hormone-signaling pathways; the discovery of new pharmaceuticals to treat human disease; the development of sensitive, representative and high-throughput endocrine-screening assays for EDCs; the analysis of the impact of global climatic change on animal populations; the elucidation of pathways and mechanisms of evolution through the study of endocrine genes and structures; and the development of more efficient means for the production of animal protein to feed the world's growing human population. Recently, the Italian Ministry of Education, University and Research has funded the project “Systems Biology”, aimed to provide a detailed picture of marine ecosystems subjected to high anthropogenic impact. The Project is articulated into two main phases: The study of the effects of toxic contaminants on “sea sentinels” and their strategic conservation; and the development and implementation of preservation and bioremediation strategies for the decontamination of marine water and sediments. Each phase is realized through a synergic collaboration among eight operative units. The unit from Federico II University of Naples (UniNA) assesses the environmental health status through the ecotoxicological impact on tissues involved in the reproductive events. UniNA defines the levels of reactive oxygen species and the antioxidants under steroid control; estimates xenobiotic effects on histones, sperm nuclear basic proteins, DNA damage and repair as well as qualitative and quantitative responses to stress of genes and proteins involved in the endocrine control of spermatogenesis. A full understanding of the capabilities of organisms to respond to environmental variation is necessary for understanding the impact of pollution on the viability of populations.
Veena Singh
PGIMS ROHTAK, India
Title: Assessment of reproductive hormones and gynaecomastia in male CML patients with imatinib therapy
Time : 16:50-17:10
Biography:
Veena Singh Ghalaut did her MBBS from J.N. Medical College Raipur, Madhya Pradesh and M.D Biochemistry from Pt. B.D.S PGIMS Rohtak. She is presently working as Senior Professor and Head in the Department of Biochemistry, Pt. B.D.S PGIMS Rohtak. She has teaching and research experience of approximately 35 years. There are more than 100 national and international publications and few biochemistry books to her credit. She has guided more than 50 MD/MS thesis projects. She is an active member of many associations and has been honored with several awards
Abstract:
It has been observed that decreased testosterone production and gynaecomastia may appear as adverse effects of imatinib therapy but vary sparse work is available in literature. In this study, we prospectively studied testosterone, LH and FSH levels at baseline and at 6 months of imatinib treatment in 34 newly diagnosed male BCR-ABL positive CML patients. While none of the patients had gynaecomastia at 6 months, the proportion of patients with low testosterone levels increased significantly from 11.8% at baseline to 58.8% (p<0.001) and those with high LH and FSH increased significantly from 26.4% and 23.5% to 82.4% and 76.4%, respectively (p<0.001 and p<0.001). Serum testosterone levels decreased significantly (p=0.002) and serum LH and FSH levels increased significantly at 6 months of imatinib therapy (p=0.001 and p=0.003) in comparison to baseline levels. The findings suggest that there may be decreasing effect of imatinib on testosterone levels in adult CML patients but there is need of further supporting studies.
P S Ghalaut
PGIMS Rohtak, India
Title: Tryptase – new diagnostic and prognostic marker for AML AND CML
Time : 17:10-17:30
Biography:
P S Ghalaut has completed his MD in Medicine 25 years ago from PGIMS Rohtak and also underwent advanced training in medical oncology at Tata Memorial Hospital Bombay and one year specially training in the field of management of leukaemias & lymphomas including Bone Marrow Transplantation at the University of Wales Cardiff (UK). He is the Director and Head of Department of medicine and hematology at PGIMS Rohtak. He has published more than 75 papers in reputed journals and has been training Postgraduate students since the past 25 years
Abstract:
Objectives: To evaluate and compare the levels of serum tryptase in patients of AML and CML before and after treatment and with controls. Methodology: The study included 25 newly diagnosed patients of AML and 20 newly diagnosed patients of CML and 40 age and sex matched healthy controls. Patients of renal failure, allergic reactions and those already taking chemotherapy were excluded. Serum tryptase levels were estimated in cases and controls by ELISA. Results: Mean age in AML and CML patients was 58.4±15.73 yrs and 51.4±13.27 yrs respectively. Severe anemia and low platelet count was observed in AML patients in comparison to controls while in CML patients, mild anemia and significantly raised TLC and Platelets was observed compared to controls. Mean value of percent of blast was 57.9±19.65 in AML patients and 7.95±5.90 in CML patients. The levels of uric acid in AML patients was (6.73±1.726 vs. 4.37±0.834), the difference in cases and control group is statistically significant (p<0.001). Serum tryptase levels were higher in AML and CML patients before Chemotherapy in comparison to controls (p value <0.001). Cases with high percentage of blasts were also found to have higher levels. Significant reduction in levels of tryptase was observed in AML and CML patients after chemotherapy (p value<0.003 and p value <0.017 respectively). Conclusion: Tryptase may be useful for diagnosis, assessment of severity of disease (leukemic cells burden), monitoring minimal residual disease and prognosis of both AML and CML patients.
A.I. Fahmi
Menoufia University,Egypt
Title: Isolation, Identification and Evaluation of Highly Cellulases Producing Trichoderma Isolates from Egypt
Time : 17:30-17:50
Biography:
Fahmi A I received his Ph.D degree in molecular genetics from Texas A&M University in May 1992. He worked as assistant and associate professor in genetics department, Faculty of Agriculture, Menoufia University. Currently, He is working as a Professor in the same department. He is teaching genetic courses for undergraduate and graduate students. Also, he has supervised many graduate students who has worked for their master and doctorate degrees he is running research programs in the areas of molecular genetics and genetic mapping. Recently, he started a research project about using of Trichoderma sp. in hydrolyzing of organic wastes to produce compost for agriculture use. The first phase of this research program needs to isolate some Trichoderma highly producing of cellulases enzymes. Therefore, the topic of my abstract is Isolation, identification and evaluation of highly cellulases producing Trichoderma isolates from Egypt.
Abstract:
The objectives of this study were: 1) to characterize some Trichoderma isolates morphologically and molecularly, 2) to evaluate the best isolates for producing cellulases enzymes. 20 Trichodrma isolates were isolated from soil samples collected from six Egyptian governorates, in addition to seven provided isolates. According to morphological and molecular identification methods, they were divided into seven aggregate groups. Selected isolates that showed high ability to degrade cellulase were further evaluated. The result of submerged fermentation technique indicated that three isolates T44, T43 and T24 showed the highest activity for cellulases enzymes FPase, CMCase and -galactosidase, respectively. Also, the isolate T31 showed the highest production of free sugar, while the isolates T14, T19 and T44 demonstrated highest total protein concentrations. As for specific enzyme activity, isolates T17 and T24 were the highest for FPase enzyme, T17 was the highest for CMase enzyme and T24 was the best for -galactosidase. Furthermore, the result of solid state fermentation technique indicated that, isolates T14 was the best for producing FPase and -galactosidase and T20 was the highest for CMCase enzyme. Also, T20 was the best for producing free sugars while five isolates T1, T17, T20, T31 and T44 were the highest in producing protein. In addition, T17 showed the best hydrolysis percentage for rice straw. Moreover, extracellular proteins were analyzed by electrophoresis SDS-PAGE method. Most of highly producing cellulases isolates showed heavy band at around 65 KDa. Finally, it was concluded T14, T17, T19, T24, T31and T44 isolates could be recommended for biotechnological applications.