Oliver Fiehn - US grants

The functions of over 1/3 of the annotated protein-coding genes of the Arabidopsis genome are still unknown, and the annotation of an even larger portion of the genome is not sufficiently accurate for unambiguous assignment of function at the biochemical and physiological levels. This project will bring together a consortium of multidisciplinary collaborators to establish pipelines for generating metabolomics data-streams and to provide statistical and computational interpretation of the resulting integrated datasets. The goal is to develop metabolite-profiling capabilities that will enhance the research community's ability to formulate testable hypotheses concerning Arabidopsis gene functions. The consortium has developed metabolomic platforms that together detect approximately 1,800 metabolites, of which 900 are chemically defined. The aim of the project is to apply these established metabolite-profiling platforms to reveal changes in the metabolome associated with knockout mutations in up to 200 Arabidopsis genes of unknown function and compare these to similar mutants in 50 genes of known function. The consortium will disseminate these data via the existing multi-functional metabolomics database: www.plantmetabolomics.org. Enhancement of this database and associated statistical and visualization toolsets will enable researchers to formulate testable computational models of the metabolic network of Arabidopsis. The successful completion of these goals and integration with other NSF-sponsored functional genomics and cyber infrastructure developments will generate transformational resources for ultimately modeling the complex metabolism of Arabidopsis.

Broader Impacts
The project will develop new resources for the research community that will enhance the capability to globally profile genome expression at the metabolite level. These metabolite resources, in collaboration with other NSF-funded resource development projects, will enable researchers in the community to formulate credible, testable hypotheses concerning gene function. The project will foster the development of the science of metabolomics as a functional genomics tool through workshops, internships and organization of national and international meetings. The project will also develop new activities to enhance the impact of science education and training in the community, by conducting workshops for researchers at consortium labs and at international biological meetings. In addition, research internships will be offered to undergraduate students, eight of whom will have the opportunity to experience international science training in a European genomics laboratory. These research-based training internships will illustrate to the students the synergy that accompanies the integrated applications of chemistry, biochemistry, genetics, bioinformatics and computational sciences to solving complex biological problems.

DESCRIPTION (provided by applicant) This proposal describes a tightly unified effort between metabolomic specialists and environmental health researchers who will collaboratively characterize the development of asthmatic-type respiratory malfunctions in newborn and young adult rats, caused by model airborne particles using (a) soot particles and (b) secondhand smoke or environmental tobacco smoke (ETS). First, the investigators will compare the effects of soot and ETS at a concentration of 1 mg/m3 on the development of respiratory malfunctions in newborn and young adult rats. These effects will be correlated by comparing these model particles with respect to physical parameters and particulate matter composition. Second, the investigators will establish routes of primary metabolization and transport of organic pollutants from pregnant mothers' lungs to blood plasma. Third, the investigators will study metabolic aberrations in fetal, newborn and young adult rat's lungs. By this, links will be established between the type of particulate matter, its organic composition, the timing of metabolic changes in lung development, and the onset and progression of airways dysfunction. This unique combination of biomedical designs with metabolomic assessments will test the hypotheses that (a) perinatal exposure to organic constituents of airborne matter cause alterations in maturation as well as cellular structure and function in the lungs of young adult rats, (b) organic components deposited in the mother 's lung are directly involved in the development of pulmonary diseases such as airway hyper-reactivity, in addition to secondary alterations in immune response or release of bronchoconstrictive mediators by neuroendocrine cells, and (c) the development of respiratory malfunctions are associated with characteristic metabolic changes and can be distinguished from non-specific stress-related changes. Testing of these hypotheses will be enabled by a combination of cutting-edge analytical and metabolomic techniques at a high throughput level. Primary analytical tools will be gas chromatograph (GC)xGC-time of flight (TOF) mass spectrometry (MS) in combination with automatic peak annotation, enabling multivariate statistical comparisons of metabolic changes as well as compositional analysis of airborne model particles. Data will be complemented by full scan liquid chromatography (LC)-ion trap mass spectrometry and unbiased biomarker detection, and multi-target characterization of metabolites as a result of organic pollutants in lung tissues by LC-triple quadrupole MS/MS.

Intellectual Merit:
Metabolomics is the large-scale study of the small molecule component (metabolites) of living organisms. These small molecules in cells represent the consequential end products of gene expression and offer a high-resolution biochemical phenotype of a cell, tissue, and/or organism. Metabolomics is now a proven high-resolution tool that is yielding advanced understanding of primary and secondary metabolism. It is providing critical insight into the molecular and biochemical events that occur during mutualistic and pathogenic plant-microbe interactions, and it is a powerful functional genomics tool for the discovery of novel metabolites and their correlated biosynthetic genes. The specific aims of this proposal are 1) to acquire funds for a workshop that will identify and prioritize key strategic areas where metabolomics offers the greatest opportunity to advance biological knowledge in the areas of energy and environment, and 2) to foster greater collaborative interactions between Japan and US scientists in these priority research areas. To achieve the above goal, a team composed of a diverse group of ~10-15 experts in the field from Japan and from the USA will meet for two days, May 6-7, 2010, at The University of California, Davis. The workshop will focus upon current technical challenges and optimum collaborator opportunities related to plant, algal, and microbial metabolomics. Specific technical challenges to be discussed include defining the Metabolome, metabolite annotation, spatially and temporally resolved sampling, flux?omics?, dynamic range and depth-of-coverage, instrumentation and infrastructure, informatics and databases, standardization, putative national plant, algal, microbial metabolomics facility(s). Collaborative application opportunities will be discussed with regards to bioenergy, environment, functional genomics & gene discovery, secondary metabolism, metabolomics & genome-wide association mapping (plant, algae, microbe), systems biology and metabolic modeling in plant, algal, and microbial metabolomics. A report of the meeting outcomes will be drafted on-site, refined through peer review, and a final report delivered to NSF and JST.

Broader Impacts: This meeting will bring together an international team of leading scientists in the field of plant, algal, and microbial metabolomics, and provide a forum to identify and prioritize the current and most critically limiting challenges in the field. The discussions should lead to a consensus opinion for the best scientific approaches to address these challenges and unify the community towards the most effective approaches to solving the challenges. Importantly, the meeting will also foster and encourage collaborative interactions between US and Japanese scientists that will provide combined resources in applying advanced metabolomics technology to the solution of major questions related to plant, microbial, and algal biology. International collaborations that result will lead to training of students and postdocs in state of the art methodology and bring a new generation of scientists to this evolving and important field.

The metabolome is the sum total of the products of chemical reactions in the cell; it comprises a diverse set of chemicals that is species-specific and that is strongly influenced by the environment. Thus, despite decades of biochemical research, metabolomics is a field that is still in development, and no comprehensive database captures the complement of species-dependent metabolites. This project will capture the metabolome of 10 photosynthetic microorganisms (algae) by identifying all detectable metabolites that are synthesized as building blocks during their growth under conditions with variable sources of carbon (for example carbon dioxide and other organic compounds, including those found in wastewater). These data will greatly increase our knowledge of algal metabolism and carbon utilization. A major goal of this project is to integrate and improve existing metabolomic databases and libraries and to build new tools for the fast identification of metabolites. The resources resulting from this project will be made freely available to foster collaborations across multiple disciplines including engineering, analytical chemistry and bioinformatics. This project will facilitate the development of more efficient technologies for biofuel production based on photosynthetic microorganisms.

Broader Impacts
This project, through collaborations with researchers at the University of Tokyo and Osaka Prefectural University, will integrate and freely disseminate information and tutorials about libraries of chemical data, chemistry software, laboratory protocols for metabolism studies and results from the algal metabolomic studies. Web-based access will enable researchers and others interested in using these tools to compare the biochemistry of different species under various environmental conditions. In addition to the international training and education of the next generation of researchers, there will be outreach by project participants to students and pupils in K-12 education through the NSF-REU and GK-12 programs. Project staff will participate in the BioTech SYSTEM biotechnology program for teachers by providing educational activities for high school students and by partnering with local schools with large populations of under-served and under-represented minority students.

DESCRIPTION (provided by applicant): We are requesting funds for a Jeol DART accuTOF mass spectrometer system including a Gerstel MPS2 robotic autosampler with a cold injection system for GC separations. NIH funded investigators pursue metabolomic analyses in order to phenotype the metabolic consequences of genetic or disease alterations in humans and animal models. This research is compromised in Davis by the lack of instrumentation combining gas chromatographic separations with high mass accuracy and good sensitivity and isotope accuracy. In the Metabolomics Facility at UC Davis, over 22,000 low- resolution GC-TOF MS chromatograms have been screened for over 1,000 identified metabolites. Data were processed and stored in the unique BinBase database for comparison of peaks (including unknown compounds) across the 345 studies that were conducted so far. For the projects of the major users, these studies have yielded important data which led to a wide range of publications. We also found many compounds that were statistically significant in comparisons of case/control studies for which we need a good annotation scheme to perform biochemical assessments. The starting point for such annotations is to obtain accurate mass data for the molecular ions. We have compared our published results from soft ionization using chemical ionization in the (loaned) Waters GCTpremier to the alternative Jeol accuTOF mass spectrometer in conjunction with gas chromatography separation and DART ionization. Mass accuracy was acceptable with average mass errors of [unreadable]3 mu, but isotope accuracy was even better in the Jeol instrument than determined by the GCTpremier MS. We are therefore convinced that the Jeol GC-DART accuTOF mass spectrometer will have a major impact on the major users of this grant and the greater UC Davis research community. The instrument will be placed in the Metabolomics Facility core unit of the UC Davis Genome Center, with dedicated and trained staff available to oversee the operation, maintenance and management of this instrument. The Metabolomics Facility has been established in 2005 to provide cutting-edge research and service in collaboration with campus faculty, specifically to advance analytical and informatics methods to study metabolic dysregulation involving small molecules. The interaction between service core unit and research unit and the integration of these laboratories to a single Facility enables a regular improvement of methods and tools used by all staff in the Facility. The UC Davis Genome Center Metabolomics Facility core unit provides these services to two campuses (Sacramento and Davis) which include over 800 biology faculty and more than 4,000 graduate students.

PROJECT SUMMARY (See instructions): The Genomics Integration Core will advance metabolomics by making improvements in tools for interpreting and using metabolic data, specifically in the context of biochemical pathways and networks, but also by integrating data generated from genomics research, such as results from SNP genotyping, microbial genomics, or transcript and protein expression studies, into metabolomics studies. The core will be pivotal for conducting regional pilot and feasibility projects and critical for the success of the training and educational mission of the Promotion and Outreach Core. The Genomics Integration Core will be comprised of four different laboratories: the Weimer metagenomics laboratory, the Karp pathway informatics research group, the Pollard statistical genomics research group and the Lin and Perroud bioinformatics services core within the UC Davis Genome Center. The core will be responsible for both advancing the content of diverse metabolomics databases and tools and integrating the use of those tools and tools from other disciplines, particularly genomics, into metabolic studies such as pathway mapping. Newly developed tools will be employed in the Genome Center's bioinformatics service core as determined in coordination with the WC3MRC's Central Service Core. Specifically, the Genomics Integration Core will provide comprehensive bioinformatics and statistical tools for interpreting metabolomic data. The Core will collaborate with regional scientists on study design, data analysis and genomic interpretation of metabolomic data. The core will test and compare existing tools for linking genomic and metabolomic data, such as pathway mapping. Specifically, scientists in this core will work to advance genomics and pathway tools for metabolomic studies. The Genomics Integration Core will focus on advancing a range of existing tools in order to connect genomic pathways and disease phenotype data. Gene-enzyme annotations in the HumanCyc pathway database, integration of text-mining results into Cytoscape representations of metabolic networks, extension of current pathway enrichment approaches to include full metabolomic network statistics, development of tools for visualizing metabolite-centric network graphs with genomic information on demand, or other appropriate technologies will be explored in these efforts. The Genomics Integration Core will develop and test improvements in such tools and validate their utility and user friendliness by collaborating with regional scientists in clinical and preclinical research projects. Finally, this core will provide training and education in conjunction with the Promotion and Outreach Core.

DESCRIPTION (provided by applicant): The West Coast Central Comprehensive Metabolomics Resource Core (WC3MRC) will offer integrated services through a Metabolomics Central Service Core, and offer advanced services, including method developments, through the Metabolomics Advanced Services Core. Combined, the WC3MRC commands over 30 mass spectrometers, 5 NMR instruments and cutting-edge imaging equipment, and computer clusters with in-house as well as open-access and commercial software for metabolomic data acquisition, data processing, and data analysis including pathway mapping. Statistical support, tool development and advanced metabolic network analysis will be conducted in the Genomics Integration Core, while pilot and feasibility projects as well as courses and workshops will be organized by the Promotion and Outreach Core, to be led by the UC Davis Clinical and Translational Science Center (CISC). Overall management and data transfer to the Data Center will be performed by the Administrative Core. Specifically, the WC3MRC will be the first center in the United States that offers quantitative targeting of over 1,000 identified metabolites over a wide variety of biochemical pathways. For all these metabolites, reference standards are available and sample preparation, mass spectra and chromatographic conditions have been validated. Additionally, the WC3MRC will use untargeted metabolomic methods by accurate mass spectrometry for discovery-driven projects, including compound identifications. Methods used in the Advanced Core laboratories will be robotized and transferred to the Central Service Core for use by recharge fee services. Novel services and tools will be developed, ranging from isotope-labeled flux analysis to image guided metabolomics that will link to the established clientele using the imaging facility. Pathway annotations will be improved through curation of HumanCyc enzymes and by using InChI structure identifiers that will be used to construct complete metabolic networks that are subsequently used for pathway over enrichment statistical analysis. Both local and regional scientists will be engaged through annual, competitive pilot and feasibility awards. Participation and award criteria have been worked out and will be conducted by the CTSC. Training programs will educate the next generation of metabolomics scientists, both on the technical and the medical level.

PROJECT SUMMARY (See instructions): The overall intent of the Outreach and Promotion Core is to provide integrated scientific, educational, administrative, and financial support for outreach efforts, training, and pilot projects related to the West Coast Central Comprehensive Metabolomics Resource Core (WC^MRC) mission. The Core will incorporates highly successful aspects of the UC Davis Clinical and Translational Science Center (CTSC) programs including the Research Education, Training, and Career Development Program, the Pilot Translational and Clinical Studies Program, and the Translational Technologies, Methodologies, and Resources Program that have been developed and thoroughly tested during 6 years of NIH funding. Offerings through the CTSC Education Program will be supplemented with new translational metabolomics core courses, seminars, and workshops that will provide crucial exposure of a spectrum of trainees to the metabolomics field. Examples for such workshops offered through this core will be Glycobiology and the Microbiome, Cancer Metabolism, Lipid Metabolism in Cardiovascular Diseases and Metabolic Pathways in Animal Models. Examples for specific metabolomic training courses are Metabolomics Basics, Advanced Metabolomics and Genomics in Metabolic Diseases, to be inclusive for all trainees in the WC^MRC with hands-on training experience opportunities. Importantly, the Core will engage regional and local scientists to participate in pilot and feasibility project programs. These programs aim to spark innovative and collaborative scientific endeavors by supporting and facilitating robust new research paradigms, technologies, and tools, and through partnerships that enhance team science. The basic principles in executing the pilot program include: (1) a requirement for submissions to represent new multidisciplinary teams; (2) the inclusion of trainees and exposure of these trainees to the WC^MRC and CTSC educational offerings; (3) active solicitation of partnerships and leveraging of funds from other UC Davis pilot programs; and (4) linking program announcements to translational workshops.

PROJECT SUMMARY (See Instructions): The Metabolomics Central Service Core will become the hub for all research projects and studies conducted in the West Coast Central Comprehensive Metabolomics Resource Core (WC3MRC). The Central Service Core will handle all service requests, receive and log samples, distribute samples to the Metabolomics Advanced Service Core, acquire data for targeted and untargeted metabolomics, process data, provide statistical analyses and report results. The Core will offer comprehensive advice on study designs and sample handling and will develop quantitative target analyses as requested. Through automated sample handling and improvement of Standard Operating Procedures (SOPs), precision and accuracy in quantifications will improve while costs are minimized and sample throughput increases. Apart from classic metabolomic and lipidomic platforms, the Core will provide services through established University recharge rates for Molecular Imaging, Nuclear Magnetic Resonance (NMR) profiling and bioinformatics. The Core will implement new protocols developed by the Metabolomics Advanced Service Core to improve the number and quality of services offered by the Central Service Core laboratories. Scientists and staff working in this core will coordinate and facilitate service requests and sample processings for metabolomic projects. Clients and collaborators will be consulted on capabilities of the Center, cost estimates and turnaround times. The central LIMS database will be used to facilitate all projects. The Core will receive all samples for the Center, log these into the LIMS system and distribute samples according to the services requested. This Core will be tasked to prepare samples, acquire and process data for targeted and untargeted metabolomics. The Central Service Core will provide a large variety of services for mass spectrometry (MS)-based targeted metabolomics as well as NMR- and MS based untargeted profiling. Services will include the entire pipeline from sample preparation steps, quality controls and data acquisition to processing raw data to provide qualitative and quantitative data according to project scopes. The Central Service Core will implement existing protocols into automation for sample handling in order to improve precision and accuracy of quantitative data, and to accelerate sample throughput in the laboratory. The Core will provide a range of services in molecular imaging of target molecules in animal tissues. The Core will also provide bioinformatics services, including analysis for network biology.

PROJECT SUMMARY (See instructions): The Metabolomics Advanced Services Core combines capabilities for metabolomic data analysis from six metabolic laboratories located at UC Davis: the Fiehn Genome Center metabolomics laboratory (primary metabolism and complex lipids), the Hammock NIEHS superfund laboratory (eicosanoids and vitamins), the Lebrilla mass spectrometry laboratory (glycans), the Newman WHNCR laboratory (lipid mediators), the Cherry laboratory (imaging) and the Gaikwad laboratory (steroids). These methods will be available for service in Pilot & Feasibility studies and through recharge-rate fee structures. The laboratories will further advance and expand these methods for cross-platform integrated metabolomic studies. All services will be promoted by the Administrative Core, with samples to be delivered through the Central Service Core and managed by the centralized LIMS software. Advanced methods that have been automated and validated to be applicable for fast, high-quality operation will be transferred to the Central Service Core to accelerate throughput and turnaround times for regional and national clients. The Advanced Services laboratories will help with metabolomics training and pilot projects administered by the Promotion & Outreach Core. The core will provide comprehensive capabilities for metabolomic studies. Faculty and staff will collaborate with regional scientists in study design, implementation and data interpretation of metabolomic projects in clinical and preclinical studies. The core will expand the scope of its current quantification capabilities of 1,069 identified metabolite targets. Using untargeted metabolomics, the core will provide discovery services that extend to novel metabolic intermediates, followed by subsequent structural annotations and validation measurements. Secondly, the Core will advance metabolomics services and transfer methods to the Central Service Core. Scientists will develop or adapt methods to accelerate sample preparation processes by automating liquid- and solid-phase handling steps using a robotic sample handling device. Data processing steps will be optimized, and final methods will be transferred to the Central Service Core for the most robustly quantifiable sets of target metabolites. Isotope-based flux analyses will be implemented and transferred to the Central Service Core on GC-MS basis. For untargeted metabolomics, generalized retention-index marker compounds will be used to enable alignment procedures across different matrices. Image-guided mass spectrometry will open a novel field in metabolomics using fluorescently labeled metabolites and drugs for spatially targeting metabolically active zones in tissues and cell types.

Intellectual Merit. Most pro- and eukaryote genomes encode hundreds of enzymes of unknown function; finding what they do is a critical task for post-genomic biology. Mounting evidence implicates many of these enzymes of unknown function in "metabolite repair", i.e. in reversing damage done to metabolites by unwanted enzymatic side-reactions or chemical degradation. Because metabolites are under constant chemical attack (e.g. by oxidation or hydrolysis) and enzymes make wasteful and toxic catalytic errors, it follows that efficient functioning of meta¬bolic networks demands a support system dedicated to meta¬bolite repair. This system has been glimpsed by classical biochemistry, genetics, and metabolomics but most of it remains hidden. This project will therefore dissect the metabolite repair system by combining chemical biology, comparative genomics, and metabolomics using bacterial models and plants. Specific aims are to: (a) identify 30-50 target metabolites that are highly prone to chemical or enzymatic damage (i.e. that need repair) by cheminformatics, genome-scale metabolic reconstruction, and data mining; (b) predict genes encoding conserved repair enzymes for target metabolites using comparative genomics, and predict chemistries for the repair reactions; (c) test 20 repair predictions by knocking out the repair gene in a model organism, analyzing metabolomic profiles in normal and stress conditions, and identifying structures by cheminformatics; (d) validate repair reactions by mass spectrometric authentication of structures, by biochemical assays of recombinant proteins, and by functional complementation of bacterial or plant mutants; and (e) incorporate validated repair functions in next-generation genome-scale metabolic models.
This project integrates modeling in two ways. First, it makes innovative use of modeling to predict a priori the metabolites most likely to need repair. Second, adding validated repair functions to genome-scale bacterial models sets up a virtuous cycle of prediction 'experiment' further prediction to drive discovery in metabolite repair. It also pioneers an essential modeling development: Models that capture the cost of uncontrolled formation and degradation of unwanted metabolites.
Research in the emerging field of metabolite repair has the potential to displace a current paradigm of metabolic routes operating with perfect precision by one where the illusion of a flawless system is maintained by a battery of unobtrusive but critical repair functions. Moreover, metabolite repair is almost surely crucial to stress adaptation, to aging, and to metabolic engineering.

Broader Impacts. This project will provide interdisciplinary training in comparative genomics, metabolomics, chemical biology, and integrative modeling to two PhD students and three postdoctorals who will spend time away from their own institution each year at another collaborating institution. Un-dergraduates will participate. In addition, there will be a training outreach component with three facets: (a) Eight two-day hands-on workshops (2 per year) at different universities to train PhD students, post¬doctorals, and faculty in comparative genomics using SEED databases and tools. At least three work¬shops will be at minority-serving institutions. Each workshop will train 10-12 people. (b) Development of a web page in which the instructional content of the workshop will be available for distance learning. (c) Instruction of project postdoctorals and students in how to organize and present workshops, culminating first in their acting as teaching assistants, and ultimately in them teaching themselves.

PROJECT SUMMARY (See instructions): . The Administrative Core, comprising the Director, the Program Coordinator and the administrative manager, will provide oversight for the program. They will prepare and administer all aspects of the budget and provide progress reports to the NIH. They will provide support to the other four cores of the Center, and oversee compliance with biosafety regulations, as well as the security of materials, data and facilities. Administrative support personnel will also perform day-to-day activities and operations of the Center such as organization of seminars, outreach activities and point of contact for questions from the scientific community and general public. Program Director Fiehn and Coordinator Wikoff will provide overall management of the West Coast Central Comprehensive Metabolomics Resource Core. They will implement monthly Steering Committee meetings as platform for core leaders to share developments and services, research progress and new applications. The Administrative Core will oversee the overall budget, technical services, outreach activities, and establish a reasonable cost-recovery fee schedule including campus approvals, and manage workflow records. The Administrative Core will coordinate data and information transfer to the Data Center (DRCC) and serve as a liaison to NIH, campus administration, other campus units, and other RCMRCs. They will monitor and regularly review the quality of services and prepare progress reports. The Center Director will assume overall responsibility for the timely conduct of research and outreach services and ensure annual milestones are met In order to maintain detailed information on all activities, the Administrative Core will implement a central LIMS system for all samples that are received in the Center for service activities or Pilot & Feasibility projects. The LIMS system will be used to summarize services and advancement projects taking place in each core. Importantly, the Core will also provide web services and metabolomic information. The Administrative Core will develop and maintain a website for the RCMRC that links to the NIH Common Fund website for the Metabolomics Program. The Center web site will be organized by a content management system to provide direct access for the Core leaders to upload content to the Core's services and projects. The web site will provide overviews on all services rendered by the Center as a one-stop site, including tutorials and videos.

US Participants: Lloyd W. Sumner (Samuel Roberts Noble Foundation); Oliver Fiehn (University of California, Davis), James Liao (University of California, Los Angeles), Georg Jander (Boyce Thompson Institute), Basil Nikolau (Iowa State University), Richard A. Dixon (University of North Texas), Jean S. VanderGheynst (University of California, Davis), John Labavitch (University of California, Davis), Tobias Kind (University of California, Davis), William Wikoff (University of California, Davis), Alisa Huffaker (USDA-ARS, Gainsville), and Eric Schmelz (USDA-ARS, Gainsville)

Japan Participants: Kazuki Saito (Riken Plant Science Center), Masanori Arita (University of Tokyo), Eiichiro Fukusaki (Osaka University), Yutaka Okumoto (Kyoto University), Kazuo Shinozaki (Riken Plant Science Center), Jun Kikuchi (Riken Plant Science Center), Mami Yamazaki (Chiba University), Hideyuki Suzuki (Kazusa DNA Research Institute), Daisaku Ohta (Osaka Prefecture University), Shigehiko Kanaya (NAIST), Hiroshi Shimizu (Osaka University), Fumio Matsuda (Osaka University), Naoki Mori (Kyoto University), Naoko Yoshinaga (Kyoto University), and Yutaka Okumoto (Kyoto University)

The primary goal of this project is to develop a Plant, Algae and Microbial Metabolomics Research Coordination Network (PAMM NET) that will promote effective communication, enhance opportunities for collaboration, build community consensus, identify key challenges in metabolomics, and facilitate coordinated community empirical efforts to meet these challenges. Participation in PAMM NET will be open to the public, and will begin with the unification of four independent and international projects funded through the joint NSF-JST Metabolomics for a Low Carbon Society program. This unification will further amplify NSF's current investment in plant, algae and microbial metabolomics, and will be achieved through regular videoconferences and annual face-to-face program meetings and workshops. This team will serve as a nucleus to build forth a more unified national and international PAMM NET that will identify and pursue solutions to the key challenges that still impede the full potential of metabolomics. This will be facilitated through the recruitment of a network coordinator who will serve as a dedicated advocate for the organization, facilitate integration, and provide logistical support for consensus reporting of the network outcomes. The initial nucleus and the network coordinator will then recruit public participants from the US and global plant, algae, and microbial metabolomics communities to build working focus groups. These focus groups will actively discuss the current grand challenges associated with metabolomics and potential solutions to these challenges. The PAMM NET recognizes that many challenges will need the involvement of the larger biology, technology, and bioinformatics communities and PAMM NET will therefore recruit feedback and active participation from these diverse groups to best formulate empirical solutions to the grand challenges. PAMM NET will further contribute to the development of a US National Chapter of the Metabolomics Society to better serve and provide long-term solutions for the needs of the US metabolomics community. PAMM NET will build a repartee with other federally funded programs such as the NIH-supported National Metabolomics Centers to better coordinate efforts across federal funding divisions and scientific disciplines. Finally, PAMM NET will initiate conversations with the global metabolomics community to identify and pursue cooperative international metabolomics funding opportunities. A coordinated community effort is expected to be more cordial, efficient, and productive, thereby leading to new and enabling scientific discoveries and innovations.

The PAMM NET RCN considers research-based education and training as an integral part of preparing young scientists for fruitful careers. Accordingly, the PAMM NET will encourage, coordinate and support the attendance of early-career scientists (young faculty, graduate students, post-docs and a limited number of undergraduate students) at annual workshops that are associated with each of the NSF-JST Metabolomics programmatic projects through a limited number of scholarships. These programmatic workshops will include hands-on demonstrations of different aspects of metabolomics research that will range from the use of sophisticated chromatographic, mass-spectrometric and NMR-based tools for metabolite analysis, to computational and statistical analyses and interpretation of metabolomics data. In addition, this RCN will organize and support an annual workshop associated with international plant biology or metabolomics meetings (e.g., Annual Meeting of the Metabolomics Society or the Annual Meeting of the American Society of Plant Biologists). Information about the activities of the PAMM NET will be housed at the project website (to be developed) and the resources generated will be transferred to the Metabolomics Society website for long-term maintenance and support.

PROJECT SUMMARY (See instructions): The Genomics Integration Core will advance metabolomics by making improvements in tools for interpreting and using metabolic data, specifically in the context of biochemical pathways and networks, but also by integrating data generated from genomics research, such as results from SNP genotyping, microbial genomics, or transcript and protein expression studies, into metabolomics studies. The core will be pivotal for conducting regional pilot and feasibility projects and critical for the success of the training and educational mission of the Promotion and Outreach Core. The Genomics Integration Core will be comprised of four different laboratories: the Weimer metagenomics laboratory, the Karp pathway informatics research group, the Pollard statistical genomics research group and the Lin and Perroud bioinformatics services core within the UC Davis Genome Center. The core will be responsible for both advancing the content of diverse metabolomics databases and tools and integrating the use of those tools and tools from other disciplines, particularly genomics, into metabolic studies such as pathway mapping. Newly developed tools will be employed in the Genome Center's bioinformatics service core as determined in coordination with the WC3MRC's Central Service Core. Specifically, the Genomics Integration Core will provide comprehensive bioinformatics and statistical tools for interpreting metabolomic data. The Core will collaborate with regional scientists on study design, data analysis and genomic interpretation of metabolomic data. The core will test and compare existing tools for linking genomic and metabolomic data, such as pathway mapping. Specifically, scientists in this core will work to advance genomics and pathway tools for metabolomic studies. The Genomics Integration Core will focus on advancing a range of existing tools in order to connect genomic pathways and disease phenotype data. Gene-enzyme annotations in the HumanCyc pathway database, integration of text-mining results into Cytoscape representations of metabolic networks, extension of current pathway enrichment approaches to include full metabolomic network statistics, development of tools for visualizing metabolite-centric network graphs with genomic information on demand, or other appropriate technologies will be explored in these efforts. The Genomics Integration Core will develop and test improvements in such tools and validate their utility and user friendliness by collaborating with regional scientists in clinical and preclinical research projects. Finally, this core will provide training and education in conjunction with the Promotion and Outreach Core.

PI Name: Jean VanderGheynst
Number: 1438211

Microalgae have remarkable potential for producing biofuels and bioproducts, and for sequestering carbon dioxide from industrial flue gases. Economical production of algal biofuels will require using nutrients from wastewater and a free source of carbon. The proposed research will involve managing wastewater treatment by mixotrophic microalgae to achieve efficient biofuel production. Mixotrophic algae can grow on either organic carbon sources or atmospheric carbon dioxide. The research has the practical goal of providing an alternative system for biofuel production leading to new industries with high global impact in the areas of energy and agriculture sustainability. Specifically, the use of biofuels produced from microalgal lipids and polysaccharides has been proposed as a potential solution to worldwide challenges related to fossil fuel scarcity and global warming. Furthermore, culturing microalgae in wastewater can improve water quality, and prevent problems associated with "food vs. fuel" competition on limited agriculture lands. This project will also train research and educational leaders who will gain knowledge of fundamental principles and applications of biological engineering and biotechnology. Students will develop an ability to work in multidisciplinary teams to achieve research goals, and gain understanding of the broader issues (global environmental and economic impacts, public/societal views) in the field, preparing them to contribute to science and policy related to energy sustainability. This research will benefit K-12 education through integration of algal research into elementary school science curriculums. It will also benefit undergraduate education through enhanced integration of algal research into an engineering design class at University of California, Davis.

Technical Description

Microalgae have been considered as a viable biofuel feedstock due to their productivity and associated higher-value by-products. Recent reports indicate that sustainable economic production of algae for bioenergy will require mixotrophic production on wastewater and waste carbon dioxide. The growth rate of microalgae, uptake of organic carbon, and accumulation of intracellular lipid and polysaccharide products resulting from mixotrophic production will likely be different from heterotrophic and autotrophic production that have been primarily studied to date. Mixotrophic conditions can improve lipid productivity by an order of magnitude. However, these cultures can exhibit low efficiency in converting the carbon source into biofuel precursors. For example, when cultured on glucose, glycerol, and acetate, a model strain of microalgae converted only 10-40% of the substrate energy into lipids and starch. In contrast, yeast cultures can convert glucose to ethanol with 70% efficiency on an energy basis. Elucidating what contributes to lower efficiency in algae is critical for designing algal-based systems for wastewater treatment and biofuel production. The goal of the proposed research is to develop algae cultivation and wastewater management systems that lead to higher substrate utilization efficiencies for biofuel production and organic matter removal from wastewater. Management options include strain selection and algae acclimation, rate and timing of substrate addition and management of wastewater composition. An additional goal is to determine bottlenecks in substrate utilization by microalgae. These goals will be achieved through three objectives. The first objective is to determine if acclimation of microalgae to organic carbon reduces substrate utilization efficiency and develop strategies to overcome this challenge. The second objective is to vary organic carbon and co-factor addition to algae using fed-batch reactors to determine if substrate supply rate impacts conversion efficiency, and the third objective is to use results from objectives 1 and 2 to tailor microalgae production variables for cultivation on food processing wastewaters. This may include application of substrate supply strategies developed in earlier objectives and supplementation of co-factors deficient in wastewaters. Metabolomic profiling techniques will be used to elucidate metabolic responses to different management strategies. This research is expected to inform the decision making process on how to manage microalgae and wastewater for achieving sustainable biofuel production.

? DESCRIPTION (provided by applicant): We propose aiding Early Career Scientists involved in the comprehensive analysis of metabolism (metabolomics) to attend next year's International Metabolomics Society Conference in San Francisco, June 29-July 02, 2015. Scientists will join with a biomedical or clinical focus, including researchers involved in developing and validating methods for improved metabolome coverage, data accuracy, genomics integration and biological context research. The conference organizers will help the Early Career Scientists' committee to arrange topical workshops, present studies and record these sessions for further educational and training use. Comprehensive analysis of metabolism is the corner stone for understanding diabetes and the metabolic syndrome, cancer metabolism and its regulation and dependency on nutrition, and the implications of metabolism in cohort studies and in lung diseases such as asthma. Sessions will focus on the impact of the microbiome on human metabolism and health, genetic variance and its implications on metabolism, different aspects of cardiovascular risk and the metabolic syndrome, integration of multi- omics data into a systems understanding of metabolic regulation, and the impact of human exposures (including food and nutrition) on metabolic health and cancer metabolism as well as drug response metabolism.

Synthetic biology enables engineering of microbes, plants, and animal cells to install new or redesigned natural biosynthetic routes to synthesize biologically-based products such as novel biofuels and pharmaceuticals. One challenge preventing synthetic biology from reaching its full potential is the need to keep in check the damage caused by unwanted chemical or enzymatic side-reactions. When unchecked, this damage can diminish yields of end-products and poisons the cells making these products, referred to as metabolites. Consequently cells must either repair the damaged metabolite or convert them into harmless compounds. Metabolite damage and its control is analogous to DNA and protein damage and repair, but is much more poorly understood. The goal of this project is to develop a better understanding of which metabolites are damaged, how cells repair damaged metabolites, and to develop computational models for predicting metabolite damage and repair. This project will contribute to the development of the next generation work-force by providing cross-disciplinary training of graduate students and post-doctoral fellows. The project will also develop a hands-on workshop on chemoinformatics for biologists of all career levels that will include participation of faculty from minority-serving institutions.

Chemical (i.e. non-enzymatic) or enzymatic side-reactions can convert metabolites to useless or toxic compounds, which requires cells to have systems to deal with these damage products. It is also clear that chemically-mediated metabolite damage can impose stress upon a cell to such an extent to influence fitness and possibly interfere with synthetic biology applications. Research suggests that there are far more metabolite damage reactions and damage-control systems than the few known so far. The goal of this project is to develop a better understanding of which metabolites are damaged and how cells repair damaged metabolites. To achieve this he goal this collaborative project will coordinate progress on: 1) building a public database of chemical reactions of metabolites with algorithms to predict such reactions analogous to what KEGG/BioCyc does for enzyme reactions; 2) development of a theory-driven approach to predict and validate damage-control genes and their mode of action; 3) developing metabolic models that predict how damage reactions potentially impact cellular physiology and synthetic biology efforts; and 4) identifying damage products among thousands of unknown peaks in metabolomics profiles, which will permit validation of predicted damaged metabolites based on computational algorithms.

Project Summary American Indians (AIs) suffer disproportionately from type 2 diabetes (T2D). Discovery of novel mechanistic biomarkers is the key to identify at-risk individuals and to develop effective preventive strategies tailored to this high risk population. In response to PA-12-165, this project leverages the wealth of unique resources collected by the Strong Heart Study (SHS), the largest longitudinal cohort study of American Indians followed over 25 years, to identify sensitive and specific metabolic markers that are predictive of T2D risk at preclinical stages above and over standard clinical factors including obesity, fasting glucose and insulin resistance. Metabolomics is an emerging technology that can simultaneously identify and accurately quantify hundreds to thousands of metabolites in biofluids. Several metabolites, such as BCAAs, acylcarnitines, and lipids, have been associated with T2D, but these results were largely derived from cross-sectional studies in almost exclusively European Caucasians. However, given the genetic regulation of metabolism, metabolites identified in Caucasians may not be generalized to AIs who may have a different genetic make-up. In addition, cross- sectional analysis cannot capture the dynamic trajectory of metabolic changes over time. Moreover, most existing studies measured a list of pre-selected metabolites on a single platform, but given the complexity of the human metabolome and the substantial diversity of metabolites, no single analytical platform can detect all metabolites in a biological sample. We hypothesize that longitudinal changes in plasma metabolites predict T2D risk independent of fasting glucose, insulin resistance (IR) and obesity, and that metabolic profiles of T2D in AIs are similar to, but distinct from, those in Caucasians. Our goal here is to identify novel and sensitive T2D predictors that are specific to AIs beyond classical T2D indicators. To achieve this, we will repeatedly measure concentrations of over 500 metabolites, including BCAAs, carbohydrates, hydroxyl acids, lipids, as well as gut microbial-derived metabolites, in fasting plasma (~5 yr apart) from normoglycemic SHS participants followed >15 years. Putative metabolites will be replicated in an independent longitudinal sample of AIs followed for 10 years. To increase coverage, we will quantify metabolites concentrations on three complementary platforms. Each assay will be performed as a dual 'targeted' and 'untargeted' analyses to provide both hypothesis-driven quantitative data and discovery-driven semi-quantitative data of unidentified metabolites. Unknown compounds will be identified by well-established workflows. Multivariate analyses will be conducted to identify novel T2D predictors above and over standard clinical factors. Our multidisciplinary team consists of experts with complementary expertise in diabetes epidemiology, metabolomics, analytical chemistry, statistics and bioinformatics. Findings of this study will greatly advance our understanding of T2D pathology, and hold promise for reducing or eliminating T2D disparity in AIs, an ethnically important but traditionally understudied minority group suffering from alarmingly high rates of T2D and obesity.

Project Summary ? Experimental Core The Experimental Core at the West Coast Metabolomics Center for Compound Identification (WCMC) is committed to the overall goals of the NIH Common Fund Metabolomics Initiative and specifically aims to greatly improve small molecule identifications. The Experimental Core is led by the WCMC director Prof. Fiehn with close support from several experienced mass spectrometrists, chromatography experts and database programmers. The following specific aims will be covered: 1) The use of deuterium exchange, specific functional group derivatization and high resolution chromatography/mass spectrometry to deduce substructural features to drastically limit the number of false isomers. The information will be incorporated into a compound identification pipeline and software tools developed in close collaboration with the Computational Core. 2) The development of a large open access reference library for LC-MS/MS and GC-MS retention times and mass spectral data. These databases serve as backbone for our LC-BinBase database that will employ high quality MS/MS spectra through our MS-DIAL software and link it to biomedical metadata through our miniX study design database. Such information will be used for the detection of presence and intensity of unknowns that can be prioritized using BinVestigate across a large diversity of biological studies. 3) The experimental validation of computational methods and confirmation of novel metabolites, the benchmark of WCMC and consortium capabilities and acquisition of data through suitable metabolomic reference studies and blinded sets of chemical standards.

The project ?Metabolomics of longevity? will partner with all projects and cores within the Longevity Consortium study. Its objective is to detail the metabolic biomarkers and biochemical mechanisms that differ betwee long- lived species or subjects, in comparison to species or subjects with shorter lifespans. We specifically investigate balancing nutritional energy, repair and prevention of metabolic damage, and metabolic responses to stress. We further hypothesize that sustaining a healthy metabolism correlates with specific molecular signatures that are acquired in other projects of the Longevity Consortium, so that metabolomics data can be integrated into context analysis in a meaningful manner. Consistent with the overall emphasis and design of the Longevity Consortium, we will focus on pathways relevant to control of metabolic regulatory capacity rather than on disease-specific signatures. According to these overarching aims, we will acquire and interpret high-quality metabolite data by using targeted and untargeted mass spectrometry. We will identify and quantify over 800 known metabolites, in addition of over 2,000 metabolic signals that lack structure identification. Metabolite classes will cover primary amines, bile acids, steroids, inflammatory oxylipins, complex lipids, biogenic amines and miscellaneous compounds, including dietary and drug exposome markers. Specifically, in aim 1, we will randomize samples from four large human cohorts to compare baseline metabolomics markers of 683 subjects who continued to live longer than 98% of subjects of the U.S. population, and compare these to 2,049 subjects who had a shorter life span. We will integrate several statistical tools to compile a panel of longevity biomarkers that will be validated by an independent cohort of 450 subjects who lived longer than 100 years. We will investigate all data in a longitudinal manner by appropriate statistical tools to predict changes in a range of age-related phenotypes such as grip strength and walking speed that ultimately may contribute to longevity. In aim 2 we will analyze cells from over 100 long-lived and short-lived species to achieve mechanistic understanding of conserved metabolic differences in cellular metabolic homeostasis. We will also compare long-lived mice against shorter-lived wild type mice, including through lifespan-extending drugs such as rapamycin and acarbose. In aim 3, we will combine literature-based food- and microbial metabolic markers into the analysis of the human cohort studies as confounding factors that . may contribute to human longevity and that are not under human genetic control. We will also contribute to efforts in the Orwoll-proteomics, the Girke- chemoinformatics and the Price-systems cores by providing comprehensive metabolite/gene and metabolite/protein annotations through integrating existing biochemical databases. We will supplement these analyses by phenotypic context information for metabolite data through text-mining association analyses.

Project Summary ? Administrative Core The Administrative Core at the West Coast Metabolomics Center for Compound Identification (WCMC) is committed to the overall goals of the NIH Common Fund Metabolomics Initiative and specifically aims to greatly improve small molecule identifications. The Administrative Core is led by the WCMC director Prof. Fiehn with support from project coordinator Dr. Barupal in coordination with Prof. Wang, Prof. Tantillo and Dr. Kind. The Administrative Core will provide leadership, organizational structure, and communication processes for the overall mission. The Core will use effective administrative structures to manage tasks and monitor achievements of the Computational Core and the Experimental Core. A steering committee and internal and an external advisory board will give strategic guidance on all project aims. The Administrative Core will actively engage in all Common Fund Metabolomics Consortium (CF-MC) activities, especially its working groups. They will help the CF-MC to draft and implement guidelines and policies towards compound identification requirements for the National Metabolomics Data Repository. The Administrative Core will take leadership in organizing internal and external benchmarking tests for compound identification and validation of in-silico libraries. They will also engage the CF-MC in efforts towards harmonizing metabolomic data reports through kits of internal chemical standards that will be distributed to all CF-MC metabolomics cores, centers and projects as donations from chemical vendors. Finally, they will assist the CF-MC Coordinating Center to reach out to key stakeholders for training opportunities and dissemination of metabolomics software tools and databases.

Project Summary ? Overall West Coast Metabolomics Center for Compound Identification (WCMC) The West Coast Metabolomics Center for Compound Identification (WCMC) is committed to the overall goals of the NIH Common Fund Metabolomics Initiative and specifically aims to largely improve small molecule identifications. Understanding metabolism is important to gain insight into biochemical processes and relevant to battle diseases such as cancer, obesity and diabetes. Compound identification in metabolomics is still a daunting task with many unknown compounds and false positive identifications. The major goal of the WCMC is therefore to develop processes and resources that accelerate and improve the accuracy of the compound identification workflow for experts and medical professionals. The WCMC for Compound Identification is structured in three different entities: the Administrative Core, the Computational Core and the Experimental Core. The Center is led by the Director Prof. Fiehn in close collaboration with quantum chemistry experts Prof. Wang and Prof. Tantillo, and metabolomics experts Dr. Barupal and Dr. Kind with broad support from mass spectrometry, computational metabolomics and programming experts. The Administrative Core will assist the Computational and Experimental Core to develop and validate large in-silico mass spectral libraries, retention time prediction models and innovative methods for constraining and ranking lists of isomers in an integrated process of cheminformatics tools and databases. The developed tools and databases will be made available to all Common Fund Metabolomics Consortium (CF-MC) members and professional working groups. The WCMC will also provide guidance for compound identification to the National Metabolomics Data Repository. The broad dissemination of developed compound identification protocols, training for compound identification workflows, databases and distribution of internal reference standard kits for metabolomic standardization will overall widely support the metabolomics community.

Project Summary_Abstract We are applying for funds for a Bruker timsTOF Pro trapped ion mobility mass spectrometer LC- MS system. This LC-ion mobility MS systems offers superior resolution to alternative systems providing much deeper metabolomics, lipidomics and exposome screening capabilities than classic LC-accurate mass MS/MS instruments. With the enhanced performance of the requested Bruker timsTOF Pro LC-MS system, structurally similar lipids and other compounds can be accurately detected and identified. Such system is urgently needed to fill gaping unmet needs by the biomedical community here at UC Davis. We have identified 8 major users for this instrument for 80% of time usage and suggest a 20% time usage by the UC Davis metabolomics core to work on recharge-based projects for minor users. By providing our infrastructure and technical expertise on LC-MS metabolomics, we project a diverse and long-term collaborations with our users. The common thread across all projects is that chemical complexities are too large to be resolved ?in depths? by current LC-accurate mass MS/MS alone. Users need to get to the next stage to solve their research problems adequately. Projects that need for ion mobility are such as: environmental health (Young: deeper MS/MS coverage for non-targeted screening, van Winkle: higher resolution for oxidized lipids), cancer metabolism (Carraway: improved sensitivity and resolution for BMP and PG lipidomics in organelles, Chen: improved resolution of oxysterol isomers), diurnal cycles (Chiu: resolving isobaric and isomeric triacylglycerides), nutritional interventions (Stanhope: resolving urinary polyphenols), methods in compound identification (Fiehn: improving confidence in structure annotations by CCS values) to gut microbiome changes (LaSalle: interaction of gut-brain axis in Rett syndrome, resolution of biogenic amines in fecal matter). All these projects have in common that they have hit roadblocks in our ongoing collaborations with respect to capabilities of our current instrumentation at the WCMC that lack the added power of ion mobility separation despite our exquisite capabilities in accurate mass analyses.