Progress 07/01/02 to 06/30/07
Outputs
OUTPUTS: Drought, and overuse of nitrogen-based fertilizer, are persistent problems in managed agriculture worldwide. An understanding of how some photosynthetic organisms can both tolerate desiccation, and also enzymatically-fix their own nitrogen from the atmosphere, are thus of consequence to the goals and programs of the USDA. Major outputs during the duration of this project include: Activities 1. Member of a task group that designed and established the multi-disciplinary Institute of Biomedical and Public Health Sciences (IBPHS) at Virginia Tech. This was a most significant investment of time and effort that included site visits, and fact-finding missions to biomedical science, food and nutrition, public health and outreach programs across the US; UC Berkeley, Purdue University, and U. Georgia. 2. Following a visit to VBI by a team of scientists from Warwick University, England, I was invited as a guest speaker and consultant to present an overview of perspectives and
suggestions to a multi-university workshop in Warwick, where a regional center of excellence in bioinformatics is being planned. 3. Design of an undergraduate Course in Genomics that is now a regular offering shared by the Departments of Biochemistry and Animal Science. Events 1. Conceived, designed and led a Workshop on "Advanced Technologies: Proteomics" at Qatar University, Doha, Qatar, 2006-7; for teaching assistants and faculty (enrollment 12) - during one week Services 1. Senior project consultant for 3M and OwensCorning (to present) on issues to do with the growth of desiccation-tolerant microorganisms on residential dwellings across the US. 2. Expert Witness; contentious litigation case in the Patent Court of the High Court of Justice, Chancery Division, London, England (retained by Bristows; http://www.Bristows.com) 3. Chair, Electorate Nominating Committee for Biological Sciences within the American Association for the Advancement of Science (AAAS), 2004-2005 Products 1.
Title: Method to preserve cells Document Type and Number: United States Patent 7150991 Inventors: Potts, Malcolm; Helm, Richard; Berninger, Mark S.; Avila, Herbert A.; Publication Date: 2006-12-19
PARTICIPANTS: Center for Biotechnology in Sfax, Tunisia.
TARGET AUDIENCES: Students and faculty of Qatar University, Doha, Qatar.
Impacts
Through this project it was possible to understand how simple microorganisms, such as cyanobacteria and yeast, withstand desiccation and other environmental stresses that are of importance to managed agriculture. This knowledge was used to study whether sensitive cells can be made more resilient. Specifically, we demonstrated that partially dried human kidney cells, under appropriate conditions, can be stored for weeks, at room temperature, in a state of suspended animation, and then recover normal function. This work is of significance to the storage and transport of biomedical products, fabrication and deployment of cell-based biosensors, as well as understanding those events in cells that may control disease states. The work is therefore of consequence to human health and safety.
Publications
- Jack, G.D., Cabrera, C.M., Manning, M.L., Slaughter, S.M., Potts, M. and R.F. Helm. 2007. Activated stress response pathways within multicellular aggregates utilize an autocrine component, Cellular Signalling. 19: 772-781.
- Kumar, D., Ramakrishnan, N., Helm, R.F. and M. Potts. 2006. Algorithms for storytelling. KDD. Proceedings of the Eleventh ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, Philadelphia. in press.
- Lim, C.-S., M. Potts and R.F. Helm. 2006. Nicotinamide extends the replicative life span of primary human cells. Mech. Ageing Dev. 127: 511-514.
- Potts, M., Slaughter, S.M., Hunneke, F.-U., Garst, J.F. and Helm, R.F. 2005. Desiccation tolerance of prokaryotes: Application of principles to human cells. J. Int. Comp. Biol.45:800 - 809.
- Wright, D.J., S.C. Smith, V. Joardar, S. Scherer, J. Jervis, A. Warren, R. F. Helm and M. Potts. UV-irradiation and desiccation modulate the 3-D extracellular matrix of Nostoc commune (Cyanobacteria). 2005. J. Biol. Chem. 280: 40271-40281.
- Singh, J., Kumar, D., Ramakrishnan, N., Singal, V., Jervis, J., Slaughter, S.M., Potts, M., and R.F. Helm. 2005. The transcriptional response of yeast (Saccharomyces cerevisiae) to desiccation and rehydration. Appl. Environ. Microbiol. 71: 8752-8763.
- Ramakrishnan, N., D. Kumar, B. Mishra, M. Potts, and R. F. Helm. 2004. Turning CARTwheels: an alternating algorithm for mining redescriptions. In Proceedings of the Tenth ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, W. Kim, R. Kohavi, J. Gehrke, W. DuMouchel (Eds.). ISBN 1-58113-888-1, pg. 266-275.
- Potts, M. 2004. Nudist colonies: Revealing glimpse of EPS synthesis. J. Phycol. 40: 1 - 3.
- Billi, D. and M. Potts. 2002. Life and death of dried prokaryotes. Res Microbiol. 153: 7-12.
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Progress 10/01/05 to 09/30/06
Outputs
The roof shingle of virtually every dwelling in the US supports a unique biofilm of cyanobacteria that is tolerant of desiccation, UV irradiation, heavy metals, extremes of temperature, acid rain and paucity of nutrients. The biofilm is a unique resource. During the reoporting period we initiated a comprehensive biochemical, biophysical and molecular analysis of the dominant cyanobacterium to understand the basis for its resistance to multiple stresses.
Impacts
A knowledge of the mechanisms of resistance in this microorganism will facilitate genetic engineering of plants, as well as the development of biosensors with consequences for national security.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
A definitive study that characterized the molecular basis for resistance to UV irradiation and drought was completed and published in J. Biol. Chem. The key aspect of the study was the central role of the extracellular matrix of cells in stress resistance. The work on drought resistance was extended to yeast - of industrial relevance - and gene expression assays were used to characterize the response. The work on drought tolerance was further extended to human cells. Specifically it was shown that human kidney cells could be stored partially dry, at room temperature for up to 6 weeks. The latter finding is of relevance to the fabrication of cell based biosensors as well as the production of biomedical reagents for civilian and military casualty care.
Impacts
The work that was developed using nitrogen-fixing cyanobacteria was extended to Saccharomyces cerevisiae (yeast; of economic significance) and also human cells. Proof of concept was shown for the storage of human cells as biomedical reagents at room temperature for 6 weeks.
Publications
- Potts, M., S.M. Slaughter, F.-U. Hunneke, J.F. Garst, and R.F. Helm. 2005. Desiccation tolerance of prokaryotes: Application of principles to human cells. J. Int. Comp. Biol. In press.
- Jack, G.D., E.A. Mead, J.F. Garst, M. C. Cabrera, S. M. Slaughter, A. I. Brooks, M. Potts, and R. F. Helm. 2005. Long term metabolic arrest and recovery of HEK293 spheroids involves NF-kappaB signaling and sustained JNK activation. J. Cell. Physiol. First published online: DOI 10.1002/jcp.20499; see commentary by S. Hurtley (below).
- Hurtley, S. (Ed.) 2005. Editors Choice; Cell Biology Long-lived cells (Jack et al. J. Cell. Physiol. 10.1002/jcp.20499). Science 310: 591.
- Singh, J., D. Kumar, N. Ramakrishnan, V. Singal, J. Jervis, S.M. Slaughter, M. Potts,and R.F. Helm. 2005. The transcriptional response of yeast (Saccharomyces cerevisiae) to desiccation and rehydration. Appl. Environ. Microbiol. In press.
- Wright, D.J., S.C. Smith, V. Joardar, S. Scherer, J. Jervis, A. Warren, R. F. Helm and M. Potts. 2005. UV-irradiation and desiccation modulate the 3-D extracellular matrix of Nostoc commune (Cyanobacteria). J. Biol. Chem. In press.
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Progress 10/01/03 to 09/30/04
Outputs
Work continued using model systems to decipher the underlying structural, physiological and molecular basis for desiccation tolerance. The long-term goal of the effort is to understand how resistance to environmental stress can be engineered in plants of significance to managed agriculture. A transcriptomics and proteomics study of desiccation tolerance was completed using the model system Saccharomyces cerevisiae (yeast) and this was used as the basis for a graduate student research project. The yeast gene Sip18 was expressed in E. coli and the recombinant protein is in the process of purification for trials in stabilization assays with other cell types. Techniques for the assay of the response of nematodes and tardigrades (arthropods) to desiccation and long-term storage were developed. A partial genomic microarray was constructed for the desiccation tolerant cyanobacterium Nostoc commune in collaboration with the University of Rochester Medical School. Signaling
pathways involved in the response of human cells to air-drying were identified and the data form the basis for further studies on longevity, aging and cell quiescence. Collaboration with colleagues at UC Davis and ORNL are involved with the annotation of the genome of the nitrogen-fixing cyanobacterium and plant symbiont Nostoc punctiforme. Study of the genome of this organism is important to understand host-symbiotic relations in plants.
Impacts
The data obtained during the past year extend the scope and significance of the research project to the areas of longevity, aging and cell transformation (cancer).
Publications
- Hunsucker, S., Klage, K., Potts, M., and Helm, R. F. 2004. A preliminary investigation of the Nostoc punctiforme proteome. Biochem. Biophys. Res. Comm. 317: 1121-1127.
- Ramakrishnan, N., Kumar, D., Mishra, B., Potts, M., and Helm, R. F. 2004. Turning CARTwheels: An alternating algorithm for mining redescriptions. KDD 266-275.
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Progress 10/01/02 to 09/30/03
Outputs
Dryophile genes that were associated with stress tolerance were isolated, and their recombinant proteins are currently under investigation in stabilization studies (a stated aim of the project): sodF from cyanobacteria encoding superoxide dismutase; spsA and spp from cyanobacteria encoding sucrose phosphate synthase and sucrose-6-phosphate phosphohydrolase, respectively; wsp from cyanobacteria encoding water stress protein; SIP18 from yeast encoding a phospholipid-binding hydrophilin. In experiments at AnhydroCyte, superoxide dismutase was shown to offer significant protection to E. coli cells and lentiviruses when they were desiccated. Also at AnhydroCyte spsA and spp (responsible for sucrose synthesis) were transfected and transiently expressed in human 293 kidney cells. Genes that were markedly upregulated in the radiation-resistant bacterium Deinococcus radiodurans when it was subjected to desiccation stress were identified. A new algorithm was developed to
uncover cryptic and subtle features of gene regulation during environmental stress. This new bioinformatics tool will be of widespread utility to the scientific community and will be freely distributed on request. We completed a comprehensive global analysis of the response of yeast to desiccation, a stated aim of the project. The most up-regulated gene was SIP18 that encodes a hydrophilin (with terminal lysine residues). sip18 protein is known to bind phospholipids and we hypothesize that it does so and acts as a compatible solute essentially replacing water molecules and stabilizing membranes. In preliminary global analyses of gene expression in human 293 kidney cells exposed to desiccation stress we observed a similar response of the cells as seen for yeast, i.e. a shift in metabolism towards fatty acid oxidation. One gene, SGK encoding serum regulated glucocorticoid kinase, was up-regulated both under desiccation stress and salt stress, and contained TonE response element (osmotic
response elements) sequences in its 5 prime region. This candidate dryophile gene encodes a protein kinase that plays an important role in activating certain potassium, sodium, and chloride channels, suggesting an involvement in the regulation of processes such as cell survival, neuronal excitability, and renal sodium excretion. SGK may be a key component of cellular stress response. GSY=3
Impacts
The data obtained during this study have direct relevance to the metabolic engineering of desiccation tolerance in sensitive cells. These findings are of relevance to the development of stress-resistant plants and other organisms.
Publications
- Slotta, D.J., L.S. Heath, N. Ramakrishnan, R. Helm and M. Potts. 2002. Computational approaches to combining predictive biological models. Proceedings of the High Performance Computing Symposium (HPC 2002), Advanced Simulation Technologies Conference, A. Tentner (Ed.), Society for Computer Simulation International, San Diego, CA, pages 75-80.
- Paerl, H.W., T.F. Steppe, K.C. Buchan, and M. Potts. 2003. Hypersaline cyanobacterial mats as indicators of elevated tropical hurricane activity and associated climate change. Ambio 32: 87-90.
- Shirkey, B., N. J. McMaster, S.C. Smith, D. J. Wright, H. Rodriguez, P. Jaruga, M. Birincioglu, R. F. Helm, and M. Potts. 2003. Genomic DNA of Nostoc commune (Cyanobacteria) becomes covalently modified during long-term (decades) desiccation but is protected from oxidative damage and degradation. Nucleic Acids Res., 31:2995-3005.
- Shaw, E., D.R. Hill, U.C. Tauber, H. Marand, R.F. Helm and M. Potts.2003. Unusual water flux in the extrapolysaccharide of Nostoc commune (Cyanobacteria). Appl. Environ. Microbiol. 69: 5679-5684.
- Slotta, G., L. S. Heath, N. Ramnakrishnan, R. Helm, and M. Potts. 2003.Clustering mass spectrometry data using order statistics. Proteomics 3: 1687-1691.
- Singh, J., Kumar, D., Ramakrishnan, N., Singal, V., Jervis, J., Steve Slaughter, S., Potts, M., and R.F. Helm.2004. Global response of yeast (Saccharomyces cerevisiae) to desiccation and subsequent rehydration. Proc. Natl. Acad. Sci. USA (Track II Submission - pending).
- 2004 - Potts, M. Nudist colonies: Revealing glimpse of EPS synthesis. J. Phycol. In press.
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Progress 10/01/01 to 09/30/02
Outputs
The current project will continue gene discovery and gene expression analyses related to desiccation tolerance, and more specifically will focus on the characterization of water stress protein (Wsp) isolated in the previous Hatch project. The scope of this work is of direct relevance to the engineering of stress tolerance in plants of agricultural significance. Objectives: The specific goals of this proposal are to: Goal 1: Identify mechanisms of desiccation tolerance using gene inactivation. Goal 2: Identify the function of water stress protein gene wsp. Goal 3: Achieve optimal desiccation tolerance in sensitive cell lines. Progress: Too early to report at this time.
Impacts
The study of the mechanisms of desiccation tolerance in cyanobacteria will provide insights into how this trait may be engineered in other organisms that are sensitive to air-drying. Such an understanding will contribute to methods for air-dry stabilization of cells, tissues and biomedical products.
Publications
- No publications reported this period
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