Detailed Research Information

Detailed project information for
Study Plan Number 01087

Branch : Fish Health Branch

Study Plan Number : 01087

Study Title : Development of Advanced Molecular Methodologies for the Identification of Rapid Response Genes in Indicator and Other Species of Interest -- Measurement of Global Gene Expression by Microarray Technology

Starting Date : 08/15/2002

Completion Date : 09/30/2005

Principal Investigator(s) : Schill, Bane (William)

Primary PI : Schill, Bane (William)

Telephone Number : (304) 724-4438

Email Address : bane_schill@usgs.gov

SIS Number :

Primary Program Element : Fisheries and Aquatic Resources

Second Program Element :

Status : Completed

Abstract : BACKGROUND
Eukaryotes have numerous mechanisms for dealing with environmental stresses and pathogens. While animals are exposed to pathogens regularly, disease is manifested only when the immune system is not able to counter the invasion and multiplication strategies of the agent. Often, the immune system succumbs as a result of pressure from perturbed environmental situations and the resulting stress. The USGS-BRD and the National Institutes of Health (NIH) have interests in understanding the mechanisms by which stress modulated immune suppression occurs and how to control it. Episodes of animal and human disease have recently increased dramatically in association with freshwater aquatic and marine coastal systems. In the Chesapeake Bay, for example, blooms of toxic algae, dinoflagellates, and fungus (Blazer et al. 2000a, Blazer et al. 2000b, Blazer et al. 2002) have been reported to have caused fish kills and lesions and perhaps human illness and currently, mycobacterial and streptococcal infections are increasing in incidence in both wild and cultured fish (Baya et al. 1990, Rhodes et al. 2001). Concern is indicated because these human pathogens may be transmitted during fish handling or preparation. Recent studies at LSC have revealed episodic immune suppression in white perch populations of impacted Chesapeake tributaries, but the cause(s) are unknown.
New technologies for gene identification and measurement of expression have exploded in recent years. Microarray methods, for example, allow for the measurement of the activity of thousands of genes simultaneously. Most of the information from these types of experiments, however, lies in teasing out patterns of gene expression rather than simply quantification of expression on a gene-by-gene basis. That is, the discoveries lie in defining networks of genes that show coordinate expression under various conditions. As is often the case in science, analytical methods familiar in one discipline are foreign to another. In the case of microarray data, statistical evaluation is best treated in a multivariate manner using methods often applied in the field of population genetics. In fact, clustering and dendrogram building have been introduced to microarray analysis only recently—the same methods applied to population genetics issues for years. Other, more advanced, data analysis methods using the artificial intelligence techniques of case based reasoning and maximum likelihood assignments successfully applied by the principal investigator to the analysis of Atlantic salmon microsatellite data (King et al. 2001) have been tested for applicability to microarray data. These methods successfully identified several key functions of human natural killer cells (Hodge et al. 2000, Hodge et al. 2002). Powerful as they may be, these global measurements of simple gene expression do not totally describe cellular activities. Posttranslational modification and control mechanisms also play significant roles as do nuclear trafficking systems. Proteomic and cellular studies are required to complete the story. We plan to use a panel of cellular/molecular biomarkers to assess fish and invertebrate health and reproductive status. The types and intensity of environmental stressors that an animal is exposed to can be ascertained by assessing the levels of a spectrum of molecules in the animal’s cells. For example, elevated levels of heat shock proteins are indicators of heat stress, while elevated levels of metallothionein are indicative of metal stress . Many of these types of important biological molecules are sufficiently conserved evolutionarily to be identifiable by hybridization and/or antibody methods with commonly available reagents. This fact will allow us to measure and quantify the levels of a variety of these molecules at the cellular and subcellular level using Quantitative Polymerase Chain Reaction (QPCR) and Laser Scanning Cytometry (LSC). We also plan to extend the use of microarray analysis (described earlier) and begin proteomic-based studies by using hybrid methodologies such as SELDI (Surface-Enhanced Laser Desorption/Ionization) mass spectroscopy to identify new biomarkers/bioindicators of fish and invertebrate health status. SELDI (www.ciphergen.com) combines affinity techniques with time of flight mass spectroscopy (MALDI-TOF) to identify novel biomarkers of health status directly. Newly identified biomarkers will be added to the panel described above to improve the robustness and resolution of the methodology.
The cooperation of the National Cancer Institute and USGS should produce benefits to both parties and the taxpayer by integrating the resources of the two agencies thus reducing duplication and enhancing scientific understanding of innate immune function in humans as well as other vertebrate and invertebrate species. Expected products include several publications describing the new approaches, methodologies, and findings resulting from the described studies. Sequences of newly discovered rapid response genes will be placed into the public databases (GenBank). The findings should help to define the interactions of the biological and non-biological elements of the environment.
OBJECTIVES
1) The new statistical approaches discussed above will be applied to microarray data sets derived from experiments using human and/or mouse immune cells to identify promising avenues for further study. These methods will then be used to examine their feasibility for the identification of expressed genes of the immune cells of fish.
2) We will test the effect of fish tissue extracts on human and/or mouse natural killer cell and leukocyte function (for example production of IFN-, cytotoxicity, and induction of apoptosis). Ongoing studies in this laboratory have documented immune suppression in fish inhabiting Chesapeake Bay tributaries where fish lesions and kills have been observed. We suspect that contaminants play a role in this problem. The study of the effect of these unknown pollutants and/or their metabolites on a (relatively) well-characterized system would aid in understanding the problem.
3) Subtraction libraries will be developed to identify human natural killer cell and fish immune function genes regulated under the influence of cytokines, chemokines, and interleukins (rapid response genes), as well as those influenced by metals, and estrogens/androgens.
HYPOTHESIS TO BE TESTED
Using massively parallel methodologies (such as microarray techniques), a panel of molecular markers (bioindicators) can be identified that will be predictive of vertebrate and invertebrate health status in multiple indicator species.

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Branch :	Fish Health Branch
Study Plan Number :	01087
Study Title :	Development of Advanced Molecular Methodologies for the Identification of Rapid Response Genes in Indicator and Other Species of Interest -- Measurement of Global Gene Expression by Microarray Technology
Starting Date :	08/15/2002
Completion Date :	09/30/2005
Principal Investigator(s) :	Schill, Bane (William)
Primary PI :	Schill, Bane (William)
Telephone Number :	(304) 724-4438
Email Address :	bane_schill@usgs.gov
SIS Number :
Primary Program Element :	Fisheries and Aquatic Resources
Second Program Element :
Status :	Completed
Abstract :	BACKGROUND Eukaryotes have numerous mechanisms for dealing with environmental stresses and pathogens. While animals are exposed to pathogens regularly, disease is manifested only when the immune system is not able to counter the invasion and multiplication strategies of the agent. Often, the immune system succumbs as a result of pressure from perturbed environmental situations and the resulting stress. The USGS-BRD and the National Institutes of Health (NIH) have interests in understanding the mechanisms by which stress modulated immune suppression occurs and how to control it. Episodes of animal and human disease have recently increased dramatically in association with freshwater aquatic and marine coastal systems. In the Chesapeake Bay, for example, blooms of toxic algae, dinoflagellates, and fungus (Blazer et al. 2000a, Blazer et al. 2000b, Blazer et al. 2002) have been reported to have caused fish kills and lesions and perhaps human illness and currently, mycobacterial and streptococcal infections are increasing in incidence in both wild and cultured fish (Baya et al. 1990, Rhodes et al. 2001). Concern is indicated because these human pathogens may be transmitted during fish handling or preparation. Recent studies at LSC have revealed episodic immune suppression in white perch populations of impacted Chesapeake tributaries, but the cause(s) are unknown. New technologies for gene identification and measurement of expression have exploded in recent years. Microarray methods, for example, allow for the measurement of the activity of thousands of genes simultaneously. Most of the information from these types of experiments, however, lies in teasing out patterns of gene expression rather than simply quantification of expression on a gene-by-gene basis. That is, the discoveries lie in defining networks of genes that show coordinate expression under various conditions. As is often the case in science, analytical methods familiar in one discipline are foreign to another. In the case of microarray data, statistical evaluation is best treated in a multivariate manner using methods often applied in the field of population genetics. In fact, clustering and dendrogram building have been introduced to microarray analysis only recently—the same methods applied to population genetics issues for years. Other, more advanced, data analysis methods using the artificial intelligence techniques of case based reasoning and maximum likelihood assignments successfully applied by the principal investigator to the analysis of Atlantic salmon microsatellite data (King et al. 2001) have been tested for applicability to microarray data. These methods successfully identified several key functions of human natural killer cells (Hodge et al. 2000, Hodge et al. 2002). Powerful as they may be, these global measurements of simple gene expression do not totally describe cellular activities. Posttranslational modification and control mechanisms also play significant roles as do nuclear trafficking systems. Proteomic and cellular studies are required to complete the story. We plan to use a panel of cellular/molecular biomarkers to assess fish and invertebrate health and reproductive status. The types and intensity of environmental stressors that an animal is exposed to can be ascertained by assessing the levels of a spectrum of molecules in the animal’s cells. For example, elevated levels of heat shock proteins are indicators of heat stress, while elevated levels of metallothionein are indicative of metal stress . Many of these types of important biological molecules are sufficiently conserved evolutionarily to be identifiable by hybridization and/or antibody methods with commonly available reagents. This fact will allow us to measure and quantify the levels of a variety of these molecules at the cellular and subcellular level using Quantitative Polymerase Chain Reaction (QPCR) and Laser Scanning Cytometry (LSC). We also plan to extend the use of microarray analysis (described earlier) and begin proteomic-based studies by using hybrid methodologies such as SELDI (Surface-Enhanced Laser Desorption/Ionization) mass spectroscopy to identify new biomarkers/bioindicators of fish and invertebrate health status. SELDI (www.ciphergen.com) combines affinity techniques with time of flight mass spectroscopy (MALDI-TOF) to identify novel biomarkers of health status directly. Newly identified biomarkers will be added to the panel described above to improve the robustness and resolution of the methodology. The cooperation of the National Cancer Institute and USGS should produce benefits to both parties and the taxpayer by integrating the resources of the two agencies thus reducing duplication and enhancing scientific understanding of innate immune function in humans as well as other vertebrate and invertebrate species. Expected products include several publications describing the new approaches, methodologies, and findings resulting from the described studies. Sequences of newly discovered rapid response genes will be placed into the public databases (GenBank). The findings should help to define the interactions of the biological and non-biological elements of the environment. OBJECTIVES 1) The new statistical approaches discussed above will be applied to microarray data sets derived from experiments using human and/or mouse immune cells to identify promising avenues for further study. These methods will then be used to examine their feasibility for the identification of expressed genes of the immune cells of fish. 2) We will test the effect of fish tissue extracts on human and/or mouse natural killer cell and leukocyte function (for example production of IFN-, cytotoxicity, and induction of apoptosis). Ongoing studies in this laboratory have documented immune suppression in fish inhabiting Chesapeake Bay tributaries where fish lesions and kills have been observed. We suspect that contaminants play a role in this problem. The study of the effect of these unknown pollutants and/or their metabolites on a (relatively) well-characterized system would aid in understanding the problem. 3) Subtraction libraries will be developed to identify human natural killer cell and fish immune function genes regulated under the influence of cytokines, chemokines, and interleukins (rapid response genes), as well as those influenced by metals, and estrogens/androgens. HYPOTHESIS TO BE TESTED Using massively parallel methodologies (such as microarray techniques), a panel of molecular markers (bioindicators) can be identified that will be predictive of vertebrate and invertebrate health status in multiple indicator species.
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