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Project Title:  Induction of Hepatocellular Carcinoma by Space Radiation: A Systems Biology Study of Causative Mechanisms Reduce
Images: icon  Fiscal Year: FY 2020 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 01/07/2015  
End Date: 01/06/2020  
Task Last Updated: 08/14/2020 
Download report in PDF pdf
Principal Investigator/Affiliation:   Emmett, Mark  Ph.D. / The University of Texas Medical Branch 
Address:  UTMB Cancer Research Center 
301 University Blvd, Rt. 1074 
Galveston , TX 77555-5302 
Email: mremmett@utmb.edu 
Phone: 409-747-1943  
Congressional District: 14 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Medical Branch 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ullrich, Robert  Ph.D. University of Texas Medical Branch 
Key Personnel Changes / Previous PI: November 2017 Report: Dr. Cheryl Lichti left UTMB (University of Texas Medical Branch) to take a position at Washington University, St. Louis. She is still a collaborator on the project, but is no longer a Co-Investigator and is not receiving salary support since 8/31/17. Ana Nia (MD/Ph.D. Graduate student, joined the project October 2017. November 2016 report: Dr. Joseph Moskal (Northwestern University) is no longer affiliated with academia nor involved with this project and is being removed as Co-I on the project. November 2015 report: Dr. Carol L. Nilsson (Co-I, 10% Effort) is no longer involved with the project. Dr. Cheryl F. Lichti has replaced Dr. Nilsson at 20% Effort. Two advanced graduate students, Brooke L. Barnette and Shinji K. Strain, will replace the TBA senior scientist (50% Effort).
Project Information: Grant/Contract No. NNX15AD65G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2013-14 HERO NNJ13ZSA002N-RADIATION 
Grant/Contract No.: NNX15AD65G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Cancer:Risk of Radiation Carcinogenesis
Human Research Program Gaps: (1) Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L)
(2) Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L)
(3) Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)
Flight Assignment/Project Notes: NOTE: Extended to 1/6/2020 per NSSC information (Ed., 2/12/19)

Task Description: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. To better understand the molecular mechanisms of HZE-induced carcinogenesis we will use a mouse model of HZE-induced hepatocellular carcinoma to study microenvironment changes after exposure to low level HZE. A comprehensive systems biology approach consisting of transcriptomics, lipidomics, proteomics, and metabolomics with novel data analysis will be used to build detailed biological pathways and identify molecular mechanisms that drive carcinogenesis. This work will further our understanding of risk at a mechanistic level and allow the development of new models for estimating human risk.

Research Impact/Earth Benefits: It is anticipated that there will be crosstalk between the molecular changes involved in HZE-induced hepatocellular carcinoma (HCC) and environmentally induced HCC seen on Earth. The Principal Investigator (PI) is actively collaborating with ground-based clinical researchers in HCC research. The computational methods being developed to analyze the vast omic data sets has the potential to revolutionize omic analyses.

Task Progress & Bibliography Information FY2020 
Task Progress: Introduction/Background

A primary concern for astronauts during deep space travels is their exposure to ionizing radiation. Galactic Cosmic Rays are primarily composed of protons (85%), helium (14%), and high charge-high energy ions (HZE) such as 56Fe, 28Si, and 16O. Earth bound humans are not normally exposed to these ions due to the shielding of Earth’s magnetosphere, but outside of this protection astronauts will be exposed to HZE. Exposure to HZE is a major risk factor for astronauts due to the possibility of HZE induced cancer as well as other potential health risks, i.e., cardiovascular and cognitive effects. This raises the question of what are the risks that will be posed to the astronauts during deep space travel such as a mission to an asteroid or to Mars and back?

HRP Relevancy

Risks: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. These risks could be compounded with other flight factors such as: microgravity, dehydration, stress, nutrition, microbial contamination, and other factors.

Gaps: This research focused on three radiation carcinogenesis gaps (Ed. note August 2020--gaps have since changed for this project to Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L); Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L); Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)).

Cancer 1: How can experimental models of tumor development for the major tissues (lung, colon, stomach, breast, liver, and leukemia’s) be developed to represent the major processes in radiation carcinogenesis and extrapolated to human risk projections?

Cancer 3: How can models of cancer risk be applied to reduce the uncertainties in radiation quality effects from SPEs and GCR?

Cancer 7: How can systems biology approaches be used to integrate research on the molecular, cellular, and tissue mechanisms of radiation damage to improve the prediction of the risk of cancer?

Final Study Hypothesis: Since indirect effects are likely a major mechanism of induction of HCC, an integrated omics approach will allow monitoring of the changes in the cellular microenvironment (lipids, mRNA and protein translation/modification) overtime resulting from low dose HZE irradiation.

Microenvironmental alterations will be used to identify: • Risk factors ; • Biomarkers for early detection ; • Mitigation targets

Specific Aims have been condensed for this final report:

1) Develop and implement an integrated omics platform utilizing transcriptomics, proteomics, lipidomics and metabolomics to identify microenvironmental-bystander responses to low dose HZE exposure in mice.

2) The integrated omics data sets will be used to predict alterations in biological/biochemical pathways induced by low dose HZE exposure. Biochemical assays will also be used as validation for the effected pathways.

3) Predictions will be made as to the risk involved for human astronauts during deep space travel who will be exposed to similar doses of HZE and potential countermeasures to combat the HZE induced changes will be presented.

Justification for a Multi-Omics Study of HZE induced HCC: Cellular signaling is known to be a very complex and eloquent process in which many levels of regulation are orchestrated to elicit the proper response (e.g., proliferation, differentiation, migration, survival). DNA contains the instructions that make these processes possible, which are transcribed into RNA to relay the message that is ultimately translated into proteins. Proteins can also be further regulated by post translational modifications. Proteins then go on to play key roles in cellular regulation. Although lipids and metabolites are products of proteins (enzymes) regulated by DNA, the lipids and metabolites themselves are not encoded within the DNA making them an excellent target for microenvironmental change. With this much interconnectivity, it is easy to understand why a multi-omics platform would be beneficial in monitoring the effects of deep space radiation especially in relation to the formation and progression of cancer where dysfunction occurs on many levels. Thus, we applied an integrated omics approach to elucidate information on the interaction between DNA/RNA, proteins, lipids and metabolites induced by low-dose, high charge, high energy (HZE) ions.

Computational Analysis of Transcriptomic Data Sets

The final Specific Aim in the original proposal of this grant had a Computational Analysis component to augment the knowledge-based Ingenuity Pathways Analysis (IPA) of these large omics data sets. Computational mathematical analysis is much less biased than IPA, but there is often still some operator bias in the selection of certain cut-off values. Pure computational analysis will enable interactions of specific species within omics data (transcripts, proteins, lipids, etc.) to be determined mathematically and not rely on previous interpretation of literature to determine these interactions. Additionally, computational analysis has the potential to identify interactions between transcripts or proteins that are not identified in the literature and thus are “assigned as undetermined.” Undetermined transcripts which are related to a specific pathway can be exploited to discover novel proteins which could prove to be valuable therapeutic targets.

The results of our computational analysis are highly complementary to our full integrated-omics analysis. The beauty of the computational analysis is that it is totally non-biased and yet still supports the results of the IPA multi-omics analysis presented early in this report. The computational analysis also provides additional information on more transcripts involved in response to HZE radiation and correlates unidentified transcripts to pathways that would have never been assigned with a knowledge-based analysis algorithm.

Two novel computational algorithms were developed using the transcriptomic data generated from the HZE radiated mice and their controls from NNX15AD65G. Both projects have been published and the publications are listed below.

The citations for these publications are (See also Bibliography section below):

1. Anna M. Nia, Tianlong Chen, Brooke L. Barnette, Kamil Khanipov, Robert L. Ullrich, Suresh K. Bhavnani, Mark R. Emmett. Efficient Identification of Multiple Pathways: RNA-Seq Analysis of Livers from 56Fe Ion Irradiated Mice. BMC Bioinformatics, 2020, 21:118-129. DOI: 10.1186/s12859-020-3446-5.

2. Anna M. Nia, Kamil Khanipov, Brooke L. Barnette, Robert L. Ullrich, George Golovko, Mark R. Emmett. Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of 56Fe, 16O, and 28Si irradiation on the induction of murine hepatocellular carcinoma. BMC Genomics, (2020) 21:453. DOI:10.1186/s12864-020-06869-4.

Final Conclusions of Integrated Omics Approach to Define the Molecular Mechanisms of Low Dose, High Charge, High Energy Irradiation in Liver

• Mitochondria are drastically affected by HZE irradiation in particularly 16O and 56Fe irradiation in C57 wild type mice.

• Mitochondria effects are exhibited in all omics datasets (transcriptomics, proteomics, lipidomics, & metabolomics) and each omics data set supports findings in the other data sets.

• Mitochondria dysfunction was also validated through biochemical assays (Complex I inhibition).

• Computational analysis independently identified mitochondrial dysfunction as a major affected pathway after low dose HZE exposure.

• Activation of Immunological Pathways is the second most affected group of pathways after exposure to low dose HZE. Computational analysis identified immunological pathways as the primary affected pathway after low dose HZE exposure.

• Mitochondria effects by HZE irradiation are novel, real, and important to the health and safety of the astronauts. Not only in liver, but these effects could also be very detrimental in brain and cardiac tissue that have high levels of mitochondria. Mitochondrial dysfunction is most likely the root cause of HZE induced cardiomyopathy and cognitive dysfunction.

• HZE induces mitochondrial effects that are of great risk for deep space flight missions. The HZE effects will be additive to conditions that are already known from low orbit studies in mice. Recent reports discuss effects seen in rodents which travelled on the Space Shuttle and on the International Space Station (ISS). After exposure to microgravity, stress, low dose/low dose rate radiation, high levels of bacterial contamination, dehydration, etc., the authors noted disruption of lipid metabolism in liver and hypothesized that mitochondrial dysfunction could be causing the detrimental effects seen in the rodents (Laiakis, E., et al., [#20337] in NASA Human Research Program Investigators’ Workshop (2020); Blaber, E., et al., Int. J. Mol. Sci, 18, 2062 (2017), doi: 10.3390/ijms18102062; and Beheshti, A., et al., Scientific Reports, 9, 19195, (2019) doi:10.1038/s41598-019-55869-2)). These low orbit biological effects will be compounded when low does HZE is added during a deep space mission.

• Premature aging may be one of the major contributing factors of HZE irradiation that results in mitochondria dysfunction which occurs earlier with HZE irradiation as compared to gamma irradiation

Future Directions

Studies That Will Require New Animal Experiments: • Delve deeper into mitochondria and immunological effects induced by HZE; • Higher n# of mice and focus on “wild type” animals; • Earlier time point (ex. Day or week post-irradiation) to better understand early ROS and drivers of early onset immune pathways; • Later time point (18 month, i.e. tumor development); • Mitochondria respiration via seahorse system (measures oxygen consumption rate and can quantify multiple parameters of mitochondria respiration); • Mitochondria specific transcriptomic, proteomics (mitochondrial formyl-peptides that drive the cytokine storm) and lipidomics; • Mitochondrial effects in other tissues: brain, cardiac; • PET scan system to monitor for potential tumor growth in animals prior to sacrifice; • Test proposed countermeasure compounds. Proposed mitochondria and inflammation centered therapeutics and supplements will be tested in vivo and specific mitochondrial, immunological, and metabolic endpoints will be monitored to determine their effectiveness at alleviating effects induced by exposure to simulated GCR; • Correlate data with cognitive behavioral studies Studies That Can Be Performed on Banked Samples; • Delve deeper into mitochondria effects induced by HZE; • Comprehensive analysis of changes in the non-polar lipid fraction (banked).

Of immediate interest: plasmologens (endogenous antioxidants) and resolvins (tightly linked with inflammation, promote normal cellular function after inflammation); • Mitochondria specific transcriptomics, proteomics (mitochondrial formyl-peptides that drive the cytokine storm), and lipidomics; • Mitochondrial effects in other tissues: brain, cardiac…(banked); • Correlate observed mitochondrial dysfunction (in liver) with cognitive changes induced by HZE (as measured by chemical LTP studies); • Further mine data at hand (enhanced computational analysis).

Proposed Countermeasures and Biomarkers: The novel countermeasures (one FDA approved drug, one in clinical trials, and four approved dietary supplements) have been identified based on the multi-omic data sets and biochemical pathway identifications based on and supported by the multi-omic data sets. The identified countermeasures are primarily targeted at improving mitochondrial function and reducing inflammatory pathways. Additionally, two potential lipid biomarkers have been identified.

Bibliography Type: Description: (Last Updated: 04/10/2021) 

Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Nia AM, Chen T, Barnette BL, Khanipov K, Ullrich RL, Bhavnani SK, Emmett MR. "Efficient identification of multiple pathways: RNA-Seq analysis of livers from 56Fe ion irradiated mice." BMC Bioinformatics. 2020 Mar 20;21(1):118. https://doi.org/10.1186/s12859-020-3446-5 ; PMID: 32192433; PMCID: PMC7082965 , Mar-2020
Articles in Peer-reviewed Journals Nia AM, Khanipov K, Barnette BL, Ullrich RL, Golovko G, Emmett MR. "Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of 56Fe, 16O, and 28Si irradiation on the induction of murine hepatocellular carcinoma." BMC Genomics. 2020 Jul 1;21(1):453. https://doi.org/10.1186/s12864-020-06869-4 ; PMID: 32611366; PMCID: PMC7329445 , Jul-2020
Articles in Peer-reviewed Journals Nia AM, Shavkunov A, Ullrich RL, Emmett MR. "137Cs γ ray and 28Si irradiation induced murine hepatocellular carcinoma lipid changes in liver assessed by MALDI-MSI combined with spatial shrunken centroid clustering algorithm: A pilot study." ACS Omega. 2020 Oct 6;5(39):25164-74. https://doi.org/10.1021/acsomega.0c03047 ; PMID: 33043195; PMCID: PMC7542585 , Oct-2020
Articles in Peer-reviewed Journals Laiakis EC, Shuryak I, Deziel A, Wang YW, Barnette BL, Yu Y, Ullrich RL, Fornace AJ Jr, Emmett MR. "Effects of low dose space radiation exposures on the splenic metabolome." Int J Mol Sci. 2021 Mar 17;22(6):3070. https://doi.org/10.3390/ijms22063070 ; PMID: 33802822; PMCID: PMC8002539 , Mar-2021
Dissertations and Theses Nia A. "Efficient Identification and Comprehension of Molecular Pathways Associated with Irradiation Induced Hepatic Carcinogenesis." Dissertation, The University of Texas Medical Branch, March 2020. , Mar-2020
Dissertations and Theses Barnette BL. "An Integrated Omics Approach to Define the Molecular Mechanisms of Low Dose, High Charge, High Energy (HZE) Irradiation in Liver." Dissertation,The University of Texas Medical Branch, July 30, 2020. , Jul-2020
Project Title:  Induction of Hepatocellular Carcinoma by Space Radiation: A Systems Biology Study of Causative Mechanisms Reduce
Images: icon  Fiscal Year: FY 2019 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 01/07/2015  
End Date: 01/06/2020  
Task Last Updated: 11/15/2018 
Download report in PDF pdf
Principal Investigator/Affiliation:   Emmett, Mark  Ph.D. / The University of Texas Medical Branch 
Address:  UTMB Cancer Research Center 
301 University Blvd, Rt. 1074 
Galveston , TX 77555-5302 
Email: mremmett@utmb.edu 
Phone: 409-747-1943  
Congressional District: 14 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Medical Branch 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ullrich, Robert  Ph.D. University of Texas Medical Branch 
Key Personnel Changes / Previous PI: November 2017 Report: Dr. Cheryl Lichti left UTMB (University of Texas Medical Branch) to take a position at Washington University, St. Louis. She is still a collaborator on the project, but is no longer a Co-Investigator and is not receiving salary support since 8/31/17. Ana Nia (MD/Ph.D. Graduate student, joined the project October 2017. November 2016 report: Dr. Joseph Moskal (Northwestern University) is no longer affiliated with academia nor involved with this project and is being removed as Co-I on the project. November 2015 report: Dr. Carol L. Nilsson (Co-I, 10% Effort) is no longer involved with the project. Dr. Cheryl F. Lichti has replaced Dr. Nilsson at 20% Effort. Two advanced graduate students, Brooke L. Barnette and Shinji K. Strain, will replace the TBA senior scientist (50% Effort).
Project Information: Grant/Contract No. NNX15AD65G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2013-14 HERO NNJ13ZSA002N-RADIATION 
Grant/Contract No.: NNX15AD65G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Cancer:Risk of Radiation Carcinogenesis
Human Research Program Gaps: (1) Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L)
(2) Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L)
(3) Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)
Flight Assignment/Project Notes: NOTE: Extended to 1/6/2020 per NSSC information (Ed., 2/12/19)

Task Description: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. To better understand the molecular mechanisms of HZE-induced carcinogenesis we will use a mouse model of HZE-induced hepatocellular carcinoma to study microenvironment changes after exposure to low level HZE. A comprehensive systems biology approach consisting of transcriptomics, lipidomics, proteomics, and metabolomics with novel data analysis will be used to build detailed biological pathways and identify molecular mechanisms that drive carcinogenesis. This work will further our understanding of risk at a mechanistic level and allow the development of new models for estimating human risk.

Research Impact/Earth Benefits: It is anticipated that there will be crosstalk between the molecular changes involved in HZE-induced hepatocellular carcinoma (HCC) and environmentally induced HCC seen on Earth. The Principal Investigator (PI) is actively collaborating with ground-based clinical researchers in HCC research. The computational methods being developed to analyze the vast omic data sets has the potential to revolutionize omic analyses.

Task Progress & Bibliography Information FY2019 
Task Progress: Year 4 Results: Project Setbacks/Modifications.

Over the past four years, several challenges have arisen that have necessitated alternative research paths to be devised. Year 4 was no exception. The PI and Co-I have worked together to circumvent these unforeseen occurrences and believe that the research and data has benefited from these changes although these set-backs have slowed progress on the project.

4th Year Specific Aims Progress.

Specific Aim 2. Determine transcriptional changes in the hepatic microenvironment of HZE- and gamma-irradiated samples, compared to controls.

RNA sequencing (UTMB sequencing core) has been completed on all samples. Transcriptomic reads were aligned to the mouse genome and Star software was used to determine expression levels. Approximately 56,000 expressed genes have been identified in each sample. Data analysis has been done to calculate the statistical significance for each gene and to determine differentially expressed transcripts in response to HZE. The amount of data obtained from the low-read RNA sequencing is staggering. There are many methods to identify significantly altered transcripts and pathways involved in each treatment group. Our group has been working with both traditional statistical R package (EdgeR) software and Ingenuity Pathway Analysis (IPA) and devising new computational methods. The traditional methods data will be presented below in Specific Aim 3. The new computational approaches results will be presented in Specific Aim 4.

Specific Aim 3. Determine comprehensive ultra high-resolution lipidomic alterations as well as high-resolution targeted proteomic microenvironment changes in hepatic tissue from tissue punches of HZE- and 137Cs gamma ray-irradiated animals as well as non-irradiated controls. Proteomic/Transcriptomic Results: Proteomic data was collected in the UTMB mass spectrometry core using Data Independent Analysis (DIA) on a Sciex 5600 Triple-TOF MS. DIA comprehensively and repeatedly samples every peptide within a protein digest (even low abundant peptides). DIA accommodates targeted data mining. If a new protein becomes of interest in the future the data can be re-mined without requiring additional analysis by MS. Ingenuity Pathway Analysis (IPA): IPA analysis predicts biological pathways that are affected by the differential expression of transcripts and proteins. Both transcriptomic and proteomic data was used to predict biological pathway changes with IPA in response to irradiation with gamma and HZE ions as compared to non-irradiated control. Pathways were identified for each time point that are key in describing the molecular mechanisms of the effects of HZE irradiations.

Specific Aim 4. Correlate large ‘omic datasets by use of Ingenuity Pathways’ Knowledge based software and unique algorithms developed by our collaborators to construct biological pathways that elucidate molecular mechanisms of HCC carcinogenesis induced by HZE irradiation.

After several different filtering steps, analysis is performed on a more manageable list of genes/transcripts, and different combinations of pairwise comparisons are used to identify transcripts that are significantly affected by different treatments. Multilevel modeling has been rarely implemented in the context of transcriptomic and lipidomic data. One focus is to use multilevel modeling on individual genes that have shown a significantly different behavior across different experimental parameters. Our goal is to identify a list of genes (using different computational approaches) and then perform a multilevel analysis. Multilevel models can allow for dynamic analysis of all data points, regardless of their behavior patterns. We believe this will provide further insight into future gene expression data based on different parameters. We hope to be able to extend the same type of analysis to the lipidomic data sets as well. These analyses are unique in that they are purely mathematically based and thus are not influenced by any external bias. The two key computational methodologies being used are: Self Organizing Maps (SOM) and Modularity Analysis.

Presentations:

During this year, there were three presentations on data from this work.

1) 29th Annual NASA Human Research Program Investigators’ Workshop, January 22-25, 2018, Galveston, TX, Brooke L. Barnette*, Anna M. Nia, Shinji K. Strain, Cheryl F. Lichti, Yongjia Yu, Robert L. Ullrich, and Mark R. Emmett, An Integrated Omics Approach to Define the Molecular Mechanisms of Hepatocellular Carcinoma (HCC) Induced by Low Dose, High-Energy, High charge Ions (HZE). *Oral presentation by my graduate student Brooke L. Barnette

2) 66th Annual American Society for Mass Spectrometry Conference (ASMS), June 3-7, 2018. San Diego, CA. Brooke L. Barnette, Anna M. Nia, Shinji K. Strain, Cheryl F. Lichti, Yu Yongjia, Robert Ullrich, Mark R. Emmett, An Integrated Omics Approach to Define the Molecular Mechanisms of Galactic Cosmic Ray Induced Hepatocellular Carcinoma. Poster Presentation.

3) 66th Annual American Society for Mass Spectrometry Conference (ASMS), June 3-7, 2018. San Diego, CA. Anna M. Nia, Brooke L. Barnette, Shinji K. Strain, Cheryl F. Lichti, Yu Yongjia, Robert Ullrich, Mark R. Emmett, Computational Mathematics Assimilation of Large Multi-Omics Datasets. Poster Presentation.

Publications:

1) Identification of Multiple Pathways: RNA-Seq Analysis of Livers from 56Fe Ion Irradiated Mice, Anna M. Nia, Tianlong Chen, Brooke L. Barnette, Robert L. Ullrich, Mark R. Emmett, and Suresh K. Bhavnani, to be submitted to BMC Bioinformatics, November 2018.

Summary of Project Status:

Despite several major setbacks, the massive multi-omic data sets are now producing informative data. The first of many manuscripts will be submitted this month. The advances in the computation reduction methods is exciting and the PI expects the Modularity based analysis to be a leap forward in multi-omics analysis. The PI and Co-I are equally excited about the prospects of identifying specific countermeasure therapeutic targets based on the multi-omics pathway analysis coupled with specific lipid/protein targeting. Additional data analysis and metabolite assays would further validate these targets.

Bibliography Type: Description: (Last Updated: 04/10/2021) 

Show Cumulative Bibliography Listing
 
Articles in Other Journals or Periodicals Nia AM, Chen T, Barnette BL, Ullrich RL, Emmett MR, Bhavnani SK. "Identification of Multiple Pathways: RNA-Seq Analysis of Livers from 56Fe Ion Irradiated Mice." Submitted to BMC Bioinformatics, November 2018 , Nov-2018
Project Title:  Induction of Hepatocellular Carcinoma by Space Radiation: A Systems Biology Study of Causative Mechanisms Reduce
Images: icon  Fiscal Year: FY 2018 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 01/07/2015  
End Date: 01/06/2019  
Task Last Updated: 11/09/2017 
Download report in PDF pdf
Principal Investigator/Affiliation:   Emmett, Mark  Ph.D. / The University of Texas Medical Branch 
Address:  UTMB Cancer Research Center 
301 University Blvd, Rt. 1074 
Galveston , TX 77555-5302 
Email: mremmett@utmb.edu 
Phone: 409-747-1943  
Congressional District: 14 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Medical Branch 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Meyer-Baese, Anke  Ph.D. Florida State University 
Ullrich, Robert  Ph.D. University of Texas Medical Branch 
Key Personnel Changes / Previous PI: November 2017 Report: Dr. Cheryl Lichti left UTMB (University of Texas Medical Branch) to take a position at Washington University, St. Louis. She is still a collaborator on the project, but is no longer a Co-Investigator and is not receiving salary support since 8/31/17. Ana Nia (MD/Ph.D. Graduate student, joined the project October 2017. November 2016 report: Dr. Joseph Moskal (Northwestern University) is no longer affiliated with academia nor involved with this project and is being removed as Co-I on the project. November 2015 report: Dr. Carol L. Nilsson (Co-I, 10% Effort) is no longer involved with the project. Dr. Cheryl F. Lichti has replaced Dr. Nilsson at 20% Effort. Two advanced graduate students, Brooke L. Barnette and Shinji K. Strain, will replace the TBA senior scientist (50% Effort).
Project Information: Grant/Contract No. NNX15AD65G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2013-14 HERO NNJ13ZSA002N-RADIATION 
Grant/Contract No.: NNX15AD65G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Cancer:Risk of Radiation Carcinogenesis
Human Research Program Gaps: (1) Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L)
(2) Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L)
(3) Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)
Task Description: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. To better understand the molecular mechanisms of HZE induced carcinogenesis we will use a mouse model of HZE-induced hepatocellular carcinoma to study microenvironment changes after exposure to low level HZE. A comprehensive systems biology approach consisting of transcriptomics, lipidomics, proteomics, and metabolomics with novel data analysis will be used to build detailed biological pathways and identify molecular mechanisms that drive carcinogenesis. This work will further our understanding of risk at a mechanistic level and allow the development of new models for estimating human risk.

Research Impact/Earth Benefits: It is anticipated that there will be crosstalk between the molecular changes involved in HZE induced hepatocellular carcinoma (HCC) and environmentally induced HCC seen on Earth. The Principal Investigator (P.I.) is actively collaborating with ground based clinical researchers in HCC research.

Task Progress & Bibliography Information FY2018 
Task Progress: Year 3 Results: Project Setbacks/Modifications: Over the past three years, several challenges have arisen that have necessitated alternative research paths to be devised. The P.I. and Co-P.I. have worked together to circumvent these unforeseen occurrences and believe that the research and data has actually benefitted from these changes. The changes will be listed in the results presented below as a “Note.”

Results Synopsis: Tissues from all time points (30, 60, 120, 270, & 360) for all groups [600 MeV/n 56Fe ions (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) and 137Cs gamma rays (1 and 3 Gy)] have been processed for all omics platforms. Comprehensive Lipid Analysis by FT-ICR LC-MS/MS has been conducted for all lipid extracts and data collected is being analyzed. RNA sequencing was performed by the UTMB sequencing core and the raw data has been received. We are actively analyzing the data to determine statistical significance for each gene. Samples have been processed (extracted) for targeted proteomic analysis, and data acquisition started last week.

Note: Due to an almost month long power outage in the Medical Research Building at UTMB where our animal tissue collection is performed (main buss bar failure), the 240 day time point had to be postponed by 30 days, thus moving the fourth time point from 240 days to 270.

Third Year Specific Aims Progress.

Specific Aim 1. Determine the microenvironmental changes in hepatic lipids by MALDI-IMS after HZE-irradiation with 600 MeV/n 56Fe ions (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) and 137Cs gamma rays (1 and 3 Gy).

Note: Due to the departure of a collaborator from UTMB, the P.I. lost access to the MALDI-IMS instrument that was to be used to collect the imaging data. The PI and Co-PI have agreed that the comprehensive lipid analysis by ultra, high-resolution FT-ICR MS supersedes the low resolution MALDI-IMS. The importance of the MALDI-IMS is primarily for monitoring the lipid microenvironment around a forming tumor. The few tumors seen in these animals were not discovered until the point of tissue harvest, thus the value of the MALDI-IMS has been deemed less important than the comprehensive lipid analysis by FT-ICR MS. In retrospect, the MALDI-IMS should be paired with PET scanning (or other available in vivo imaging technique) of each animal to allow monitoring of tumor formation over time.

Specific Aim 2. Determine transcriptional changes in the hepatic microenvironment of HZE- and gamma-irradiated samples, compared to controls. RNA was isolated from left lobe liver tissue samples from all time points and all treatment groups and submitted to the UTMB Next Generation sequencing core for low-read RNA sequencing. All samples have been analyzed and raw data has been received from the core. Approximately 50,000 expressed genes have been identified in each sample. The amount of data obtained from the low-read RNA sequencing is staggering. Analysis is underway to determine significantly altered genes and pathways involved in each treatment group. (See Specific Aim 4).

Note: The P.I. transitioned to collecting transcriptomic data with low-read RNA sequencing. The collaborators at Northwestern University, Evanston, IL, who were to provide the targeted transcriptomic gene array analysis withdrew from academic research and thus from this research project. Low read RNA sequencing provides much more transcriptomic data than could have been obtained by targeted transcriptomic analysis as originally proposed. Although RNA sequencing was not in the budget for this project, the P.I. had to find a replacement for the targeted transcriptomics after the collaborators withdrew. To cover the costs of the low-read RNA sequencing, the P.I. committed non-NASA funds along with other re-budgeting to obtain the transcriptomic data for this NASA project.

Preliminary Transcriptomic Data: Transcriptomic analysis was performed on RNA extracted from two 40 micron left lobe liver slices which were sliced on a cryotome at -20°C. Isolated RNA was sequenced with an Illumina HiSeq 1500 Analyzer in the UTMB Molecular Genomics Core Facility. Transcriptomic reads were aligned to the mouse genome and Star software was used to determine expression levels. Principle component analysis (PCA) plots were used to visualize the initial data.

Specific Aim 3. Determine comprehensive ultra high-resolution lipidomic alterations as well as high-resolution targeted proteomic microenvironment changes in hepatic tissue from tissue punches of HZE- and 137Cs gamma ray-irradiated animals as well as non-irradiated controls. All samples have been processed for both lipidomic and proteomic analysis. FT-ICR LC-MS/MS spectra have been collected for all lipid samples and data analysis of all the files is currently underway. The proteomic analysis started last week and should be completed by December 2017.

Note: The ultra, high-resolution 12T FT-ICR MS system used to collect the lipidomic and targeted proteomic data was down for over six months during the last year (December 2016-May 2017). The loss of this crucial instrument during this time necessitated that the P.I. devise alternative analysis strategies to complete the analysis outlined in the proposal. The targeted proteomics analysis has been moved to a Sciex 5600 triple-TOF instrument in the UTMB proteomics core. For our targeted proteomics analysis, we will be using a novel application of SWATH data independent acquisition. Preliminary Lipidomic Data: Preliminary lipid data interpretation based on the distribution of lipid classes in each treatment show that the lipid classes identified are dependent upon radiation type. This was true for both mice strains. Chi Squared tests were then used to look at the frequency of the different lipid classes compared to non-irradiated control group of the respective strain. At a P value of .05 frequencies for 56Fe, 3Gy gamma, & 16O were all significantly different compared to the control for C3H strain of mice. For the C57 mice, the frequencies of control lipid classes compared to 1Gy & 3 Gy gamma irradiation were not significantly different, whereas they were for the 56Fe, 16O, and 28Si irradiated animals. Progress on Targeted Proteomic Data: Since RNA sequencing has been completed we are moving toward the targeted proteomic analysis of the samples. All samples have been processed for proteomic analysis and the first LC-MS/MS analysis began last week.

Specific Aim 4. Correlate large ‘omic datasets by use of Ingenuity Pathways’ Knowledge based software and unique algorithms developed by our collaborators to construct biological pathways that elucidate molecular mechanisms of HCC carcinogenesis induced by HZE irradiation. We have continued to evaluate different software packages for data analysis regimes of lipid identification and quantification which greatly surpass the manual lipid data analysis. We have determined that the peak picking algorithm used within Elements does not perform well enough with our data files, and has trouble distinguishing between the isotopic distributions from isobaric lipids within the same family. We have also experimented with the program mzMine, and determined that while the program seemed promising, it could not handle the size of our data files. Our group is also in contact with developers of new lipid analysis software and is in the process of gaining access to the software for interfacing with our in-house developed lipid database. We have also been working toward writing our own script for lipid data analysis.

We have currently collected transcriptomic and lipidomic data. Anna Nia (M.D.-Ph.D. student) joined the PI’s group in October 2017. Miss Nia is a computational biologist and has started working on the large data set analysis of both the lipidomic and transcriptomic data sets. After several different filtering steps, analysis will be performed on a more manageable list of genes/transcripts, and different combinations of pairwise comparison will be used to identify transcripts that are significantly affected by different treatments. Multilevel modeling has been rarely implemented in the context of transcriptomic and lipidomic data. Our focus in this type of modeling would be on individual genes that have shown a significantly different behavior across different experimental parameters. Specifically, Miss Nia is working to identify a list of genes (using different computational approaches) and then perform a multilevel analysis. Multilevel models can allow for dynamic analysis of all data points, regardless of their behavior patterns. We believe this will provide further insight into future gene expression data based on different parameters. We hope to be able to extend the same type of analysis to the lipidomic data set as well. These analysis are unique in that they are purely mathematically based and thus are not influenced by any external bias.

Another computational Ph.D. student, John Miller, is currently rotating through the P.I.’s laboratory. Mr. Miller is pursuing a more traditional approach to analysis of the large transcriptomic data sets. The analysis of transcriptomic data will be done using a statistical R package (EdgeR). EdgeR is one of the most commonly used packages for differential gene expression analysis. EdgeR enables the comparison of the variation in the read counts from the different RNA-seq sample groups to determine which genes are differential expressed (genes that have a log fold change = 2, and an FDR value < 0.10). Using these parameters we will be able to analyze the differential gene expression through individual group analysis (i.e., comparison of individual groups), multi-group analysis (i.e., comparison of a treatment group across all time points), and whole-system analysis (i.e., comparison of the entire dataset). The three levels of analysis will allow for maximum data collection from the dataset given. Our goal is to determine how the changes in mRNA levels are affected based on the time, treatment, and strain of the sample groups used in the experiment. Finally, once a list of significantly altered genes for the model(s) has been determined we will import the list into IPA along with other data sets to determine affected pathways.

Presentations: During this year, preliminary data from this work was presented at the 65th ASMS Conference in Indianapolis, Indiana, in a poster entitled “Systems Biology Approach to Define the Molecular Mechanisms of Galactic Cosmic Ray Induced Hepatocellular Carcinoma.” Data was also presented at 28th Annual NASA Human Research Program Investigators’ Workshop Integrated Pathways to Mars in Galveston, TX, and will be presented at the 29th Annual NASA Human Research Program Investigators’ Workshop in January 2018. Finally, it is anticipated that the first publications from this work will be submitted during 2018.

Summary of Project Status:

Despite several major setbacks, the PI and Co-PI are confident that the project is on-track and on schedule. The final year of primarily large “omics” data set analysis are already in progress. The addition of a computational mathematician MD/Ph.D. student and our Florida State University collaborator (computational mathematician) are crucial to integrating these omics data sets into a full systems biological analysis of the effects of HZE irradiation on induction of hepatocellular carcinoma.

Bibliography Type: Description: (Last Updated: 04/10/2021) 

Show Cumulative Bibliography Listing
 
 None in FY 2018
Project Title:  Induction of Hepatocellular Carcinoma by Space Radiation: A Systems Biology Study of Causative Mechanisms Reduce
Images: icon  Fiscal Year: FY 2017 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 01/07/2015  
End Date: 01/06/2019  
Task Last Updated: 11/08/2016 
Download report in PDF pdf
Principal Investigator/Affiliation:   Emmett, Mark  Ph.D. / The University of Texas Medical Branch 
Address:  UTMB Cancer Research Center 
301 University Blvd, Rt. 1074 
Galveston , TX 77555-5302 
Email: mremmett@utmb.edu 
Phone: 409-747-1943  
Congressional District: 14 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Medical Branch 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Meyer-Baese, Anke  Ph.D. Florida State University 
Ullrich, Robert  Ph.D. University of Texas Medical Branch 
Lichti, Cheryl  Ph.D. University of Texas Medical Branch 
Key Personnel Changes / Previous PI: November 2016 report: Dr. Joseph Moskal (Northwestern University) is no longer affiliated with academia nor involved with this project and is being removed as Co-I on the project. November 2015 report: Dr. Carol L. Nilsson (Co-I, 10% Effort) is no longer involved with the project. Dr. Cheryl F. Lichti has replaced Dr. Nilsson at 20% Effort. Two advanced graduate students, Brooke L. Barnette and Shinji K. Strain, will replace the TBA senior scientist (50% Effort).
Project Information: Grant/Contract No. NNX15AD65G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2013-14 HERO NNJ13ZSA002N-RADIATION 
Grant/Contract No.: NNX15AD65G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Cancer:Risk of Radiation Carcinogenesis
Human Research Program Gaps: (1) Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L)
(2) Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L)
(3) Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)
Task Description: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. To better understand the molecular mechanisms of HZE induced carcinogenesis we will use a mouse model of HZE-induced hepatocellular carcinoma to study microenvironment changes after exposure to low level HZE. A comprehensive systems biology approach consisting of transcriptomics, lipidomics, proteomics, and metabolomics with novel data analysis will be used to build detailed biological pathways and identify molecular mechanisms that drive carcinogenesis. This work will further our understanding of risk at a mechanistic level and allow the development of new models for estimating human risk.

Research Impact/Earth Benefits: It is anticipated that there will be crosstalk between the molecular changes involved in HZE induced hepatocellular carcinoma (HCC) and environmentally induced HCC seen on Earth. The Principal Investigator (P.I.) is actively collaborating with ground based clinical researchers in HCC research.

Task Progress & Bibliography Information FY2017 
Task Progress: Year 2 Results: Tissues have been harvested from all time points (30, 60, 120, 270, & 360) for all groups (600 MeV/n 56Fe ions (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) and 137Cs gamma rays (1 and 3 Gy). Due to an almost month long power outage in the Medical Research Building at University of Texas Medical Branch (UTMB) where our animal sacrifice is performed (main buss bar failure), the 240 day time point had to be postponed by 30 days, thus moving the fourth time point from 240 days to 270. The final 360 day tissues were recently harvested on October 10, 11, and 17 of 2016. NOTE: The first liver tumors were observed in some of the 270 and 360 day, 3 Gy Gamma, and HZE irradiated mice. Based on previous work done by the Ullrich Group, it is expected that more tumors will develop with time, but there is no budget to extend the studies out another six months (total of 540 days post irradiation). The progress on the specific aims for this project are below.

Specific Aim 2. Determine transcriptional changes in the hepatic microenvironment of HZE- and gamma-irradiated samples, compared to controls. All tissue samples have been collected. Extraction of RNA for transcription analysis is underway. The P.I. has transitioned to collecting transcriptomic data from these samples with low read RNA sequencing. Low read RNA sequencing will provide much more transcriptomic data than could have been obtained by targeted transcriptomic analysis as originally proposed. The main drawback of RNA sequencing is the high cost of analysis. Although RNA sequencing was not in the budget for this project, the P.I. had to find a replacement for the targeted transcriptomics that was going to be performed in the laboratories of collaborators at Northwestern University in Evanston, IL. These collaborators started a small pharmaceutical company and withdrew from academic research.

RNA sequencing is a “cutting edge” technology, but is also an expensive technology. The RNA sequencing will be performed at the UTMB Molecular Genomics Core. The RNA sequencing originally was projected to come at no cost to the project, but due to cost increases there will be costs for these analysis which will require some re-budgeting by the P.I. The P.I.’s laboratory is working with the UTMB Molecular Genomics Core to determine the quality and quantity of RNA produced from small samples of liver tissue. Preliminary results demonstrated that the RNA was of high enough quality and quantity to perform low read RNA sequencing. To keep RNA sequencing costs to a minimum, all samples are being processed in the P.I.’s laboratory and will be analyzed in blocks to maximize the efficient use of the RNA Libraries with the Illumina HiSeq 1500 Analyzer in the UTMB Genomics Core Facility.

Because not all transcripts are translated into protein, this data will be validated by targeted proteomic studies. Pilot proteomic studies have verified that FULL proteomic data sets can be obtained from small samples of liver tissue.and thus, will provide adequate protein concentrations for our targeted proteomic analysis. Once the RNA sequencing studies have been completed, we are ready to proceed with the targeted proteomics (Targeted Proteomics is part of Specific Aim 3).

Specific Aim 3. Determine comprehensive ultra high-resolution lipidomic alterations as well as high-resolution targeted proteomic microenvironment changes in hepatic tissue from tissue punches of HZE- and 137Cs gamma ray-irradiated animals as well as non-irradiated controls The lipid extraction methodology has been optimized for small samples of liver tissue without having to thaw the sample multiple times. All sampling for lipidomics, transcriptomics, and proteomics are being collected by this method, which preserves sample integrity. This provides a more uniform representation of what is occurring on the molecular level across the whole tissue instead of being localized to a specific area, i.e., from a tissue punch. Lipid samples are homogenized in 155mM ammonium acetate which allows a total protein concentration can be determined using a Bradford protein assay before lipid extraction, which denatures the protein pellet. Determination of total protein concentration permits a uniform sample load for each sample and allows for consistent quantification. Lipid extractions have been conducted on the 30, 60, and 120 day time point and are currently underway for the 270 and 360 day time points. A data dependent method for automated collection of MS/MS spectra has been optimized using a combination of lipid standards such as GM1, GD1a, sphingosine-1-phosphate, 3-sn-phosphatidic acid sodium salt, etc., along with biological samples obtained from murine synaptosomes. MS/MS data was obtained for GM1 at 45.0V. Other standards such as GD1a also required 45.0V to produce adequate fragmentation, whereas lipids, such as 3-sn-phosphatidic acid sodium salt, only required 20.0V. The automated MS/MS with collision energies 45.0V and 20.0V has also been optimized with samples from biological samples (synaptosomes derived from mouse brain). Since these collision energies have been successful with both lipid standards and biological samples, they are currently being applied for the starting points for MS/MS analysis of the liver lipids in this project. MS/MS data for the first three time points of this study have been collected and data analysis is underway.

Specific Aim 4. Correlate large ‘omic datasets by use of Ingenuity Pathways’ Knowledge based software and unique algorithms developed by our collaborators to construct biological pathways that elucidate molecular mechanisms of HCC carcinogenesis induced by HZE irradiation. New data analysis regimes have continued to be evaluation and progress has been made on efficient lipid identification and quantification which greatly surpass the manual lipid data analysis and identifications used previously by the P.I. Dr. Cheryl Lichti and Brooke Barnette have continued to evaluate new software packages for lipidomic data analysis, and have been working closely with some of the developers. These software packages are primarily designed for peak picking and identification of small molecule metabolites not large lipid ions. Dr. Lichti has also adapted available proteomic software packages to aid in peak picking of the higher m/z lipids. Software packages currently being evaluated and modified are:

1) Bruker Daltonics Target Analysis. This software is proposed to be used with the P.I.’s lipid library (>25,000 entries). Lipid identifications will be made based on accurate mass and ms/ms fragmentation patterns.

2) Skyline (MacCoss Lab Software) is an open source program. This software is currently only able to import transition lists which will allow it to be used for quantification, but the species identification must be completed elsewhere. We are working with the developers to establish a library import feature to work with the P.I.’s lipid library for identification.

3) Elements for Metabolomics (Proteome Software). This software has the capability of searching the LipidMaps database which is the gold standard for lipid database searching. A drawback of this software is that there is relatively no control over chromatographic alignment. Our lab is working directly with the programmers of the software and the program now accepts raw .d data files. The programmers are also addressing issues with data size and these changes are to be included in the next updated release.

4) Progenesis QI (Nonlinear Dynamics, Waters Corporation). This software utilizes the raw .d format of the data files, and control of chromatographic alignment. This software can search LipidMaps for lipid identification. We have encountered problems with the peak picking algorithm in this software which does not find the high m/z lipids such as gangliosides. We are working with Nonlinear Dynamics to update their algorithms to correct this problem. In the interim, Dr. Lichti has cleverly used the Progenesis proteomics software to find these peaks in the data.

Discussion: Even with multiple setbacks, the P.I. and Co-I are confident that the project is on-track and on-schedule. The P.I.’s lab has made several proposal changes that were necessitated by loss of our transcriptomics collaborator which dictated the move to low read RNA sequencing, and a major power outage (lab was out of power for almost one month), which necessitated a change from 240 to 270 day time point. The transcriptomic change dictated that new methodology needed to be developed. The advent of, optimization of, and use of the small samples of liver tissue all sample processing (lipid, RNA, and protein) has greatly enhanced our flexibility and preserved sample integrity across all sample analysis platforms. The move to RNA sequencing also greatly increased costs of the crucial Aim 2 project, there will be costs for these analysis that will require re-budgeting. Even with this re-budgeting, the P.I. is confident that the project can stay within the original budget.

During this year, preliminary data from this work was presented at the 64th American Society for Mass Spectrometry Conference in San Antonio, TX in a poster entitled “The application of lipidomics to the study of Heptocellular Carcinoma (HCC) induced by low dose, high-energy, high-charge ions (HZE).” Data was also presented at 27th Annual NASA Human Research Program Investigators’ Workshop Integrated Pathways to Mars in Galveston, TX and will be presented at the 28th Annual NASA Human Research Program Investigators’ Workshop in January 2017. Finally, it is anticipated that the first publications from this work will be submitted during 2017.

Bibliography Type: Description: (Last Updated: 04/10/2021) 

Show Cumulative Bibliography Listing
 
 None in FY 2017
Project Title:  Induction of Hepatocellular Carcinoma by Space Radiation: A Systems Biology Study of Causative Mechanisms Reduce
Images: icon  Fiscal Year: FY 2016 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 01/07/2015  
End Date: 01/06/2019  
Task Last Updated: 11/06/2015 
Download report in PDF pdf
Principal Investigator/Affiliation:   Emmett, Mark  Ph.D. / The University of Texas Medical Branch 
Address:  UTMB Cancer Research Center 
301 University Blvd, Rt. 1074 
Galveston , TX 77555-5302 
Email: mremmett@utmb.edu 
Phone: 409-747-1943  
Congressional District: 14 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Medical Branch 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Meyer-Baese, Anke  Ph.D. Florida State University 
Moskal, Joseph  Ph.D. Falk Center for Molecular Therapeutics 
Ullrich, Robert  Ph.D. University of Texas Medical Branch 
Lichti, Cheryl Fae Ph.D. University of Texas Medical Branch 
Key Personnel Changes / Previous PI: November 2015 report: Dr. Carol L. Nilsson (Co-I, 10% Effort) is no longer involved with the project. Dr. Cheryl F. Lichti has replaced Dr. Nilsson at 20% Effort. Two advanced graduate students, Brooke L. Barnette and Shinji K. Strain, will replace the TBA senior scientist (50% Effort).
Project Information: Grant/Contract No. NNX15AD65G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2013-14 HERO NNJ13ZSA002N-RADIATION 
Grant/Contract No.: NNX15AD65G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Cancer:Risk of Radiation Carcinogenesis
Human Research Program Gaps: (1) Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L)
(2) Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L)
(3) Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)
Task Description: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. To better understand the molecular mechanisms of HZE induced carcinogenesis we will use a mouse model of HZE-induced hepatocellular carcinoma to study microenvironment changes after exposure to low level HZE. A comprehensive systems biology approach consisting of transcriptomics, lipidomics, proteomics, and metabolomics with novel data analysis will be used to build detailed biological pathways and identify molecular mechanisms that drive carcinogenesis. This work will further our understanding of risk at a mechanistic level and allow the development of new models for estimating human risk.

Research Impact/Earth Benefits:

Task Progress & Bibliography Information FY2016 
Task Progress: Although this grant has only been operational for approximately seven months, the Principal Investigator (P.I.) is happy to report significant progress. The official start date on the grant is listed as 01/06/2015, but funds did not arrive at University of Texas Medical Branch (UTMB) until ~03/22/2015. The P.I. had usable funds for this project starting approximately 04/01/2015. There have been major staffing changes to the project. The P.I. and Co-I (Dr. Robert L. Ullrich) are confident that these changes will enhance the project. In the resubmission of the proposal, the proteomics focus was shifted to targeted proteomics based on the transcriptomic data instead of broad based shotgun proteomics. The targeted proteomics can easily be performed with the P.I.’s ultra high-resolution 12T Fourier transform ion cyclotron mass spectrometer (FT-ICR MS). This change no longer warranted an expert in global proteomics. To enhance productivity, Dr. Cheryl F. Lichti has been added at 20% Effort. Dr. Lichti is highly qualified and has over 10 years of experience in proteomics, mass spectrometry instrumentation and is highly skilled in mass spectrometry data analysis. In the original budget, the P.I. had budgeted to bring in Dr. Lichti at 10% effort in years 3 and 4. The personnel change now allows Dr. Lichti to participate in the project from the beginning. Dr. Lichti is currently applying her expertise in proteomics data analysis to enhance the throughput and efficiency of the lipid analysis, which is a major advance over the manual identification used previously by the P.I.

Two graduate students have joined this project: Brooke L. Barnette (Ph.D. student) and Shinji K. Strain (MD/Ph.D. student). Both of these students are highly advanced. Ms. Barnette joined the P.I.’s lab May of 2015 with a Master’s Degree and a great deal of mass spectrometry experience. She already has the nano-LC chromatography for lipids interfaced to the 12T FT-ICR MS. She has optimized the lipid extraction from samples and moved to a much smaller sample size (20 micron tissue slices vs. 5 mm tissue punches) and has demonstrated reproducible, high sensitivity analysis of these samples on the 12T FT-ICR MS. She is also working with Dr. Lichti to develop enhanced data analysis of the global lipid profiling from the data that she is collecting on the 12T FT-ICR MS. Shinji K. Strain was in his third year of his Ph.D. in physics at Rice University (Houston) when he decided to pursue a degree in medicine. Mr. Strain came to UTMB’s MD/Ph.D. program and joined the P.I.’s lab in July 2015. Mr. Strain’s background in physics and instrumentation has allowed him to master the mass spectrometry instrumentation used in this project faster than any student the P.I. has ever encountered. His two years of medical school training has provided him with the biological/biochemical background to be a great asset in the interpretation of the biochemical mechanisms involved in the molecular changes in the irradiated vs. control samples in this project. The P.I. is FULLY confident that these two advanced graduate students can easily replace the TBA senior scientist (50% effort) in this project. They are both fully trained on the instrumentation and currently working on the project.

The P.I. travelled to the NASA Space Research Laboratory (NSRL) at Brookhaven National Laboratory (BNL) in October, 2015 and was able to irradiate all the mice for this project. Mice were irradiated with HZE-irradiation with 600 MeV/n 56Fe ions (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) and 137Cs gamma rays (1 and 3 Gy). These mice have been received at UTMB in good health from Brookhaven Laboratory Animal Facilities (BLAF) and the first samples will be taken for analysis beginning on 11/16/2015.

The progress on the specific aims for this project is:

Specific Aim 1. Determine the microenvironmental changes in hepatic lipids by MALDI-IMS after HZE-irradiation with 600 MeV/n 56Fe ions (0.2 Gy), 1 GeV/n 16O (0.2 Gy) and 350 MeV/n 28Si (0.2 Gy) and 137Cs gamma rays (1 and 3 Gy). All irradiations HZE and gamma on all mice have been completed. First time point sample collection will begin 11/16/2015.

Specific Aim 2. Determine transcriptional changes in the hepatic microenvironment of HZE- and gamma-irradiated samples, compared to controls. Transcriptional analysis will begin when enough samples have been collected to establish a full chip for efficient analysis.

Specific Aim 3. Determine comprehensive ultra high-resolution lipidomic alterations as well as high-resolution targeted proteomic microenvironment changes in hepatic tissue from tissue punches of HZE- and 137Cs gamma ray-irradiated animals as well as non-irradiated controls The lipid extraction methodology has been optimized. The nano-LC chromatographic separation has been optimized and interfaced to the 12T FT-ICR MS. Data collection has been demonstrated and reproducibility established with control samples. The assay for global analysis is fully functional and ready for application as soon as samples are produced (beginning 11/16/2015).

Specific Aim 4. Correlate large ‘omic datasets by use of Ingenuity Pathways’ Knowledge based software and unique algorithms developed by our collaborators to construct biological pathways that elucidate molecular mechanisms of HCC carcinogenesis induced by HZE irradiation. New data analysis regimes have been under evaluation and progress has been made on efficient lipid identification and quantification which greatly surpass previous methodology used by the P.I.

In summary, the project is on-track and on-schedule.

Bibliography Type: Description: (Last Updated: 04/10/2021) 

Show Cumulative Bibliography Listing
 
 None in FY 2016
Project Title:  Induction of Hepatocellular Carcinoma by Space Radiation: A Systems Biology Study of Causative Mechanisms Reduce
Images: icon  Fiscal Year: FY 2015 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 01/07/2015  
End Date: 01/06/2019  
Task Last Updated: 02/27/2015 
Download report in PDF pdf
Principal Investigator/Affiliation:   Emmett, Mark  Ph.D. / The University of Texas Medical Branch 
Address:  UTMB Cancer Research Center 
301 University Blvd, Rt. 1074 
Galveston , TX 77555-5302 
Email: mremmett@utmb.edu 
Phone: 409-747-1943  
Congressional District: 14 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Medical Branch 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Meyer-Baese, Anke  Ph.D. Florida State University 
Moskal, Joseph  Ph.D. Falk Center for Molecular Therapeutics 
Nilsson, Carol  M.D., Ph.D. University of Texas Medical Branch, Galveston 
Ullrich, Robert  Ph.D. University of Texas Medical Branch 
Project Information: Grant/Contract No. NNX15AD65G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2013-14 HERO NNJ13ZSA002N-RADIATION 
Grant/Contract No.: NNX15AD65G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:  
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Cancer:Risk of Radiation Carcinogenesis
Human Research Program Gaps: (1) Cancer-102:Determine the role of radiation quality on carcinogenesis and shared biology with other degenerative diseases (IRP Rev L)
(2) Cancer-302:Identify tissue-specific surrogate end-points for space radiation induced pre-malignancy and shared biology with other degenerative diseases (IRP Rev L)
(3) Cancer-401:Identify biomarkers for estimating individual susceptibility, risk assessment, and health monitoring (IRP Rev L)
Task Description: Exposure to high-energy heavy ions (HZE) during space travel is a health risk for astronauts. Even at low doses, exposure to HZE can lead to cancer. To better understand the molecular mechanisms of HZE induced carcinogenesis we will use a mouse model of HZE-induced hepatocellular carcinoma to study microenvironment changes after exposure to low level HZE. A comprehensive systems biology approach consisting of transcriptomics, lipidomics, proteomics, and metabolomics with novel data analysis will be used to build detailed biological pathways and identify molecular mechanisms that drive carcinogenesis. This work will further our understanding of risk at a mechanistic level and allow the development of new models for estimating human risk.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2015 
Task Progress: New project for FY2015.

Bibliography Type: Description: (Last Updated: 04/10/2021) 

Show Cumulative Bibliography Listing
 
 None in FY 2015