Medical Countermeasures for Radiation Induced Health Effects: Reports of an Interagency Panel Session Held at the NASA Human Research Program Investigator's Workshop, January 26, 2017

An Interagency Panel Session organized by the NASA Human Research Program Space Radiation Program Element (SRPE) was held during the NASA Human Research Program (HRP) Investigators Workshop (IWS) in Galveston, Texas on January 26, 2017 to identify complementary research areas that will advance the testing and development of medical countermeasures (MCM) in support of radioprotection and radiation mitigation on the ground and in space. There were several areas of common interest identified among the various participating agencies. This report provides a summary of the topics discussed by each agency along with potential areas of intersection for mutual collaboration opportunities. Common goals included repurposing of pharmaceuticals, neutraceuticals for use as radioprotectors and/or mitigators, low-dose/chronic exposure paradigms, late effects post-radiation exposure, mixed-field exposures of gamma-neutron, performance decrements, and methods to determine individual exposure levels

Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences

Correction: Taliaferro, L. et al. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection. Viruses 2014, 6, 1876-1896

We have noted some inaccuracies in Table 1 and Table 2 of our article “Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection” (Viruses 2014, 6, 1876–1896) [1].[...

Protein-Protein Interactions of Rob and SoxS and their role in stress defense response systems in Escherichia coli.

Escherichia coli Rob, SoxS and MarA are monomeric members of the AraC/XylS family of transcription activators, possessing two helix-turn-helix motifs that are used for DNA binding and transcription activation. SoxS and MarA are synthesized de novo in response to superoxide stress and salicylate. Rob is unique, being constitutively expressed at 5,000-10,000 molecules per cell, but maintained in an inactive form by being sequestered into 3-4 intracellular aggregates. Dipyridyl (DIP) and bile salts (DEC) induce Rob activity, by dispersing the aggregates into monomers that activate transcription of 40-50 genes that mediate the cell's defense response against the above stresses. Rob's activity is controlled by sequestration-dispersal (S-D), a new mechanism of induction where under normal conditions Rob resides in an inactive, sequestered state, primed to be dispersed in response to environmental stress. Our goal is to determine how Rob is sequestered and whether a cytoplasmic factor converts dispersed, active Rob into its inactive, sequestered form. In this study, Rob was tagged with Protein A or His₁₀ and protein complex immunoprecipitation was carried out to isolate complexes between Rob and the putative factor (Chapter IV). Potential protein partners of Rob were then identified by mass spectrometry. In addition, we conducted alanine-scanning mutagenesis of Rob's C-terminal domain (CTD), the portion of the protein shown previously to be necessary and sufficient for S-D (Chapter II). In previous work, several regions important in aggregation and induction were identified using quad alanine-scanning mutagenesis. In this work, single alanine mutants of these regions were made in order to identify the amino acids important to the formation or maintenance of the aggregates or to inducer binding. Several mutant phenotypes of Rob were observed: constitutive, uninducible, partially constitutive/inducible and super-aggregated/inducible. Furthermore, direct protein-protein interactions between Rob and RNAP were identified at class II promoters (Chapter V). Moreover, we determined Rob's orientation at class II promoters and demonstrated that Rob occludes σ⁷⁰ region 4 from binding the -35 hexamer at class II promoters (Chapter V). Finally, Rob was shown to form in vivo binary complexes with RNAP, which supports pre-recruitment as Rob's mechanism of transcription activation (Chapter III)

Protein-Protein Interactions of Rob and SoxS and their role in stress defense response systems in Escherichia coli.

Escherichia coli Rob, SoxS and MarA are monomeric members of the AraC/XylS family of transcription activators, possessing two helix-turn-helix motifs that are used for DNA binding and transcription activation. SoxS and MarA are synthesized de novo in response to superoxide stress and salicylate. Rob is unique, being constitutively expressed at 5,000-10,000 molecules per cell, but maintained in an inactive form by being sequestered into 3-4 intracellular aggregates. Dipyridyl (DIP) and bile salts (DEC) induce Rob activity, by dispersing the aggregates into monomers that activate transcription of 40-50 genes that mediate the cell's defense response against the above stresses. Rob's activity is controlled by sequestration-dispersal (S-D), a new mechanism of induction where under normal conditions Rob resides in an inactive, sequestered state, primed to be dispersed in response to environmental stress. Our goal is to determine how Rob is sequestered and whether a cytoplasmic factor converts dispersed, active Rob into its inactive, sequestered form. In this study, Rob was tagged with Protein A or His₁₀ and protein complex immunoprecipitation was carried out to isolate complexes between Rob and the putative factor (Chapter IV). Potential protein partners of Rob were then identified by mass spectrometry. In addition, we conducted alanine-scanning mutagenesis of Rob's C-terminal domain (CTD), the portion of the protein shown previously to be necessary and sufficient for S-D (Chapter II). In previous work, several regions important in aggregation and induction were identified using quad alanine-scanning mutagenesis. In this work, single alanine mutants of these regions were made in order to identify the amino acids important to the formation or maintenance of the aggregates or to inducer binding. Several mutant phenotypes of Rob were observed: constitutive, uninducible, partially constitutive/inducible and super-aggregated/inducible. Furthermore, direct protein-protein interactions between Rob and RNAP were identified at class II promoters (Chapter V). Moreover, we determined Rob's orientation at class II promoters and demonstrated that Rob occludes σ⁷⁰ region 4 from binding the -35 hexamer at class II promoters (Chapter V). Finally, Rob was shown to form in vivo binary complexes with RNAP, which supports pre-recruitment as Rob's mechanism of transcription activation (Chapter III)

A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection

ABSTRACT The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. IMPORTANCE Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms

Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences

Metabolomics in Radiation Biodosimetry: Current Approaches and Advances

Triage and medical intervention strategies for unanticipated exposure during a radiation incident benefit from the early, rapid and accurate assessment of dose level. Radiation exposure results in complex and persistent molecular and cellular responses that ultimately alter the levels of many biological markers, including the metabolomic phenotype. Metabolomics is an emerging field that promises the determination of radiation exposure by the qualitative and quantitative measurements of small molecules in a biological sample. This review highlights the current role of metabolomics in assessing radiation injury, as well as considerations for the diverse range of bioanalytical and sampling technologies that are being used to detect these changes. The authors also address the influence of the physiological status of an individual, the animal models studied, the technology and analysis employed in interrogating response to the radiation insult, and variables that factor into discovery and development of robust biomarker signatures. Furthermore, available databases for these studies have been reviewed, and existing regulatory guidance for metabolomics are discussed, with the ultimate goal of providing both context for this area of radiation research and the consideration of pathways for continued development