Human space exploration missions will continue the development of sustainable plant cultivation in what are obviously novel habitat settings. Effective pathology mitigation strategies are needed to cope with plant disease outbreaks in any space-based plant growth system. However, few technologies currently exist for space-based diagnosis of plant pathogens. Therefore, we developed a method of extracting plant nucleic acid that will facilitate the rapid diagnosis of plant diseases for future spaceflight applications. The microHomogenizer™ from Claremont BioSolutions, originally designed for bacterial and animal tissue samples, was evaluated for plant-microbial nucleic acid extractions. The microHomogenizer™ is an appealing device in that it provides automation and containment capabilities that would be required in spaceflight applications. Three different plant pathosystems were used to assess the versatility of the extraction process. Tomato, lettuce, and pepper plants were respectively inoculated with a fungal plant pathogen, an oomycete pathogen, and a plant viral pathogen. The microHomogenizer™, along with the developed protocols, proved to be an effective mechanism for producing DNA from all three pathosystems, in that PCR and sequencing of the resulting samples demonstrated clear DNA-based diagnoses. Thus, this investigation advances the efforts to automate nucleic acid extraction for future plant disease diagnosis in space.

Recent studies regarding the origin of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: The Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly-related to life on Earth indicating a shared-ancestry due to lithological exchange, or (2) is unrelated to life on Earth suggesting a convergent origin of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results equivalent to 1 ppb life-detection sensitivity achieved by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier.

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.

Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies.

Due to meteoritic transfer between Earth and Mars, if life on Mars exists, it may be related to life on Earth and utilize nucleic acids as informational polymers. Thus, a Search for Extra-Terrestrial Genomes (SETG) could detect and sequence (deoxy) ribonucleic acids (DNA/RNA) utilized by any extant or recently dead life on Mars. The abiotic synthesis of common organic building blocks, such as nucleobases, sugars, and amino acids, in the solar nebula and potentially in diverse habitable environments could also bias a second genesis of life towards utilizing informational polymers similar to life as we know it. Here we build on prior work and describe the advancement of a SETG instrument to technology readiness level 4 through sample-to-sequence processing with limited manual handling. Another advance includes validation of nucleic acid extraction from Mars analogs at cell counts down to 10(4) per 50 mg sample, equivalent to a limit of detection of approximately 1 part per billion. In addition, we demonstrate that biological nanopore-based single molecule sequencing can be used to detect non-standard bases. Finally, we link sequence data to a statistical test to distinguish between any forward contamination and putative life beyond Earth. Nanopore-based sensing may ultimately enable characterization of nonstandard polymers and other molecules, highlighting the potential for nanopore-based life detection and sequencing on Mars or other words such as the icy moons Enceladus or Europa.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths. There are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have less than a 7% 5-year survival rate and the median survival from the time of diagnosis is less than 12 months. Next-Generation Sequencing (NGS) together with in vitro/in vivo functional studies is likely to identify and validate new biomarkers of PDAC onset, progression and therapy resistance. Importantly, the substantial archives of formalin-fixed, paraffin-embedded (FFPE) samples from PDAC patients are likely to be a rich resource for linking molecular signatures to relevant clinical data. However, NGS methods on FFPE samples are severely hindered because extracting high-quality nucleic acid material from these samples is time-consuming and inefficient. We have sought to develop novel methods for improving the extraction of high-quality RNA from FFPE samples within the context of commercially-available FFPE RNA kits and protocols. Together with researchers and clinicians at Claremont BioSolutions, AntiCancer Inc. and UCSD our laboratory has developed a novel nucleic acid extraction method that significantly increases RNA yield and integrity from PDAC cell line and Patient-Derived Xenograft FFPE samples. By briefly (<5 minutes) integrating the newly designed Claremont BioSolutions’ microhomogenizer (mH) tool within the commercially available Qiagen FFPE RNA extraction protocol, RNA recovery from these samples is increased by approximately 3-fold while maintaining standard 260/280 ratios (2.03 +/- 0.02 w/ mH step) and high RNA Quality Index (RQI) values (7.3 +/- 0.6 w/ mH step). Bioanalyzer testing further demonstrated that the mH-purified FFPE RNA was longer. Previous studies have revealed that PDAC cell gene expression signatures vary significantly when cells are propagated in vitro versus in vivo as subcutaneous or orthotopic xenografts. Notably, we found that the previously published expression patterns for KRas dependency genes within these three microenvironments were most accurately reproduced when extracting PDAC FFPE RNA with our mH-based method. Finally, we used our mH-based method to test the effects of the in vivo tumor microenvironment (TME) on the expression trends of a panel of novel PDAC biomarkers. In this regard, we demonstrate that PEAK1 and MST1R expression levels are decreased and increased, respectively, by over 100 fold in the orthotopic microenvironment relative to the subcutaneous microenvironment. These results reveal the critical nature of the tumor microenvironment when evaluating the clinical relevance of new biomarkers in cell lines or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to positively impact the FFPE-RNA-NGS workflow for cancer biomarker identification/validation. Citation Format: Malachia Hoover, Yvess Adamian, Mark Brown, Ali A. Maawy, Robert Hoffman, Michael Bouvet, Robert Doebler, Jonathan A. Kelber. Analysis of microenvironment effects on pancreatic cancer biomarker expression using a novel method for FFPE RNA extraction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-263.

Widespread synthesis of complex organics, including nucleobases and ribose precursors, occurred early in the history of the solar system in the solar nebula. These organics, delivered to multiple potentially habitable zones, may have biased the evolution of life towards utilization of similar informational polymers. Meteoritic exchange might also have produced shared ancestry, most plausible for Earth and Mars. To test this hypothesis, we are developing the Search for Extra-Terrestrial Genomes (SETG), a life detection instrument for in-situ isolation and sequencing of nucleic acids. Our mission focus area is astrobiology and the search for life beyond Earth. Our science goal for Mars is to search for related or unrelated nucleic acid-based life, particularly life that has the potential to interact with life on Earth; this may also inform sample selection for Mars Sample Return (MSR) and reduce the risks of false positives through the first in-situ measurement of forward contamination. Our science goal for Enceladus is to search for a second genesis based on nucleic acids in the plumes emanating from the South Polar Region. Life detection may also be possible in Europa orbit but the availability of a suitable plume is tenuous and it is a challenge for biological reagents to survive intense radiation there. Here we describe advancements in SETG geared towards in-situ sequencing during a future Mars mission, including extraction of nucleic acids coupled with proof of principle for in-situ single-molecule nanopore-based sequencing. We briefly describe plans to advance SETG from Technology Readiness Level 3 to 6 in preparation for future flight definition and show that under realistic assumptions, a sensitivity of parts per billion or better is feasible.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths. Because there are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have a dismal prognosis. Substantial archives of formalin‐fixed, paraffin‐embedded (FFPE) samples from PDAC tissue have been established and linked to clinical data. However, identifying biomarkers from these tumors is severely hindered because extracting high‐quality nucleic acid material from FFPE samples is time‐consuming and inefficient. Thus, developing improved methods for extracting high‐quality RNA/DNA from FFPE samples represents an unmet need in the fields of cancer biology and Next‐Generation Sequencing (NGS). We have tested Claremont BioSolutions’ microhomogenization (mH) tools in combination with commercial FFPE RNA extraction kits for their ability to improve high‐quality RNA recovery from PDAC xenograft samples. Our results demonstrate that the addition of brief (< 5min) mH steps improves total RNA yield up to 3‐fold over current methods, as well as RNA purity and length, as characterized by 260/280 ratios (2.03 +/‐ 0.02 w/ mH step) and chip‐based electrophoresis (RNA Quality Index = 7.3 w/ mH step). These results have important implications for streamlining the FFPE‐RNA‐NGS workflow as a realistic approach for cancer biomarker identification.Grant Funding Source: n/a