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42 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Role of Tensile Stress in DNA Nanoresonators for Epigenetic Studies

The evaluation of epigenetic features such as DNA methylation is becoming increasingly important in many biochemical processes like gene expression and transcription as well as in several diseases like schizophrenia or diabetes. Here, in this work, we report that self-assembled nanomechanical resonators entirely composed of DNA molecules can be used to explore gross changes in DNA methylation levels (0–25–50%), while careful control of tensile stress is needed to reduce the variability of resonance frequency for rigorous quantification. The effect of the tensile stress retained by the suspended DNA nanoresonators on the application of the technique is extensively explored using a combination of laser Doppler vibrometry and atomic force spectroscopy. DNA nanoresonators are real-time, label-free sensors and could avoid chemical functionalization and sample amplification. Therefore, they may represent in the future a key complementary routine tool for global DNA methylation analysis needed to evaluate the consequences of environmental stresses on the human genome.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2017

Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

microgravity↗
NASA NTRS2001

Focal adhesion kinase is involved in mechanosensing during fibroblast migration

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

NASA Discipline Cell Biology↗
NASA NTRS1998

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

NASA Discipline Plant Biology↗
NASA NTRS1997

Three-dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics

The Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogeneous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system:(a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

Mixed Tumor, Mullerian/ultrastructure↗
NASA NTRS2023

Automated Fluidics Device for Extraction and Quantification of miRNA Biomarkers From Blood

Radiation Assessment DuRing Exposure And long-Duration Spaceflight (RADREADS) demonstrates space-compatible point-of-care technology for quantitative biological monitoring of blood miRNA biomarkers in response to long-term low dose radiation exposure. This individualized monitoring approach will inform targeted treatment strategies to maximize medical resource utilization by accounting for individual susceptibility to radiation-related illnesses. As human spaceflight progresses beyond Earth’s magnetic shielding, radiation exposure poses a significant risk to astronaut health and safety. Extended operation in this environment comes with an increased risk of radiation exposure, leading to higher risks of radiation sickness, cancer, central nervous system effects, and degenerative diseases. While conventional physical dosimetry techniques capture radiation dose, individualistic susceptibility to radiation damage is varied. Multiple characteristics, including age, body weight, sex, genetics, and immune status, have been found to influence radiosensitivity (Liu et al. 2011, and Bouffler 2016). This differential response necessitates individualized monitoring and targeted treatment strategies to maximize medical resource utilization; however, a practical diagnostic platform for quantifying long-term, low dose radiation-induced tissue damage does not currently exist. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by mediating the degradation of messenger RNA. The levels of particular miRNAs are influenced by biological processes such as inflammation and serve as biomarkers for a variety of conditions including cancer (Singh et al. 2017). MicroRNAs are found in various bodily fluids and are amenable to collection via liquid biopsies, providing a minimally invasive and easily quantifiable readout for a variety of radiosensitive reporters. A preliminary signature of 15 spaceflight sensitive miRNA has been identified in rodent and human studies, including miR-21-5p, miR-24-3p, miR-92a-3p, miR-17-5p, miR-16a-3p, miR-34a-3p, and miR-223-3p. These targets generally increased expression with radiation dose and linear energy transfer, though variation between individuals is not yet described. Current gaps in the field include a lack of understanding of longitudinal biological responses to long-term, low dose radiation exposure and the absence of space-compatible point-of-care technology for quantitative biological monitoring. In this body of work, we aim to develop an automated bleed-to-read system to process whole blood for the detection of miRNA biomarkers in order to monitor individualistic responses to radiation exposure. This will be achieved via separating serum (or plasma) from whole blood, followed by extraction, amplification, and quantification of the miRNA using a RT-qPCR reaction. Previously, the WetLab-2 hardware enabled execution of a RT-qPCR reaction aboard ISS; however, it is a manual system that requires crew manipulation and bulky components (Parra et al. 2017). To address these issues, automated fluid handling hardware was developed for each stage of sample preparation. Extraction of total RNA is achieved by sequentially pumping reagents through an off-the-shelf nucleic acid binding column (miRNeasy Serum/Plasma Advanced Kit, Qiagen). This approach eliminates several manual pipetting and centrifuging steps and limits the use of toxic chemicals commonly found in other sample processing techniques. The resulting elution will then be automatically dispensed for RT-qPCR analysis using a compact rotary qPCR (Mic qPCR Cycler, Bio Molecular Systems) that will improve spaceflight compatibility by removing bubbles from the detection region, another challenge highlighted by WetLab-2 (Parra et al. 2017). Efforts are also being made to simplify the RT-qPCR reaction to a 1-step air-dryable mix to improve long-term reagent stability at room temperature and reduce system complexity. By automating the RT-qPCR processes via microfluidic manipulation, RADREADS will reduce crewmember hands-on time and enable the personalized detection of radiation-induced tissue damage during long duration missions. Minimally invasive, longitudinal monitoring of individual’s response to radiation exposure will inform how the physiological system responds to long-term low dose space radiation and enables development of targeted countermeasures by the medical team. Ultimately, this portable technology will require minimal technical expertise and can also be used to monitor miRNA biomarkers associated with other diseases.

Tristen Head↗