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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Antitoxin control of optimal transcriptional repression in the atypical HigB–HigA toxin–antitoxin system from Proteus vulgaris

Abstract Bacterial toxin–antitoxin (TA) pairs transcriptionally autoregulate their expression via a repression/derepression mechanism in response to changing environmental conditions. The structural diversity of TA systems influences the mechanisms of transcriptional regulation. Here, we define the molecular mechanism for the plasmid-encoded HigB–HigA TA pair originally identified in a post-operative infection with antibiotic-resistant Proteus vulgaris. We determine DNA binding and promoter activity by the HigB–HigA complex supported by structural biology and molecular dynamics simulations of an elusive DNA operator–TA repressor complex. To define the optimal oligomeric TA repressor–DNA operator complex required for derepression, we engineered a dedicated trimeric HigB–HigA2 complex that represses transcription more than 26-fold as compared to the tetrameric HigB2–HigA2. These results expand the known diversity of how the HigB–HigA TA family is autoregulated.

Biochemistry & Molecular Biology↗
NASA NTRS2002

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

Non-NASA Center↗
NASA NTRS2010

Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules (~ 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

Mangala, Lingegowda S.↗
NASA NTRS2014

Rapid Polymer Sequencer

Solid-state nanopore-based analysis of nucleic acid polymers is revolutionary. No other technique can determine information content in single molecules of genetic material at the speed of 1 subunit per microsecond. Because individual molecules are counted, the output is intrinsically quantitative. The nanopore approach is more generalized than any other method and in principle may be used to analyze any polymer molecule, including proteins. The approach to the development of a solid-state nanopore device is novel in the use of nanofabrication, nanoelectronic components, and high-speed signal acquisition. A novel geometry of the solid-state nanopore (less than 5 nm in length and 5 nm in diameter) will enable 1 to 5 nucleotide resolution measurements. This means that maximum resolution will be improved at least 100-fold compared to biological ion-channel measurements. The solid-state nanopore sensor will be made to enable sequencing DNA at a much faster rate than presently possible without the need for extensive sample preparation procedures, such as enzymatic amplification and labeling reactions. It will analyze electronic properties of individual subunits of DNA or RNA, to obtain linear composition of each genetic polymer molecule.

technology opportunity↗
DOE OSTI2024

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genetic circuit design automation for yeast

Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, a process that requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120 base pairs), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, in this work we constructed nine NOT/NOR gates with nearly identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of messenger RNAs resulting from RNA polymerase readthrough. Based on these gates, Cello 2.0 was used to build circuits with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Genetic circuit design automation for eukaryotes simplifies the construction of regulatory networks as part of cellular engineering projects, whether it be to stage processes during bioproduction, serve as environmental sentinels or guide living therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

NASA Discipline Musculoskeletal↗
DOE OSTI2023

A coarse-grained simulation model for colloidal self-assembly via explicit mobile binders

Colloidal particles with mobile binding molecules constitute a powerful platform for probing the physics of self-assembly. Binding molecules are free to diffuse and rearrange on the surface, giving rise to spontaneous control over the number of droplet–droplet bonds, i.e., valence, as a function of the concentration of binders. This type of valence control has been realized experimentally by tuning the interaction strength between DNA-coated emulsion droplets. Optimizing for valence two yields droplet polymer chains, termed ‘colloidomers’, which have recently been used to probe the physics of folding. To understand the underlying self-assembly mechanisms, here we present a coarse-grained molecular dynamics (CGMD) model to study the self-assembly of this class of systems using explicit representations of mobile binding sites. Further, we explore how valence of assembled structures can be tuned through kinetic control in the strong binding limit. More specifically, we optimize experimental control parameters to obtain the highest yield of long linear colloidomer chains. Subsequently tuning the dynamics of binding and unbinding via a temperature-dependent model allows us to observe a heptamer chain collapse into all possible rigid structures, in good agreement with recent folding experiments. Our CGMD platform and dynamic bonding model (implemented as an open-source custom plugin to HOOMD-Blue) reveal the molecular features governing the binding patch size and valence control, and opens the study of pathways in colloidomer folding. This model can therefore guide programmable design in experiments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2012

Changes in Liver Metabolic Gene Expression from Radiation Exposure

Increased exposure to radiation is one physiological stressor associated with spaceflight. While known to alter normal physiological function, how radiation affects metabolism of administered medications is unclear. Crew health could be affected if the actions of medications used in spaceflight deviated from expectations formed during terrestrial medication use. Three different doses of gamma radiation (50 mGy - 6.05 Gy) and a sham were administered to groups of 6 mice each, and after various intervals of recovery time, liver gene expression was measured with RT-qPCR arrays for drug metabolism and DNA repair enzymes. Results indicated approx.65 genes of the 190 tested were significantly affected by at least one of the radiation doses. Many of the affected genes are involved in the metabolism of drugs with hydrophobic or steroid-like structures, maintenance of redox homeostasis and repair of DNA damage. Most affected genes returned to near control expression levels by 7 days post-treatment. With 6 Gy exposure, metallothionein expression was 132-fold more than control at the 4 hr time point, and fell at each later time point (11-fold at 24 hrs, and 8-fold at 7 days). In contrast, Cyp17a1 showed a 4-fold elevation at 4 hrs after exposure and remained constant for 7 days.

Peters, C. P.↗
NASA NTRS2003

Impact of low-temperature plasmas on Deinococcus radiodurans and biomolecules

The effects of cold plasma on Deinococcus radiodurans, plasmid DNA, and model proteins were assessed using microbiological, spectrometric, and biochemical techniques. In low power O(2) plasma (approximately 25 W, approximately 45 mTorr, 90 min), D. radiodurans, a radiation-resistant bacterium, showed a 99.999% reduction in bioburden. In higher power O(2) plasma (100 W and 500 mTorr), the reduction rate increased about 10-fold and observation by atomic force microscopy showed significant damage to the cell. Damage to cellular lipids, proteins, and chromosome was indicated by losses of infrared spectroscopic peaks at 2930, 1651, 1538, and 1245 cm(-1), respectively. In vitro experiments show that O(2) plasmas induce DNA strand scissions and cross-linking as well as reduction of enzyme activity. The observed degradation and removal of biomolecules was power-dependent. Exposures to 200 W at 500 mTorr removed biomolecules to below detection limits in 60 s. Emission spectroscopy indicated that D. radiodurans cells were volatilized into CO(2), CO, N(2), and H(2)O, confirming that these plasmas were removing complex biological matter from surfaces. A CO(2) plasma was not as effective as the O(2) plasma, indicating the importance of plasma composition and the dominant role of chemical degradation. Together, these findings have implications for NASA planetary protection schemes and for the contamination of Mars.

Evaluation Studies↗
NASA NTRS2019

Using DNA Damage to Investigate the Individual Variability of Human Sensitivity to Ionizing Radiation

High-LET ionizing radiation is a major occupational health hazard for astronauts, but risk assessment remains elusive due to limited epidemiological data. Identifying genetic factors modulating the individual radiation response may be the most effective strategy to provide individualized risk management for long-duration high-radiation missions. We have started tackling the challenge of predicting individual risks by identifying human genetic loci associated with various radiation sensitivity phenotypes in primary blood mononuclear cells from a relatively large healthy human cohort. To date, we have performed the isolation of PBMCs from 768 subjects of the same ethnicity, and irradiated PBMCs from 576 subjects with 1 and 3 particles/100µm2 of 600 MeV/n 56Fe, 350 MeV/n 40Ar and 350 MeV/n 28Si ions. The phenotypes of interest were: number of radiation-induced foci (or RIFs), CellROX oxidative stress responses and cell death, at 4h and 24h following irradiation. We have observed a significant inter-individual variability at 0 Gy between the 576 studied subjects, with a mean fold difference between the 10% lowest and highest responders of 5.6 of RIFs/cell, 7.9 in mean CellRox intensity, and 9.3 in percentage of dead cells. In order to better assess genetic factors influencing DNA repair, we used a metric previously introduced by our group to sort out radiation sensitivity phenotypes in mice: i.e. the ratio of the first to the second slope of RIFs/cell (between 0 and 1, and between 1 and 3 particle/100µm2). Preliminary data on 192 individuals showed a distribution of low-dose responders (ratio > 1) to high-dose responders (ratio < 1) at 4h of 12%, 55% and 52% respectively for Fe, Ar and Si. The average value for the first and the second slopes was very similar for the two lowest LET (0.10 [-0.26;0.58] and 0.09 [-0.45;0.41] for Ar, 0.07 [-0.27;0.38] and 0.08 [-0.19;0.42] for Si), indicating a linear dose response across both fluence. Fe showed clear saturation for the highest dose with a slope of -0.09 [-0.86;1.51] against 0.68 [-2.21;2.20] for the low dose range, which probably reflects that many PBMCs are beyond repair at the high dose. Note that other significances were found for additional factors such as BMI and age whereas none were found for sex. GWAS will be performed on all phenotypes upon completion of measurements.

Pariset, E.↗
NASA NTRS2019

Using DNA Damage to Investigate the Individual Variability of Human Sensitivity to Ionizing Radiation

High-LET ionizing radiation is a major occupational health hazard for astronauts, but risk assessment remains elusive due to limited epidemiological data. Identifying genetic factors modulating the individual radiation response may be the most effective strategy to provide individualized risk management for long-duration high-radiation missions. We have started tackling the challenge of predicting individual risks by identifying human genetic loci associated with various radiation sensitivity phenotypes in primary blood mononuclear cells from a relatively large healthy human cohort. To date, we have performed the isolation of PBMCs from 768 subjects of the same ethnicity, and irradiated PBMCs from 576 subjects with 1 and 3 particles/100µm2 of 600 MeV/n 56Fe, 350 MeV/n 40Ar and 350 MeV/n 28Si ions. The phenotypes of interest were: number of radiation-induced foci (or RIFs), CellROX oxidative stress responses and cell death, at 4h and 24h following irradiation. We have observed a significant inter-individual variability at 0 Gy between the 576 studied subjects, with a mean fold difference between the 10% lowest and highest responders of 5.6 of RIFs/cell, 7.9 in mean CellRox intensity, and 9.3 in percentage of dead cells. In order to better assess genetic factors influencing DNA repair, we used a metric previously introduced by our group to sort out radiation sensitivity phenotypes in mice: i.e. the ratio of the first to the second slope of RIFs/cell (between 0 and 1, and between 1 and 3 particle/100µm2). Preliminary data on 192 individuals showed a distribution of ?low-dose responders? (ratio > 1) to ?high-dose responders? (ratio < 1) at 4h of 12%, 55% and 52% respectively for Fe, Ar and Si. The average value for the first and the second slopes was very similar for the two lowest LET (0.10 [-0.26;0.58] and 0.09 [-0.45;0.41] for Ar, 0.07 [-0.27;0.38] and 0.08 [-0.19;0.42] for Si), indicating a linear dose response across both fluence. Fe showed clear saturation for the highest dose with a slope of -0.09 [-0.86;1.51] against 0.68 [-2.21;2.20] for the low dose range, which probably reflects that many PBMCs are beyond repair at the high dose. Note that other significances were found for additional factors ? such as BMI and age ? whereas none were found for sex. GWAS will be performed on all phenotypes upon completion of measurements.

Pariset, E.↗
NASA NTRS2002

Biomolecular Effects of Cold Plasma Exposure

The effects of cold plasma exposure on Deinococcus radiodurans, plasmid DNA and model proteins were assessed using microbiological, spectrometric and biochemical techniques. Exposure of D. radiodurans, an extremely radiation resistant microbe, to O2 plasma (less than or equal to 25 W, approx. 45 mTorr, 90 min) yielded a approx. 99.999 % sterilization and the sterilization rate was increased approx. 10-fold at 100 W and 500 mTorr. AFM images shows that the exposed cells are significantly deformed and possess 50-70 nm concavities. IR analysis indicates the chemical degradation of lipids, proteins and carotenoids of the cell wall and membrane. Intracellular damage was indicated by major absorbance loss at 1245, 1651 and 1538/cm corresponding to degradation of DNA and proteins, respectively. Biochemical experiments demonstrate that plasmas induce strand scissions and crosslinking of plasmid DNA, and reduction of enzyme activity; the degradation is power dependent with total sample loss occurring in 60 s at 200 W and 500 mTorr. Emission spectroscopy shows that D. radiodurans is volatilized into CO2, CO, N2 and H2O confirming the removal of biological matter from contaminated surfaces. The O2 plasma impacts several cellular components predominantly through chemical degradation by atomic oxygen. A CO2, plasma, however, was not effective at degrading D. radiodurans, revealing the importance of plasma composition, which has implications for planetary protection and the contamination of Mars.

Mogul, Rakesh↗
DOE OSTI2024

Novel Microbial Routes to Synthesize Industrially Significant Precursor Compounds

Ethylene is the most widely employed organic precursor compound in industry. The potential to impact ethylene formation via recently discovered microbial processes is tenable using plentiful CO2 feedstocks. The overall long-term objective of this project was to develop an industrially compatible microbial process to synthesize ethylene in high yields. The key objective of this project was to fully define and initially characterized a recently discovered and genetically regulated anaerobic pathway to produce high levels of ethylene called the Dihydroxyacetone Phosphate - Ethylene Pathway in phototrophic bacteria. This was addressed through the following specific aims: 1. Fully probe the catalytic potential of all enzymes of the DHAP ethylene pathway and determine the regulatory mechanism of DHAP-ethylene pathway gene expression. 2. Discover effective and active ethylene enzymes encoded in cultured and uncultured organisms from anoxic environments. 3.Model the thermodynamics and kinetics of ethylene synthetic pathways to guide engineering efforts in integrating best performing DHAP-ethylene pathway enzymes into model bacteria chassis for enhance ethylene yields. Through this project we discovered the initially missing genetic and enzyme component of the DHAP-ethylene pathway that directly synthesized ethylene and other important industrial compounds like methane and ethane from specific substrates. We uncovered and partially characterized a nitrogenase-like reductase that functions in DHAP-ethylene pathway specifically and in methionine synthesis in general. This nitrogenase-like system is called the Methylthio-Alkane Reductase (MAR) for its ability to cleave volatile organic sulfur compounds into methanethiol (CH3-SH) for methionine synthesis and a hydrocarbon byproduct. Key to the DHAP-ethylene pathway, MAR is the essential enzyme that cleaves 2-methylthioethanol (CH3-S-CH2-CH2-OH) into ethylene. Coordinately, we uncovered that the MAR genes and genes associated with conversion of methanethiol (CH3-SH) to methionine are under genetic control of a LysR Type Transcriptional Regulator called SalR, whose activity is dependent upon the amount of sulfate available to the cell. When sulfate as the preferred sulfur source for cell growth drops below 200 micromolar, SalR become active for expressing the MAR and methionine biosynthesis genes to enable the cell to grow from volatile organic sulfur compounds and make ethylene. Metabolic thermos-kinetic modeling revealed that these MAR reactions for ethylene and other hydrocarbon production are highly thermodynamically favorable and are one of the largest driving forces for ethylene production by the DHAP-ethylene pathway for high ethylene yields. Modeling also indicated that a key aldolase and to a lesser extent an isomerase of the DHAP-ethylene pathway for production of the ethylene precursor, 2-methylthioethanol, also would increase ethylene yields. Through metagenomic mining and gene synthesis by the JGI DNA synthesis program, over 500 aldolase and isomerase homologs were synthesized and screened. From this, variants were uncovered with substantially higher activity that increased ethylene yields 5-fold via the aldolase reaction and 1.5-fold via the isomerase reaction. Each of these elements that increase ethylene production were integrated together via plasmid under appropriate gene promoter elements in the phototrophic bacterium, Rhodospirillum rubrum, resulting in at least 3 orders of magnitude increase in ethylene yield from carbon dioxide feedstock.

10 SYNTHETIC FUELS↗
NASA NTRS2012

Late Effects of Heavy Ion Irradiation on Ex Vivo Osteoblastogenesis and Cancellous Bone Microarchitecture

Prolonged spaceflight causes degeneration of skeletal tissue with incomplete recovery even after return to Earth. We hypothesize that heavy ion irradiation, a component of Galactic Cosmic Radiation, damages osteoblast progenitors and may contribute to bone loss during long duration space travel beyond the protection of the Earth's magnetosphere. Male, 16 week old C57BL6/J mice were exposed to high LET (56 Fe, 600MeV) radiation using either low (5 or 10cGy) or high (50 or 200cGy) doses at the NASA Space Radiation Lab and were euthanized 3 - 4, 7, or 35 days later. Bone structure was quantified by microcomputed tomography (6.8 micron pixel size) and marrow cell redox assessed using membrane permeable, free radical sensitive fluorogenic dyes. To assess osteoblastogenesis, adherent marrow cells were cultured ex vivo, then mineralized nodule formation quantified by imaging and gene expression analyzed by RT PCR. Interestingly, 3 - 4 days post exposure, fluorogenic dyes that reflect cytoplasmic generation of reactive nitrogen/oxygen species (DAF FM Diacetate or CM H2DCFDA) revealed irradiation (50cGy) reduced free radical generation (20-45%) compared to sham irradiated controls. Alternatively, use of a dye showing relative specificity for mitochondrial superoxide generation (MitoSOX) revealed an 88% increase compared to controls. One week after exposure, reactive oxygen/nitrogen levels remained lower(24%) relative to sham irradiated controls. After one month, high dose irradiation (200 cGy) caused an 86% decrement in ex vivo nodule formation and a 16-31% decrement in bone volume to total volume and trabecular number (50, 200cGy) compared to controls. High dose irradiation (200cGy) up regulated expression of a late osteoblast marker (BGLAP) and select genes related to oxidative metabolism (Catalase) and DNA damage repair (Gadd45). In contrast, lower doses (5, 10cGy) did not affect bone structure or ex vivo nodule formation, but did down regulate iNOS by 0.54 - 0.58 fold. Thus, both low and high doses of heavy ion irradiation cause time dependent, adaptive changes in redox state within marrow cells but only high doses (50, 200cGy) inhibit osteoblastogenesis and cause cancellous bone loss. We conclude space radiation has the potential to cause persistent damage to bone marrow derived stem and progenitor cells for osteoblasts despite adaptive changes in cellular redox state.

Tran, Luan Hoang↗
DOE OSTI2024

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Abstract Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2018

Late Effects of Heavy-Ion Irradiation on Ex Vivo Osteoblastogenesis and Cancellous Bone Microarchitecture

Prolonged spaceflight causes degeneration of skeletal tissue with incomplete recovery even after return to Earth. We hypothesize that heavy-ion irradiation, a component of Galactic Cosmic Radiation, damages osteoblast progenitors and may contribute to bone loss during long duration space travel beyond the protection of the Earth's magnetosphere. Male, 16 week-old C57BL6/J mice were exposed to high-LET (56-Fe, 600MeV) radiation using either low (5 or 10cGy) or high (50 or 200cGy) doses at the NASA Space Radiation Lab and were euthanized 3-4, 7, or 35 days later. Bone structure was quantified by microcomputed tomography (6.8 μm pixel size) and marrow cell redox assessed using membrane permeable, free radical-sensitive fluorogenic dyes. To assess osteoblastogenesis, adherent marrow cells were cultured ex vivo, then mineralized nodule formation quantified by imaging and gene expression analyzed by RT-PCR. Interestingly, 3-4 days post-exposure, fluorogenic dyes that reflect cytoplasmic generation of reactive nitrogen/oxygen species (DAF-FM Diacetate or CM-H2DCFDA) revealed irradiation (50cGy) reduced free radical generation (20-45%) compared to sham-irradiated controls. Alternatively, use of a dye showing relative specificity for mitochondrial superoxide generation (MitoSOX) revealed an 88% increase compared to controls. One week after exposure, reactive oxygen/nitrogen levels remained lower (24%) relative to sham-irradiated controls. After one month, high dose irradiation (200 cGy) caused an 86% decrement in ex vivo nodule formation and a 16-31% decrement in bone volume to total volume and trabecular number (50, 200cGy) compared to controls. High dose irradiation (200cGy) up-regulated expression of a late osteoblast marker (BGLAP) and select genes related to oxidative metabolism (Catalase) and DNA damage repair (Gadd45). In contrast, lower doses (5, 10cGy) did not affect bone structure or ex vivo nodule formation, but did down-regulate iNOS by 0.54-0.58 fold. Thus, both low- and high-doses of heavy-ion irradiation cause time-dependent, adaptive changes in redox state within marrow cells but only high doses (50, 200cGy) inhibit osteoblastogenesis and cause cancellous bone loss. We conclude space radiation has the potential to cause persistent damage to bone marrow-derived stem and progenitor cells for osteoblasts despite adaptive changes in cellular redox state.

LATE EFFECTS OF HEAVY-ION IRRADIATION↗
DOE OSTI2020

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3′ terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

59 BASIC BIOLOGICAL SCIENCES↗