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DOE OSTI2023

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. Furthermore, by activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Low dose rate γ-irradiation protects fruit fly chromosomes from double strand breaks and telomere fusions by reducing the esi-RNA biogenesis factor Loquacious

Abstract It is still continuously debated whether the low-dose/dose-rate (LDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute γ-irradiation, is considerably reduced when flies are previously exposed to a protracted dose of 0.4 Gy delivered at a dose rate of 2.5 mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a reduced expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDR-mediated chromosome protection as it decreases the IR-induced CBs and TFs frequency. Thus, our molecular characterization of RAR identifies Loqs as a key factor in the cellular response to LDR and in the epigenetic routes involved in radioresistance.

Porrazzo, A.↗

DOE OSTI2021

Laboratory Evaluation of the Thermal Breakout Method for Maximum Horizontal Stress Measurement

Measuring in situ stress is essential for many problems in geomechanics, and the maximum horizontal stress is the most difficult to constrain. We are developing an extension of the breakout method to measure maximum horizontal stress in regions where natural breakouts do not occur. In the novel thermal breakout method, additional compression which leads to breakout development is induced by heating the borehole wall. In the present study, we validated the method experimentally in a true-triaxial apparatus on samples with predrilled boreholes. Two rocks were selected for laboratory testing: high-porosity Berea sandstone and low-porosity Niagaran dolomite. Prior to main true-triaxial tests, we carried out standard testing to characterize the strength, elasticity and thermal properties. The true-triaxial experiments consisted of: (1) room-temperature tests where samples were first loaded mechanically until the breakout formed, and (2) elevated-temperature tests where samples were loaded mechanically within the elastic range with additional compression induced thermally. Breakout initiation was monitored by acoustic emission sensors mounted on the pistons that applied horizontal stresses. Furthermore, the magnitude of induced thermal stress was calculated from temperature measurements around the borehole wall. In both rock types, we created thermally induced breakouts and examined analytical expressions to constrain maximum horizontal stress based on strength, elastic and thermal properties of the rocks.

58 GEOSCIENCES↗

DOE OSTI2020

Escherichia coli Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability

CRISPR-based interference has become common in various applications from genetic circuits to dynamic metabolic control. In E. coli, the native CRISPR Cascade system can be utilized for silencing by deletion of the cas3 nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of spacer sequences from guide arrays utilized for dynamic silencing. Here, we report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of spacers from guide arrays. Subsequently, mutant guide arrays can be amplified through selection. Replacing a constitutive promoter driving Cascade complex expression with a tightly controlled inducible promoter improves guide array stability, while minimizing leaky gene silencing. Additionally, these results demonstrate the potential of Cas1/2 mediated guide deletion as a mechanism to avoid CRISPR based autoimmunity.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Interdependent iron and phosphorus availability controls photosynthesis through retrograde signaling

Abstract Iron deficiency hampers photosynthesis and is associated with chlorosis. We recently showed that iron deficiency-induced chlorosis depends on phosphorus availability. How plants integrate these cues to control chlorophyll accumulation is unknown. Here, we show that iron limitation downregulates photosynthesis genes in a phosphorus-dependent manner. Using transcriptomics and genome-wide association analysis, we identify two genes, PHT4;4 encoding a chloroplastic ascorbate transporter and bZIP58 , encoding a nuclear transcription factor, which prevent the downregulation of photosynthesis genes leading to the stay-green phenotype under iron-phosphorus deficiency. Joint limitation of these nutrients induces ascorbate accumulation by activating expression of an ascorbate biosynthesis gene, VTC4 , which requires bZIP58. Furthermore, we demonstrate that chloroplastic ascorbate transport prevents the downregulation of photosynthesis genes under iron-phosphorus combined deficiency through modulation of ROS homeostasis. Our study uncovers a ROS-mediated chloroplastic retrograde signaling pathway to adapt photosynthesis to nutrient availability.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

High-throughput feedback-enabled optogenetic stimulation and spectroscopy in microwell plates

Abstract The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli . We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Biosensors for the detection of chorismate and cis,cis -muconic acid in Corynebacterium glutamicum

Abstract Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate–pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism. One-Sentence Summary High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

De novo transcriptome in roots of switchgrass ( Panicum virgatum L. ) reveals gene expression dynamic and act network under alkaline salt stress

Background: Soil salinization is a major limiting factor for crop cultivation. Switchgrass is a perennial rhizomatous bunchgrass that is considered an ideal plant for marginal lands, including sites with saline soil. Here we investigated the physiological responses and transcriptome changes in the roots of Alamo (alkaline-tolerant genotype) and AM314/MS-155 (alkaline-sensitive genotype) under alkaline salt stress. Results: Alkaline salt stress significantly affected the membrane, osmotic adjustment and antioxidant systems in switchgrass roots, and the ASTTI values between Alamo and AM-314/MS-155 were divergent at different time points. A total of 108,319 unigenes were obtained after reassembly, including 73,636 unigenes in AM-314/MS-155 and 65,492 unigenes in Alamo. A total of 10,219 DEGs were identified, and the number of upregulated genes in Alamo was much greater than that in AM-314/MS-155 in both the early and late stages of alkaline salt stress. The DEGs in AM-314/MS-155 were mainly concentrated in the early stage, while Alamo showed greater advantages in the late stage. These DEGs were mainly enriched in plant-pathogen interactions, ubiquitin-mediated proteolysis and glycolysis/gluconeogenesis pathways. We characterized 1480 TF genes into 64 TF families, and the most abundant TF family was the C2H2 family, followed by the bZIP and bHLH families. A total of 1718 PKs were predicted, including CaMK, CDPK, MAPK and RLK. WGCNA revealed that the DEGs in the blue, brown, dark magenta and light steel blue 1 modules were associated with the physiological changes in roots of switchgrass under alkaline salt stress. The consistency between the qRT-PCR and RNA-Seq results confirmed the reliability of the RNA-seq sequencing data. A molecular regulatory network of the switchgrass response to alkaline salt stress was preliminarily constructed on the basis of transcriptional regulation and functional genes. Conclusions: Alkaline salt tolerance of switchgrass may be achieved by the regulation of ion homeostasis, transport proteins, detoxification, heat shock proteins, dehydration and sugar metabolism. These findings provide a comprehensive analysis of gene expression dynamic and act network induced by alkaline salt stress in two switchgrass genotypes and contribute to the understanding of the alkaline salt tolerance mechanism of switchgrass and the improvement of switchgrass germplasm.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Full solution of the medium-induced radiation spectrum

New measurements of jet quenching observables at RHIC and at the LHC, such as jet substructure observables, demand an increased precision in the theory calculations describing medium-induced radiation of gluons. Closed expressions for the gluon spectrum including a full resummation of multiple scatterings have been known for the past 20 years. Still they have only been evaluated in specific limits either taking a few terms in an opacity expansion or by employing a Gaussian approximation for the interaction potential -- which misses essential physical effects. We present here a new flexible method to compute the full spectrum for a realistic interaction potential, thus allowing us for the first time to properly quantify the effect of the all-order resummation of multiple scatterings. This new approach paves the way for precision phenomenological studies including multiple scattering effects such as coherence phenomena.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

NASA NTRS1977

Photovoltaic, gravitationally-stabilized solid-state, satellite solar power station /GSS4PS/

The salient feature of a gravitationally-stabilized satellite solar power system (SSPS) for a photovoltaic system is that the large solar array is separated into smaller segments or subunits, joined together in a linear array oriented along the local vertical. Applicability of gravitational stabilization to the design of photovoltaic SSPS is demonstrated. Both actively controlled systems using multiple solar panels oriented to the sun and passively controlled systems requiring no sun orientation are considered and determined to be feasible. Expressions for gravity-gradient induced tension and restoring torques about the local vertical are assessed. Particular attention is directed to the applicability of solid-state approaches to the design of SSPS. It is shown that an all solid-state SSPS utilizing the LITOMIC (light-to-microwave conversion) concept is feasible but that a hybrid system employing a combination of both solid-state and cryogenic cooled low-voltage techniques to the design of the power management and distribution subsystem may also provide a solution.

Cantafio, L. J.↗

NASA NTRS1980

Delta V errors due to attitude correction maneuvers of a spinning spacecraft

A statistical error analysis has been performed to evaluate the change in velocity of a spin stabilized spacecraft due to thruster induced attitude correction maneuvers. Analytical expressions were developed to determine the mean and variance of the velocity in terms of the nominal values and uncertainties of key spacecraft parameters. These parameters include thrust, thruster timing, thruster alignment, plume impingement force and estimated spacecraft mass. Typical values of these parameters and their uncertainties for NASA's Galileo spacecraft were used to evaluate these expressions. The results of the analysis allow the effect of attitude correction maneuvers to be evaluated with respect to the functional requirements on nongravitational acceleration. Other results include the identification of major sources of delta V error and studies of the sensitivity of delta V to variations in the error sources.

Zipursky, H. J.↗

NASA NTRS1984

Discrete orthogonal function expansions for non-uniform grids using the fast Fourier transform

A technique for applying discrete Fourier series to infinite domains is presented. The technique uses mappings designed to minimize truncation error and can be applied to solve mixed initial boundary value problems among others. The method is alias-free and yields consistent differentiation and integration operators. The mapping-induced truncation error is explicitly expressible and small in nearly all cases of interest. The method is illustrated for three problems involving convection, diffusion, and vortex interaction.

Cain, A. B.↗

NASA NTRS1988

Alfven propulsion at Jupiter

The operation of an electrodynamic tether at Jupiter is investigated. The pertinent Jupiter environments are investigated, and analytic expressions are derived for the induced electric field and the voltage, current, and force for an electrodynamic tether as a function of the radial distance from Jupiter. The formulas are combined with environmental models to arrive at quantitative values for the above parameters. The results are used to investigate propulsion applications.

Gabriel, S. B.↗

NASA NTRS1993

Thermal shock resistance of ceramic matrix composites

The experimental and analytical investigation of the thermal shock phenomena in ceramic matrix composites is detailed. The composite systems examined were oxide-based, consisting of an aluminosilicate matrix with either polycrystalline aluminosilicate or single crystal alumina fiber reinforcement. The program was divided into three technical tasks; baseline mechanical properties, thermal shock modeling, and thermal shock testing. The analytical investigation focused on the development of simple expressions for transient thermal stresses induced during thermal shock. The effect of various material parameters, including thermal conductivity, elastic modulus, and thermal expansion, were examined analytically for their effect on thermal shock performance. Using a simple maximum stress criteria for each constituent, it was observed that fiber fracture would occur only at the most extreme thermal shock conditions and that matrix fracture, splitting parallel to the reinforcing fiber, was to be expected for most practical cases. Thermal shock resistance for the two material systems was determined experimentally by subjecting plates to sudden changes in temperature on one surface while maintaining the opposite surface at a constant temperature. This temperature change was varied in severity (magnitude) and in number of shocks applied to a given sample. The results showed that for the most severe conditions examined that only surface matrix fracture was present with no observable fiber fracture. The impact of this damage on material performance was limited to the matrix dominated properties only. Specifically, compression strength was observed to decrease by as much as 50 percent from the measured baseline.

Carper, D. M.↗

NASA NTRS1999

Is skeletal muscle ready for long-term spaceflight and return to gravity?

It is now clear that prevention of muscle debilitation during spaceflight will require a broader approach than simple exercise aimed at strengthening of the muscle fibers. The levels of several hormones and receptors are altered by unloading and must be returned to homeostasis. Pharmacotherapy and gene transfer strategies to raise the relative level of structural proteins may minimize the problems faced by astronauts in readapting to Earth-gravity. Up to now, we have only minimally exploited microgravity for advancing our understanding of muscle biology. A research laboratory in the space station with a centrifuge facility (gravity control) is essential for conducting basic research in this field. Microgravity has proven an excellent tool for noninvasively perturbing the synthesis of muscle proteins in the search for molecular signals and gene regulatory factors influencing differentiation, growth, maintenance and atrophy of muscle. Understanding the relation between blood flow and interstitial edema and between workload and subsequent structural failure are but two important problems that require serious attention. The roles of hormones and growth factors in regulating gene expression and their microgravity-induced altered production are other urgent issues to pursue. These types of studies will yield information that advances basic knowledge of muscle biology and offers insights into countermeasure design. This knowledge is likely to assist rehabilitation of diseased or injured muscles in humans on Earth, especially individuals in the more vulnerable aging population and persons participating in strenuous sports. Will the skeletal muscle system be prepared for the increased exposure to microgravity and the return to gravity loading without injury when space station is operational? The answer depends in large part on continued access to space and funding of ground-based models and flight experiments. The previous two decades of spaceflight research have described the effects of microgravity on multiple systems. The next generation of experiments promises to be even more exciting as we are challenged to define the cellular and molecular mechanisms of microgravity-induced changes.

manned↗

NASA NTRS2019

A Possible Second Large Subglacial Impact Crater in Northwest Greenland

Following the discovery of the Hiawatha impact crater beneath the northwest margin of the Greenland Ice Sheet, we explored satellite and aerogeophysical data in search of additional such craters. Here we report the discovery of a possible second subglacial impact crater that is 36.5‐km wide and 183 km southeast of the Hiawatha impact crater. Although buried by 2 km of ice, the structure's rim induces a conspicuously circular surface expression, it possesses a central uplift, and it causes a negative gravity anomaly. The existence of two closely spaced and similarly sized complex craters raises the possibility that they formed during related impact events. However, the second structure's morphology is shallower, its overlying ice is conformal and older, and such an event can be explained by chance. We conclude that the identified structure is very likely an impact crater, but it is unlikely to be a twin of the Hiawatha impact crater.

Greenland↗

NASA NTRSDate not supplied

Surface Instability of Liquid Propellants in Microgravity During Pulsed Settling Operations

Pulsing reaction control system (RCS) thrusters, vent valves, or other propulsion devices can preserve propellant resources in space, but this operation also effectively introduces a vibration to the vehicle. When the vibration is perpendicular to the liquid propellant surface, Faraday waves may be generated at the liquid-vapor interface. These Faraday instabilities can perturb or break up the liquid surface of cryogenic tanks, leading to inefficiencies in thermal management or even ullage collapse. Drawing from theory and experiments, an engineering model defining the allowable design regions for pulsed settling in microgravity was assembled and verified with computational fluid dynamics (CFD) simulations. A traditional settling metric, the Bond number, was also overlaid in the model to indicate which duty cycles were insufficient to overcome surface tension and aggregate propellant. Mission planners and engineers can consult the tool to rapidly evaluate the stability of a liquid-vapor interface given the pulse frequency and the excitation acceleration. Expressions developed for Faraday waves induced by a sinusoidal forcing input at standard gravity were found to provide excellent predictive capabilities for pulsed, or rectangular, waveforms in the absence of a consistent gravitational acceleration. This study extends the usage of these equations to an alternative forcing function and microgravity environments for the purpose of estimating natural frequencies, surface mode shapes, surface wave amplitudes, and the onset of droplet ejection. CFD simulations with the Loci/STREAM-VoF (Volume of Fluid) solver were initially validated against experimental results in standard gravity. Discrete points on the design map were then investigated with CFD and confirmed that the engineering model reliably indicates surface stability and most Faraday wave characteristics without requiring higher-fidelity tools. The engineering model is highly extensible and can be adapted for various propellant fill fractions, fluid properties, and tank sizes.

Faraday Waves↗

NASA NTRSDate not supplied

Surface Instability of Liquid Propellants in Microgravity During Pulsed Settling Operations

Faraday Waves↗