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At least 487 records · Page 27
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Abstract RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1997

Lewis Investigates Frequency Sharing Between Future NASA Space Systems and Local Multipoint Distribution Systems in the 27-GHz Band

At the request of the Federal Communications Commission (FCC), the NASA Lewis Research Center undertook an intensive study to examine the feasibility of frequency sharing between future NASA space services and proposed Local Multipoint Distribution Systems (LMDS) in the 25.25- to 27.5-GHz band. This follows NASA's earlier involvement in the FCC's 1994 Negotiated Rule Making Committee which studied frequency sharing between Ka-band Fixed Satellite Services and LMDS in the 27.5- to 29.5-GHz band. LMDS is a terrestrial, cellular, wireless communication service primarily intended to provide television distribution from hub stations located within relatively small cells to fixed subscriber receivers. Some proposed systems, however, also plan to offer interactive services via subscriber-to-hub transmissions. LMDS providers anticipate that their systems will be a cost-effective alternative to cable television systems, especially in urban areas. LMDS proponents have expressed an interest in using frequencies below 27.5 GHz. NASA, however, plans to operate three types of space systems below 27.5 GHz. The H, I, and J follow-on satellites for the Tracking and Data Relay Satellite System (TDRSS), which are planned for launch beginning in 1999, are designed to receive high-data-rate transmissions (up to 800 Mbps) from low-Earth orbiting "user" spacecraft in the 25.25- to 27.5-GHz band. In this case, the potential interference is the aggregate interference from LMDS transmitters (both hubs and subscribers) into the TDRSS tracking receive beams as they sweep over the Earth's surface while tracking lower altitude user spacecraft.

Source record↗
DOE OSTI2022

Planar, longitudinal, compressive waves in solids: Thermodynamics and uniaxial strain restrictions

A new tri-axial pressure-based constitutive expression has been found using Cauchy's stress tensor. This stress state emphasizes pressure and shear stress. The description is a pressure plus an effective shear stress allowing for a constitutive law based on atomic solid-state phase changes in crystalline cells due to pressure plus shear-based dislocation motion commonly associated with plasticity. Pressure has a new role in the material's constitutive response as it is separated from plasticity. The thermo-mechanical system describes third-order Gibbs’ expressions without specific volume restrictions placed upon the material. Isothermally, the ratio of heat to shear work in elastic copper is shown to approach zero at a very low temperature and become larger than one as temperature approaches melting. Wave compression models investigated are elastic and plastic: in fully elastic materials, the planar wave is restricted by Poisson's effect although plastic shear changes this constraint. Plastic deformation, dominated by dissipative shear stresses in uniaxial strain, heats the material while excluding phase changes from hydrostatic pressures. The material properties per se across Hugoniot shocks are described with entropy concepts. Shock waves are exceedingly complex since the constitutive laws are linked at extreme temperatures, pressures, and shear stresses. Isothermal, isentropic, isochoric, and iso-shear conditions are used throughout with Jacobian algebra.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2020

Creating multifunctional synthetic lichen platforms for sustainable biosynthesis of biofuel precursors

In this project, we were creating a sustainable platform for biofuel production, utilizing carbon-fixing autotrophs to supply oxygen and organic substrates to heterotrophic partners, which in turn produce carbon dioxide to feed the autotrophs. This symbiotic lichen community could lower the input cost, optimize metabolic exchanges and improve the generation of biofuel precursors through multi-omics driven genetic engineering. The cyanobacteria Synechococcus elongatus (S. elongatus) was used as the primary autotroph to provide oxygen and organic substrates, especially sucrose, to a co-culture system. The strain with overexpression of sucrose transporter cscB demonstrated a significant increase in sucrose production under salt stress as what we expected. We also implemented 13C metabolic flux analysis on the sucrose secreting strain S. elongatus cscB-NaCl. Next, transporters proteins like glutamate exporter mscCG from Corynebacterium glutamicum was overexpressed in S. elongatus to improve metabolite exchange. We provided sucrose and glutamate to filamentous fungi to enable their growth and production of biochemicals using substrates from the cyanobacterium. Two fungi ( Aspergillus nidulans and Aspergillus niger ) and two yeast strains ( Rhodotorula toruloides and Lipomyces starkeyi ) served as heterotrophs in this system. They were co-cultured with S.elongatus under different pH condition, and growth on different carbon source to understand the symbiotic system and optimize the parameters for production. The two fungi strained grew much better at pH 7, 8 and 9 with Yeast Nitrogen Base (YNB ) supplementation. Two yeast strains grew well on pH 7 and 8 with YNB supplementation. All four species achieved the highest biomass level when utilizing sucrose as the main carbon sources, which demonstrates a great pairing with S.elongatus . In addition to investigate the metabolite exchange and growth condition of the co-culture system, we incorporated an Aspergillus strain expressing three genes (PAND, BAPAT and HPDH). Expression of these three genes enabled production of 3-hydroxypropionic acid (3HP) with a titer of 3-6 g/L regardless of pH and other nutrient conditions. Moreover, we developed a computational model with different heterotrophic fungi symbiosing with S.elongatus to evaluate the exchange of hundreds of metabolites. The accuracy and sensitivity had been optimized by high-throughput phenotyping assays. This project demonstrated ways to enhance a synthetic lichen platform’s efficiency and scalability to create a robust system for sustainable bioproduct synthesis. The project has enabled us to explore the technological and commercial potential of sustainable lichen co-culture. The project has also trained the next generation of scientists to address sustainability, carbon fixation, and design solutions to contemporary global challenges. The lichen platform represented a novel approach to sustainably producing bioproducts with a reduced carbon footprint and lower costs.

09 BIOMASS FUELS↗
DOE OSTI2021

Approximation of the Frequency-Amplitude Curve Using the Homotopy Analysis Method

The Frequency-Amplitude (F-A) curve on power system oscillation under a large disturbance characterizes how a natural oscillation mode transitions to nonlinear oscillations with growing amplitudes and decaying frequencies. The existing formulation of the F-A curve is derived by solving elliptical integrals on oscillation of a single-machine-infinite-bus equivalent about the targeted oscillation mode. The formula is in a form of infinite series and needs to sum a large number of terms for satisfactory accuracy. This paper introduces an explicit, approximate expression obtained from the Homotopy Analysis Method on the F-A curve. The proposed F-A curve expression is derived from an SMIB system and verified on the IEEE 3-machine 9-bus system to show how the oscillation frequency of a dominant mode varies with oscillation amplitude under large disturbances.

frequency-amplitude (F-A) curve↗
DOE OSTI2026

Advances in genetic tools for metabolic engineering of non-conventional yeasts

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Analysis and design of DC-link capacitor in cascaded H-bridge multilevel active front-end converter

Medium-voltage grid-tied systems often use a cascaded H-bridge multi-level active front-end. In this converter, dc link bus capacitors play an important role in stabilizing the converter and enabling both active and reactive power injections. The present work provides analytical expressions for the capacitor current, which are essential for optimizing system design (especially capacitor size vs. lifetime). Then, the expression is incorporated into the grid connected bidirectional power system model. Consequently, this work contributes to the guiding principles to choose accurate dc link capacitor ratings against grid-side power delivery requirements. DC-link capacitors come with an expiration date which is dependent on the operating power and voltage values. Lack of information about the lifetime of the dc-link capacitor creates uncertainty in the duration of online operation of the converter, thus increasing the probability of a contingency outage. The lifetime of the capacitor can range from only a few seconds of online operation of the station due to under-sizing, to a compromised quality of power flowing through other solid state devices which may not have been designed for the transients that are caused by over-sizing of the dc-link capacitor. A better estimate of the lifetime of these capacitors can help maximize usage and maintain scheduled outages more efficiently, without disruption in service.

24 POWER TRANSMISSION AND DISTRIBUTION↗
DOE OSTI2022

Identification of a Fusarium ear rot resistance gene in maize by QTL mapping and RNA sequencing

Fusarium ear rot (FER) caused by Fusarium verticillioides is a prevalent maize disease. To comprehensively characterize the genetic basis of the natural variation in FER resistance, a recombinant inbred line (RIL) population was used to map quantitative trait loci (QTL) for FER resistance. A total of 17 QTL were identified by linkage mapping in eight environments. These QTL were located on six chromosomes and explained 3.88–15.62% of the total phenotypic variation. Moreover, qFER1.03 had the strongest effect and accounted for 4.98–15.62% of the phenotypic variation according to analyses of multiple environments involving best linear unbiased predictions. The chromosome segment substitution lines (CSSLs) derived from a cross between Qi319 (donor parent) and Ye478 (recurrent parent) were used to verify the contribution of qFER1.03 to FER resistance. The line CL171, which harbored an introgressed qFER1.03 , was significantly resistant to FER. Further fine mapping of qFER1.03 revealed that the resistance QTL was linked to insertion/deletion markers InDel 8 and InDel 2, with physical distances of 43.55 Mb and 43.76 Mb, respectively. Additionally, qFER1.03 differed from the previous resistance QTL on chromosome 1. There were three annotated genes in this region. On the basis of the RNA-seq data, which revealed the genes differentially expressed between the FER-resistant Qi319 and susceptible Ye478, GRMZM2G017792 (MPK3) was preliminarily identified as a candidate gene in the qFER1.03 region. The Pr-CMV-VIGS system was used to decrease the GRMZM2G017792 expression level in CL171 by 34–57%, which led to a significant decrease in FER resistance. Using RIL and CSSL populations combined with RNA-seq and Pr-CMV-VIGS, the candidate gene can be dissected effectively, which provided important gene resource for breeding FER-resistant varieties.

Xia, Yusheng↗
NASA NTRS1980

Survival probabilities for interstellar hydrogen flowing into the interplanetary system from far regions of the heliosphere

The expressions for 'survival' probabilities are presented for an atomic hydrogen particle moving on a trajectory from far regions of the heliosphere to the vicinity of the sun. Three 'destroying' processes have been considered; photoionization, charge transfer and electron ionization. The solar wind has been assumed to be a two-flux steady stream radially expanding with constant flow velocity. Recent profiles of solar-wind electron temperature have been used. The results can be useful for theoretical analyses as well as for analysis of spaceflight observations.

Kunc, J. A.↗
NASA NTRS1980

A new method of measuring radio source parameters of a partially polarized distributed source from spacecraft observations

An experimental system is analyzed which is designed to determine the angular and polarization properties of low frequency radio sources from measurements made on a spinning spacecraft. The system has been optimized to provide high accuracies for sources at high as well as low elevation angles. Theoretical expressions are derived for the response of this system to a partially polarized point source. Integrations are then carried out to get the system response to a uniform circular distributed source. Data processing techniques are derived so that computer simulations can be carried out to investigate the accuracy of this technique. It is shown that using 24 measurements of a partially polarized source (with Q = U = V = 0.5), taken in one spacecraft rotation, the resulting rms errors in angular position are less than one degree and the errors in determining the Stokes parameters are generally 1-10% for a wide range of source elevations.

Manning, R.↗
NASA NTRS2017

R2U2: Tool Overview

R2U2 (Realizable, Responsive, Unobtrusive Unit) is an extensible framework for runtime System HealthManagement (SHM) of cyber-physical systems. R2U2 can be run in hardware (e.g., FPGAs), or software; can monitorhardware, software, or a combination of the two; and can analyze a range of different types of system requirementsduring runtime. An R2U2 requirement is specified utilizing a hierarchical combination of building blocks: temporal formula runtime observers (in LTL or MTL), Bayesian networks, sensor filters, and Boolean testers. Importantly, the framework is extensible; it is designed to enable definitions of new building blocks in combination with the core structure. Originally deployed on Unmanned Aerial Systems (UAS), R2U2 is designed to run on a wide range of embedded platforms, from autonomous systems like rovers, satellites, and robots, to human-assistive ground systems and cockpits. R2U2 is named after the requirements it satisfies; while the exact requirements vary by platform and mission, the ability to formally reason about realizability, responsiveness, and unobtrusiveness is necessary for flight certifiability, safety-critical system assurance, and achievement of technology readiness levels for target systems. Realizability ensures that R2U2 is suficiently expressive to encapsulate meaningful runtime requirements while maintaining adaptability to run on different platforms, transition between different mission stages, and update quickly between missions. Responsiveness entails continuously monitoring the system under test, real-time reasoning, reporting intermediate status, and as-early-as-possible requirements evaluations. Unobtrusiveness ensures compliance with the crucial properties of the target architecture: functionality, certifiability, timing, tolerances, cost, or other constraints.

Rozier, Kristin Y.↗
NASA NTRS1978

Critical fault patterns determination in fault-tolerant computer systems

The method proposed tries to enumerate all the critical fault-patterns (successive occurrences of failures) without analyzing every single possible fault. The conditions for the system to be operating in a given mode can be expressed in terms of the static states. Thus, one can find all the system states that correspond to a given critical mode of operation. The next step consists in analyzing the fault-detection mechanisms, the diagnosis algorithm and the process of switch control. From them, one can find all the possible system configurations that can result from a failure occurrence. Thus, one can list all the characteristics, with respect to detection, diagnosis, and switch control, that failures must have to constitute critical fault-patterns. Such an enumeration of the critical fault-patterns can be directly used to evaluate the overall system tolerance to failures. Present research is focused on how to efficiently make use of these system-level characteristics to enumerate all the failures that verify these characteristics.

Mccluskey, E. J.↗
DOE OSTI2021

Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (P lac , P tac , P tetR/tetA and P bad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1992

The orbital record in stratigraphy

Orbital signals are being discovered in pre-Pleistocene sediments. Due to their hierarchical nature these cycle patterns are complex, and the imprecision of geochronology generally makes the assignment of stratigraphic cycles to specific orbital cycles uncertain, but in sequences such as the limnic Newark Group under study by Olsen and pelagic Cretaceous sequence worked on by our Italo-American group the relative frequencies yield a definitive match to the Milankovitch hierarchy. Due to the multiple ways in which climate impinges on depositional systems, the orbital signals are recorded in a multiplicity of parameters, and affect different sedimentary facies in different ways. In platform carbonates, for example, the chief effect is via sea-level variations (possibly tied to fluctuating ice volume), resulting in cycles of emergence and submergence. In limnic systems it finds its most dramatic expression in alternations of lake and playa conditions. Biogenic pelagic oozes such as chalks and the limestones derived from them display variations in the carbonate supplied by planktonic organisms such as coccolithophores and foraminifera, and also record variations in the aeration of bottom waters. Whereas early studies of stratigraphic cyclicity relied mainly on bedding variations visible in the field, present studies are supplementing these with instrumental scans of geochemical, paleontological, and geophysical parameters which yield quantitative curves amenable to time-series analysis; such analysis is, however, limited by problems of distorted time-scales. My own work has been largely concentrated on pelagic systems. In these, the sensitivity of pelagic organisms to climatic-oceanic changes, combined with the sensitivity of botton life to changes in oxygen availability (commonly much more restricted in the Past than now) has left cyclic patterns related to orbital forcing. These systems are further attractive because (1) they tend to offer depositional continuity, and (2) presence of abundant microfossils yields close ties to geochronology. A tantalizing possibility that stratigraphy may yield a record of orbital signals unrelated to climate has turned up in magnetic studies of our Cretaceous core. Magnetic secular variations here carry a strong 39 ka periodicity, corresponding to the theoretical obliquity period of that time - Does the obliquity cycle perhaps have some direct influence on the magnetic field?

Fischer, Alfred G.↗
NASA NTRS1980

A solution of the variational equations for elliptic orbits in rotating coordinates

For elliptic reference orbits, formulas are given for the perturbation state transition matrix of the two-body problem. The formulas relate perturbations expressed in a local vertical rotating coordinate system and are valid for motion in the linear neighborhood of reference orbits with e in the range of 0 to 1. The elements of the state transition matrix are expressed in terms of natural parameters (horizontal and radial velocity, radius, eccentricity, true anomaly, etc.) at the initial and final points. In addition to the general form, a simplified version, valid for small eccentricity orbits, is given.

Jones, J. B.↗
NASA NTRS2002

Regulation of period 1 expression in cultured rat pineal

The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

NASA Discipline Space Human Factors↗
DOE OSTI2023

Shaping Nanobodies and Intrabodies against Proteoforms

Proteoforms expand genomic diversity and direct developmental processes. While high-resolution mass spectrometry has accelerated characterization of proteoforms, molecular techniques working to bind, and disrupt the function of specific proteoforms have lagged behind. In this study, we worked to develop intrabodies capable of binding specific proteoforms. Herein we employed a synthetic camelid nanobody library expressed in yeast to identify nanobody binders of different SARS-CoV-2 receptor binding domain (RBD) proteoforms. Importantly, employment of the positive and negative selection mechanism inherent to the synthetic system allowed for amplification of nanobody-expressing yeast that bind to the original (Wuhan strain RBD) but not the E484K (Beta variant) mutation. Nanobodies raised against specific RBD proteoforms were validated by yeast-2-hybrid analysis and sequence comparisons. These results provide a clear framework for development of proteoform-specific nanobodies and intrabodies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1993

Evolving fuzzy rules in a learning classifier system

The fuzzy classifier system (FCS) combines the ideas of fuzzy logic controllers (FLC's) and learning classifier systems (LCS's). It brings together the expressive powers of fuzzy logic as it has been applied in fuzzy controllers to express relations between continuous variables, and the ability of LCS's to evolve co-adapted sets of rules. The goal of the FCS is to develop a rule-based system capable of learning in a reinforcement regime, and that can potentially be used for process control.

Valenzuela-Rendon, Manuel↗