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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Experimental investigation into coal wettability changes caused by reactions with scCO 2 -H 2 O

Geological CO 2 sequestration (GCS) can help mitigate global warming and enhance methane recovery from coal beds. However, few studies have linked the effects of CO 2 to surface chemistry changes controlling wetting behavior in deep coal beds. Contact angles (CAs) of CO 2 /N 2 -high volatile bituminous coal-water systems were measured under different temperatures and pressures. The surface chemistry and physical structure of coals were characterized to investigate changes in physicochemical properties and their relations with wettability after reactions. For N 2 treatment, the time-dependence of static and dynamic CAs were insignificant, ranging within 4°. For gaseous CO 2 treatment, the static CAs and the average advancing angles increased slightly. With supercritical (sc) CO 2 , both the static and dynamic CAs increased significantly, and θ adv changed to intermediate-wet (92°). Reactions with minerals exposed to scCO 2 resulted in greater surface roughness and heterogeneity, greater contact angle hysteresis and more surface sites occupied by scCO 2 rather than H 2 O. Increases in hydrophobic functional groups and decreases in hydrophilicity were shown by FTIR spectra, reflecting the shedding of polar oxygen-containing functional groups, reduction of hydrogen bonds, and increasing percentage of hydrocarbons. XRD patterns obtained following scCO 2 -treatment showed that crystallite growth and molecular polymerization were higher toward graphite-like. The calculated structural parameters of functional groups and crystallites both showed elevated coal rank. Changes in crystallite structure, notably higher carbon content and decreased negative surface charge, are unfavorable for water-wetting. Finally, this study contributes to understanding surface chemistry changes responsible for decreased wettability during CO 2 -enhanced coal bed methane recovery and GCS in coal reservoirs.

01 COAL, LIGNITE, AND PEAT↗
DOE OSTI2021

Capillary absorption spectroscopy for high temporal resolution measurements of stable carbon isotopes in soil and plant-based systems

Capillary Absorption Spectroscopy (CAS) is a relatively new analytical technique for performing stable isotope analysis. Here, we demonstrate the utility of CAS by recording and quantifying variation in 13C in controlled and biologically relevant applications. We calibrated CAS system response to increased 13CO2, with an observed ~4‰ increase in measured Δ13C for each 0.03 ppm shift in 13CO2 concentration. We leveraged this calibration to quantify rates of biogeochemical processes using a 13C tracer. For example, we monitored microbial respiration of 13C-glucose within an agricultural soil at 10 s quantification intervals and results demonstrated 8.6% ± 0.4 of added glucose was converted to 13CO2 within 1.5 h of incubation. We expanded the demonstration by adapting a rhizobox to permit continuous monitoring of 13CO2 in a soil (as distinct from plant) headspace to track the timing and quantify respiration rates of fresh plant photosynthate and observed a 3.5 h delay between plant exposure to a13CO2 tracer and the first signs of respiration by soil biota. These experiments highlight CAS is effective in producing high temporal resolution quantification of 13CO2 and demonstrate potential applications.

13CO2↗
DOE OSTI2022

Identifying candidate structured RNAs in CRISPR operons

Noncoding RNAs with secondary structures play important roles in CRISPR-Cas systems. Many of these structures likely remain undiscovered. We used a large-scale comparative genomics approach to predict 156 novel candidate structured RNAs from 36,111 CRISPR-Cas systems. A number of these were found to overlap with coding genes, including palindromic candidates that overlapped with a variety of Cas genes in type I and III systems. Among these 156 candidates, we identified 46 new models of CRISPR direct repeats and 1 tracrRNA. This tracrRNA model occasionally overlapped with predicted cas9 coding regions, emphasizing the importance of expanding our search windows for novel structure RNAs in coding regions. We also demonstrated that the antirepeat sequence in this tracrRNA model can be used to accurately assign thousands of predicted CRISPR arrays to type II-C systems. This study highlights the importance of unbiased identification of candidate structured RNAs across CRISPR-Cas systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid–binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Closure Report for Corrective Action Unit 116: Area 25 Test Cell C Facility, Nevada National Security Site, Nevada with ROTC-1

CR loaded to this OSTI record. Just adding new file which includes CR plus new ROTC 1 and update the metadata to the following: This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 116, Area 25 Test Cell C Facility. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 [as amended March 2010]). CAU 116 consists of the following two Corrective Action Sites (CASs), located in Area 25 of the Nevada National Security Site: (1) CAS 25-23-20, Nuclear Furnace Piping and (2) CAS 25-41-05, Test Cell C Facility. CAS 25-41-05 consisted of Building 3210 and the attached concrete shield wall. CAS 25-23-20 consisted of the nuclear furnace piping and tanks. Closure activities began in January 2007 and were completed in August 2011. Activities were conducted according to Revision 1 of the Streamlined Approach for Environmental Restoration Plan for CAU 116 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2008). This CR provides documentation supporting the completed corrective actions and provides data confirming that closure objectives for CAU 116 were met. Site characterization data and process knowledge indicated that surface areas were radiologically contaminated above release limits and that regulated and/or hazardous wastes were present in the facility. The Record of Technical Change 1 updated the use restriction information.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur–Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Novel adaptive immune systems in pristine Antarctic soils

Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Chapter 7 - CRISPR-Based Tools for Microbial Cell Factories

The development of microbial chassis for the production of a variety of biochemicals and biofuels is a growing area of research. How to efficiently manipulate genetic information to achieve optimal production of these compounds is a key area of focus in the field. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become a popular strategy for gene editing and regulation in many organisms due to its versatility and efficacy. Here, we describe methods developed utilizing CRISPR-Cas systems for engineering microbial cell factories (e.g., bacteria and yeast) at the single gene to genome scale for mutagenesis and transcriptional regulation of target genes. Finally, we provide a perspective on the challenges and opportunities for the applications of advanced CRISPR-Cas-based tools for engineering microbial cell factories.

BIOMASS FUELS↗
DOE OSTI2024

Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase

Abstract Carbonic Anhydrases (CAs) have been a target forde novoprotein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes’ first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d 7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H 2 O/OH − )] N (TRIL2WL23H) 3 n+ that behaves similarly to native Co(II) substituted human‐CAs. Like the Zn(II) analogue, the cobalt‐derivative at slightly basic pH is incapable of hydrolyzing p‐nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH − )] N (TRIL2WL23H) 3 + peptide complex. X‐ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5‐coordinate species by pH 11. UV‐vis spectroscopy can monitor this transition, giving a pK a for the conversion of 10.3. We assign this conversion to the formation of a 5‐coordinate Co(II)(N imid ) 3 (OH)(H 2 O) species. The pH dependent kinetic analysis indicates the maximal rate (k cat ), and thus the catalytic efficiency (k cat /K m ), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

Chemistry↗
DOE OSTI2022

Spectroscopy-based isotopic (δ 13 C) analysis for high spatial resolution of carbon exchange in the rhizosphere

The rhizosphere is a highly dynamic zone bridging plant roots with needed nutrient resources in soil. While the rhizosphere may be small, it has a disproportionally large impact on plant success and biomass production. A suite of rhizosphere-hosted microbial and geochemical interactions facilitate nutrient acquisition by plant roots, and, in turn, the roots stimulate these processes by supplying organic carbon into the rhizosphere. The small physical dimensions of the rhizosphere, however, can constrain efforts to elucidate key carbon exchange processes and their spatial extent and localization. We present a method for spatially resolved δ 13 C analysis of rhizosphere samples by coupling laser ablation (LA) sampling with isotopic analysis using capillary absorption spectroscopy (CAS) which differs from conventional mass spectrometer (MS) approaches. The CAS system has high sensitivity (requires fewer nanomoles of CO 2 per analysis) than comparable MS systems, which enables reduced sample size requirements to thereby improve spatial resolution (from 25 μm to as low as a projected 5 μm spatial resolution). We demonstrate the utility of CAS using rhizosphere samples from switchgrass plants exposed to 13 CO 2 . As a result, this technique will provide a capability for tracking the extent and spatial distribution of root exudate into the rhizosphere at highly detailed spatial scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Hexagonal Lattices of HIV Capsid Proteins Explored by Simulations Based on a Thermodynamically Consistent Model

HIV capsid proteins (CAs) may self-assemble into a variety of shapes under in vivo and in vitro conditions. Here, we employed simulations based on a residue-level coarse-grained (CG) model with full conformational flexibility to investigate hexagonal lattices, which are the underlying structural pattern for CA aggregations. Facilitated by enhanced sampling simulations to rigorously calculate CA dimerization and polymerization affinities, we calibrated our model to reproduce the experimentally measured affinities. Here using the calibrated model, we performed unbiased simulations on several large systems consisting of 1512 CA subunits, allowing reversible binding and unbinding of the CAs in a thermodynamically consistent manner. In one simulation, a preassembled hexagonal CA sheet developed spontaneous curvatures reminiscent of those observed in experiments, and the edges of the sheet exhibited local curvatures larger than those of the interior. In other simulations starting with randomly distributed CAs at different concentrations, existing CA assemblies grew by binding free capsomeres to the edges and by merging with other assemblies. At high CA concentrations, rapid establishment of predominant aggregates was followed by much slower adjustments toward more regular hexagonal lattices, with increasing numbers of intact CA hexamers and pentamers being formed. Our approach of adapting a general CG model to specific systems by using experimental binding data represents a practical and effective strategy for simulating and elucidating intricate protein aggregations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

3D Printed Parahydrophobic Surfaces as Multireaction Platforms

We report Parahydrophobic surfaces (PHSs) composed of arrays of cubic μ-pillars with a double scale of roughness and variable wettability were systematically obtained in one step and a widely accessible stereolithographic Formlabs 3D printer. The wettability control was achieved by combining the geometrical parameters (H = height and P = pitch) and the surface modification with fluoroalkyl silane compounds. Homogeneous distribution of F and Si atoms onto the pillars was observed by XPS and SEM-EDAX. A nano-roughness on the heads of the pillars was achieved without any post-treatment. The smallest P values lead to surfaces with static contact angles (CAs) >150° regardless of the H utilized. Interestingly, the relationship 0.6 ≤ H/P ≤ 2.6 obtained here was in good agreement with the H/P values reported for nano- and submicron pillars. Furthermore, experimental CAs, advancing and receding CAs, were consistent with the theoretical prediction from the Cassie–Baxter model. Structures covered with perfluorodecyltriethoxysilane with high H and short P lead to PHSs. Conversely, structures covered with perfluorodecyltrimethoxysilane exhibited a superhydrophobic behavior. Finally, several aqueous reactions, such as precipitation, coordination complex, and nanoparticle synthesis, were carried out by placing the reactive agents as microdroplets on the parahydrophobic pillars, demonstrating the potential application as chemical multi-reaction array platforms for a large variety of relevant fields in microdroplet manipulation, microfluidics systems, and health monitoring, among others.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Clades of huge phages from across Earth’s ecosystems

Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is—to our knowledge—the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR–Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR–Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR–Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth’s ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Metal ion dopant-induced famatinite to chalcostibite phase transformation of copper antimony sulphide colloidal nanostructures: effect on photophysical and pseudocapacitance properties

Here, the strategic doping of transition metal ions into copper antimony sulphide (CAS) semiconducting nanostructures can significantly influence their photophysical and pseudocapacitive properties, for which there are few reports and therefore is the focus of this study. Accordingly, highly crystalline metal ion (Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ and Zn 2+ ) doped off-stoichiometric copper rich/poor, antimony-rich/poor, and sulphur-poor CAS nanostructures (10–23 nm) were grown via colloidal (hot-injection) synthesis using metal diethyldithiocarbamate precursors. Importantly, metal ion doping significantly influences the structure and composition of the off-stoichiometric CAS nanostructures. Data from powder X-ray diffraction, Raman spectroscopy, high-resolution scanning/transmission electron microscopy, and energy dispersive X-ray spectroscopy confirm that the heavier metal ion dopants induce a novel phase transformation from famatinite (fCAS) to chalcostibite (cCAS) nanostructures. The influence of the metal ion dopants is also observed in the optical properties of as-synthesized nanostructures. This involves blue-shifted ultraviolet-visible absorption, reduced Urbach tailing, and tunable bandgaps between 2.17 and 2.38 eV. Also, the doped nanostructures display broad visible-near infrared photoluminescence via a triple radiative pathway with relatively short decay lifetimes between 0.2 and 6.1 ns. This is mediated by electronic transitions involving intrinsic (copper/antimony/sulphur interstitial) and extrinsic (metal ion interstitial) defect states. Additionally, electrodes prepared from Mn 2+ -doped fCAS nanostructures show enhanced pseudocapacitance via Na+ surface adsorption and intercalation relative to undoped fCAS electrodes, while Zn 2+ -doped cCAS electrodes exhibit pseudocapacitance via a combination of Na+ surface adsorption, intercalation, and redox reactions. These electrodes exhibit reduced charge transfer resistance, improved electronic conductivity and notably enhanced specific capacitance (∼222 F g −1 ), and charge transport, as measured in 1 M Na 2 SO 4 electrolyte via cyclic voltammetry and electrochemical impedance spectroscopy. To this end, the discovery of the metal ion-induced phase transformation presents a new avenue for optimizing the functional properties of fCAS and cCAS nanostructures, highlighting the critical role of metal ion-related defects in controlling the optical and electrochemical properties, towards potential solar absorption and energy storage applications.

25 ENERGY STORAGE↗
DOE OSTI2022

Decorating chromatin for enhanced genome editing using CRISPR-Cas9

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Functional characterization of diverse type I-F CRISPR-associated transposons

CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli : bioinformatic and experimental approaches

Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO 2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO 2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗