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At least 163 records · Page 9

SEM petrography of dispersed organic matter in black shales: A review

Organic matter (OM)-hosted pores are important constituents of the pore system of black shales and play a crucial role in determining their methane adsorption capacity and porosity. OM-hosted pores are generally observed and described with scanning electron microscope (SEM) on Ar ion-milled surfaces. However, SEM imaging is not able to reliably distinguish OM types and relate the observed pores to specific macerals. Partly because of this inability to relate organic pores to macerals, the evolution of organic porosity during thermal maturation remains poorly understood. In this paper, we review the petrographic characteristics of dispersed organic matter (DOM) in black shales under the SEM. Organic petrographic classification of DOM developed for reflected-light microscopy is so far the most practical method when describing DOM in black shales under the SEM because this classification has information on the origin of DOM. Therefore, correlative microscopy (combination of reflected-light and electron microscopy) is the most effective method to identify both OM types and OM-hosted pores. This method, however, is not readily available to most researchers. Although identifying OM on the basis of SEM observations is a challenging task, it is achievable provided there is a good understanding of the studied shales, especially their thermal maturity and original OM composition. Therefore, the overall objective of this paper is to review petrographic characteristics of DOM in black shales under the SEM to provide some guidelines for identifying DOM from SEM observations. We also review factors that control the formation and preservation of OM-hosted pores. OM-hosted pores consist of primary and secondary organic pores. Primary organic pores are pores inherited from the biological structure of the original OM. Secondary organic pores develop during hydrocarbon generation and expulsion from oil-prone OM and are hosted by solid bitumen or pyrobitumen. The development of secondary organic pores is controlled by thermal maturity and OM type, and their preservation is subject to thermal maturity, OM content, and mineralogical composition. The presented view of the evolution of micropore and mesopore characteristics of OM with thermal maturity is based on data from the literature. The specific surface area and pore volume of OM in black shales follow a parabolic pattern with increasing thermal maturity (quantified via vitrinite reflectance, R o ). The initial increase reflects development of OM-hosted pores, and the subsequent decrease is due to denser stacking of aromatic units in the macromolecular structure of OM, with maximum values (specific surface area ~ 300 m 2 /g and pore volume ~ 0.3 cm 3 /g) reached at R o values in the 2.5-3.5% range. The contribution of OM-hosted pores to the pore characteristics of black shales depends on OM content, OM type, and thermal maturity.

03 NATURAL GAS↗

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro . The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

59 BASIC BIOLOGICAL SCIENCES↗

Raman Microscopy Analysis of Wyoming CarbonSAFE Pilot Well Thin Sections for Mineralogy and Organic Matter Characterization

Scanning confocal Raman microspectroscopy (RMS) was used to analyze thin sections for inorganic mineral content and for evaluation of organic material (OM). Thin sections were selected from a stratigraphic test well core in the Powder River Basin, Wyoming under the Wyoming CarbonSAFE project. The well was analyzed with a variety of rock and fluid characterization techniques to determine the feasibility of a commercial-scale CO2 storage site. RMS complements other analyses, including traditional petrography, SEM, porosity and permeability, and XRD For this aspect of the study, RMS is especially important in the evaluation of OM in sealing lithologies. At prospective geologic CO2 storage sites, it is imperative to assess the unconventional oil and gas potential of seals to ensure that any future development would not compromise the integrity of the seals. Whole-slide mineralogical surveys were performed on thin sections from various shale and sandstone formations. Surveys were analyzed with Direct Classical Least Squares to identify and quantify minerals. The location and concentration of minerals was color-coded and overlaid on optical images for visualization of the distribution of minerals. Dense hyperspectral Raman mapping of OM was performed on five thin sections. Eleven spectral parameters diagnostic of organic type and thermal maturity were used to train a Partial Least Squares (PLS) calibration against a set of artificially matured samples spanning the pre- to mid-oil window. The PLS was applied to the study set and a post-mature set. Additionally, the PLS was applied to each point in hyperspectral maps for visualization of trends in maturity across sample sets and discrimination of organic matter types within a given map. In inorganic surveys on thin sections, a total of 14 unique inorganic minerals were identified in Raman spectra including quartz, dolomite, calcite, hematite and anhydrite. Shale thin sections tended to be dominated by organic material. OM was often observed mixed with inorganic minerals. Sand- and mudstones were dominated by inorganic minerals. The PLS extrapolated the post-mature set to reflectances >1.2%. The study set ranged from very immature to postmature in the median of map fit-peak parameters. However, point maturity maps indicate that matrix OM in all study samples is immature and that discreet organic particles selected for mapping, which may be inertinites, bias medians towards more-mature. The work demonstrates the capabilities of RMS to perform both whole-slide mineralogy and OM analysis with applications to formation evaluation in oil & gas, carbon sequestration and mining. Here, analysis of sealing formations in the well indicates high levels of immature organic matter that would not be a viable target for future oil production that could compromise the CO2 storage site. The work brings together diverse disciplines from geology and petrography to analytical chemistry, big data and microscopy.

Myers, Grant↗

Cybersecurity vulnerability mitigation framework

Systems, methods, and computer media for mitigating cybersecurity vulnerabilities of systems are provided herein. A current cybersecurity maturity of a system can be determined based on maturity criteria. The maturity criteria can be ranked based on importance. Solution candidates for increasing the cybersecurity maturity of the system can be determined based on the ranking. The solution candidates specify cybersecurity levels for the maturity criteria. A present state value reflecting the current cybersecurity maturity of the system can be calculated. For the solution candidates, an implementation state value and a transition state value can be determined. The implementation state value represents implementation of the maturity levels of the solution candidate, and the transition state value represents a transition from the present state value to the implementation state value. Based on the transition state values, a solution candidate can be selected for the system, and the system can be modified accordingly.

Gourisetti, Sri Nikhil Gupta↗

Comparing Life Histories of the Shortest-Lived Turtle Known (Chicken Turtles, Deirochelys reticularia ) with Long-Lived Blanding's Turtles ( Emydoidea blandingii )

Evolutionary theories predict major differences in life-history trait values of long- and short-lived organisms. Such comparisons have not been possible for chelonians because no shortlived turtle was known until research revealed that chicken turtles (Deirochelys reticularia; DR) have a maximum longevity of 21 yrs. Life-history trait values of DR females are 1) age at maturity of females = 5–6 yrs; 2) clutches per season = 1.6; 3) annual fecundity = 6–8 female eggs per female; 4) average juvenile survivorship from age 1 to maturity = 0.60; and 5) low average annual adult survivorship = 0.66. We compared DR with the very long-lived Blanding’s turtles (Emydoidea blandingii; EB) in Michigan. Over 14 yrs with no mortality (the minimum age at maturity of EB), the maximum potential fecundity produced by a single female embryo and her mature female offspring was 5 female eggs for EB and 1040 eggs for DR. Comparisons of life table output for approximately stable populations of DR and EB resulted in cohort generation times of 7 and 37 yrs, respectively. The life-history prediction that short-lived organisms should produce smaller offspring was not supported. Average wet mass of eggs is 10 g (8.4–11.3 g) for DR and 12 g (10–14 g) for EB; and average wet mass of hatchlings is 7.3 g (6–9 g) for DR and 9.3 g (6–13 g) for EB. Both differences are smaller than expected based on the difference in longevity. Here, short-lived female DR have an unusual tactic of investing in high fecundity and making substantial body sizespecific investment in large eggs, which may reflect why juvenile survivorship had greater influence on population change rates than did adult survivorship. In contrast, adult survivorship had the greatest influence on population change rates of EB. Comparison of cohorts of 1000 female DR and EB hatchlings highlights the differences in life histories of short- and long-lived turtles: all DR would be dead by the time the last female EB had reached maturity at 21 yrs of age.

59 BASIC BIOLOGICAL SCIENCES↗

Photosynthetic decline in aging perennial grass is not fully explained by leaf nitrogen

Abstract Aging in perennial plants is traditionally observed in terms of changes in end-of-season biomass; however, the driving phenological and physiological changes are poorly understood. We found that 3-year-old (mature) stands of the perennial grass Miscanthus×giganteus had 19–30% lower Anet than 1-year-old M.×giganteus (juvenile) stands; 10–34% lower maximum carboxylation rates of Rubisco and 34% lower light-saturated Anet (Asat). These changes could be related to nitrogen (N) limitations, as mature plants were larger and had 14–34% lower leaf N on an area basis (Na) than juveniles. However, N fertilization restored Na to juvenile levels but compensated only 50% of the observed decline in leaf photosynthesis with age. Comparison of leaf photosynthesis per unit of leaf N (PNUE) showed that mature stands had at least 26% lower PNUE than juvenile stands across all N fertilization rates, suggesting that other factors, besides N, may be limiting photosynthesis in mature stands. We hypothesize that sink limitations in mature stands could be causing feedback inhibition of photosynthesis which is associated with the age-related decline in photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗

Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

59 BASIC BIOLOGICAL SCIENCES↗

Propagation method and planting density influence canopy developmental transition and biomass productivity in Miscanthus × giganteus

Understanding how establishment practices influence the mechanisms underlying Miscanthus × giganteus (miscanthus) productivity and canopy development is critical for optimizing management. Data was collected during the juvenile (2011–2013) and mature (2024) phases of a long-term field experiment established in Urbana, Illinois, to evaluate the effects of propagation method (plug propagation [PP] and rhizome propagation [RP]), planting density (1.0, 0.75, and 0.25 plants m⁻²), and nitrogen application (0 and 67 kg N ha⁻¹) on end-of-season biomass yield, tiller mass, tiller density, and tiller height. Linear regression models identified the dominant predictors of yield across stand ages and management regimes. Planting density, nitrogen (N) application, and propagation method significantly influenced early yield and canopy development. During the juvenile phase, biomass yield was driven by tiller density due to canopy expansion; in the mature phase, yield became driven by tiller mass. The PP plots produced higher tiller density than the RP plots, resulting in faster canopy closure and higher juvenile-phase yields. Rhizome-propagated (RP) plots produced lower tiller density, but individual tillers were 3.3–6.4 g tiller −1 heavier than PP tillers. After the canopy reached equilibrium, the PP and RP yields were similar because greater RP tiller mass compensated for its lower tiller density. Higher planting density resulted in greater yield and tiller density during the second year (2012), but this effect was absent from the third year (2013) onward. In the juvenile phase, N fertilization enhanced yield by 1.6–3.4 Mg ha −1 . Initiating fertilization in 2013 on unfertilized plots produced biomass similar to that in fertilized plots, suggesting yield recovery in the mature phase. These findings revealed that establishment strategies, including propagation method and planting density, influence juvenile miscanthus canopy development and productivity, transitioning from tiller-density- to mass-dominated yields, but not mature phase productivity.

09 BIOMASS FUELS↗

Dl-3-n-butylphthalide promotes neurite outgrowth of primary cortical neurons by Sonic Hedgehog signaling via upregulating Gap43

Highlights: • NBP promoted neurite outgrowth in immature primary cortical neurons. • NBP increased axon and dendrite extension in mature primary cortical neurons. • NBP promoted neuritogenesis and neuronal plasticity via Shh signaling pathway. • The Shh signaling pathway increased Gap43 expression in primary cortical neuron. • NBP promoted neurite outgrowth by Shh signaling via upregulating Gap43 expression. Neurite outgrowth is the basis for wiring during the development of the nervous system. Dl-3-n-butylphthalide (NBP) has been recognized as a promising treatment to improve behavioral, neurological and cognitive outcomes in ischemic stroke. However, little is known about the effect and mechanism of NBP on the neurite outgrowth. In this study, we used different methods to investigate the potential effects of NBP on the neurite extension and plasticity of immature and mature primary cortical neurons and explored the underlying mechanisms. Our results demonstrated that in immature and mature cortical neurons, NBP promoted the neurite length and intersections, increased neuritic arborization, elevated numbers of neurite branch and terminal points and improved neurite complexity and plasticity of neuronal development processes. Besides, our data revealed that NBP promoted neurite extension and branching partly by activating Shh signaling pathway via increasing Gap43 expression both in immature and mature primary cortical neurons. The present study provided new insights into the contribution of NBP in neuronal plasticity and unveiled a novel pathway to induce Gap43 expression in primary cortical neurons.

60 APPLIED LIFE SCIENCES↗

Probing the subcellular nanostructure of engineered human cardiomyocytes in 3D tissue

Abstract The structural and functional maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential for pharmaceutical testing, disease modeling, and ultimately therapeutic use. Multicellular 3D-tissue platforms have improved the functional maturation of hiPSC-CMs, but probing cardiac contractile properties in a 3D environment remains challenging, especially at depth and in live tissues. Using small-angle X-ray scattering (SAXS) imaging, we show that hiPSC-CMs matured and examined in a 3D environment exhibit a periodic spatial arrangement of the myofilament lattice, which has not been previously detected in hiPSC-CMs. The contractile force is found to correlate with both the scattering intensity ( R 2 = 0.44) and lattice spacing ( R 2 = 0.46). The scattering intensity also correlates with lattice spacing ( R 2 = 0.81), suggestive of lower noise in our structural measurement than in the functional measurement. Notably, we observed decreased myofilament ordering in tissues with a myofilament mutation known to lead to hypertrophic cardiomyopathy (HCM). Our results highlight the progress of human cardiac tissue engineering and enable unprecedented study of structural maturation in hiPSC-CMs.

36 MATERIALS SCIENCE↗

Structural basis for breadth development in the HIV-1 V3-glycan targeting DH270 antibody clonal lineage

Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza has therefore focused on recapitulating the natural affinity maturation process. Here, we determine structures of antibodies in complex with HIV-1 Envelope for all observed members and ancestral states of the broadly neutralizing HIV-1 V3-glycan targeting DH270 antibody clonal B cell lineage. These structures track the development of neutralization breadth from the unmutated common ancestor and define affinity maturation at high spatial resolution. By elucidating contacts mediated by key mutations at different stages of antibody development we identified sites on the epitope-paratope interface that are the focus of affinity optimization. Thus, our results identify bottlenecks on the path to natural affinity maturation and reveal solutions for these that will inform immunogen design aimed at eliciting a broadly neutralizing immune response by vaccination.

60 APPLIED LIFE SCIENCES↗

Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

The maturation and installation of the active site metal cluster (FeMo-co, Fe 7 S 9 CMo- R -homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe 8 S 9 C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDK RS contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK RS ). Apo-NifDK RS could be activated for N 2 reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK RS plus E. coli produced NifB RS and NifH RS were sufficient to yield active NifDK RS when incubated with the necessary substrates (homocitrate, molybdate, and S -adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifH RS and NifB RS proteins were independently shown to be functional. The reconstituted NifDK RS demonstrated reduction of N 2 , protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH RS , NifB RS , and apo-NifDK RS .

Payá-Tormo, Lucía↗

A Maize GT14 Family Glycosyltransferase Affects Cell Wall Composition and Carbohydrate Export from Source Leaves

Sucrose translocation from photosynthetic leaves to distant parts of a plant, such as seeds and roots, is a critical aspect of plant growth and development and a major determinant of crop yield. To identify genes contributing to this process in maize (Zea mays), we isolated four allelic mutants, carbohydrate partitioning defective7, 48, 49 (cpd7, cpd48, cpd49) and a UniformMu insertion (mu1049954), all of which exhibited reduced growth and fertility and hyperaccumulation of starch and soluble sugars in mature leaves. Consistent with carbohydrate accumulation, cpd7 mutants exhibited reduced sucrose export from mature leaves. Cpd7 encodes a Golgi-resident glucuronosyltransferase belonging to the Glycosyltransferase14 (GT14) family, which is involved in decoration of type II arabinogalactan proteins. No previously described GT14 mutants exhibit reduced sucrose transport or carbohydrate partitioning defects. Additionally, we show that mature leaves of cpd7 mutants have reduced cellulose content and an altered cell wall composition. Further, cpd7 mutants exhibit ectopic phloem lignification likely as a compensatory mechanism for reduced cell wall integrity. Collectively, our data suggest that Cpd7 functions to facilitate cell wall development in the phloem, which is required for efficient sucrose export from mature maize leaves.

59 BASIC BIOLOGICAL SCIENCES↗

Differential early–life survival underlies the adaptive significance of temperature–dependent sex determination in a long–lived reptile

1. Many ectotherms rely on temperature cues experienced during development to determine offspring sex. The first descriptions of temperature-dependent sex determination (TSD) were made over 50 years ago, yet an understanding of its adaptive significance remains elusive, especially in long-lived taxa. 2. One novel hypothesis predicts that TSD should be evolutionarily favoured when two criteria are met—(a) incubation temperature influences annual juvenile survival and (b) sexes mature at different ages. Under these conditions, a sex-dependent effect of incubation temperature on offspring fitness arises through differences in age at sexual maturity, with the sex that matures later benefiting disproportionately from temperatures that promote juvenile survival. 3. The American alligator (Alligator mississippiensis) serves as an insightful model in which to test this hypothesis, as males begin reproducing nearly a decade after females. Here, through a combination of artificial incubation experiments and mark-recapture approaches, we test the specific predictions of the survival-to-maturity hypothesis for the adaptive value of TSD by disentangling the effects of incubation temperature and sex on annual survival of alligator hatchlings across two geographically distinct sites. 4. Hatchlings incubated at male-promoting temperatures (MPTs) consistently exhibited higher survival compared to those incubated at female-promoting temperatures. This pattern appears independent of hatchling sex, as females produced from hormone manipulation at MPT exhibit similar survival to their male counterparts. 5. Additional experiments show that incubation temperature may affect early-life survival primarily by affecting the efficiency with which maternally transferred energy resources are used during development. 6. Results from this study provide the first explicit empirical support for the adaptive value of TSD in a crocodilian and point to developmental energetics as a potential unifying mechanism underlying persistent survival consequences of incubation temperature.

59 BASIC BIOLOGICAL SCIENCES↗

Soil net nitrogen mineralization and leaching under Miscanthus × giganteus and Zea mays

Abstract The winter fallow period common in annual cropping systems leaves soils vulnerable to erosion and nutrient loss, especially to nitrogen (N) leaching. This vulnerability can be mitigated with perennial crops that have living roots in the ground year‐round. The mechanisms, magnitude, and consistency with which perennial crops retain N are not clear. We used an experiment to test whether a perennial crop, miscanthus ( Miscanthus × giganteus Greef et Deu.), would leach less N than continuous maize ( Zea mays L.) and how soil net N mineralization (N min ) may explain observed leaching under varied environment and management conditions. The experiment included three crossed factors: (1) cropping system (maize, juvenile miscanthus = 1–2 years old, mature miscanthus = 3–4 years old); (2) N fertilization (0 and 224 kg N ha −1 ); and (3) environment (four site‐years at two locations in Iowa, USA, that differed in climate and soil fertility). We measured N cycling dynamics, including: inorganic soil N (ammonium + nitrate), in situ N min , N leaching, crop N uptake, and calculated system N use efficiency. There were many complex interactions among factors. On average, cumulative N min under juvenile miscanthus was 111% greater than maize, but as miscanthus matured, there was no difference in N min between the perennial crop and maize. There was no difference in N leaching between juvenile miscanthus and maize, but mature miscanthus decreased N leaching by 42% and 88% compared to maize (with and without N fertilization, respectively). Across all treatments, there was no relationship between N min and N leaching, suggesting other mechanisms are regulating N leaching. Overall, mature miscanthus shows promise as a tool to reduce N losses in areas dominated by annual row‐crops.

54 ENVIRONMENTAL SCIENCES↗

Temperature dependence of viscoelastic Poisson’s ratio of cement mortar

Abstract Concrete creep research has focused primarily on uniaxial response. However, biaxially prestressed concrete structures are common, resulting in a multiaxial stress state that can complicate the behavior of a viscoelastic material like concrete. Significant creep strains may be induced in directions transverse to each principle stress due to Poisson’s effect. Past research is unclear regarding the viscoelastic or viscoplastic properties of concrete outside of uniaxial response. It has been reported in separate studies that concrete viscoelastic/viscoplastic Poisson’s ratio (VPR) is an increasing, decreasing and constant function with time, with all reported measurements performed at room temperature. In this paper, the 3D basic creep response of mature cement mortar is examined using a confined compression experiment that allows direct determination of the full stress and infinitesimal strain tensors in a single test, which enables the determination of VPR under a multiaxial stress state. For this purpose, a unique, miniature version of the standardized concrete creep frame is designed that is amenable to placing in climate chambers and temperature ovens. The experimental results indicate that the VPR of sealed, mature cement mortar is nearly constant and equal to the elastic at room temperature, while the VPR gradually increases with time when measured at 60 °C.

Construction & Building Technology↗

Unbalancing Symbiotic Nitrogen Fixation: Can We Make Effectiveness More Effective?

One of the most critical uses of carbon fuels is in generating nitrogen fertilizer. Atmospheric dinitrogen is too stable to be used directly by plants, so plants need other chemical forms of nitrogen. Nitrogen fertilizer production uses ~4% of the world’s natural gas. Making N fertilizers leverages methane’s energy content by enabling additional energy to be captured into food and fiber through increased photosynthesis. Manufacturing fertilizer is essential—without it there would only be enough combined nitrogen to feed ~3 billion of the world’s ~7 billion people. How to alter this addiction is not obvious, but the best chance to secure substantial and sustainable amounts of future N might be through symbiotic nitrogen fixation (SNF). SNF describes mutualistic interactions where nitrogen-fixing bacteria provide fixed nitrogen to their plant hosts, freeing them from the need for nitrogen fertilizers. SNF already occurs on a large scale; "biological nitrogen” contributes about 5 Tg N/yr to American agriculture, a quarter of the nitrogen needed. Improving SNF and replacing inorganic fertilizers are both important goals in establishing sustainable and more energy-efficient farming. SNF has been used in agriculture for millennia, largely by using legumes like beans or alfalfa in mixed cropping systems. Legume SNF occurs in root nodules, organs that develop after a growing root is infected by bacteria called rhizobia. In the plant cells of a legume nodule, specialized forms of rhizobia fix nitrogen and can produce enough ammonia to supply the growing plant. The exchange of photosynthetically derived plant carbon compounds for nitrogen reduced by the bacteria is the engine that drives the symbiosis. Important details about the organization of development and metabolism in SNF remain to be determined, such as how plant and bacterial metabolism are coordinated as nitrogen is being fixed and what governs the overall level of nitrogen fixation. Most bacterial mutations that affect SNF completely block fixation (e.g. are Fix – ), providing a limited window into the feedback interactions that must be part of the process. We have investigated an unusual mutant of Sinorhizobium meliloti, a symbiotic partner of alfalfa, that fixes dinitrogen at a normal rate (i.e., it is Fix + ) but is not effective in supporting plant growth (i.e., it is ineffective or Eff – ). We have shown that the mutant has a specific mutation in the glnD gene, which codes for a protein that regulates the bacterial response to nitrogen stress, among other key pathways. A Fix + Eff – phenotype contains an unavoidable puzzle—how is it possible for the bacteria to fix nitrogen at a normal rate and without benefiting the nitrogen starved plant host? After eliminating other possibilities, we proposed that the bacteria synthesize a nitrogen-containing compound that the plant can’t use so that acquiring this compound does not relieve plant nitrogen stress. Extracts of nodules made by glnD mutant strains have high concentrations of pyruvate canaline oxime (PCO), a derivative of the plant defensive compound canaline, a toxic analog of ornithine, an amino acid. Many legumes produce canaline and the related arginine mimic canavanine. For reasons we do not yet understand, the mutation in glnD appears to trigger significant overproduction of canaline in a defense response that leads to substantial synthesis of PCO. We expected the bacteria to be able to use PCO, but we have not been able to grow S. meliloti on PCO under several conditions. PCO thus appears to be a metabolic dead end for both the plant and bacteria. We also examined bacterial catabolic pathways that degrade arginine and canavanine and showed that mutation of these did not alter the symbiosis in an obvious way. In related experiments to examine the metabolic development of root nodules, we carried out an ambitious experiment to simultaneously measure metabolites and protein changes during nodule maturation. For these experiments we used a related symbiotic interaction between S. medicae and Medicago truncatula. M. truncatula has a simpler genome than alfalfa so we could identify plant proteins based on predictions from the DNA sequence. Nodules formed on Medicago grow linearly, with the least mature tissues at the tip. Because development of nitrogen fixation is accompanied by the production of the pigmented plant oxygen carrier protein leghemoglobin, we were able to locate and manually separate the tip, transitional, and mature nitrogen-fixing regions from each other. Advances in proteomic and metabolomic techniques allowed us to analyze these tissues from a pool of as few as 10 nodules. We observed correlated transitions of various enzymes in the plant and bacterial proteomes from those characteristic of free-living metabolism to the microaerobic metabolism of the nitrogen-fixing tissues. The metabolome could not be separated into plant and bacterial domains but was consistent with this overall transition. A subsequent paper examined proteolysis in nodule bacteria. Protein turnover in mature regions of the nodule is significant. We were able to show complexes between various proteins and important proteases, using precipitation capture techniques to isolate the proteases and sensitive proteomic analysis to reveal the associated proteins.

09 BIOMASS FUELS↗

Active High Assurance Authentication Protocol (AHAAP)

The AHAAP Maturation Project involves maturation and evaluation of a patented zero-trust tamper-resistant high-assurance session-less dynamic and active device authentication protocol that simultaneously authenticates identity and provides integrity verification in a single step, substantially reducing the risk of cyberattack, and eliminating the need for costly and complex conventional communication security systems requirements (i.e., cryptography, Public Key Infrastructure (PKI), and key management). These cybersecurity attributes of the technology must be preserved when applying the technology to different cybersecurity solutions, including Command & Control (C&C), Over-the-Air (OTA) update, Common Access Card (CAC), and distributed energy resource (DER) implementations, among others. The technology research objective is to test and verify that the cybersecurity attributes of the technology are not degraded in different cybersecurity applications. The primary technology development objective is to build minimum viable products to demonstrate the technology addresses today’s cybersecurity threats so that prospective investors, strategic partners, regulatory agencies, and commercial customers can interact with and assess the protection assured by the technology. The AHAAP Maturation Project goal is to develop, test, and validate one or more AHAAP implementations. The AHAAP Maturation Project tasks are: (i) engineer AHAAP implementation software, (ii) build a functional prototype that implements the AHAAP software for demonstration, testing, analysis, and evaluation purposes, and (iii) generate a report detailing the results of the AHAAP C&C software and hardware implementation. The final project deliverables are: (i) AHAAP software implementation and prototype, (ii) a report from Sandia National Laboratories detailing the results of the AHAAP implementations.

97 MATHEMATICS AND COMPUTING↗