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

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At least 1,081 records · Page 60
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

An Artificial Intelligence-Assisted Method for Dementia Detection Using Images from the Clock Drawing Test

Background: Widespread dementia detection could increase clinical trial candidates and enable appropriate interventions. Since the Clock Drawing Test (CDT) can be potentially used for diagnosing dementia-related disorders, it can be leveraged to develop a computer-aided screening tool. Objective: To evaluate if a machine learning model that uses images from the CDT can predict mild cognitive impairment or dementia. Methods: Images of an analog clock drawn by 3,263 cognitively intact and 160 impaired subjects were collected during in-person dementia evaluations by the Framingham Heart Study. We processed the CDT images, participant’s age, and education level using a deep learning algorithm to predict dementia status. Results: When only the CDT images were used, the deep learning model predicted dementia status with an area under the receiver operating characteristic curve (AUC) of 81.3% ± 4.3%. A composite logistic regression model using age, level of education, and the predictions from the CDT-only model, yielded an average AUC and average F1 score of 91.9% ±1.1% and 94.6% ±0.4%, respectively. Conclusion: Our modeling framework establishes a proof-of-principle that deep learning can be applied on images derived from the CDT to predict dementia status. When fully validated, this approach can offer a cost-effective and easily deployable mechanism for detecting cognitive impairment.

Neurosciences & Neurology↗
DOE OSTI2023

Optimized purification methods for metallic contaminant removal from directly recycled Li-ion battery cathodes

Metallic contaminants pose a significant challenge to the viability of directly recycling Li-ion batteries. To date, few strategies exist to selectively remove metallic impurities from mixtures of shredded end-of-life material (black mass; BM) without concurrently damaging the structure and electrochemical performance of the target active material. We herein present tailored methods to selectively ionize two major contaminants—Al and Cu—while retaining a representative cathode (LiNi 0.33 Mn 0.33 Co 0.33 O 2 ; NMC-111) intact. This BM purification process is conducted at moderate temperatures in a KOH-based solution matrix. We rationally evaluate approaches to increase both the kinetic corrosion rate and the thermodynamic solubility of Al 0 and Cu 0 , and evaluate the impact of these treatment conditions on the structure, chemistry, and electrochemical performance of NMC. Specifically, we explore the impacts of chloride-based salts, a strong chelating agent, elevated temperature, and sonication on the rate and extent of contaminant corrosion, while concurrently evaluating the effects on NMC. The reported BM purification process is then demonstrated on samples of “simulated BM” containing a practically relevant 1 wt% concentration of Al or Cu. Increasing the kinetic energy of the purifying solution matrix through elevated temperature and sonication accelerates the corrosion of metallic Al and Cu, such that ∼100% corrosion of 75 μm Al and Cu particles is achieved within 2.5 hr. Further, we determine that effective mass transport of ionized species critically impacts the efficacy of Cu corrosion, and that saturated Cl – hinders rather than accelerates Cu corrosion by increasing solution viscosity and introducing competitive pathways for Cu surface passivation. The purification conditions do not induce bulk structural damage to NMC, and electrochemical capacity is maintained in half-cell format. Testing in full cells suggests that a limited quantity of residual surface species are present after treatment, which initially disrupt electrochemical behavior at the graphite anode but are subsequently consumed. Process demonstration on simulated BM suggests that contaminated samples—which prior to treatment show catastrophic electrochemical performance—can be recovered to pristine electrochemical capacity. The reported BM purification method offers a compelling and commercially viable solution to address contamination, particularly in the “fine” fraction of BM where contaminant sizes are on the same order of magnitude as NMC and where traditional separation approaches are unfeasible. Thus, this optimized BM purification technique offers a pathway towards viable direct recycling of BM feedstocks that would otherwise be unusable.

25 ENERGY STORAGE↗
DOE OSTI2019

Effects of Extractive Ammonia Pretreatment on the Ultrastructure and Glycan Composition of Corn Stover

Lignocellulosic biomass is highly recalcitrant and requires a pretreatment step to improve the enzyme accessibility and fermentable sugar yields during enzymatic hydrolysis. Our previous studies demonstrated the rearrangement of the hydrogen bond network within CIII, makes it “amorphous-like” and facilitates easier glucan chain extraction by enzyme. Also, these changes increase the number of solvent-exposed glucan chain hydrogen bonds with water ~50% lowering the surface-bound cellulase by 60–70%. Also, major chemical modifications to lignin occur via ammonolysis of ester-linked ferulate and coumarate linkage. These apparent ultrastructural changes help the enhancement of cellulase activity resulting in higher production of fermentable sugars during enzyme hydrolysis of EA pretreated corn stover relative to Ammonia Fiber Expansion (AFEX) pretreatment. To understand ultra-structural modifications that occur during EA pretreatment, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) were used to examine untreated and EA-pretreated corn stover in an effort to visualize changes in the biomass resulting from the pretreatment. In addition, Immunofluorescence Microscopy was applied to both untreated and pretreated plant cell walls using glycan-directed monoclonal antibodies to reveal possible changes in the spatial distributions of wall glycan epitopes resulting from EA pretreatment. This evaluation was complemented with glycome profiling to determine the glycan epitope compositions of EA-pretreated cell walls relative to untreated and AFEX pretreated corn stover, where lignin and carbohydrates are not extracted. Distinct differences could be observed in the case of xyloglucan, unsubstituted and substituted pectin- and pectic-arabinogalactan-epitope levels in the plant cell wall after EA pretreatment compared with untreated and AFEX-pretreated walls. Liquid ammonia induced de-lignification of cell wall has helped to clearly identify the glucans that are intact after pretreatment. These studies support some of our hypothesis that liquid ammonia cleaves lignin–carbohydrate ester linkages, partially solubilizing lignin and its associated carbohydrates. Specifically, the imaging studies clearly show that some of the cell wall components are extracted as a separate liquid stream during the EA process, thereby creating porous, interconnected tunnel-like networks in the plant cell walls thereby providing better access of carbohydrate polymers to enzymes and thereby increasing the sugar yield from the EA-pretreated biomass.

09 BIOMASS FUELS↗
DOE OSTI2022

We Must Stop Fossil Fuel Emissions to Protect Permafrost Ecosystems

Climate change is an existential threat to the vast global permafrost domain. The diverse human cultures, ecological communities, and biogeochemical cycles of this tenth of the planet depend on the persistence of frozen conditions. The complexity, immensity, and remoteness of permafrost ecosystems make it difficult to grasp how quickly things are changing and what can be done about it. Here, we summarize terrestrial and marine changes in the permafrost domain with an eye toward global policy. While many questions remain, we know that continued fossil fuel burning is incompatible with the continued existence of the permafrost domain as we know it. If we fail to protect permafrost ecosystems, the consequences for human rights, biosphere integrity, and global climate will be severe. The policy implications are clear: the faster we reduce human emissions and draw down atmospheric CO 2 , the more of the permafrost domain we can save. Emissions reduction targets must be strengthened and accompanied by support for local peoples to protect intact ecological communities and natural carbon sinks within the permafrost domain. Some proposed geoengineering interventions such as solar shading, surface albedo modification, and vegetation manipulations are unproven and may exacerbate environmental injustice without providing lasting protection. Conversely, astounding advances in renewable energy have reopened viable pathways to halve human greenhouse gas emissions by 2030 and effectively stop them well before 2050. We call on leaders, corporations, researchers, and citizens everywhere to acknowledge the global importance of the permafrost domain and work towards climate restoration and empowerment of Indigenous and immigrant communities in these regions.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Chronic Physical Disturbance Substantially Alters the Response of Biological Soil Crusts to a Wetting Pulse, as Characterized by Metatranscriptomic Sequencing

Biological soil crusts (biocrusts) are microbial communities that are a feature of arid surface soils worldwide. In drylands where precipitation is pulsed and ephemeral, the ability of biocrust microbiota to rapidly initiate metabolic activity is critical to their survival. Community gene expression was compared after a short duration (1 h) wetting pulse in both intact and soils disturbed by chronic foot trampling. Across the metatranscriptomes the majority of transcripts were cyanobacterial in origin, suggesting that cyanobacteria accounted for the bulk of the transcriptionally active cells. Chronic trampling substantially altered the functional profile of the metatranscriptomes, specifically resulting in a significant decrease in transcripts for nitrogen fixation. Soil depth (biocrust and below crust) was a relatively small factor in differentiating the metatranscriptomes, suggesting that the metabolically active bacteria were similar between shallow soil horizons. The dry samples were consistently enriched for hydrogenase genes, indicating that molecular hydrogen may serve as an energy source for the desiccated soil communities. The water pulse was associated with a restructuring of the metatranscriptome, particularly for the biocrusts. Biocrusts increased transcripts for photosynthesis and carbon fixation, suggesting a rapid resuscitation upon wetting. In contrast, the trampled surface soils showed a much smaller response to wetting, indicating that trampling altered the metabolic response of the community. Finally, several biogeochemical cycling genes in carbon and nitrogen cycling were assessed for their change in abundance due to wetting in the biocrusts. Different transcripts encoding the same gene product did not show a consensus response, with some more abundant in dry or wet biocrusts, highlighting the challenges in relating transcript abundance to biogeochemical cycling rates. These observations demonstrate that metatranscriptome sequencing was able to distinguish alterations in the function of arid soil microbial communities at two varying temporal scales, a long-term ecosystems disturbance through foot trampling, and a short term wetting pulse. Thus, community metatranscriptomes have the potential to inform studies on the response and resilience of biocrusts to various environmental perturbations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus’s ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity

Thermococcus kodakarensis (T. kodakarensis), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N 6 position of adenine on one strand of the recognition sequences GTGAAG and TTCAAG, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N) 20 /(N) 18 and TTCAAG(N) 10 /(N) 8 , respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus

Introduction African Swine Fever (ASF) is a highly infectious disease of pigs, caused by African swine fever virus (ASFV). The lack of vaccines and drugs makes strict disinfection practices to be one of the main measurements to curb the transmission of ASF. Therefore, it is important to assess if all viruses are inactivated after disinfection or after long time exposure in their natural conditions. Currently, the infectivity of ASFV is determined by virus isolation and culture in a biosafety level 3 (BSL-3) laboratory. However, BSL-3 laboratories are not readily available, need skilled expertise and may be time consuming. Methods In this study, a Triton X-100 assisted PMAxx-qPCR method was developed for rapid assessment of infectious ASFV in samples. PMAxx, an improved version of propidium monoazide (PMA), can covalently cross-link with naked ASFV-DNA or DNA inside inactivated ASFV virions under assistance of 0.1% (v/v) TritonX-100, but not with ASFV-DNA inside live virions. Formation of PMAxx-DNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, the limit of detection of the PMAxx-qPCR assay was 2.32log 10 HAD 50 /mL of infectious ASFV. Testing different samples showed that the PMAxx-qPCR assay was effective to evaluate intact ASFV virions after treatment by heat or chemical disinfectants and in simulated samples such as swine tissue homogenate, swine saliva swabs, and environmental swabs. However, whole-blood and saliva need to be diluted before testing because they may inhibit the PCR reaction or the cross-linking of PMAxx with DNA. Conclusion The Triton X-100 assisted PMAxx-qPCR assay took less than 3 h from sample to result, offering an easier and faster way for assessing infectious ASFV in samples from places like pig farms and pork markets.

Liu, Huan↗
DOE OSTI2023

BONCAT-FACS-Seq reveals the active fraction of a biocrust community undergoing a wet-up event

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Stomatal Responses to Light, CO 2 , and Mesophyll Tissue in Vicia faba and Kalanchoë fedtschenkoi

The responses of stomatal aperture to light intensity and CO 2 concentration were studied in both Vicia faba (C 3 ) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C 3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C 3 in response to light and CO 2 . By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C 3 . This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C 3 and CAM species.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Duration of Contagion of Foot-And-Mouth Disease Virus in Infected Live Pigs and Carcasses

Data-driven modeling of incursions of high-consequence, transboundary pathogens of animals is a critical component of veterinary preparedness. However, simplifying assumptions and excessive use of proxy measures to compensate for gaps in available data may compromise modeled outcomes. The current investigation was prospectively designed to address two major gaps in current knowledge of foot-and-mouth disease virus (FMDV) pathogenesis in pigs: the end (duration) of the infectious period and the viability of FMDV in decaying carcasses. By serial exposure of sentinel groups of pigs to the same group of donor pigs infected by FMDV A24 Cruzeiro, it was demonstrated that infected pigs transmitted disease at 10 days post infection (dpi), but not at 15 dpi. Assuming a latent period of 1 day, this would result in a conservative estimate of an infectious duration of 9 days, which is considerably longer than suggested by a previous report from an experiment performed in cattle. Airborne contagion was diminished within two days of removal of infected pigs from isolation rooms. FMDV in muscle was inactivated within 7 days in carcasses stored at 4°C. By contrast, FMDV infectivity in vesicle epithelium harvested from intact carcasses stored under similar conditions remained remarkably high until the study termination at 11 weeks post mortem. The output from this study consists of experimentally determined data on contagion associated with FMDV-infected pigs. This information may be utilized to update parameterization of models used for foot-and-mouth disease outbreak simulations involving areas of substantial pig production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Acute Exposure to Aerosolized Nanoplastics Modulates Redox-Linked Immune Responses in Human Airway Epithelium

Micro- and nanoplastics (MPs and NPs) are pervasive environmental pollutants detected in aquatic ecosystems, with emerging evidence suggesting their presence in airborne particles generated by water body motion. Inhalation exposure to airborne MPs and NPs remains understudied despite documented links between occupational exposure to these particles and adverse respiratory outcomes, including airway inflammation, oxidative stress, and chronic respiratory diseases. This study explored the effects of acute NP exposure on a fully differentiated 3D human airway epithelial model derived from 14 healthy donors. Airway epithelium was exposed to aerosolized 50 nm polystyrene NPs at concentrations ranging from 2.5 to 2500 µg/mL for three minutes per day over three days. Functional assays revealed no significant alterations in tissue integrity, cell survival, mucociliary clearance, or cilia beat frequency, suggesting intact epithelial function post-exposure. However, cytokine and chemokine profiling identified a significant five-fold increase in CCL3 (MIP-1α), a neutrophilic chemoattractant, in NP-exposed samples compared to controls. This was corroborated by increased neutrophil chemotaxis in response to conditioned media from NP-exposed tissues, indicating a pro-inflammatory neutrophilic response. Conversely, levels of interleukins (IL-21, IL-2, IL-15), CXCL10, and TGF-β were significantly reduced, suggesting immunomodulatory effects that may impair adaptive immune responses and tissue repair mechanisms. These findings demonstrate that short-term exposure to NP-containing aerosols induces a distinct pro-inflammatory response in airway epithelium, characterized by enhanced neutrophil recruitment and reduced secretion of key immune modulators. These findings underscore the potential for aerosolized NPs to induce oxidative and inflammatory stress, raising concerns about their long-term impact on respiratory health and redox regulation.

Biochemistry & Molecular Biology↗
DOE OSTI2018

Systematic Quantum Cluster Typical Medium Method for the Study of Localization in Strongly Disordered Electronic Systems

Great progress has been made in recent years towards understanding the properties of disordered electronic systems. In part, this is made possible by recent advances in quantum effective medium methods which enable the study of disorder and electron-electronic interactions on equal footing. They include dynamical mean-field theory and the Coherent Potential Approximation, and their cluster extension, the dynamical cluster approximation. Despite their successes, these methods do not enable the first-principles study of the strongly disordered regime, including the effects of electronic localization. The main focus of this review is the recently developed typical medium dynamical cluster approximation for disordered electronic systems. This method has been constructed to capture disorder-induced localization and is based on a mapping of a lattice onto a quantum cluster embedded in an effective typical medium, which is determined self-consistently. Unlike the average effective medium-based methods mentioned above, typical medium-based methods properly capture the states localized by disorder. The typical medium dynamical cluster approximation not only provides the proper order parameter for Anderson localized states, but it can also incorporate the full complexity of Density-Functional Theory (DFT)-derived potentials into the analysis, including the effect of multiple bands, non-local disorder, and electron-electron interactions. After a brief historical review of other numerical methods for disordered systems, we discuss coarse-graining as a unifying principle for the development of translationally invariant quantum cluster methods. Together, the Coherent Potential Approximation, the Dynamical Mean-Field Theory and the Dynamical Cluster Approximation may be viewed as a single class of approximations with a much-needed small parameter of the inverse cluster size which may be used to control the approximation. We then present an overview of various recent applications of the typical medium dynamical cluster approximation to a variety of models and systems, including single and multiband Anderson model, and models with local and off-diagonal disorder. We then present the application of the method to realistic systems in the framework of the DFT and demonstrate that the resulting method can provide a systematic first-principles method validated by experiment and capable of making experimentally relevant predictions. We also discuss the application of the typical medium dynamical cluster approximation to systems with disorder and electron-electron interactions. Most significantly, we show that in the limits of strong disorder and weak interactions treated perturbatively, that the phenomena of 3D localization, including a mobility edge, remains intact. However, the metal-insulator transition is pushed to larger disorder values by the local interactions. We also study the limits of strong disorder and strong interactions capable of producing moment formation and screening, with a non-perturbative local approximation. Here, we find that the Anderson localization quantum phase transition is accompanied by a quantum-critical fan in the energy-disorder phase diagram.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2020

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Small-Angle Neutron Scattering for Studying Lipid Bilayer Membranes

Small-angle neutron scattering (SANS) is a powerful tool for studying biological membranes and model lipid bilayer membranes. The length scales probed by SANS, being from 1 nm to over 100 nm, are well-matched to the relevant length scales of the bilayer, particularly when it is in the form of a vesicle. However, it is the ability of SANS to differentiate between isotopes of hydrogen as well as the availability of deuterium labeled lipids that truly enable SANS to reveal details of membranes that are not accessible with the use of other techniques, such as small-angle X-ray scattering. In this work, an overview of the use of SANS for studying unilamellar lipid bilayer vesicles is presented. The technique is briefly presented, and the power of selective deuteration and contrast variation methods is discussed. Approaches to modeling SANS data from unilamellar lipid bilayer vesicles are presented. Finally, recent examples are discussed. While the emphasis is on studies of unilamellar vesicles, examples of the use of SANS to study intact cells are also presented.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Tetranuclear Polypyridylruthenium(II) Complexes as Selective Nucleic Acid Stains for Flow Cytometric Analysis of Monocytic and Epithelial Lung Carcinoma Large Extracellular Vesicles

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

A High-Throughput MEMS-Based Differential Scanning Calorimeter for Direct Thermal Characterization of Antibodies

Calorimeters, which can be used for rapid thermal characterization of biomolecules, are getting intense attention in drug development. This paper presents a novel MEMS-based differential scanning calorimeter (DSC) for direct thermal characterization of protein samples. The DSC consisted of a pair of temperature sensors made by vanadium oxide (VOx) film with a temperature coefficient of resistivity of –0.025/K at 300 K, a microfluidic device with high thermal insulation (2.8 K/mW), and a Peltier heater for linear temperature scanning. The DSC exhibited high sensitivity (6.1 µV/µW), low noise (0.4 µW), high scanning rate (45 K/min), and low sample consumption volume (0.63 µL). The MEMS DSC was verified by measuring the temperature-induced denaturation of lysozyme at different pH, and then used to study the thermal stability of a monoclonal antibody (mAb), an antigen-binding fragment (Fab), and a dual variable domain immunoglobulin (DVD-Ig) at pH = 6. The results showed that lysozyme is a stable protein in the pH range of 4.0–8.0. The protein stability study revealed that the transition temperatures of the intact Fab fragment, mAb, and DVD proteins were comparable with conformational stability results obtained using conventional commercial DSC. These studies demonstrated that the MEMS DSC is an effective tool for directly understanding the thermal stability of antibodies in a high-throughput and low-cost manner compared to conventional calorimeters.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Exposed Phosphatidylserine as a Biomarker for Clear Identification of Breast Cancer Brain Metastases in Mouse Models

Brain metastasis is the most common intracranial malignancy in adults. The prognosis is extremely poor, partly because most patients have more than one brain lesion, and the currently available therapies are nonspecific or inaccessible to those occult metastases due to an impermeable blood–tumor barrier (BTB). Phosphatidylserine (PS) is externalized on the surface of viable endothelial cells (ECs) in tumor blood vessels. In this study, we have applied a PS-targeting antibody to assess brain metastases in mouse models. Fluorescence microscopic imaging revealed that extensive PS exposure was found exclusively on vascular ECs of brain metastases. The highly sensitive and specific binding of the PS antibody enables individual metastases, even micrometastases containing an intact BTB, to be clearly delineated. Furthermore, the conjugation of the PS antibody with a fluorescence dye, IRDye 800CW, or a radioisotope, 125I, allowed the clear visualization of individual brain metastases by optical imaging and autoradiography, respectively. In conclusion, we demonstrated a novel strategy for targeting brain metastases based on our finding that abundant PS exposure occurs on blood vessels of brain metastases but not on normal brain, which may be useful for the development of imaging and targeted therapeutics for brain metastases.

Oncology↗