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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
NASA NTRS2002

Persistent Ehrlichia chaffeensis infection occurs in the absence of functional major histocompatibility complex class II genes

Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis. We investigated the impact of two genes that control macrophage and T-cell function on murine resistance to E. chaffeensis. Congenic pairs of wild-type and toll-like receptor 4 (tlr4)- or major histocompatibility complex class II (MHC-II)-deficient mice were used for these studies. Wild-type mice cleared the infection within 2 weeks, and the response included macrophage activation and the synthesis of E. chaffeensis-specific Th1-type immunoglobulin G response. The absence of a functional tlr4 gene depressed nitric oxide and interleukin 6 secretion by macrophages and resulted in short-term persistent infections for > or =30 days. In the absence of MHC-II alleles, E. chaffeensis infections persisted throughout the entire 3-month evaluation period. Together, these data suggest that macrophage activation and cell-mediated immunity, orchestrated by CD4(+) T cells, are critical for conferring resistance to E. chaffeensis.

NASA Discipline Cell Biology↗
NASA NTRS1989

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Non-NASA Center↗
NASA NTRS1988

Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

NASA Discipline Number 40-10↗
DOE OSTI2020

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

Abstract A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2‐µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single‐use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥10 4 colony‐forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 10 4 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Model acetylcholinesterase‐Fc fusion glycoprotein biotechnology system for the manufacture of an organophosphorus toxicant bioscavenging countermeasure

Abstract Organophosphate (OP) toxicants remain an active threat to public health and to warfighters in the military. Current countermeasures require near immediate administration following OP exposure and are reported to have controversial efficacies. Acetylcholinesterase (AChE) fused to the human immunoglobulin 1 (IgG1) Fc domain (AChE‐Fc) is a potential bioscavenger for OP toxicants, but a reproducible AChE‐Fc biomanufacturing strategy remains elusive. This report is the first to establish a comprehensive laboratory‐scale bioprocessing strategy that can reproducibly produce AChE‐Fc and AChE(W86A)‐Fc which is a mutated AChE protein with reduced enzymatic activity. Characterization studies revealed that AChE‐Fc and AChE(W86A)‐Fc are N ‐glycosylated dimeric fusion glycoproteins but only AChE‐Fc had the capability to bind to paraoxon (a model OP). This AChE‐Fc fusion glycoprotein bioprocessing strategy can be leveraged during industrial biomanufacturing development, while the research‐grade AChE‐Fc proteins can be used to determine the potential clinical relevance of the countermeasure against OP toxicants.

Biel, Thomas G.↗
DOE OSTI2023

Crystal Structures of Diaryl Hydrazone and Sulfone Stabilizers in Complex with an Amyloidogenic Light Chain Reveal an Alternate Ligand‐Binding Cavity

Abstract Stabilization of amyloidogenic immunoglobulin light chains (LCs) by binding of small molecule “kinetic stabilizers” is under development as a novel treatment for light chain amyloidosis. From a high‐throughput screen, we previously identified 16 full‐length (FL) LC stabilizers from five distinct chemotypes. We then obtained structural biological information on two classes of hits, coumarins and hydantoins, revealing that both chemotypes bind to a pocket at the V L −V L interface of the FL LC dimer. Here, we report crystal structures of three screening hits from two other chemotypes, diaryl hydrazones and sulfones, in complex with an amyloidogenic FL LC. While two of these hits bind to the previously identified pocket, one diaryl hydrazone binds to a different pocket bisected by the C 2 symmetry axis of the dimer. These data further expand on the FL LC stabilizer‐binding surface that could be used in design of more potent FL LC aggregation inhibitors.

Chemistry↗
DOE OSTI2025

Integrated structural model of the palladin–actin complex using XL ‐ MS , docking, NMR , and SAXS

Abstract Palladin is an actin‐binding protein that accelerates actin polymerization and is linked to the metastasis of several types of cancer. Previously, three lysine residues in an immunoglobulin‐like domain of palladin have been identified as essential for actin binding. However, it is still unknown where palladin binds to F‐actin. Evidence that palladin binds to the sides of actin filaments to facilitate branching is supported by our previous study showing that palladin was able to compensate for Arp2/3 in the formation of Listeria actin comet tails. Here, we used chemical crosslinking to covalently link palladin and F‐actin residues based on spatial proximity. Samples were then enzymatically digested, separated by liquid chromatography, and analyzed by tandem mass spectrometry. Peptides containing the crosslinks and specific residues involved were then identified for input to the HADDOCK docking server to model the most likely binding conformation. Small‐angle x‐ray scattering was used to provide further insight into palladin flexibility and the binding interface, and NMR spectra identified potential interactions between palladin's Ig domains. Our final structural model of the F‐actin:palladin complex revealed how palladin interacts with and stabilizes F‐actin at the interface between two actin monomers. Three actin residues that were identified in this study also appear commonly in the actin‐binding interface with other proteins such as myotilin, myosin, and tropomodulin. An accurate structural representation of the complex between palladin and actin extends our understanding of palladin's role in promoting cancer metastasis through the regulation of actin dynamics.

Sargent, Rachel [Department of Chemistry and Bioch↗
DOE OSTI2020

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/ neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1 A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryoelectron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Molecular and structural basis of olfactory sensory neuron axon coalescence by Kirrel receptors

Projections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here, we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal immunoglobulin (IG) domains is necessary and sufficient to create homodimers and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3 –/– animals. Our results provide evidence for Kirrel3 homodimerization controlling axonal coalescence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular determinants of cross-reactivity and potency by VH3-33 antibodies against the Plasmodium falciparum circumsporozoite protein

IGHV3-33 -encoded antibodies are prevalent in the human humoral response against the Plasmodium falciparum circumsporozoite protein (PfCSP). Among VH3-33 antibodies, cross-reactivity between PfCSP major repeat (NANP), minor (NVDP), and junctional (NPDP) motifs is associated with high affinity and potent parasite inhibition. However, the molecular basis of antibody cross-reactivity and the relationship with efficacy remain unresolved. Here, we perform an extensive structure-function characterization of 12 VH3-33 anti-PfCSP monoclonal antibodies (mAbs) with varying degrees of cross-reactivity induced by immunization of mice expressing a human immunoglobulin gene repertoire. We identify residues in the antibody paratope that mediate cross-reactive binding and delineate four distinct epitope conformations induced by antibody binding, with one consistently associated with high protective efficacy and another that confers comparably potent inhibition of parasite liver invasion. Our data show a link between molecular features of cross-reactive VH3-33 mAb binding to PfCSP and mAb potency, relevant for the development of antibody-based interventions against malaria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Designer molecules of the synaptic organizer MDGA1 reveal 3D conformational control of biological function

MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs), which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs. In crystal structures, the six immunoglobulin (Ig) and single fibronectin III domains of MDGA1 reveal a striking compact, triangular shape, both alone and in complex with NLGNs. Whether this unusual domain arrangement is required for biological function or other arrangements occur with different functional outcomes is unknown. Here, we show that WT MDGA1 can adopt both compact and extended 3D conformations that bind NLGN2. Designer mutants targeting strategic molecular elbows in MDGA1 alter the distribution of 3D conformations while leaving the binding affinity between soluble ectodomains of MDGA1 and NLGN2 intact. In contrast, in a cellular context, these mutants result in unique combinations of functional consequences, including altered binding to NLGN2, decreased capacity to conceal NLGN2 from NRXN1β, and/or suppressed NLGN2-mediated inhibitory presynaptic differentiation, despite the mutations being located far from the MDGA1-NLGN2 interaction site. Thus, the 3D conformation of the entire MDGA1 ectodomain appears critical for its function, and its NLGN-binding site on Ig1-Ig2 is not independent of the rest of the molecule. As a result, global 3D conformational changes to the MDGA1 ectodomain via strategic elbows may form a molecular mechanism to regulate MDGA1 action within the synaptic cleft.

MDGAs↗
DOE OSTI2022

Anatomy of Protein Electrospray Ionization Mass Spectra by Superconducting Tunnel Junction Mass and Energy Spectrometry

Cryogenic superconducting tunnel junction (STJ) detectors have the advantage of single-particle sensitivity, high quantum efficiency, low noise, and the ability to detect the time and relative impact energy of deposited ions. This makes them attractive for use in mass spectrometry (MS) and as a form of energy spectrometry. STJ cryodetectors have been coupled to time-offlight (TOF) mass spectrometers equipped with a matrix-assisted laser desorption ionization (MALDI) source and to an electrospray ionization (ESI) TOF mass spectrometer. Here, a lab-made linear ion trap (LIT) mass spectrometer system was coupled to an ESI source and a 16-channel Nb-STJ array with improved readout electronics. The goal was to investigate fundamentals of ESI-generated protein ions by further exploiting the advantage of resolving these ions in a third dimension of the relative energy deposited into the STJs. The proteins equine cytochrome c, bovine carbonic anhydrase, bovine serum albumin, and murine immunoglobulin G were studied using this ESI-LIT-STJ-MS instrument. Multiply charged monomers, multimers, and fragments from metastable ions were resolved from monomer peaks by differences in ion deposition energy even when these ions have the same mass-to-charge ratio as the corresponding monomer. The determination of fragment mass from metastable decomposition is accomplished without knowing the charge state of the fragment. The average charge state of the multimers is reduced with each addition of a protein which is a direct reflection of the surface area available for charging. Multiply charged in-source fragments have also been observed and distinguished in the mass spectrum of carbonic anhydrase by using the differences in the energy deposited in the STJs.

42 ENGINEERING↗
DOE OSTI2021

Microbial production of megadalton titin yields fibers with advantageous mechanical properties

Manmade high-performance polymers are typically non-biodegradable and derived from petroleum feedstock through energy intensive processes involving toxic solvents and byproducts. While engineered microbes have been used for renewable production of many small molecules, direct microbial synthesis of high-performance polymeric materials remains a major challenge. Here we engineer microbial production of megadalton muscle titin polymers yielding high-performance fibers that not only recapture highly desirable properties of natural titin (i.e., high damping capacity and mechanical recovery) but also exhibit high strength, toughness, and damping energy — outperforming many synthetic and natural polymers. Structural analyses and molecular modeling suggest these properties derive from unique inter-chain crystallization of folded immunoglobulin-like domains that resists inter-chain slippage while permitting intra-chain unfolding. These fibers have potential applications in areas from biomedicine to textiles, and the developed approach, coupled with the structure-function insights, promises to accelerate further innovation in microbial production of high-performance materials.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Mechanism of glycoform specificity and in vivo protection by an anti-afucosylated IgG nanobody

Abstract Immunoglobulin G (IgG) antibodies contain a complex N -glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N -glycan and acts as a ‘glycan sensor’, forming hydrogen bonds with the afucosylated IgG N -glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Mapping brucellosis risk in Kenya and its implications for control strategies in sub-Saharan Africa

In Sub-Saharan Africa (SSA), effective brucellosis control is limited, in part, by the lack of long-term commitments by governments to control the disease and the absence of reliable national human and livestock population-based data to inform policies. Therefore, we conducted a study to establish the national prevalence and develop a risk map for Brucella spp. in cattle to contribute to plans to eliminate the disease in Kenya by the year 2040. We randomly generated 268 geolocations and distributed them across Kenya, proportionate to the area of each of the five agroecological zones and the associated cattle population. Cattle herds closest to each selected geolocation were identified for sampling. Up to 25 cattle were sampled per geolocation and a semi-structured questionnaire was administered to their owners. We tested 6,593 cattle samples for Brucella immunoglobulin G (IgG) antibodies using an Enzyme-linked immunosorbent assay (ELISA). We assessed potential risk factors and performed spatial analyses and prevalence mapping using approximate Bayesian inference implemented via the integrated nested Laplace approximation (INLA) method. The national Brucella spp. prevalence was 6.8% (95% CI: 6.2–7.4%). Exposure levels varied significantly between agro-ecological zones, with a high of 8.5% in the very arid zone with the lowest agricultural potential relative to a low of 0.0% in the agro-alpine zone with the highest agricultural potential. Additionally, seroprevalence increased with herd size, and the odds of seropositivity were significantly higher for females and adult animals than for males or calves. Similarly, animals with a history of abortion, or with multiple reproductive syndromes had higher seropositivity than those without. At the herd level, the risk of Brucella spp. transmission was higher in larger herds, and herds with a history of reproductive problems such as abortion, giving birth to weak calves, or having swollen testes. Geographic localities with high Brucella seroprevalence occurred in northern, eastern, and southern regions of Kenya all primarily characterized by semi-arid or arid agro-ecological zones dominated by livestock pastoralism interspersed with vast areas with mixed livestock-wildlife systems. The large spatial extent of our survey provides compelling evidence for the widespread geographical distribution of brucellosis risk across Kenya in a manner easily understandable for policymakers. Our findings can provide a basis for risk-stratified pilot studies aiming to investigate the cost-effectiveness and efficacy of singular and combined preventive intervention strategies that seek to inform Kenya’s Brucellosis Control Policy.

60 APPLIED LIFE SCIENCES↗