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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 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain

Ca 2+ /calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin

Here, in this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure–function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM–D166V) and dilated (DCM–D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I 11 /I 10 ) together with the force-pCa relationship over a full range of [Ca 2+ ] and at a sarcomere length of 2.1 μm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM–D94A mice, HCM–D166V significantly increased the Ca 2+ sensitivity of force and left shifted the I 11 /I 10 -pCa relationship, indicating an apparent movement of HCM–D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca 2+ activation. The HCM–D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM–D166V, the DCM–D94A model favored the energy-conserving SRX state, but the structure/function–pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM–D166V and DCM–D94A mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural and physical features that distinguish tumor-controlling from inactive cancer neoepitopes

Neoepitopes arising from amino acid substitutions due to single nucleotide polymorphisms are targets of T cell immune responses to cancer and are of significant interest in the development of cancer vaccines. However, understanding the characteristics of rare protective neoepitopes that truly control tumor growth has been a challenge, due to their scarcity as well as the challenge of verifying true, neoepitope-dependent tumor control in humans. Taking advantage of recent work in mouse models that circumvented these challenges, here, we compared the structural and physical properties of neoepitopes that range from fully protective to immunologically inactive. As neoepitopes are derived from self-peptides that can induce immune tolerance, we studied not only how the various neoepitopes differ from each other but also from their wild-type counterparts. We identified multiple features associated with protection, including features that describe how neoepitopes differ from self as well as features associated with recognition by diverse T cell receptor repertoires. In conclusion, we demonstrate both the promise and limitations of neoepitope structural analysis and predictive modeling and illustrate important aspects that can be incorporated into neoepitope prediction pipelines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

CACNA1D is a circadian gene and causes familial advanced sleep phase

Familial advanced sleep phase (FASP) is a heritable human sleep trait characterized by early sleep onset and offset times. We have identified five variants in five different families in the human voltage-gated calcium channel subunit alpha1 D (CACNA1D) that cosegregate with FASP. The variants inCACNA1Dlead to altered channel dynamics in vitro. A mouse model of the E427K variant has a normal circadian period under constant darkness but displays altered phase shifts in response to light in the subjective night at circadian time (CT) 16 and CT22. Overall, these experiments establishCACNA1Das an FASP gene with altered entrainment, highlighting the ability of human genetics to uncover novel aspects of human circadian regulation.

Science & Technology - Other Topics↗
DOE OSTI2020

Efficient generation of bone morphogenetic protein 15-edited Yorkshire pigs using CRISPR/Cas9†

Abstract Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta (TGF-β) superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in poly-ovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in poly-ovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at pre-antral stages, with formation of numerus structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in non-rodent poly-ovulatory species may be as important as that in mono-ovulatory species.

Shi, Xuan↗
DOE OSTI2021

DNA Breaks-Mediated Fitness Cost Reveals RNase HI as a New Target for Selectively Eliminating Antibiotic-Resistant Bacteria

Abstract Antibiotic resistance often generates defects in bacterial growth called fitness cost. Understanding the causes of this cost is of paramount importance, as it is one of the main determinants of the prevalence of resistances upon reducing antibiotics use. Here we show that the fitness costs of antibiotic resistance mutations that affect transcription and translation in Escherichia coli strongly correlate with DNA breaks, which are generated via transcription–translation uncoupling, increased formation of RNA–DNA hybrids (R-loops), and elevated replication–transcription conflicts. We also demonstrated that the mechanisms generating DNA breaks are repeatedly targeted by compensatory evolution, and that DNA breaks and the cost of resistance can be increased by targeting the RNase HI, which specifically degrades R-loops. We further show that the DNA damage and thus the fitness cost caused by lack of RNase HI function drive resistant clones to extinction in populations with high initial frequency of resistance, both in laboratory conditions and in a mouse model of gut colonization. Thus, RNase HI provides a target specific against resistant bacteria, which we validate using a repurposed drug. In summary, we revealed key mechanisms underlying the fitness cost of antibiotic resistance mutations that can be exploited to specifically eliminate resistant bacteria.

Balbontín, Roberto↗
DOE OSTI2024

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP’s enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP’s role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Gram-positive bacterial cell wall components inhibit herpes simplex virus infection

The role of the mucosal microbiome in viral infections remains unclear. Genital herpes, caused by herpes simplex virus 1 and 2 (HSV-1 and HSV-2), is among the most prevalent sexually transmitted infections worldwide. Despite evidence linking vaginal Lactobacillus to protection against sexually transmitted viruses, the specific microbial components and mechanisms that mediate this defense are not well understood. Here, we show that multiple cell wall components from diverse gram-positive bacteria, including lactobacilli, inhibit HSV-1 and HSV-2 infection in cells and in a mouse model of genital herpes infection. Peptidoglycan (PG) and lipoteichoic acid (LTA), both major components of the gram-positive bacterial cell wall, significantly reduced HSV infectivity in vitro and improved survival and disease outcomes in mice. We further showed that Lactobacillus crispatus surface layer proteins SlpA and SlpB bind HSV-1 and inhibit infection. Antiviral effects of cell wall components were dose-dependent, relied on intact PG structure, and, in the case of PG and LTA, were independent of TLR2-mediated host signaling. Collectively, our findings identify a species-independent antiviral function for gram-positive bacterial cell wall components against HSV and suggest that the composition of the mucosal microbiome may play an underappreciated role in suppressing mucosal herpes infection in humans.

Adams↗
DOE OSTI2021

Sepsis induces muscle atrophy by inhibiting proliferation and promoting apoptosis via PLK1‐AKT signalling

Abstract Sepsis and sepsis‐induced skeletal muscle atrophy are common in patients in intensive care units with high mortality, while the mechanisms are controversial and complicated. In the present study, the atrophy of skeletal muscle was evaluated in sepsis mouse model as well as the apoptosis of muscle fibres. Sepsis induced atrophy of skeletal muscle and apoptosis of myofibres in vivo and in vitro. In cell‐based in vitro experiments, lipopolysaccharide (LPS) stimulation also inhibited the proliferation of myoblasts. At the molecular level, the expression of polo‐like kinase 1 (PLK1) and phosphorylated protein kinase B (p‐AKT) was decreased. Overexpression of PLK1 partly rescued LPS‐induced apoptosis, proliferation suppression and atrophy in C2C12 cells. Furthermore, inhibiting the AKT pathway deteriorated LPS‐induced atrophy in PLK1‐overexpressing C2C12 myotubes. PLK1 was found to participate in regulating apoptosis and E3 ubiquitin ligase activity in C2C12 cells. Taken together, these results indicate that sepsis induces skeletal muscle atrophy by promoting apoptosis of muscle fibres and inhibiting proliferation of myoblasts via regulation of the PLK1‐AKT pathway. These findings enhance understanding of the mechanism of sepsis‐induced skeletal muscle atrophy.

Cao, Ying‐Ya↗
DOE OSTI2022

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble angiotensin-converting enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses using ACE2 as their receptor. Using structure-guided approaches, we developed a series of bivalent ACE2-Fcs harboring functionally and structurally validated mutations that enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain recognition by up to ~12-fold and remove angiotensin enzymatic activity. The lead variant M81 potently cross-neutralized SARS-CoV-2 variants of concern (VOCs), including Omicron, at subnanomolar half-maximal inhibitory concentration and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. When tested in a stringent K18-hACE2 mouse model, Fc-enhanced ACE2-Fc delayed death by 3 to 5 days or effectively resolved lethal SARS-CoV-2 infection in both prophylactic and therapeutic settings via the combined effects of neutralization and Fc-effector functions. These data add to the demonstrated utility of soluble ACE2 as a valuable SARS-CoV-2 antiviral and indicate that Fc-effector functions may constitute an important component of ACE2-Fc therapeutic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury

The signaling of cells by scaffolds of synthetic molecules that mimic proteins is known to be effective in the regeneration of tissues. Here, we describe peptide amphiphile supramolecular polymers containing two distinct signals and test them in a mouse model of severe spinal cord injury. One signal activates the transmembrane receptor β1-integrin and a second one activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomers in nonbioactive domains, we intensified the motions of molecules within scaffold fibrils. This resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. Here, we hypothesize that the signaling of cells by ensembles of molecules could be optimized by tuning their internal motions.

36 MATERIALS SCIENCE↗
DOE OSTI2022

A noncoding single-nucleotide polymorphism at 8q24 drives IDH1 -mutant glioma formation

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)–mutant low-grade glioma (LGG). Here, we reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV–infected mice

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

NF-κB perturbation reveals unique immunomodulatory functions in Prx1 + fibroblasts that promote development of atopic dermatitis

Skin is composed of diverse cell populations that cooperatively maintain homeostasis. Up-regulation of the nuclear factor κB (NF-κB) pathway may lead to the development of chronic inflammatory disorders of the skin, but its role during the early events remains unclear. Here, through analysis of single-cell RNA sequencing data via iterative random forest leave one out prediction, an explainable artificial intelligence method, we identified an immunoregulatory role for a unique paired related homeobox-1 (Prx1) + fibroblast subpopulation. Disruption of Ikkb–NF-κB under homeostatic conditions in these fibroblasts paradoxically induced skin inflammation due to the overexpression of C-C motif chemokine ligand 11 (CCL11; or eotaxin-1) characterized by eosinophil infiltration and a subsequent T H 2 immune response. Because the inflammatory phenotype resembled that seen in human atopic dermatitis (AD), we examined human AD skin samples and found that human AD fibroblasts also overexpressed CCL11 and that perturbation of Ikkb–NF-κB in primary human dermal fibroblasts up-regulated CCL11. Monoclonal antibody treatment against CCL11 was effective in reducing the eosinophilia and T H 2 inflammation in a mouse model. Together, the murine model and human AD specimens point to dysregulated Prx1 + fibroblasts as a previously unrecognized etiologic factor that may contribute to the pathogenesis of AD and suggest that targeting CCL11 may be a way to treat AD-like skin lesions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A

Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (V H 3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific V H 3-IgM via a mechanism requiring CD4 + T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Pseudomonas aeruginosa C-Terminal Processing Protease CtpA Assembles into a Hexameric Structure That Requires Activation by a Spiral-Shaped Lipoprotein-Binding Partner

Pseudomonas aeruginosa CtpA is a carboxyl-terminal processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer of dimers and an N-terminal helix of LbcA are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of the LbcA-CtpA proteolytic system and reveals some striking differences from the arrangements found in some other bacterial CTPs.

structural biology↗
DOE OSTI2025

Early immune response to Coccidioides is characterized by robust neutrophil and fibrotic macrophage recruitment and differentiation

Coccidioidomycosis, or Valley fever, is an emerging respiratory disease caused by soil-dwelling fungi of the Coccidioides genus that is expected to spread from the southwest into the central U.S. by 2050. While 60% of infections are asymptomatic, the other 40% of patients experience a range of symptoms, from self-limiting pneumonia to life-threatening disseminated disease. The immunological events that underlie the progression to severe disease remain underdefined. Here, we probed the early immune response to Coccidioides using a high dose of an attenuated strain of Coccidioides posadasii in a mouse model of infection coupled with single-cell RNA sequencing. At 24 h post-infection, robust immune infiltration is detected in the lung, marked by high levels of inflammatory PD-L1 + neutrophils and fungal-contact-dependent pro-fibrotic Spp1 + macrophages. These findings elucidate the early dynamics of the host response to Coccidioides and provide a deeper understanding of host-pathogen interactions in the lung.

Coccidioides↗
DOE OSTI2025

Metabolomic and transcriptomic remodeling of bone marrow myeloid cells in response to maternal obesity

Maternal obesity puts the offspring at high risk of developing obesity and cardiometabolic diseases in adulthood. Here, we utilized a mouse model of maternal high-fat diet (HFD)-induced obesity that recapitulates metabolic perturbations seen in humans. We show increased adiposity in the offspring of HFD-fed mothers (Off-HFD) when compared with the offspring of regular diet-fed mothers (Off-RD). We have previously reported significant immune perturbations in the bone marrow of newly weaned Off-HFD. Here, we hypothesized that lipid metabolism is altered in the bone marrow of Off-HFD versus Off-RD. To test this hypothesis, we investigated the lipidomic profile of bone marrow cells collected from 3-week-old Off-RD and Off-HFD. Diacylglycerols (DAGs), triacylglycerols (TAGs), sphingolipids, and phospholipids were remarkably different between the groups, independent of fetal sex. Levels of cholesteryl esters were significantly decreased in Off-HFD, suggesting reduced delivery of cholesterol. These were accompanied by age-dependent progression of mitochondrial dysfunction in bone marrow cells. We subsequently isolated CD11b+ myeloid cells from 3-wk-old mice and conducted metabolomic, lipidomic, and transcriptomic analyses. The lipidomic profiles of myeloid cells were similar to those of bone marrow cells and included increases in DAGs and decreased TAGs. Transcriptomics revealed altered expression of genes related to immune pathways, including macrophage alternative activation, B-cell receptors, and transforming growth factor-β signaling. All told, this study revealed lipidomic, metabolomic, and gene expression abnormalities in bone marrow cells broadly, and in bone marrow myeloid cells particularly, in the newly weaned offspring of mothers with obesity, which might at least partially explain the progression of metabolic and cardiovascular diseases in their adulthood.

RNA sequencing↗