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At least 1,099 records · Page 61
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Precise transcript targeting by CRISPR-Csm complexes

Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90–99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Activation of human STING by a molecular glue-like compound

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages

Here, diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Characterization of the effects of immunomodulatory drug fingolimod (FTY720) on human T cell receptor signaling pathways

Immune responses against gene therapy products limit its therapeutic efcacy and present a safety risk. Identification of agents that blunt immune reactions may aid in developing novel immunomodulatory therapies. Fingolimod (FTY720) is an FDA approved immunomodulatory drug for treating multiple sclerosis that inhibits lymphocyte egress from lymphoid tissues by down regulating sphingosine-1 phosphate receptor (S1PR). Recent studies found that FTY720 inhibits T cell activation (TCA) in a S1PRindependent manner; however, the mechanism is incompletely understood. Here we characterized the effects of FTY720 on human T cell receptor (TCR) signaling pathways. FTY720 inhibited both the TCR-dependent and independent activation of primary human T cells. FTY720 did not affect proximal TCR signaling events as measured by phosphorylation of Lck, ZAP-70 and LAT; however, inhibited PMA/Ionomycin induced distal TCR signaling as measured by IL-2, IFN-γ release and CD25 expression. FTY720 induced aberrant NFAT1, AP1 and NFκB activation which were associated with increased acetylation of histone (H3K9). Phosphorylated FTY720 did not inhibit TCA, and arachidonic acid did not rescue FTY720 mediated inhibition of TCA. These data suggest that FTY720 mediated inhibition of TCA is due to inhibition of distal TCR signaling. Understanding FTY720-mediated inhibition of TCA may aid in developing novel FTY720-based immunomodulatory agents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Modeling the Effects of Morphine-Altered Virus Specific Antibody Responses on HIV/SIV Dynamics

Abstract Drugs of abuse, such as opiates, have been widely associated with enhancing HIV replication, accelerating disease progression and diminishing host-immune responses, thereby making it harder to effectively manage HIV infection. It is thus important to study the effects of drugs of abuse on HIV-infection and immune responses. Here, we develop mathematical models that incorporate the effects of morphine-altered antibody responses on HIV/SIV dynamics. Based on fitting the model to experimental data from simian immunodeficiency virus (SIV) infections in control and morphine-addicted macaques, we found that two of the most significant effects of virus specific antibodies are neutralizing viral particles and enhancing viral clearance. Using our model, we quantified how morphine alters virus-specific antibody responses, and how this alteration affects the key components of virus dynamics such as infection rate, virus clearance, viral load, CD4 + T cell count, and CD4 + T cell loss in SIV-infected macaques under conditioning with morphine. We found that in a subpopulation of SIV-infected morphine addicted macaques, the presence of drugs of abuse may cause significantly diminished antibody responses, resulting in more severe infection with increased SIV infectivity, a decreased viral clearance rate, increased viral load, and higher CD4 + T cell loss.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Spatial organization of FcγR and TLR2/1 on phagosome membranes differentially regulates their synergistic and inhibitory receptor crosstalk

Abstract Many innate immune receptors function collaboratively to detect and elicit immune responses to pathogens, but the physical mechanisms that govern the interaction and signaling crosstalk between the receptors are unclear. In this study, we report that the signaling crosstalk between Fc gamma receptor (FcγR) and Toll-like receptor (TLR)2/1 can be overall synergistic or inhibitory depending on the spatial proximity between the receptor pair on phagosome membranes. Using a geometric manipulation strategy, we physically altered the spatial distribution of FcγR and TLR2 on single phagosomes. We demonstrate that the signaling synergy between FcγR and TLR2/1 depends on the proximity of the receptors and decreases as spatial separation between them increases. However, the inhibitory effect from FcγRIIb on TLR2-dependent signaling is always present and independent of receptor proximity. The overall cell responses are an integration from these two mechanisms. This study presents quantitative evidence that the nanoscale proximity between FcγR and TLR2 functions as a key regulatory mechanism in their signaling crosstalk.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Forecasting and modeling of the COVID-19 pandemic in the USA with a timed intervention model

We propose a novel Timed Intervention S, P, E, I, Q, R, D model for projecting the possible futures of the COVID-19 pandemic in the USA. The proposed model introduces a series of timed interventions that can account for the influence of real time changes in government policy and social norms. We consider three separate types of interventions: (i) Protective interventions: Where population moves from susceptible to protected corresponding to mask mandates, stay-at-home orders and/or social distancing. (ii) Release interventions: Where population moves from protected to susceptible corresponding to social distancing mandates and practices being lifted by policy or pandemic fatigue. (iii) Vaccination interventions: Where population moves from susceptible, protected, and exposed to recovered (meaning immune) corresponding to the mass immunization of the U.S. Population. By treating the pandemic with timed interventions, we are able to model the pandemic extremely effectively, as well as directly predicting the course of the pandemic under differing sets of intervention schedules. We show that without prompt effective protective/vaccination interventions the pandemic will be extended significantly and result in many millions of deaths in the U.S.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Theoretical modeling of hepatitis C acute infection in liver-humanized mice support pre-clinical assessment of candidate viruses for controlled-human-infection studies

Designing and carrying out a controlled human infection (CHI) model for hepatitis C virus (HCV) is critical for vaccine development. However, key considerations for a CHI model protocol include understanding of the earliest viral-host kinetic events during the acute phase and susceptibility of the viral isolate under consideration for use in the CHI model to antiviral treatment before any infections in human volunteers can take place. Humanized mouse models lack adaptive immune responses but provide a unique opportunity to obtain quantitative understanding of early HCV kinetics and develop mathematical models to further understand viral and innate immune response dynamics during acute HCV infection. We show that the models reproduce the measured HCV kinetics in humanized mice, which are consistent with early acute HCV-host dynamics in immunocompetent chimpanzees. Our findings suggest that humanized mice are well-suited to support development of a CHI model. In-silico and in-vivo modeling estimates provide a starting point to characterize candidate viruses for testing in CHI model studies.

Agent-based modeling↗
DOE OSTI2021

Structures of the ApoL1 and ApoL2 N-terminal domains reveal a non-classical four-helix bundle motif

Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Human antibodies to SARS-CoV-2 with a recurring YYDRxG motif retain binding and neutralization to variants of concern including Omicron

Studying the antibody response to SARS-CoV-2 informs on how the human immune system can respond to antigenic variants as well as other SARS-related viruses. Here, we structurally identified a YYDRxG motif encoded by IGHD3-22 in CDR H3 that facilitates antibody targeting to a functionally conserved epitope on the SARS-CoV-2 receptor binding domain. A computational search for a YYDRxG pattern in publicly available sequences uncovered 100 such antibodies, many of which can neutralize SARS-CoV-2 variants and SARS-CoV. Thus, the YYDRxG motif represents a common convergent solution for the human humoral immune system to target sarbecoviruses including the Omicron variant. These findings suggest an epitope-targeting strategy to identify potent and broadly neutralizing antibodies for design of pan-sarbecovirus vaccines and antibody therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The co-crystal structure of Cbl-b and a small-molecule inhibitor reveals the mechanism of Cbl-b inhibition

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound’s interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A bipartite bacterial virulence factor targets the complement system and neutrophil activation

Abstract The complement system and neutrophils constitute the two main pillars of the host innate immune defense against infection by bacterial pathogens. Here, we identify T-Mac, a novel virulence factor of the periodontal pathogen Treponema denticola that allows bacteria to evade both defense systems. We show that T-Mac is expressed as a pre-protein that is cleaved into two functional units. The N-terminal fragment has two immunoglobulin-like domains and binds with high affinity to the major neutrophil chemokine receptors FPR1 and CXCR1, blocking N -formyl-Met-Leu-Phe- and IL-8-induced neutrophil chemotaxis and activation. The C-terminal fragment functions as a cysteine protease with a unique proteolytic activity and structure, which degrades several components of the complement system, such as C3 and C3b. Murine infection studies further reveal a critical T-Mac role in tissue damage and inflammation caused by bacterial infection. Collectively, these results disclose a novel innate immunity-evasion strategy, and open avenues for investigating the role of cysteine proteases and immunoglobulin-like domains of gram-positive and -negative bacterial pathogens.

Kurniyati, Kurni↗
DOE OSTI2016

Preliminary analysis of salivary microbiome and their potential roles in oral lichen planus

Several studies have explored the origin and development mechanism of oral lichen planus (OLP) with limited attention to the role of bacteria in the progression of this common oral disease. Here we utilized MiSeq sequencing of 16S rRNA gene amplicons to identify complex oral microbiota associated with OLP from saliva samples of two subtypes (reticular and erosive) of OLP patients and healthy controls. Our analyses indicated that the overall structure of the salivary microbiome was not significantly affected by disease status. However, we did observe evident variations in abundance for several taxonomic groups in OLP. Porphyromonas and Solobacterium showed significantly higher relative abundances, whereas Haemophilus, Corynebacterium, Cellulosimicrobium and Campylobacter showed lower abundances in OLP patients, as compared with healthy controls. In addition, we explored specific microbial co-occurrence patterns in OLP, and revealed significantly fewer linkers of Streptococcus comprising species in erosive OLP. Furthermore, the disease severity and immune dysregulation were also genus-associated, including with Porphyromonas that correlated to disease scores and salivary levels of interleukin (IL)-17 and IL-23. Overall, this study provides a general description of oral microbiome in OLP, and it will be useful for further investigation of their potential roles in the initiation and immune modulation of OLP.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Graphene-extracted membrane lipids facilitate the activation of integrin α v β 8

Despite the remarkable electrochemical properties of graphene, strong van der Waals attraction between graphene and biomolecules often causes cytotoxicity, which hinders its applications in the biomedical field. Unfortunately, surface passivation of graphene might stimulate undesired immune response as the nanomaterial triggers cytokine production through membrane receptor activation. In this work, we use all-atom Molecular Dynamics (MD) simulations to unravel the underlying mechanism of graphene-induced inside-out activation of integrin αvβ8, a prominent membrane receptor expressed in immune cells. We model the transmembrane (TM) domains of integrin αvβ8 in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer and observe the structural changes in the integrin–membrane complex when interacting with a graphene nanosheet across the membrane. We find that the β8 TM domain interacts with the graphene nanosheet directly or indirectly through extracted lipids, facilitating the pulling of a β8 subunit away from an αv subunit and thus leading to the disruption of the TM domain association by breaking the hydrophobic cluster in the cytoplasmic domains of the αv and β8 subunits. Alanine substitution of two conserved phenylalanine residues on the αv subunit at this hydrophobic cluster further reveals the importance of a stable T-shaped structure in retaining integrin in its inactive state. Our results agree with previous studies on the interactions between other integrin subtypes and their endogenous activators, suggesting an intriguing role that the graphene nanosheet may play in the integrin-related signal transduction during its interaction with the membrane.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

A skyrmionic topology perspective on Lehmann clusters

When two parallel coplanar dislocations with opposite Burgers vectors are brought into collision by the action of stress in solid crystals, they annihilate each other. In cholesterics with pitch p, under certain conditions, colliding dislocations with Burgers vectors b = p and b = − p are immune against annihilation and form stable twin-like pairs known as Lehmann clusters. We point out that this immunity is due to the escape of the director field into the third dimension. We show that from a topological point of view, Lehmann clusters can be seen as skyrmion tubes embedded in a helicoidal background, and their extremities have the topology of monopoles.

Pieranski, Pawel [Univ. Paris-Saclay, Orsay (Franc↗
DOE OSTI2021

Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice

Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). Here, we investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11 +/+ and Cdh11 –/– mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11 +/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11 +/– and KPC/Cdh11 –/– mice survived significantly longer than KPC/Cdh11 +/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11 +/+ mice and incompletely in KPC/Cdh11 +/– and KPC/Cdh11 –/– mice, whose lesions also contained fewer FOXP3 + cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11 +/+ mice, tumors of KPC/Cdh11 +/– mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11 +/– and KPC/Cdh11 –/– mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11 +/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Two-color, intracavity pump–probe, cavity ringdown spectroscopy

Here, we report a proof-of-principle demonstration of intracavity pump–probe, cavity ringdown (CRD) detection in a three-mirror, traveling-wave cavity. With cavity-enhanced pump power and probe absorption path length, the technique is a generally applicable, high-sensitivity, high-selectivity detection method. In our experiments, the pump radiation is switched off during every other probe ringdown, which allows uncorrelated measurements of analyte and background cavity decay rates. The net, two-color signal from the difference between the pump-on and pump-off decay rates is immune to empty-CRD drifts and spectral overlaps from non-target molecular transitions. The immunity to the ringdown drifts allows longer signal-averaging and, thus, higher detection sensitivity. The ability to compensate for the background absorption enhances the detection selectivity in spectrally congested regions. Our technique is well-suited for trace-detection in the mid-IR region, where pump–probe schemes based on strong rovibrational transitions can be applied. In this work, two-color CRD detection is implemented on a ladder-type, three-level system based on the N 2 O, ν 3 = 1 ← 0, P(19) (pump) and ν 3 = 2 ← 1, R(18) (probe), rovibrational transitions. By frequency-locking two-quantum cascade lasers to the p-polarization (pump, Finesse = 5280) and s-polarization (probe, Finesse = 67 700) cavity modes, we achieve high intracavity pump power (36 W) and high probe ringdown rates (>2 kHz). The observed two-color spectra are simulated by a density-matrix, three-level system model that is solved under the constraints of the cavity resonance conditions. In addition to its background compensation capability, experimental flexibility in the selection of pump–probe schemes and signal insensitivity to intracavity laser power are further features that enhance the utility of our technique for mid-IR trace-detection.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗