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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Large Eddy Simulation of Rotationally Induced Ingress and Egress around an Axial Seal between Rotor and Stator Disks

In gas turbines, the hot gas exiting the combustor can have temperatures as high as 2000 °C, and some of this hot gas enter into the space between the stator and rotor disks (wheelspace). Since the entering hot gas could damage the disks, its ingestion must be minimized. This is carried out by rim seals and by introducing a cooler flow from the compressor (sealing flow) into the wheelspace. Ingress and egress into rim seals are driven by the stator vanes, the rotor and its rotation, and the rotor blades. This study focuses on the ingress and egress driven by the rotor and its rotation. This is carried out by performing wall-resolved large eddy simulation (LES) around an axial seal in a rotor–stator configuration without vanes and blades. Results obtained show the mechanisms by which the rotor and its rotation induce ingress, egress, and flow trajectories. Kelvin–Helmholtz instability was found to create a wavy shear layer and displacement thickness that produces alternating regions of high and low pressures around the rotor side of the seal. Vortex shedding on the backward-facing side of the seal and its impingement on the rotor side of the seal also produces alternating regions of high and low pressures. The locations of the alternating regions of high and low pressures were found to be statistically stationary and to cause ingress to start on the rotor side of the seal. Vortex shedding and recirculating flow in the seal clearance also cause ingress by entrainment. With the effects of the rotor and its rotation on ingress and egress isolated, this study enables the effects of stator vanes and rotor blades to be assessed.

42 ENGINEERING↗
DOE OSTI2024

The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress–budding at the inner nuclear membrane–is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

membrane curvature↗
DOE OSTI2016

Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials. - Highlights: • Cell penetrable human scFvs (transbodies) to Ebolavirus (EBOV) VP40 were produced. • The transbodies inhibited egress of EBOV-like particles (VLPs) from human hepatocytes. • They interacted with VP40 CTD basic residues important for plasma membrane binding. • And hence interfere with viral matrix assembly and viral progeny budding. • This is the first report on human antibodies that target intracellular EBOV VP40.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion. -- Highlights: •Phosphorylated nuclear lamins were inefficiently remodeled in p53KO cells. •p53KO cells expressed UL50, but it was not efficiently targeted to the nuclear rim. •UL53 was not expressed in the large majority of p53KO cells. •Cells failing to express UL53 did not localize UL50 to the nucleus. •NEC puncta/infoldings of the inner nuclear membrane were scarce in p53KO cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Egress of murine regulatory T cells from the thymus requires TIPE2

Highlights: • TIPE2 deficiency leads to the accumulation of Tregs in the thymus. • TIPE2 deficiency doesn't affect the development and apoptosis of tTregs. • TIPE2 promotes the chemotaxis of tTregs in vitro. • TIPE2 promotes the polarization of pAKT and F-actin in tTregs undergoing directed migration. Regulatory T cells (Tregs) can be divided into thymus-derived Treg (tTregs) and peripheral induced Tregs (pTregs) in vivo according to their origins and are essential for the maintenance of immune hemostasis and immune tolerance. Tumor necrosis factor-α-induced protein 8 like 2 (TIPE2) is expressed primarily by immune cells and is a negative regulator of the innate and adaptive immune response. Previous studies indicate that TIPE2 is required for the expression of Treg signature genes and promotes leading-edge formation in neutrophils through cytoskeleton remodeling. In the current study, we showed that TIPE2 deficient mice accumulate more Treg cells in the thymus. Further studies revealed that TIPE2 deficiency doesn't affect the development and apoptosis of tTregs. Instead, TIPE2 promotes the chemotaxis of tTregs in vitro, which may account for the accumulation of Tregs in the thymus of TIPE2 deficient mice. Mechanistic study revealed that TIPE2 promotes the polarization of pAKT and F-actin in tTregs undergoing directed migration. Taken together, these results demonstrated that TIPE2 enhances the cytoskeleton remodeling and promotes the thymus egress of tTregs, which may play an important role in the maintenance of self-tolerance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Chromatin organization regulates viral egress dynamics

Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes simplex virus 1–sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Herpes simplex virus 1 induces egress channels through marginalized host chromatin

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Gas Ingress and Egress of MEMS Multi-Chip Modules and MEMS Devices

Hermetic microcircuit packaging was the dominant method of protecting semiconductor devices in the 1960s and 1970s. After losing majority market sectors to plastic encapsulated microelectronics over the last a few decades, hermetic packaging remains the preferred method of protecting semiconductor devices for critical applications such as in military, space, and medical fields, where components and systems are required to serve for several decades. MEMS devices impose additional challenges to packaging by requiring specific internal cavity pressures to function properly or deliver the needed quality (Q) factors. In MEMS multichip modules, internal pressure requirement conflicts arise when different MEMS devices require different internal gases and pressures. The authors developed a closed-formed equation to model pressure changes of hermetic enclosures due to gas ingression. This article expands the authors mathematical model to calculate gas pressure of a MEMS multichip module package as well as those of MEMS devices inside the multichip module package. These equations are not only capable of calculating service lifetimes of MEMS devices and multi-chip modules but can also help develop MEMS device packaging strategies to extend the service life of MEMS multi-chip modules.

36 MATERIALS SCIENCE↗
DOE OSTI2016

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm. -- Highlights: •The majority of p53KO cells release fewer functional virions than wt cells. •Nucleocapsids do not efficiently exit the nucleus in p53KO cells. •Infoldings of the inner nuclear membrane are not efficiently formed in p53KO cells. •Cytoplasmic capsids are not efficiently re-enveloped in p53KO cells. •Reintroduction of p53 largely ameliorates these phenotypes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Nipah Virus-Like Particle Egress Is Modulated by Cytoskeletal and Vesicular Trafficking Pathways: a Validated Particle Proteomics Analysis

Classified as a biosafety level 4 (BSL4) select agent, Nipah virus (NiV) is a deadly henipavirus in the Paramyxoviridae family, with a nearly 75% mortality rate in humans, underscoring its global and animal health importance. Elucidating the process of viral particle production in host cells is imperative both for targeted drug design and viral particle-based vaccine development. However, little is understood concerning the functions of cellular machinery in paramyxoviral and henipaviral assembly and budding. Recent studies showed evidence for the involvement of multiple NiV proteins in viral particle formation, in contrast to the mechanisms understood for several paramyxoviruses as being reliant on the matrix (M) protein alone. Further, the levels and purposes of cellular factor incorporation into viral particles are largely unexplored for the paramyxoviruses. To better understand the involvement of cellular machinery and the major structural viral fusion (F), attachment (G), and matrix (M) proteins, we performed proteomics analyses on virus-like particles (VLPs) produced from several combinations of these NiV proteins. Our findings indicate that NiV VLPs incorporate vesicular trafficking and actin cytoskeletal factors. The involvement of these biological processes was validated by experiments indicating that the perturbation of key factors in these cellular processes substantially modulated viral particle formation. These effects were most impacted for NiV-F-modulated viral particle formation either autonomously or in combination with other NiV proteins, indicating that NiV-F budding relies heavily on these cellular processes. These findings indicate a significant involvement of the NiV fusion protein, vesicular trafficking, and actin cytoskeletal processes in efficient viral particle formation. Nipah virus is a zoonotic biosafety level 4 agent with high mortality rates in humans. The genus to which Nipah virus belongs, Henipavirus , includes five officially recognized pathogens; however, over 20 species have been identified in multiple continents within the last several years. As there are still no vaccines or treatments for NiV infection, elucidating its process of viral particle production is imperative both for targeted drug design as well as for particle-based vaccine development. Developments in high-throughput technologies make proteomic analysis of isolated viral particles a highly insightful approach to understanding the life cycle of pathogens such as Nipah virus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Passive filtration of air egressing from nuclear containment

A nuclear reactor includes a reactor core comprising fissile material disposed in a reactor pressure vessel. A radiological containment contains the nuclear reactor. A containment compartment contains the radiological containment. A heat sink includes a chimney configured to develop an upward-flowing draft in response to heated fluid flowing into a lower portion of the chimney. A fluid conduit is arranged to receive fluid from the containment compartment and to discharge into the chimney. A filter may be provided, with the fluid conduit including a first fluid conduit arranged to receive fluid from the containment compartment and to discharge into an inlet of the filter, and a second fluid conduit arranged to receive fluid from an outlet of the filter and to discharge into the chimney. As the draft is developed passively, there is no need for a blower or pump configured to move fluid through the fluid conduit.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗