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At least 127 records · Page 7

All sources NASA NTRS DOE OSTI

DOE OSTI2022

Deletion of the EP296R Gene from the Genome of Highly Virulent African Swine Fever Virus Georgia 2010 Does Not Affect Virus Replication or Virulence in Domestic Pigs

African swine fever virus (ASFV) causes a lethal disease (ASF) in domestic pigs, African swine fever (ASF). ASF is currently producing a pandemic affecting pig production across Eurasia, leading to a shortage of food accessibility. ASFV is structurally complex, harboring a large genome encoding over 150 genes. One of them, EP296R, has been shown to encode for an endonuclease that is necessary for the efficient replication of the virus in swine macrophages, the natural ASFV target cell. Here, we report the development of a recombinant virus, ASFV-G-ΔEP296R, harboring the deletion of the EP296R gene from the genome of the highly virulent field isolate ASFV Georgia 2010 (ASFV-G). The recombinant ASFV-G-ΔEP296R replicates in primary swine macrophages with similar kinetics as the parental virus ASFV-G. Pigs experimentally infected by the intramuscular route with 102 HAD50 show a slightly protracted, although lethal, presentation of the disease when compared to that of animals inoculated with parental ASFV-G. Viremia titers in the ASFV-G-ΔEP296R-infected animals closely followed the kinetics of presentation of clinical disease. Results presented here demonstrate that ASFV-G-ΔEP296R is not essential for the processes of ASFV replication in swine macrophages, nor is it radically involved in the process of virus replication or disease production in domestic pigs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Deletion of an African Swine Fever Virus ATP-Dependent RNA Helicase QP509L from the Highly Virulent Georgia 2010 Strain Does Not Affect Replication or Virulence

African swine fever virus (ASFV) produces a lethal disease (ASF) in domestic pigs, which is currently causing a pandemic deteriorating pig production across Eurasia. ASFV is a large and structurally complex virus with a large genome harboring more than 150 genes. ASFV gene QP509L has been shown to encode for an ATP-dependent RNA helicase, which appears to be important for efficient virus replication. Here, we report the development of a recombinant virus, ASFV-G-ΔQP509L, having deleted the QP509L gene in the highly virulent field isolate ASFV Georgia 2010 (ASFV-G). It is shown that ASFV-G-ΔQP509L replicates in primary swine macrophage cultures as efficiently as the parental virus ASFV-G. In addition, the experimental inoculation of pigs with 10 2 HAD 50 by the intramuscular route produced a slightly protracted but lethal clinical disease when compared to that of animals inoculated with virulent parental ASFV-G. Viremia titers in animals infected with ASFV-G-ΔQP509L also had slightly protracted kinetics of presentation. Therefore, ASFV gene QP509L is not critical for the processes of virus replication in swine macrophages, nor is it clearly involved in virus replication and virulence in domestic pigs.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1986

Replication of grazing incidence optics

The replication of grazing incidence optics is reviewed. Electroform and epoxy replication are described and compared. It is concluded that for light weight and deep nesting, replication has a distinct advantage over direct production. The resolution of optics produced in this manner is however, limited to about 10 arc seconds; a typical value is 40 arc seconds. Epoxy replicated pieces tend to have better optical figures than electroformed optics, but the latter can be made thinner to make more deeply nested systems.

Ulmer, Melville P.↗

NASA NTRS1989

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

Weber, Arthur L.↗

NASA NTRS1990

Low cost management of replicated data in fault-tolerant distributed systems

Many distributed systems replicate data for fault tolerance or availability. In such systems, a logical update on a data item results in a physical update on a number of copies. The synchronization and communication required to keep the copies of replicated data consistent introduce a delay when operations are performed. A technique is described that relaxes the usual degree of synchronization, permitting replicated data items to be updated concurrently with other operations, while at the same time ensuring that correctness is not violated. The additional concurrency thus obtained results in better response time when performing operations on replicated data. How this technique performs in conjunction with a roll-back and a roll-forward failure recovery mechanism is also discussed.

Joseph, Thomas A.↗

NASA NTRS1989

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

Weber, Arthur L.↗

NASA NTRS1992

Self-replication of chemical systems based on recognition within a double or a triple helix - A realistic hypothesis

A scenario is proposed for the non-enzymatic self-replication of short RNA molecules. The self-replication of an oligopyrimidine strand is considered and the process of template-directed synthesis based on recognition within a double helix is discussed. Replication mechanisms are suggested for selected oligonucleotides. The mechanisms are based on Watson-Crick base pairing between complementary nucleotides as well as Hoogsteen base pairing between a duplex and the complementary third strand. It is suggested that self-replication based on these mechanisms may be accomplished but may result in a substantial amount of misinformation transfer when mixed oligonucleotides are used.

Kanavarioti, Anastassia↗

NASA NTRS2002

Who or What? Self-Replication and Function-Reproduction in the Origin of Life

In this presentation, we will present results on the fundamental properties of two classes of replicating systems: autocatalytic replicators that reproduce exact copies of a template molecule, and function reproducers that maintain a set of essential functions without replicating the identities of the functional moieties. We will describe the stability and behavior in-the-large of autocatalytic replicators. Most importantly, we have found no sharp distinction between an autocatalytic and a non-autocatalytic domain. We will also present a new derivation of von Kiedrowski's square-root rate law. Function - reproducers are proposed as an important component of protocells and we will present theoretical results on a simple model system that incorporates known peptide biophysics. For a wide range of parameters, we have shown that this type of system can improve its overall performance, even in the absence of any method for information storage. This type of system improvement is defined to be non-genomic evolution.

New, Michael H.↗

NASA NTRS2019

Deterministic Polishing of Replicating Grazing-Incidence Mandrels

In an effort to manufacture high-angular-resolution, grazing-incidence, x-ray optics, Marshall Space Flight Center (MSFC) is taking measures to improve its electroformed replicated optics. A key development is the use of computer-numerical control (CNC) polishing to deterministically improve the surface of electroless nickel mandrels used to replicate grazing- incidence optics. Metrology, control software and polishing parameters must function together seamlessly to reach the specifications required to replicate sub-arcsecond optics. Each change in polishing parameters effects the wear pattern of the polishing head. Using Richardson-Lucy deconvolution, the controller software fits the wear pattern to metrology data to calculate the changing feedrates across the mandrel. Here we present an overview of our process, and early results showing the effectiveness of deterministic polishing for replicated optics.

Davis, Jacqueline↗

DOE OSTI2018

High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair

Abstract Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2′-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs.

Vallabhaneni, Haritha↗

DOE OSTI2018

Trametinib suppresses HIV-1 replication by interfering with the disassembly of human immunodeficiency virus type 1 capsid core

Highlights: • Phosphorylated/activated MEK1/2 as well as ERK2 are incorporated into the virion. • Trametinib treatment results in a decrease in ERK2 phosphorylation level in the virion. • ERK2-phosphorylation-defective virion shows an attenuated HIV-1 replication. • Capsid core dissociation is attenuated by Trametinib treatment. Our previous study showed that the phosphorylation of a highly conserved serine residue, Ser{sup 16} in the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is promoted by virion-incorporated extracellular signal-regulated kinase 2 (ERK2) and required for proper peptidyl-prolyl isomerase (Pin1)-mediated uncoating. Interestingly, western blot analysis demonstrated that phosphorylated/activated mitogen-activated protein kinase kinase 1/2 (MEK1/2), the upstream activator of ERK2, as well as ERK2 are incorporated into virions. Here, we show that the MEK1/2 selective allosteric inhibitor Trametinib reduces HIV-1 infectivity via the decrease in virion-incorporated ERK2 phosphorylation. The treatment of chronic HIV-1-infected T-cell line, CEM/LAV-1 cells with Trametinib results in a decrease in ERK2 phosphorylation in the virions. The viruses have relatively low infectivity and impaired reverse transcription. Cell-based fate-of-capsid uncoating assay showed that the reduction in infectivity was caused by a functional impairment of the uncoating process. Furthermore, the viruses from Trametinib-treated CEM/LAV-1 cells also showed decreased reverse transcription efficiency and attenuated multiple rounds of replication in human peripheral blood mononuclear cells (PBMCs). Taken together, these findings suggest that Trametinib suppresses HIV-1 replication by abrogating the proper disassembly of CA core.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

HBx-elevated SIRT2 promotes HBV replication and hepatocarcinogenesis

Highlights: • HBx upregulates the expression of SIRT2 in HCC cell lines. • SIRT2 promotes the transcription and replication of HBV. • SIRT2 facilitates HBx-mediated hepatocarcinogenesis. Sirtuin 2 (SIRT2) is a class III histone deacetylase that has been implicated to promote HCC development. However, the functional role of SIRT2 in HBV is still unclear. In this study, we found that HBV could upregulate SIRT2 expression. Additionally, HBx could activate SIRT2 promoter to upregulate the mRNA and protein level of SIRT2. Furthermore, we found that SIRT2 could facilitate HBV transcription and replication. Finally, we demonstrated that upregulation of SIRT2 by HBx promoted hepatocarcinogenesis. In summary, our findings revealed a novel function of SIRT2 in HBV and HBV-mediated HCC. First, SIRT2 could promote HBV replication. And then HBx-elevated SIRT2 could enhance the transformation of HBV-mediated HCC. Those findings highlight the potential role of SIRT2 in HBV and HBV-mediated HCC by interaction with HBx.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2026

Origin of replication discovery for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Leveraging predicted origin sequences from a previously characterized groundwater plasmidome, we constructed a barcoded plasmid library to screen for previously unknown origins. Testing this library against a panel of representative bacterial strains led to the identification of 3 previously unknown origins that replicate in gram-negative bacteria not previously associated with these origin sequences. Experimental validation confirmed that a plasmid bearing origin 6911 as the sole origin could replicate with a copy number of 9 (±2) in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium. Plasmids based on this new origin were used to express the reporter protein GFP, and non-native metabolite pathways for the natural product indigoidine and the terpenoid compound isoprenol. Functional previously unknown origins of replication in such non-model organisms can expand the toolkit for genetic manipulations of both model and less-studied bacteria.

molecular biology↗

DOE OSTI2024

A comparison of model validation approaches for echo state networks using climate model replicates

As global temperatures continue to rise, climate mitigation strategies such as stratospheric aerosol injections (SAI) are increasingly discussed, but the downstream effects of these strategies are not well understood. As such, there is interest in developing statistical methods to quantify the evolution of climate variable relationships during the time period surrounding an SAI. Feature importance applied to echo state network (ESN) models has been proposed as a way to understand the effects of SAI using a data-driven model. This approach depends on the ESN fitting the data well. If not, the feature importance may place importance on features that are not representative of the underlying relationships. Typically, time series prediction models such as ESNs are assessed using out-of-sample performance metrics that divide the times series into separate training and testing sets. However, this model assessment approach is geared towards forecasting applications and not scenarios such as the motivating SAI example where the objective is using a data driven model to capture variable relationships. Here, in this paper, we demonstrate a novel use of climate model replicates to investigate the applicability of the commonly used repeated hold-out model assessment approach for the SAI application. Simulations of an SAI are generated using a simplified climate model, and different initialization conditions are used to provide independent training and testing sets containing the same SAI event. The climate model replicates enable out-of-sample measures of model performance, which are compared to the single time series hold-out validation approach. For our case study, it is found that the repeated hold-out sample performance is comparable, but conservative, to the replicate out-of-sample performance when the training set contains enough time after the aerosol injection.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2015

Human keratinocytes restrict chikungunya virus replication at a post-fusion step

Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5{sup Δ84-96} (aa 84-96 deletion), and GP5{sup Δ97-119} (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5{sup Δ97-119}, but not full-length or GP5{sup Δ84-96}, induced a cell cycle arrest at the G2/M phase resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5{sup Δ84-96} inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology. - Highlights: • Marc-145 cell lines stable expression PRRSV GP5 or truncated GP5 were constructed. • GP5{sup Δ97-119} expression in Marc-145 cell induced cell cycle arrest at G2/M phase. • Expression of GP5 and truncated GP5 could not induce Marc-145 cells apoptosis. • PRRSV replication in Marc-145-GP5{sup Δ84-96} was significantly inhibited.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.

60 APPLIED LIFE SCIENCES↗