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DOE OSTI2020

Single Amino Acid Mutations Affect Zika Virus Replication In Vitro and Virulence In Vivo

The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1993

SURPHEX (tm): New dry photopolymers for replication of surface relief diffractive optics

High efficiency, deep groove, surface relief Diffractive Optical Elements (DOE) with various optical functions can be recorded in a photoresist using conventional interferometric holographic and computer generated photolithographic recording techniques. While photoresist recording media are satisfactory for recording individual surface relief DOE, a reliable and precise method is needed to replicate these diffractive microstructures to maintain the high aspect ratio in each replicated DOE. The term 'high aspect ratio' means that the depth of a groove is substantially greater, i.e. 2, 3, or more times greater, than the width of the groove. A new family of dry photopolymers SURPHEX was developed recently at Du Pont to replicate such highly efficient, deep groove DOE's. SURPHEX photopolymers are being utilized in Du Pont's proprietary Dry Photopolymer Embossing (DPE) technology to replicate with very high degree of precision almost any type of surface relief DOE. Surfaces relief microstructures with width/depth aspect ratio of 1:20 (0.1 micron/2.0 micron) were faithfully replicated by DPE technology. Several types of plastic and glass/quartz optical substrates can be used for economical replication of DOE.

Shvartsman, Felix P.↗

DOE OSTI2020

Reproducibility and Replication of Experimental Particle Physics Results

Recently, much attention has been focused on the replicability of scientific results, causing scientists, statisticians, and journal editors to examine closely their methodologies and publishing criteria. Experimental particle physicists have been aware of the precursors of non-replicable research for many decades and have many safeguards to ensure that the published results are as reliable as possible. The experiments require large investments of time and effort to design, construct, and operate. Large collaborations produce and check the results, and many papers are signed by more than three thousand authors. This paper gives an introduction to what experimental particle physics is and to some of the tools that are used to analyze the data. It describes the procedures used to ensure that results can be computationally reproduced, both by collaborators and by non-collaborators. It describes the status of publicly available data sets and analysis tools that aid in reproduction and recasting of experimental results. It also describes methods particle physicists use to maximize the reliability of the results, which increases the probability that they can be replicated by other collaborations or even the same collaborations with more data and new personnel. Examples of results that were later found to be false are given, both with failed replication attempts and one with alarmingly successful replications. While some of the characteristics of particle physics experiments are unique, many of the procedures and techniques can be and are used in other fields.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

NASA NTRS2020

Perspectives on Data Reproducibility and Replicability in Paleoclimate and Climate Science

This paper summarizes the current state of reproducibility and replicability in the fields of climate and paleoclimate science, including brief histories of their development and applications in climate science, new and recent approaches towards improvement of reproducibility and replicability, and challenges. Recommendations for addressing those challenges include: development of searchable, auto-updated, interlinked, multi-archive public paleoclimate repositories for raw and processed digital datasets; cross-center standardized code base cases, improved data storage techniques, and a focus on replicability for climate simulation storage and access; and support of the development and community awareness of findable, accessible, interoperable and reusable (FAIR) principles by funding agencies and publishers. This paper is largely based on the May 2018 presentations of a panel of researchers to the Committee on Reproducibility and Replicability in Science, part of the National Academies of Science, Engineering, and Medicine. The commentary and recommendations made here are in alignment with those of its Consensus Study Report on Reproducibility and Replicability in Science (2019).

data repositories↗

NASA NTRSDate not supplied

Techniques in Assessing Random Uncertainty of Wind Tunnel Replicate Test Data

The preferred method to determine the random uncertainty of variables of interest during wind tunnel tests is by direct analysis of replicate data. However, current information is limited to analysis of a single replicate set condition that does not consider data covariance. This paper provides additional information on considerations for quantifying random uncertainty including the evaluation and correction of replicate data covariance, the selection of replicate set conditions for the test, and the interpolation of random uncertainty at other than replicate set conditions. The analysis and techniques are supported by data from a conventional aircraft configuration test at the Oran Nicks Low-Speed Wind Tunnel and an ASME nozzle flow characterization at the Propulsion Systems Laboratory.

Uncertainty↗

DOE OSTI2022

Identification of Novel, Replicable Genetic Risk Loci for Suicidal Thoughts and Behaviors Among US Military Veterans

Importance: Suicide is a leading cause of death; however, the molecular genetic basis of suicidal thoughts and behaviors (SITB) remains unknown. Objective: To identify novel, replicable genomic risk loci for SITB. Design, Setting, and Participants: This genome-wide association study included 633 778 US military veterans with and without SITB, as identified through electronic health records. GWAS was performed separately by ancestry, controlling for sex, age, and genetic substructure. Cross-ancestry risk loci were identified through meta-analysis. Study enrollment began in 2011 and is ongoing. Data were analyzed from November 2021 to August 2022. Main Outcome and Measures: SITB. Results: A total of 633 778 US military veterans were included in the analysis (57 152 [9%] female; 121 118 [19.1%] African ancestry, 8285 [1.3%] Asian ancestry, 452 767 [71.4%] European ancestry, and 51 608 [8.1%] Hispanic ancestry), including 121 211 individuals with SITB (19.1%). Meta-analysis identified more than 200 GWS (P < 5 × 10 -8 ) cross-ancestry risk single-nucleotide variants for SITB concentrated in 7 regions on chromosomes 2, 6, 9, 11, 14, 16, and 18. Top single-nucleotide variants were largely intronic in nature; 5 were independently replicated in ISGC, including rs6557168 in ESR1, rs12808482 in DRD2, rs77641763 in EXD3, rs10671545 in DCC, and rs36006172 in TRAF3. Associations for FBXL19 and AC018880.2 were not replicated. Gene-based analyses implicated 24 additional GWS cross-ancestry risk genes, including FURIN, TSNARE1, and the NCAM1-TTC12-ANKK1-DRD2 gene cluster. Cross-ancestry enrichment analyses revealed significant enrichment for expression in brain and pituitary tissue, synapse and ubiquitination processes, amphetamine addiction, parathyroid hormone synthesis, axon guidance, and dopaminergic pathways. Seven other unique European ancestry–specific GWS loci were identified, 2 of which (POM121L2 and METTL15/LINC02758) were replicated. Two additional GWS ancestry-specific loci were identified within the African ancestry (PET112/GATB) and Hispanic ancestry (intergenic locus on chromosome 4) subsets, both of which were replicated. Further, no GWS loci were identified within the Asian ancestry subset; however, significant enrichment was observed for axon guidance, cyclic adenosine monophosphate signaling, focal adhesion, glutamatergic synapse, and oxytocin signaling pathways across all ancestries. Within the European ancestry subset, genetic correlations (r > 0.75) were observed between the SITB phenotype and a suicide attempt-only phenotype, depression, and posttraumatic stress disorder. Additionally, polygenic risk score analyses revealed that the Million Veteran Program polygenic risk score had nominally significant main effects in 2 independent samples of veterans of European and African ancestry.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli

Escherichia coli cell cycle features two critical cell-cycle checkpoints: initiation of replication and the onset of constriction. While the initiation of DNA replication has been extensively studied, it is less clear what triggers the onset of constriction and when exactly it occurs during the cell cycle. Here, using high-throughput fluorescence microscopy in microfluidic devices, we determine the timing for the onset of constriction relative to the replication cycle in different growth rates. Our single-cell data and modeling indicate that the initiation of constriction is coupled to replication-related processes in slow growth conditions. Furthermore, our data suggest that this coupling involves the mid-cell chromosome blocking the onset of constriction via some form of nucleoid occlusion occurring independently of SlmA and the Ter linkage proteins. This work highlights the coupling between replication and division cycles and brings up a new nucleoid mediated control mechanism in E. coli.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Structured sequences emerge from random pool when replicated by templated ligation

The central question in the origin of life is to understand how structure can emerge from randomness. The Eigen theory of replication states, for sequences that are copied one base at a time, that the replication fidelity has to surpass an error threshold to avoid that replicated specific sequences become random because of the incorporated replication errors [M. Eigen, Naturwissenschaften 58 (10), 465–523 (1971)]. Here, we showed that linking short oligomers from a random sequence pool in a templated ligation reaction reduced the sequence space of product strands. We started from 12-mer oligonucleotides with two bases in all possible combinations and triggered enzymatic ligation under temperature cycles. Surprisingly, we found the robust creation of long, highly structured sequences with low entropy. At the ligation site, complementary and alternating sequence patterns developed. However, between the ligation sites, we found either an A-rich or a T-rich sequence within a single oligonucleotide. Our modeling suggests that avoidance of hairpins was the likely cause for these two complementary sequence pools. What emerged was a network of complementary sequences that acted both as templates and substrates of the reaction. This self-selecting ligation reaction could be restarted by only a few majority sequences. The findings showed that replication by random templated ligation from a random sequence input will lead to a highly structured, long, and nonrandom sequence pool. This is a favorable starting point for a subsequent Darwinian evolution searching for higher catalytic functions in an RNA world scenario.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

An enzyme-based approach for highly efficient self-replication of DNA origami dimers

Self-replication and exponential growth are essential to all living things, the driving force for Darwinian evolution, and potentially useful in nanotechnology for large-scale production of nanoscopic materials. An artificial (nonliving) self-replication system has been shown to exhibit exponential growth and selection using DNA monomer origami tiles templated on a dimer seed. That system purposefully avoided the use of enzymes to get a hint of how self-replication might have evolved in a prebiotic world by using CNV K and UV light to crosslink complementary DNA single strands. For further investigations into competition and extinction and for potential applications involving biocompatibility, we wanted to investigate enzymatic ligation to replace the chemical photo crosslinking step. Here, we present a system which uses thermotolerant T4 DNA ligase and no UV. This system has several additional advantages including a much faster cycling time, yielding 2,000,000 amplifications in 12 h. We also introduce competition to study the possibility of Darwinian-like evolution. Two pairs of DNA origami tiles compete for the same connection strands and show different growth rates under different connection strand concentrations. This system has the potential to combine with other enzymes, such as RNA polymerase to support feedback, allowing us to fine-tune replication dynamics and achieve sophisticated, life-like behaviors. The highly efficient self-replication and exponential growth of DNA origami dimers demonstrated in this work not only enhances our understanding of Darwinian evolution in nature but also opens the door to applications ranging from synthetic biology to smart materials.

Science & Technology - Other Topics↗

DOE OSTI2026

EEPD1 evolved a unique DNA clamping dimer protecting reversed replication forks

Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family domain containing protein 1 (EEPD1) acts in DNA stress responses, its proposed nuclease activities are enigmatic. Here, we integrate hybrid structural methods, evolution, biochemistry, cancer genomics, plus molecular and cell biology to define EEPD1 structure, assembly, and function at stalled DNA replication forks. Results imply EEPD1 surprisingly requires both unique EEP domain dimer and distinctive tandem Helix-hairpin-Helix [(HhH) 2 ] domains to clamp double-stranded (ds) DNA at reversed DNA replication forks for fork protection. Small-angle X-ray Scattering (SAXS), crystal, and cryo-EM structures unveil an unprecedented tryptophan handshake dimer, conserved interface di-Trp-Pro pocket, and adjustable “wrist” enabling an open-closed conformational switch. EEPD1 dimer cooperatively binds complex dsDNA replication fork intermediates but alone lacks nuclease activity due to loss of key EEP catalytic residues during Metazoan evolution and atmospheric oxygen buildup. Instead, EEPD1 prevents nucleolytic degradation of reversed replication forks by MRE11. Furthermore, cancer bioinformatics support oxidative damage-dependent EEPD1 association as a significant modulator of overall patient survival. Collective findings uncover unexpected EEP dimer and fork protection function in clamping, not cleaving, reversed replication forks for metazoan oxidative stress responses controlling genome stability and cancer outcomes.

Shen, Runze [Univ. of Texas, Houston, TX (United S↗

DOE OSTI2020

Swine Host Protein Coiled-Coil Domain-Containing 115 (CCDC115) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2 during Virus Replication

Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2–CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2–CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Evaluation of an ASFV RNA Helicase Gene A859L for Virus Replication and Swine Virulence

African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-ΔA859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-ΔA859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-ΔA859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1980

A theory for the origin of a self-replicating chemical system. I - Natural selection of the autogen from short, random oligomers

A general theory is presented for the origin of a self-replicating chemical system, termed an autogen, which is capable of both crude replication and translation (protein synthesis). The theory requires the availability of free energy and monomers to the system, a significant background low-yield synthesis of kinetically stable oligopeptides and oligonucleotides, the localization of the oligomers, crude oligonucleotide selectivity of amino acids during oligopeptide synthesis, crude oligonucleotide replication, and two short peptide families which catalyze replication and translation, to produce a localized group of at least one copy each of two protogenes and two protoenzymes. The model posits a process of random oligomerization, followed by the random nucleation of functional components and the rapid autocatalytic growth of the functioning autogen to macroscopic amounts, to account for the origin of the first self-replicating system. Such a process contains steps of such high probability and short time periods that it is suggested that the emergence of an autogen in a laboratory experiment of reasonable time scale may be possible.

White, D. H.↗

NASA NTRS1993

Simple systems that exhibit self-directed replication

Biological experience and intuition suggest that self-replication is an inherently complex phenomenon, and early cellular automata models support that conception. More recently, simpler computational models of self-directed replication called sheathed loops have been developed. It is shown here that 'unsheathing' these structures and altering certain assumptions about the symmetry of their components leads to a family of nontrivial self-replicating structures some substantially smaller and simpler than those previously reported. The dependence of replication time and transition function complexity on initial structure size, cell state symmetry, and neighborhood are examined. These results support the view that self-replication is not an inherently complex phenomenon but rather an emergent property arising from local interactions in systems that can be much simpler than is generally believed.

Reggia, James A.↗

NASA NTRS1993

Mobile and replicated alignment of arrays in data-parallel programs

When a data-parallel language like FORTRAN 90 is compiled for a distributed-memory machine, aggregate data objects (such as arrays) are distributed across the processor memories. The mapping determines the amount of residual communication needed to bring operands of parallel operations into alignment with each other. A common approach is to break the mapping into two stages: first, an alignment that maps all the objects to an abstract template, and then a distribution that maps the template to the processors. We solve two facets of the problem of finding alignments that reduce residual communication: we determine alignments that vary in loops, and objects that should have replicated alignments. We show that loop-dependent mobile alignment is sometimes necessary for optimum performance, and we provide algorithms with which a compiler can determine good mobile alignments for objects within do loops. We also identify situations in which replicated alignment is either required by the program itself (via spread operations) or can be used to improve performance. We propose an algorithm based on network flow that determines which objects to replicate so as to minimize the total amount of broadcast communication in replication. This work on mobile and replicated alignment extends our earlier work on determining static alignment.

Chatterjee, Siddhartha↗

NASA NTRS1996

Design and Implementation of Replicated Object Layer

One of the widely used techniques for construction of fault tolerant applications is the replication of resources so that if one copy fails sufficient copies may still remain operational to allow the application to continue to function. This thesis involves the design and implementation of an object oriented framework for replicating data on multiple sites and across different platforms. Our approach, called the Replicated Object Layer (ROL) provides a mechanism for consistent replication of data over dynamic networks. ROL uses the Reliable Multicast Protocol (RMP) as a communication protocol that provides for reliable delivery, serialization and fault tolerance. Besides providing type registration, this layer facilitates distributed atomic transactions on replicated data. A novel algorithm called the RMP Commit Protocol, which commits transactions efficiently in reliable multicast environment is presented. ROL provides recovery procedures to ensure that site and communication failures do not corrupt persistent data, and male the system fault tolerant to network partitions. ROL will facilitate building distributed fault tolerant applications by performing the burdensome details of replica consistency operations, and making it completely transparent to the application.Replicated databases are a major class of applications which could be built on top of ROL.

Koka, Sudhir↗

NASA NTRS2002

A Self-Replicating Ligase Ribozyme

A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

Paul, Natasha↗

NASA NTRS1992

Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

Non-NASA Center↗