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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

PrimerDesign-M: A multiple-alignment based multiple-primer design tool for walking across variable genomes

Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. In addition, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-M finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

Background: Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results: Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions: PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next generation sequencing, and other experimental protocols targeting highly variable DNA samples.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Importance of appropriate genome information for the design of mating type primers in black and yellow morel populations

Abstract Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type ( MAT ) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit ( RPB2 ) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Additive Manufacturing for Designers: A Primer

Additive Manufacturing, also known as AM or 3D printing, is a class of manufacturing processes that create objects by shaping material layer by layer. Having demonstrated the ability to produce miraculously complex geometries, it is broadly claimed that AM will have endless applications as the technology improves. However, underneath the hype surrounding this technology is a world of nuance and constraints as well as highly strategic applications.Additive Manufacturing for Designers: A Primer, written by Dr. Amy Elliott from Oak Ridge National Laboratory and Dr. Cynthia K. Waters from North Carolina A&T State University discusses the topics needed for a holistic understanding of the many micro and macro components of the world of 3D printing.Additive Manufacturing for Designers: A Primer takes the reader on a journey beginning with important aspects of AM part design and process dependence, including resolution and tolerance issues of interest to any manufacturer. It also offers useful insights into how to build especially complex parts and avoid expensive mistakes during and after the manufacturing process is completed.

Elliott, Amy↗
DOE OSTI2016

Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.

Walters, William↗
DOE OSTI2020

BigDNA

SAND2020-13696 M BigDNA takes a user input fast DNA sequence and outputs overlapping long PCR primers for Gibson Assembly. The software can design primers that remove gene products, insert new gene products, or completely circularize long DNAs. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.

Porter, Kelley↗
DOE OSTI2021

Diagnostic Testing for COVID-19 Bridging Study for CDC EUA assays vs CDC Multiplex N1 FAM, N2 SUN, RNAse P ATTO 647 Assays

This report describes testing performed by LANL’s Biological Agent Testing Laboratory (BATL) to validate modifications to the CDC EUA 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (EUA-CDC-nCoV-IFU). BATL intends to implement the modification to increase thoughput for daily testing. BATL validated the original component, CDC designed primers and probes purchased from IDT (Cat # 10006770) and the new component, CDC assays designed with new Reporter dyes allowing the assays to be multiplexed, IDT (Cat # 10006830, 10006831, 10006823, 10006833, 10006834, 10007050, 10006836, 10006837, 10007062)

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions

Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics (i.e., chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer–probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 10 4 colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells (i.e., without PMAxx) from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx (i.e., live cells). Furthermore, in 50–500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4–459.3 RFU) and total cells (207.82–684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Populus VariantDB v3.2 facilitates CRISPR and functional genomics research

The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Association of Vampirovibrio chlorellavorus with decline and death of Chlorella sorokiniana in outdoor reactors

The outdoor ARID raceway was established for optimizing the cultivation of microalgae for biofuel production. During the summers of 2014 and 2015, discoloration was observed in cultures of Chlorella sorokiniana , which shifted from a vibrant green color to yellow, followed by cell clumping, decline in density, and rapid death, resulting in 40–60% reduced biomass production. To investigate, total DNA was purified from the raceway samples and subjected to polymerase chain reaction (PCR) amplification using degenerate primers that amplify the 16S rRNA gene of eubacteria. BLASTn analysis of the cloned amplicon sequences revealed the presence of the Gram-negative, predatory bacterium, Vampirovibrio chlorellavorus . Scanning electron microscopic examination showed an abundance of coccoid cells, 0.3–0.6 μm in diameter, some of which were attached to C. sorokiniana cells. PCR amplification indicated the presence of V. chlorellavorus in raceway vessels, water lines, connective tubing, and in early, scaled-up DOE1412 cultures used to inoculate the raceway. Based on PCR detection, the decontamination of the equipment and water line with "Wal-Clean" more effectively eliminated V. chlorellavorus and delayed the onset of attack, compared to the chlorine disinfectant, trichloromelamine (TCM). Total DNA was isolated from soil samples collected monthly from the nearby Rillito River during 2014–2015 and subjected to PCR amplification using primers designed to amplify the 16S rRNA and 18S rRNA gene of V. chlorellavorus and C. sorokiniana , respectively. Results indicated that V. chlorellavorus and Chlorella spp. were present in most of the riverbed samples nearly year round, suggesting a possible naturally occurring reservoir of the predatory bacterium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57–60% GC-content at a rate of 50–100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

PUNCS: Towards Predictive Understanding of Nitrogen Cycling in Soils

In anoxic environments, the major nitrate/nitrite-consuming processes are respiratory ammonification (also known as dissimilatory nitrate reduction to ammonium) and denitrification (i.e., the formation of the gaseous products N 2 O and N 2 ). Respiratory ammonification oxidizes more carbon per mole of nitrate than denitrification and generates a cation (NH 4+ ), which is retained in soils and bioavailable for plants. Thus, these processes have profoundly different impacts on N retention and greenhouse gas (CO 2 , N 2 O) emissions. Microbes capable of respiratory ammonification or denitrification coexist but the environmental controls over these competing nitrate/nitrite-reducing processes are largely unknown. With the current level of understanding, predictions under what environmental conditions respiratory ammonification activity predominates leading to N-retention rather than N-loss are tenuous. Further, the diversity of genes encoding the ammonium-forming nitrite reductase NrfA is poorly defined hampering the development of tools to assess and monitor this activity in environmental systems. Incomplete denitrification leads to N 2 O, a gas implicated in ozone layer destruction and climate change. The conversion of the greenhouse gas N 2 O to benign N 2 is catalyzed by N 2 O reductase, the characteristic enzyme system of complete denitrifiers. Thus, efforts to estimate N 2 O conversion to N 2 have focused on the well-characterized denitrifier nosZ genes; however, our understanding of the diversity of genes and organisms contributing to N 2 O consumption is incomplete. This paucity of information limits the development of more accurate, predictive models for C- and N-fluxes and greenhouse gas emissions. A comprehensive analysis of the key catalyst of respiratory ammonification, ammonia-forming nitrite reductase NrfA, revealed the evolutionary history of nrfA and identified novel diagnostic features, allowing optimized primer design for nrfA monitoring. Further, a novel group of functional “atypical” nosZ genes was found indicating that a much broader diversity of genes and organisms contribute to consumption of N 2 O. The atypical nosZ genes are distributed in soil ecosystems and often outnumber their typical counterparts, emphasizing their potential role in N 2 O consumption in soils and possibly other environments. Kinetic studies revealed that organisms with atypical NosZ exhibit significantly higher affinity to N 2 O, indicating that the relative activity of bacteria with typical versus atypical NosZ control N 2 O emissions and determine a soil’s N 2 O sink capacity. Collectively, the discoveries made under the PUNCS project improve understanding of N- and associated C-cycling processes in soils, enable the design of enhanced monitoring tools, and allow a larger research community to generate comprehensive datasets required to generate Earth System Models with higher predictive power.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Establishment of a reverse transcription real-time quantitative PCR method for Getah virus detection and its application for epidemiological investigation in Shandong, China

Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus Alphavirus of the family Togaviridae . Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) method combined with plaque assay was established for GETV with specific primers designed for the highly conserved region of GETV Nsp1 gene. The results showed that after optimizing the condition of RT-qPCR reaction, the minimum detection limit of the assay established in this study was 7.73 PFU/mL, and there was a good linear relationship between viral load and Cq value with a correlation coefficient ( R 2 ) of 0.998. Moreover, the method has good specificity, sensitivity, and repeatability. The established RT-qPCR is 100-fold more sensitive than the conventional RT-PCR. The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.956. Meanwhile, we collected 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens in Shandong Province in 2022. The positive detection rates by RT-qPCR were 1%, 1%, 0.2%, 0%, and 3%, respectively. In conclusion, the method was used for epidemiological investigation, which has extensive application prospects.

Cao, Xinyu↗
DOE OSTI2024

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa , which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa . The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

Zhu, Xiao-Xuan↗
DOE OSTI2020

KENO-VI Primer: Performing Calculations using SCALE’s Criticality Safety Analysis Sequence (CSAS6) with Fulcrum

The SCALE code system developed at Oak Ridge National Laboratory is widely used and accepted around the world for criticality safety analysis. The well-known KENO-VI three-dimensional Monte Carlo criticality computer code is one of the primary criticality safety analysis tools in SCALE. The KENO-VI primer is designed to help a new user understand and use the SCALE/KENO-VI Monte Carlo code for nuclear criticality safety analysis. It assumes that the user has a college education in a technical field. There is no assumption of familiarity with Monte Carlo codes in general or with SCALE/KENO-VI in particular. The primer is designed to teach by example, with each example illustrating two or three features of SCALE/KENO-VI that are useful in criticality analysis. The primer is based on SCALE 6.2 and 6.3, which includes the Fulcrum graphical user interface. Each example uses Fulcrum to provide the framework for preparing input data and viewing output results. Starting with a Quickstart section, the primer gives an overview of the basic requirements for SCALE/KENO-VI input and allows the user to quickly run a simple criticality problem with SCALE/KENO-VI. Each following section begins with a list of basic objectives identifying the goal of the section and the individual SCALE/KENO-VI features covered in detail in the section’s sample problems. Upon completion of the primer, a new user should be comfortable using Fulcrum to set up criticality problems in SCALE/KENO-VI. The primer provides a starting point for the criticality safety analyst who uses SCALE/KENO-VI. Complete descriptions are provided in the SCALE/KENO-VI manual. Although the primer is self-contained, it is intended as a companion volume to the SCALE/KENO-VI training and documentation. The SCALE manual and training schedule are available at https://scale.ornl.gov. The primer provides specific examples of using SCALE/KENO-VI for criticality analysis; the SCALE/KENO-VI manual provides information on the use of SCALE/KENO-VI and all its modules. The primer also contains an appendix with sample input files. In addition, this primer, its errata, and sample inputs are also available at https://code.ornl.gov/scale/primers/kenovi/.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗
DOE OSTI2020

KENO V.a Primer: Performing Calculations using SCALE’s Criticality Safety Analysis Sequence (CSAS5) with Fulcrum

The SCALE code system developed at Oak Ridge National Laboratory is widely used and accepted around the world for criticality safety analyses. The well-known KENO V.a three-dimensional Monte Carlo criticality computer code is one of the primary criticality safety analysis tools in SCALE. The KENO V.a primer is designed to help a new user understand and use the SCALE/KENO V.a Monte Carlo code for nuclear criticality safety analyses. It assumes that the user has a college education in a technical field. There is no assumption of familiarity with Monte Carlo codes in general or with SCALE/KENO V.a in particular. The primer is designed to teach by example, with each example illustrating two or three features of SCALE/KENO V.a that are useful in criticality analyses. The primer is based on SCALE 6.2 and 6.3, which includes the Fulcrum graphical user interface (GUI). Each example uses Fulcrum to provide the framework for preparing input data and viewing output results. Starting with a Quickstart section, the primer gives an overview of the basic requirements for SCALE/KENO V.a input and allows the user to quickly run a simple criticality problem with SCALE/KENO V.a. The sections that follow Quickstart include a list of basic objectives at the beginning that identifies the goal of the section and the individual SCALE/KENO V.a features that are covered in detail in the sample problems in that section. Upon completion of the primer, a new user should be comfortable using Fulcrum to set up criticality problems in SCALE/KENO V.a. The primer provides a starting point for the criticality safety analyst who uses SCALE/KENO V.a. Complete descriptions are provided in the SCALE/KENO V.a manual. Although the primer is self-contained, it is intended as a companion volume to the SCALE/KENO V.a training and documentation. The SCALE manual and training schedule are available at https://scale.ornl.gov. The primer provides specific examples of using SCALE/KENO V.a for criticality analyses; the SCALE/KENO V.a manual provides information on the use of SCALE/KENO V.a and all its modules. The primer also contains an appendix with sample input files. In addition, this primer, its errata and sample inputs are also available at https://code.ornl.gov/scale/primers/kenova.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗