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At least 181 records · Page 10

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 hours post-fertilization after exposure to eight retene concentrations. The concentrations were selected to produce effects ranging from no phenotype to mortality and malformations in 100% of animals at 5 days post-fertilization. We identified a concentration-response relationship between retene teratogenicity and differential gene expression in both number of DEGs and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of AHR-dependent teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-ß (TGF-ß) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module 7 genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module 7 genes are largely responsible for retene toxicity.

Toxin, Zebrafish, Retene, Transcriptomics, network↗

DN/DG Screening of Environmental Swipe Samples: FY2016 Report

The Delayed Neutron Delayed Gamma (DNDG) technique provides a new analytical capability to the International Atomic Energy Agency (IAEA) for detecting undeclared nuclear activities. IAEA’s Long Term R&D (LTRD) plan has a stated high urgency need to develop elemental and isotopic signatures of nuclear fuel cycle activities and processes (LTRD 2.2). The new DNDG capability is used to co-detect both uranium and plutonium as an extension of a DN only method that is already being utilized by the IAEA for the analysis of swipes to inform on undeclared nuclear activities. Analytical method involving irradiation of swipe samples potentially containing trace quantities of fissile material in a thermal neutron field, followed by the counting of delayed neutrons, is a well-known technique in the field of safeguards and nonproliferation. It is used for detecting the presence of microscopic amounts of fissile material, (typically a linear combination of 233 U, 235 U, 239 Pu, and 241 Pu)and quantifying it in terms of the equivalent mass of 235U. The delayed neutron (DN) technique is very sensitive and is been routinely employed at the High Flux Isotope Reactor (HFIR) facility at Oak Ridge National Laboratory (ORNL). Both uranium and plutonium are of high safeguards value. However, the DN technique is not well suited for distinguishing between U and Pu isotopes since the decay curves overlap closely. The delayed gamma (DG) technique will help detect the presence of 239Pu in a mixture of U and Pu. Thus the DNDG approach combines the best of both worlds; the sensitivity of DN counting and the isotopic specificity of DG counting. The present work seeks to build on the delayed neutron and delayed gamma methods that have been developed at ORNL. It is recognized that the distribution profile of heavy fission products remains fairly invariant for the fissile nuclides whereas the distribution of light fission products varies from one isotope to another. That is, the ratio of the yield of a light fission fragment to a heavy fission fragments is isotope specific. Measurement of the ratio of the net full energy peak (FEP) from low/high mass fission products is an elegant way to characterize the fraction of fissile materials present in a mixture. By empirically calibrating the ratio of the net FEP as a function of known concentration of the binary mixture, one can determine the fraction of fissile isotopes in an unknown sample. In the work done in fiscal year (FY) 2016, samples of single fissile material isotopes as well as binary mixtures were irradiated in a well thermalized irradiation field in the HFIR. Delayed neutron counting was performed using the neutron counter at the HFIR Neutron Activation Analysis (NAA) laboratory. Delayed gamma counting was performed using a shielded high purity germanium (HPGe) detector. Delayed neutron decay curve results highlighted the difficulty of distinguishing between U and Pu isotopes, and the need for including the delayed gamma component. Based on delayed gamma spectrometry, twelve ratios of low mass/high fission product gamma ray FEP have been identified as valid candidates. Linearity of the ratios, as a function of 239 Pu fraction in 235 U+ 239 Pu mixtures, was confirmed for the low mass/high mass candidates that were selected. The DNDG method we are spearheading allows not only the presence of total fissile content to be detected, but whether the material is predominantly U or predominantly Pu, or a mixture. This provides additional SG relevant information.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Analysis of Supercritical-Extracted Chelated Metal Ions From Mixed Organic-Inorganic Samples

Organic and inorganic contaminants of an environmental sample are analyzed by the same GC-MS instrument by adding an oxidizing agent to the sample to oxidize metal or metal compounds to form metal ions. The metal ions are converted to chelate complexes and the chelate complexes are extracted into a supercritical fluid such as CO2. The metal chelate extract after flowing through a restrictor tube is directly injected into the ionization chamber of a mass spectrometer, preferably containing a refractory metal filament such as rhenium to fragment the complex to release metal ions which are detected. This provides a fast, economical method for the analysis of metal contaminants in a sample and can be automated. An organic extract of the sample in conventional or supercritical fluid solvents can be detected in the same mass spectrometer, preferably after separation in a supercritical fluid chromatograph.

Sinha, Mahadeva P.↗

Evaluating the performance of a high resolution SLIM IM module integrated with an Agilent 6560 IM-QTOF MS - CRADA 413 (Final Report)

Developing new technologies that enhance our abilities to identify molecular signatures is important for the health of the public, the environment, and national security. Deploying these novel technologies requires multiple stages of validation and evaluation. This project focused on evaluating a technology developed at PNNL named Structures for Lossless Ion Manipulations (SLIM) in the context of a commercially available ion mobility mass spectrometer instrument from Agilent Technologies. There is a benefit to integrating the recently developed SLIM module into a real-world instrument platform. Data from the combined instrument will provide new standards for characterizing samples using ion mobility. These standards, related to both the drift time of ions and their resulting collisional cross sections (CCS) will be of value in characterizing samples from both humans and environmental samples, such as soil microbiomes. Because no previous instrument has been designed and implemented to simultaneously analyze the same sample in both constant and oscillating drift fields, the project will provide a new research capability that will support research in a wide variety of applications. The evaluation approach included measuring the resolving power, mass range, sensitivity, and accuracy of two SLIM variations (constant and oscillating fields). Although SLIM performed similarly to the commercial instrument in terms of sensitivity, mass range, and accuracy the resolving power of SLIM far exceeded that of the commercially-available instrument. This validation will provide critically useful data to our commercial partners which they can use to reach informed decisions on the advantages of the SLIM technology such as the outstanding performance and the low cost of manufacturing.

42 ENGINEERING↗

Next Generation Microbiology Requirements

As humans continue to explore deep into space, microorganisms will travel with them. The primary means to mitigate the risk of infectious disease are a combination of prudent spacecraft design and rigorous operational controls. The effectiveness of these methods are evaluated by microbiological monitoring of spacecraft, food, water, and the crew that is performed preflight, in-flight, and post-flight. Current NASA requirements associated with microbiological monitoring are based on culture-based methodology where microorganisms are grown on a semi-solid growth medium and enumerated. Subsequent identification of the organisms requires specialized labor and large equipment, which historically has been performed on Earth. Requirements that rely strictly on culture-based units limit the use of non-culture based monitoring technology. Specifically, the culture-based "measurement criteria" are Colony Forming Units (CFU, representing the growth of one microorganism at a single location on the agar medium) per a given volume, area, or sample size. As the CFU unit by definition is culture-based, these requirements limit alternative technologies for spaceflight applications. As spaceflight missions such as those to Mars extend further into space, culture-based technology will become difficult to implement due to the (a) limited shelf life of the culture media, (b) mass/volume necessary to carry these consumables, and (c) problems associated with the production of biohazardous material in the habitable volume of the spacecraft. In addition, an extensive amount of new knowledge has been obtained during the Space Shuttle, NASA-Mir, and International Space Station Programs, which gave direction for new or modified microbial control requirements for vehicle design and mission operations. The goal of this task is to develop and recommend a new set of requirements for vehicle design and mission operations, including microbiological monitoring, based upon "lessons learned" and new technology. During 2011, this study focused on evaluating potable water requirements by assembling a forum of internal and external experts from NASA, other federal agencies, and academia. Key findings from this forum included: (1) Preventive design and operational strategies should be stringent and the primary focus of NASA's mitigation efforts, as they are cost effective and can be attained with conventional technology. (2) Microbial monitoring hardware should be simple and must be able to measure the viability of microorganisms in a sample. Multiple monitoring technologies can be utilized as long as at the microorganisms being identified can also be confirmed as viable. (3) Evidence showing alterations in the crew immune function and microbial virulence complicates risk assessments and creates the need for very conservative requirements. (4) One key source of infectious agents will always be the crew, and appropriate preventative measures should be taken preflight. (5) Water systems should be thoroughly disinfected (sterilized if possible) preflight and retain a residual biocide throughout the mission. Future forums will cover requirements for other types of samples, specifically spaceflight food and environmental samples, such as vehicle air and vehicle and cargo surfaces. An interim report on the potable water forum has been delivered to the Human Research Program with a final report on the recommendations for all sample types being delivered in September 2013.

Ott, C. M.↗

Moving beyond the van Krevelen diagram: A new stoichiometric approach for compound classification in organisms

van Krevelen diagrams (O:C vs H:C ratios of elemental formulas) have been widely used in studies to obtain an estimation of the main compound categories present in environmental samples. However, the limits defining a specific compound category based solely on O:C and H:C ratios of elemental formulas have never been accurately listed or proposed to classify metabolites in biological samples. Furthermore, while O:C vs. H:C ratios of elemental formulas can provide an overview of the compound categories, such classification is inefficient because of the large overlap among different compound categories along both axes. We propose a more accurate compound classification for biological samples analyzed by high-resolution mass spectrometry-based on an assessment of the C:H:O:N:P stoichiometric ratios of over 130,000 elemental formulas of compounds classified in 6 main categories: lipids, peptides, amino-sugars, carbohydrates, nucleotides and phytochemical compounds (oxy-aromatic compounds). Our multidimensional stoichiometric compound classification (MSCC) constraints showed a highly accurate categorization of elemental formulas to the main compound categories in biological samples with over 98% of accuracy representing a substantial improvement over any classification based on the classic van Krevelen diagram. This method represents a significant step forward in environmental research, especially ecological stoichiometry and eco-metabolomics studies, by providing a novel and robust tool to further our understanding the ecosystem structure and function through the chemical characterization of different biological samples.

Compound Classification, Ecological Stoichiometry,↗

Training in Tbilisi nuclear facility provides new sampling perspectives for IAEA inspectors

Office of Nonproliferation and Arms Control- (NPAC-) sponsored training in a “cold” nuclear facility in Tbilisi, Georgia provides International Atomic Energy Agency (IAEA) inspectors with a new perspective on environmental sampling strategies. Sponsored by the Nuclear Safeguards program under the NPAC, Pacific Northwest National Laboratory (PNNL) experts have been conducting an annual weeklong class for IAEA inspectors in a closed nuclear facility since 2011. The Andronikashvili Institute of Physics and the Republic of Georgia collaborate with PNNL to provide the training, and the U.S. Department of State, the U.S. Embassy in Tbilisi and the U.S. Mission to International Organizations in Vienna provide logistical support.

environmental sampling↗

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the importance and diversity of the microbial world contrasts starkly with our limited understanding of its fundamental structure. Despite remarkable advances in DNA sequence generation, a lack of standardized protocols and common analytical framework impede useful comparison between studies, hindering development of global inferences about microbial life on Earth. Here, we show that with coordinated protocols, exact microbial 16S rRNA gene sequences can be followed across scores of individual studies, revealing patterns of diversity, community structure, and life history strategy at a planetary scale. Using 27,751 crowdsourced environmental samples comprising more than 2.2 billion reads, we find sharp divides between host-associated and free-living communities. We show that the distribution of taxonomic and sequence diversity follows consistent trends across samples types and along gradients of environmental parameters, highlighting some of the global evolutionary patterns and ecological principles that underpin Earth’s microbiome. Here, this dataset provides the most complete environmental survey of our microbial world to date, and serves as a growing reference to provide immediate global context to future microbial surveys.

54 ENVIRONMENTAL SCIENCES↗

Solid phase sequencing of biopolymers

This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

Cantor, Charles↗

Environmental Persistence of Monkeypox Virus on Surfaces in Household of Person with Travel-Associated Infection, Dallas, Texas, USA, 2021

In July 2021, we conducted environmental sampling at the residence of a person in Dallas, Texas, USA, who had travel-associated human West African monkeypox virus (MPXV-WA). Targeted environmental swab sampling was conducted 15 days after the person who had monkeypox left the household. Results indicate extensive MPXV-WA DNA contamination, and viable virus from 7 samples was successfully isolated in cell culture. There was no statistical difference (p = 0.94) between MPXV-WA PCR positivity of porous (9/10, 90%) vs. nonporous (19/21, 90.5%) surfaces, but there was a significant difference (p<0.01) between viable virus detected in cultures of porous (6/10, 60%) vs. nonporous (1/21, 5%) surfaces. These findings indicate that porous surfaces (e.g., bedding, clothing) may pose more of a MPXV exposure risk than nonporous surfaces (e.g., metal, plastic). Viable MPXV was detected on household surfaces after at least 15 days. However, low titers (<102 PFU) indicate a limited potential for indirect transmission.

60 APPLIED LIFE SCIENCES↗

Hanford Site Environmental Surveillance Master Sampling Schedule for Calendar Year 2011

This document contains the calendar year 2011 schedule for the routine collection of samples for the Surface Environmental Surveillance Project and the Drinking Water Monitoring Project. Each section includes sampling locations, sampling frequencies, sample types, and analyses to be performed. In some cases, samples are scheduled on a rotating basis. If a sample will not be collected in 2011, the anticipated year for collection is provided. Maps showing approximate sampling locations are included for media scheduled for collection in 2011.

54 ENVIRONMENTAL SCIENCES↗

Multiplex gas chromatography for use in space craft

Gas chromatography is a powerful technique for the analysis of gaseous mixtures. Some limitations in this technique still exist which can be alleviated with multiplex gas chromatography (MGC). In MGC, rapid multiple sample injections are made into the column without having to wait for one determination to be finished before taking a new sample. The resulting data must then be reduced using computational methods such as cross correlation. In order to efficiently perform multiplexgas chromatography, experiments in the laboratory and on board future space craft, skills, equipment, and computer software were developed. Three new techniques for modulating, i.e., changing, sample concentrations were demonstrated by using desorption, decomposition, and catalytic modulators. In all of them, the need for a separate gas stream as the carrier was avoided by placing the modulator at the head of the column to directly modulate a sample stream. Finally, the analysis of an environmental sample by multiplex chromatography was accomplished by employing silver oxide to catalytically modulate methane in ambient air.

Valentin, J. R.↗