Environmental Molecular Sciences Laboratory

A DOE Office of Science User Facility

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Virtual Tour

Welcome to EMSL's virtual tours. Here you will be able to virtually facilities within EMSL's user program.

Easy Navigation
You can zoom and pan 360-degrees at each tour stop. Major stops include symbols indicating different types of information that provide greater detail about the location.

The symbols include:
• A "star" – provides an overview of the capability of that lab.
• A "video camera" – indicates a video featuring an EMSL expert explaining that laboratory and capabilities available to EMSL users.
• A "paragraph" – takes you to additional materials, such as science highlights and brochures.

EMSL EMSL building
This tour includes an overview from former EMSL Director Allison Campbell and features four laboratories within EMSL. You can see firsthand 11 of its state-of-the-art instruments and hear from EMSL experts.

The laboratories featured include:
• Nuclear Magnetic Resonance
• Surface Science
• High-Sensitivity Laser Imaging
• Ion Mobility Mass Spectrometry

This tour includes six laboratories. On this tour, you will hear from Nancy Hess, EMSL Science Theme lead for Terrestrial Subsurface Ecosystems, and EMSL experts.

The laboratories featured include:
• Nuclear Magnetic Resonance and Electron Magnetic Resonance – NMR/EPR
• Transmission Electron Microscopy – TEM
• Scanning Electron Microscopy – SEM
• Electron Microprobe and Scanning Probe Microscopy – EMP/SPM
• X-ray Photoelectron Spectrometry – XPS
• Sample Receiving and Preparation/Analytical Chemistry

Instruments

Highlighted Research Applications EMSL’s suite of NMR metabolomics and metabolic flux analysis capabilities enables EMSL users to probe the...
Custodian(s): David Hoyt

Science Highlights

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Instruments

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Related Videos

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Welcome to EMSL's virtual tours. Here you will be able to virtually facilities within EMSL's user program.

Easy Navigation
You can zoom and pan 360-degrees at each tour stop. Major stops include symbols indicating different types of information that provide greater detail about the location.

The symbols include:
• A "star" – provides an overview of the capability of that lab.
• A "video camera" – indicates a video featuring an EMSL expert explaining that laboratory and capabilities available to EMSL users.
• A "paragraph" – takes you to additional materials, such as science highlights and brochures.

EMSL EMSL building
This tour includes an overview from former EMSL Director Allison Campbell and features four laboratories within EMSL. You can see firsthand 11 of its state-of-the-art instruments and hear from EMSL experts.

The laboratories featured include:
• Nuclear Magnetic Resonance
• Surface Science
• High-Sensitivity Laser Imaging
• Ion Mobility Mass Spectrometry

This tour includes six laboratories. On this tour, you will hear from Nancy Hess, EMSL Science Theme lead for Terrestrial Subsurface Ecosystems, and EMSL experts.

The laboratories featured include:
• Nuclear Magnetic Resonance and Electron Magnetic Resonance – NMR/EPR
• Transmission Electron Microscopy – TEM
• Scanning Electron Microscopy – SEM
• Electron Microprobe and Scanning Probe Microscopy – EMP/SPM
• X-ray Photoelectron Spectrometry – XPS
• Sample Receiving and Preparation/Analytical Chemistry

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15

Abstract: 

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 µM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer’s therapy, was shown to have antiviral activity between 10 to 66 µM, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.

Citation: 
Choi R., M. Zhou, R. Shek, J.W. Wilson, L. Tillery, J.C. Craig, and I.A. Salukhe, et al. 2021. "High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity." <i>PLoS One</i> 16, no. 4:e0250019. PNNL-SA-159108. doi:10.1371/journal.pone.0250019
Authors: 
Shek
Roger;Salukhe
Indraneel A;Wu
Ruilian;Nguyen
Tu-Trinh;Choi
Ryan;Craig
Justin C;Kumar
Neeraj;Hurst
Brett;Barrett
Lynn;Hyde
Jennifer L;Zhou
Mowei;Wilson
Jesse W;Tillery
Logan;Hickson
Sarah E;James
Rhema Mary;Van Voorhis
Wesley C;Buchko
Garry W;Huff
Sydney;Cherry
Sara
Facility: 

Quantitative cardiac phosphoproteomics profiling during ischemia-reperfusion in an immature swine model

Abstract: 

Ischemia-reperfusion (I/R) results in altered metabolic and molecular responses, and phosphorylation is one of the most noted regulatory mechanisms mediating signaling mechanisms during physiological stresses. To expand our knowledge of the potential phosphoproteomic changes in the myocardium during I/R, we used Isobaric Tags for Relative and Absolute Quantitation-based analyses in left ventricular samples obtained from porcine hearts under control or I/R conditions. The data are available via ProteomeXchange with identifier PXD006066. We identified 1,896 phosphopeptides within left ventricular control and I/R porcine samples. Significant differential phosphorylation between control and I/R groups was discovered in 111 phosphopeptides from 86 proteins. Analysis of the phosphopeptides using Motif-x identified five motifs: (..R..S..), (..SP..), (..S.S..), (..S…S..), and (..S.T..). Semiquantitative immunoblots confirmed site location and directional changes in phosphorylation for phospholamban and pyruvate dehydrogenase E1, two proteins known to be altered by I/R and identified by this study. Novel phosphorylation sites associated with I/R were also identified. Functional characterization of the phosphopeptides identified by our methodology could expand our understanding of the signaling mechanisms involved during I/R damage in the heart as well as identify new areas to target therapeutic strategies.

Citation: 
Ledee D.R., M.A. Kang, M. Kajimoto, S.O. Purvine, H.M. Brewer, L. Pasa Tolic, and M.A. Portman. 2017. "Quantitative cardiac phosphoproteomics profiling during ischemia-reperfusion in an immature swine model." <i>American Journal of Physiology. Heart and Circulatory Physiology</i> 313, no. 1:H125-H137. PNNL-SA-128530. doi:10.1152/ajpheart.00842.2016
Authors: 
Heather Brewer,
Pasa Tolic, Ljiljana
Purvine, Samuel O
Ledee
Dolena R;Kajimoto
Masaki;Portman
Michael Alan;Brewer
Heather M;Pasa Tolic
Ljiljana;Purvine
Samuel O;Kang
Min A
Facility: 

Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms – us) dynamics in the C

Abstract: 

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein’s backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone 1H, 13C, and 15N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of a ~70% deuterated sample and residue-specific, 15N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the “missing” amide resonances for N96-L106 in the 1H-15N HSQC spectrum, a region that comprises almost the complete C-terminal alpha-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to us timescale and/or is heterogenous. These “missing” amide resonances do not appear in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.007 mM. At this concentration, native mass spectrometry indicates the protein is almost exclusively in the monomer state, suggesting the intermediate dynamics in the C-terminal of Nsp9 is not due to intermolecular interactions. Perhaps this intermediate ms to us timescale dynamics is responsible for a previously suggested “fluidity” of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.

Citation: 
Buchko G.W., M. Zhou, J.C. Craig, W.C. Van Voorhis, and P.J. Myler. 2021. "Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms – us) dynamics in the C-terminal helix at the dimer interface." <i>Biomolecular NMR Assignments</i> 15, no. 1:107-116. PNNL-SA-157114. doi:10.1007/s12104-020-09992-1
Authors: 
Buchko
Garry W;Zhou
Mowei;Craig
Justin C;Van Voorhis
Wesley C;Myler
Peter J
Facility: 

Metabolic effects of vitamin B12 on physiology, stress resistance, growth rate and biomass productivity of Cyanobacterium

Abstract: 

Although synthesized only by bacteria and archaea, cobalamin (vitamin B12) is essential for virtually all living cells. One major function is its role in methionine synthesis, as a co-factor in for the B12-dependent methionine synthase MetH. However, large number of microbes avoid requirements for B12 by encoding cobalamin-independent enzymes, such as the B12-independent methionine synthase MetE. Ineterestingly, many such microbes retain transporters for exogenous B12, produced by neighboring microbes. We hypothesise that selection for retention of B12 transport suggests preservation of unrevealed but critical roles for cobalamin in photoautotroph fitness. To identify the impacts of B12 on photoautorophic metabolism, we studied the physiological and transcriptional adaptation of Cyanobacterium stanieri HL-69 to varying irradiance and oxidative stress in the presence and absence of B12. The metabolic flexibility of C. stanieri, which possesses both MetH and MetE, alows comparative analysis of cobalamin impacts on its global metabolism. As anticipated, B12 availability governed transcription of cobalamin transporter btuB, metH and a number of genes involved in the methionine-folate cycle. Surprisingly, however, B12 impacted the cell integrity and growth rate of C. stanieri under conditions of likely oxidative stress due to biofilm growth or under high partial pressures of O2. Furthermore, C. stanieri response to B12 globally rewired cellular metabolic networks, including nitrogen metabolism, energy metabolism, and redox homeostasis and oxidative stress response. These findings demonstrate previously-unappreciated roles for B12 metabolism beyond methionine synthesis and reveal how interactions with cobalamin-producing heterotrophs may affect phytoplankton function and dynamics in natural microbial communities.
Importance
Cobalamin cross-feeding is recognised as a key factor promoting establishment of complex microbial systems. However, the lack of understanding regarding B12 impacts on photoautotroph metabolism hinders our ability to predict structure-function relationships in phytoplankton communities. Our data suggesting B12’s irrelevance to C. stanieri’s growth rate in the absence of oxygen stress may explain the loss of B12 synthesis genes from its genome. However, B12 impacts on fitness during periodic exposures to elevated pO2 in the diffusion-limited environment of a phototrophic microbial mat suggests a rationale for retention of B12-dependent processes and transport in C. stanieri. Furthermore, this study reveals that B12 availability exerts far-reaching impacts on C. stanieri metabolism, such as in redox homeostasis and oxidative stress response. Finally, understanding the mechanisms underlying the protective effect of cobalamin against oxidative stress may help explain the high robustness of phototrophic microbial communities and suggests strategies for engineering more efficient bioprocesses.

Citation: 
Bohutskyi P., R.S. McClure, E.A. Hill, W.C. Nelson, W.B. Chrisler, J. Nunez, and R.S. Renslow, et al. 2019. "Metabolic effects of vitamin B12 on physiology, stress resistance, growth rate and biomass productivity of Cyanobacterium stanieri planktonic and biofilm cultures." <i>Algal Research</i> 42. PNNL-SA-136164. doi:10.1016/j.algal.2019.101580
Authors: 
Bohutskyi
Pavlo;McClure
Ryan S;Hill
Eric A;Nelson
William C;Chrisler
William B;Nunez
Jamie;Renslow
Ryan S;Charania
M.;Lindemann
Steve;Beliaev
Alex S
Facility: 

A Phenotarget Approach for Identifying an Alkaloid Interacting with the Tuberculosis Protein Rv1466

Abstract: 

In recent years, there has been a revival of interest in phenotypic-based drug discovery (PDD) due to target-based drug discovery (TDD) falling below expectations. Both PDD and TDD have their unique advantages and should be used as complementary methods in drug discovery. The PhenoTarget approach combines the strengths of the PDD and TDD approaches. Phenotypic screening is conducted initially to detect cellular active components and the hits are then screened against a panel of putative targets. This PhenoTarget protocol can be equally applied to pure compound libraries as well as natural product fractions. Here we described the use of the PhenoTarget approach to identify an anti-tuberculosis lead compound. Fractions from Polycarpa aurata were identified with activity against Mycobacterium tuberculosis H37Rv. Native magnetic resonance mass spectrometry (MRMS) against a panel of 37 proteins from Mycobacterium proteomes showed that a fraction from a 95% ethanol re-extraction specifically formed a protein-ligand complex with Rv1466, a putative uncharacterized Mycobacterium tuberculosis protein. The natural product responsible was isolated and characterized to be polycarpine. The molecular weight of the ligand bound to Rv1466, 233 Da, was half the molecular weight of polycarpine less one proton, indicating that polycarpine formed a covalent bond with Rv1466.

Citation: 
Xie Y., Y. Feng, A. Di Capua, T. Mak, G.W. Buchko, P.J. Myler, and M. Lui, et al. 2020. "A Phenotarget Approach for Identifying an Alkaloid Interacting with the Tuberculosis Protein Rv1466." <i>Marine Drugs</i> 18, no. 3:149. PNNL-SA-150251. doi:10.3390/md18030149
Authors: 
Buchko
Garry W;Myler
Peter J;Quinn
Ronald J;Lui
Miaomiao;Mak
Tin;Feng
Yunjiang;Xie
Yan;Di Capua
Angela
Facility: 

Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c

Abstract: 

Pathogenic and opportunistic mycobacteria have a distinct class of non-heme di-iron hemerythrin-like proteins (HLPs). The first to be isolated was the Rv2633c protein, which plays a role in infection by Mycobacterium tuberculosis (Mtb), but could not be crystallized. This work presents the first crystal structure of an ortholog of Rv2633c, the mycobacterial HLP from Mycobacterium kansasii (Mka). This structure differs from those of hemerythrins and other known HLPs. It is comprised of five ??-helices, whereas all other HLP domains have four. In contrast to other HLPs, the HLP domain is not fused to an additional protein domain. The residues ligating and surrounding the di-iron site are also unique among HLPs. Notably; a tyrosine occupies the position normally held by one of the histidine ligands in hemerythrin. This structure was used to construct a homology model of Rv2633c. The structure of five ??-helices is conserved and the di-iron site ligands are identical in Rv2633c. Two residues near the ends of helices in the Mka HLP structure are replaced with prolines in Rv2633c model. This may account for structural perturbations that decrease the solubility of Rv2633c relative to Mka HLP. Clusters of residues that differ in charge or polarity between Rv2633c and Mka HLP that point outward from the helical core could reflect a specificity for potential differential interactions with other protein partners in vivo, which are related to function. The Mka HLP exhibited weaker catalase activity than Rv2633c. Evidence was obtained for interaction of Mka HLP irons with nitric oxide.

Citation: 
Ma Z., J. Abendroth, G.W. Buchko, K.H. Rohde, and V.L. Davidson. 2020. "Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c protein of M. tuberculosis." <i>Biochemical Journal</i> 477, no. 2:567-581. PNNL-SA-147766. doi:10.1042/BCJ20190827
Authors: 
Ma
Zhongxin;Abendroth
Jan;Buchko
Garry W;Rohde
Kyle H;Davidson
Victor L
Facility: 

Temporospatial shifts in the human gut microbiome and metabolome after gastric bypass surgery

Abstract: 

Although the etiology of obesity is not well-understood, genetic, environmental, and microbiome elements are recognized as contributors to this rising pandemic. For morbid obesity, Roux-en-Y gastric bypass (RYGB) surgery alters the fecal microbiome, but data are sparse on temporal and spatial changes in the microbiome and metabolome. We characterized the structure and metabolism of the microbial communities in the gut lumen and on mucosal surfaces in morbidly obese individuals before and after RYGB surgery, and we compared our longitudinal cohort to a previously studied cross-sectional one. RYGB concurrently changed the gut microbiome and led to improvements of obesity comorbidities. Changes in the gut microbiome were reflected in the metabolome, including fermentation products and bile acids. The effects persisted 12 months post-surgery, and the microbiomes and metabolomes were similar to those found for the cross-sectional RYGB cohort. Thus, RYGB surgery had profound and persistent impacts on the intestinal microbiome and metabolome.

Citation: 
Ilhan Z.E., J.K. DiBaise, S.E. Dautel, N.G. Isern, Y. Kim, D.W. Hoyt, and A.A. Schepmoes, et al. 2020. "Temporospatial shifts in the human gut microbiome and metabolome after gastric bypass surgery." <i>NPJ Biofilms and Microbiomes</i> 6, no. 1:Article No. 12. PNNL-SA-133750. doi:10.1038/s41522-020-0122-5
Authors: 
Heather Brewer,
Hoyt, David W
Ilhan
Zehra E;Dautel
Sydney E;Kim
Young-Mo;DiBaise
John K;Brewer
Heather M;Weitz
Karl K;Kang
Dae Wook;Isern
Nancy G;Hoyt
David W;Metz
Thomas O;Crowell
Michael;Schepmoes
Athena A;Rittmann
Bruce E;Krajmalnik-Brown
Rosa
Facility: 

Deep-Subsurface Pressure Stimulates Metabolic Plasticity in Shale-Colonizing Halanaerobium spp.

Abstract: 

Bacterial Halanaerobium strains become the dominant persisting microbial
community member in produced fluids across geographically distinct hydraulically
fractured shales. Halanaerobium is believed to be inadvertently introduced into
this environment during the drilling and fracturing process and must therefore tolerate
large changes in pressure, temperature, and salinity. Here, we used a Halanaerobium
strain isolated from a natural gas well in the Utica Point Pleasant formation to
investigate metabolic and physiological responses to growth under high-pressure
subsurface conditions. Laboratory incubations confirmed the ability of Halanaerobium
congolense strain WG8 to grow under pressures representative of deep shale
formations (21 to 48 MPa). Under these conditions, broad metabolic and physiological
shifts were identified, including higher abundances of proteins associated with
the production of extracellular polymeric substances. Confocal laser scanning microscopy
indicated that extracellular polymeric substance (EPS) production was associated
with greater cell aggregation when biomass was cultured at high pressure.
Changes in Halanaerobium central carbon metabolism under the same conditions
were inferred from nuclear magnetic resonance (NMR) and gas chromatography
measurements, revealing large per-cell increases in production of ethanol, acetate,
and propanol and cessation of hydrogen production. These metabolic shifts were associated
with carbon flux through 1,2-propanediol in response to slower fluxes of
carbon through stage 3 of glycolysis. Together, these results reveal the potential for
bioclogging and corrosion (via organic acid fermentation products) associated with
persistent Halanaerobium growth in deep, hydraulically fractured shale ecosystems,
and offer new insights into cellular mechanisms that enable these strains to dominate
deep-shale microbiomes.
IMPORTANCE The hydraulic fracturing of deep-shale formations for hydrocarbon recovery
accounts for approximately 60% of U.S. natural gas production. Microbial activity
associated with this process is generally considered deleterious due to issues
associated with sulfide production, microbially induced corrosion, and bioclogging in
the subsurface. Here we demonstrate that a representative Halanaerobium species,
frequently the dominant microbial taxon in hydraulically fractured shales, responds
to pressures characteristic of the deep subsurface by shifting its metabolism to generate
more corrosive organic acids and produce more polymeric substances that
cause “clumping” of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly
impact hydrocarbon recovery, these data also offer new insights for microbial control
in these ecosystems.

Citation: 
Booker A.E., D.W. Hoyt, T. Meulia, E.K. Eder, C.D. Nicora, S.O. Purvine, and R. Daly, et al. 2019. "Deep-Subsurface Pressure Stimulates Metabolic Plasticity in Shale-Colonizing Halanaerobium spp." <i>Applied and Environmental Microbiology</i> 85, no. 12:e00018-19. PNNL-SA-144542. doi:10.1128/AEM.00018-19
Authors: 
Booker
Anne Elizabeth;Hoyt
David W;Meulia
Tea;Eder
Elizabeth K;Nicora
Carrie D;Purvine
Samuel O;Daly
Rebecca;Moore
Joseph D;Wunch
Kenneth;Pfiffner
Susan M;Lipton
Mary S;Mouser
Paula J;Wrighton
Kelly C;Wilkins
Michael James
Facility: 

Spatiotemporal Transformation in the Alkaloid Profile of Pinus Roots in Response to Mycorrhization

Abstract: 

The development of ectomycorrhizae (EM), a symbiotic association between roots of woody gymnosperms (e.g., Pinaceae) and ectomycorrhizal fungi (EMF), involves dramatic changes in root and hyphal morphology and biochemistry which are tightly regulated in response to molecular signals (Martin & Hilbert, 1991; Isidorov et al., 2008; Liao et al., 2016). There is emerging evidence that secondary metabolites (SM), in particular flavonoids, terpenes, phytohormones, and sterols can affect EM colonization (Hanna & Patrycja, 2011).

Citation: 
Velickovic D., H. Liao, R. Vilgalys, R.K. Chu, and C.R. Anderton. 2019. "Spatiotemporal Transformation in the Alkaloid Profile of Pinus Roots in Response to Mycorrhization." <i>Journal of Natural Products</i> 82, no. 5:1382-1386. PNNL-SA-140336. doi:10.1021/acs.jnatprod.8b01050
Authors: 
Velickovic
Dusan;Liao
Hui-Ling;Vilgalys
Rytas;Chu
Rosalie K;Anderton
Christopher R
Facility: 

In Situ and Ex Situ NMR for Battery Research

Abstract: 

A rechargeable battery stores readily convertible chemical energy to operate a variety of devices such as mobile phones, laptop computers, and electric automobiles, etc. A battery generally consists of four components, i.e., a cathode, an anode, a separator and electrolytes. The properties of these components jointly determine the safety, the lifetime, and the electrochemical performance, include but not limited to the power density and the charge, recharge time/rate associated with a battery system. Extensive amount of research is thus dedicated to understand the physical and chemical properties associated with each of the four components aimed at developing new generations of battery systems with greatly enhanced safety and electrochemical performance while at significantly reduced cost for large scale applications. Advanced characterization tools are prerequisite to fundamentally understand battery materials. Considering that some of the key electrochemical processes can only exist under in situ conditions, thus, can only be captured under a working battery conditions when electric wires are attached and current and voltage applied, in situ detection is critical. NMR, a non-invasive and atomic specific tool, is capable of detecting all phases, including crystalline, amorphous, liquid and gaseous phases simultaneously and is ideal for in situ detection on a working battery system. Ex situ NMR on the other hand can provide more detailed molecular or structural information on stable species with better spectral resolution and sensitivity. The combination of in situ and ex situ NMR, thus, offers a powerful tool for investigating the detailed electrochemistry in batteries.

Citation: 
Hu J.Z., N.R. Jaegers, M.Y. Hu, and K.T. Mueller. 2018. "In Situ and Ex Situ NMR for Battery Research." <i>Journal of Physics: Condensed Matter</i> 30, no. 46:Article No. 463001. PNNL-SA-134425. doi:10.1088/1361-648X/aae5b8
Authors: 
Hu
Jian Z;Jaegers
Nicholas R;Hu
Mary Y;Mueller
Karl T
Facility: 

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