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.

The question of how Zika virus (ZIKV) changed from a seemingly mild virus to a human pathogen capable of microcephaly and sexual transmission remains unanswered. The unexpected emergence of ZIKV's pathogenicity and capacity for sexual transmission may be due to genetic changes, and future changes in phenotype may continue to occur as the virus expands its geographic range. Alternatively, the sheer size of the 2015-16 epidemic may have brought attention to a pre-existing virulent ZIKV phenotype in a highly susceptible population. Thus, it is important to identify patterns of genetic change that may yield a better understanding of ZIKV emergence and evolution. However, because ZIKV has an RNA genome and a polymerase incapable of proofreading, it undergoes rapid mutation which makes it difficult to identify combinations of mutations associated with viral emergence. As next generation sequencing technology has allowed whole genome consensus and variant sequence data to be generated for numerous virus samples, the task of analyzing these genomes for patterns of mutation has become more complex. However, understanding which combinations of mutations spread widely and become established in new geographic regions versus those that disappear relatively quickly is essential for defining the trajectory of an ongoing epidemic. In this study, multiscale analysis of the wealth of genomic data generated over the course of the epidemic combined with in vivo laboratory data allowed trends in mutations and outbreak trajectory to be assessed. Mutations were detected throughout the genome via deep sequencing, and many variants appeared in multiple samples and in some cases become consensus. Similarly, amino acids that were previously consensus in pre-outbreak samples were detected as low frequency variants in epidemic strains. Protein structural models indicate that most of the mutations associated with the epidemic transmission occur on the exposed surface of viral proteins. At the macroscale level, consensus data was organized into large and interactive databases to allow the spread of individual mutations and combinations of mutations to be visualized and assessed for temporal and geographical patterns. Thus, the use of multiscale modeling for identifying mutations or combinations of mutations that impact epidemic transmission and phenotypic impact can aid the formation of hypotheses which can then be tested using reverse genetics.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

[This corrects the article DOI: 10.1371/journal.pone.0146251.].

In vivo serial passage of non-pathogenic viruses has been shown to lead to increased viral virulence, and although the precise mechanism(s) are not clear, it is known that both host and viral factors are associated with increased pathogenicity. Under- or overnutrition leads to a decreased or dysregulated immune response and can increase viral mutant spectrum diversity and virulence. The objective of this study was to identify the role of viral mutant spectra dynamics and host immunocompetence in the development of pathogenicity during in vivo passage. Because the nutritional status of the host has been shown to affect the development of viral virulence, the diet of animal model reflected two extremes of diets which exist in the global population, malnutrition and obesity. Sendai virus was serially passaged in groups of mice with differing nutritional status followed by transmission of the passaged virus to a second host species, guinea pigs. Viral population dynamics were characterized using deep sequence analysis and computational modeling. Histopathology, viral titer and cytokine assays were used to characterize viral virulence. Viral virulence increased with passage and the virulent phenotype persisted upon passage to a second host species. Additionally, nutritional status of mice during passage influenced the phenotype. Sequencing revealed the presence of several non-synonymous changes in the consensus sequence associated with passage, a majority of which occurred in the hemagglutinin-neuraminidase and polymerase genes, as well as the presence of persistent high frequency variants in the viral population. In particular, an N1124D change in the consensus sequences of the polymerase gene was detected by passage 10 in a majority of the animals. In vivo comparison of an 1124D plaque isolate to a clone with 1124N genotype indicated that 1124D was associated with increased virulence.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater than 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.

Cyanobacterial organic matter excretion is crucial to carbon cycling in many microbial communities, but the nature and bioavailability of this C depend on unknown physiological functions. Cyanobacteria-dominated hypersaline laminated mats are a useful model ecosystem for the study of C flow in complex communities, as they use photosynthesis to sustain a more or less closed system. Although such mats have a large C reservoir in the extracellular polymeric substances (EPSs), the production and degradation of organic carbon is not well defined. To identify extracellular processes in cyanobacterial mats, we examined mats collected from Elkhorn Slough (ES) at Monterey Bay, California, for glycosyl and protein composition of the EPS. We found a prevalence of simple glucose polysaccharides containing either α or β (1,4) linkages, indicating distinct sources of glucose with differing enzymatic accessibility. Using proteomics, we identified cyanobacterial extracellular enzymes, and also detected activities that indicate a capacity for EPS degradation. In a less complex system, we characterized the EPS of a cyanobacterial isolate from ES, ESFC-1, and found the extracellular composition of biofilms produced by this unicyanobacterial culture were similar to that of natural mats. By tracing isotopically labeled EPS into single cells of ESFC-1, we demonstrated rapid incorporation of extracellular-derived carbon. Taken together, these results indicate cyanobacteria reuse excess organic carbon, constituting a dynamic pool of extracellular resources in these mats.

This material is based upon work supported by the National Science Foundation through the Robert Noyce Teacher Scholarship Program under Grant No. DUE 1340110. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The STAR program is facilitated by the Cal Poly Center for Excellence in Science and Mathematics Education (CESaME) on behalf of the California State University (CSU). REFERENCES Understanding the very nature of infectious diseases allows for the prevention of new outbreaks as well as the development of drugs and vaccines. Three fourths of newly identified human pathogens are viruses, highlighting a need to better understand these rapidly changing pathogens. At the heart of these are mostly RNA viruses, the fastest when it comes to evolution [5]. RNA viruses replicate and may recombine quickly, due mostly to their error-prone RNA-dependent RNA polymerase (RdRp) which produces mutations in each replication cycle [1]. The RdRp also leads to frame shifts due to the secondary structure of the RNA as well as template swapping, starting on the genome of one strain and “jumping” to the genome of another co-infecting strain [4]. The lack of proofreading mechanisms that many other organisms rely on to prevent lethal mutations allows RNA viruses to balance on the edge of extinction in order to fully maximize their variability [1]. All of this leads up to the production of a highly variable and responsive population that scientists have dubbed “quasispecies” [6]. These large RNA virus populations hold many different variations of their genome, maintained by the very nature of their reproduction and the ability for the collective gene pool of the population to shift to accommodate host environment. It’s this very reason that of all the emerging infectious diseases identified since 1940 (about 400), more than 60% have been zoonotic [3]. In other words, in the past 75 years over 240 diseases have been able to swap hosts. Fig. 1: Human Rectal Tumor cells (HRT18) were grown in wells to produce monolayers (seen in the right image). Fig. 2: Cells were infected with virus at MOI 0.001. Above image shows cytopathic effect (CPE) caused by the virus. Fig. 3: RNA was extracted from the supernatant (using a Trizol extraction method) and converted to cDNA (using Invitrogen’s SuperScript III kit)

Abstract Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs vs. delivery with free MPLA induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the species-matched NLP scaffold protein were detected. Using the ovalbumin/TCR transgenic model, MPLA-NLPs enhanced antigen-specific T cell expansion in lung-draining lymph nodes. In summary, the NLP platform enhances cellular, humoral, and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Nanoparticles hold great promise for the delivery of therapeutics, yet limitations remain with regards to the use of these nanosystems for efficient long-lasting targeted delivery of therapeutics, including imparting functionality to the platform, in vivo stability, drug entrapment efficiency and toxicity. To begin to address these limitations, we evaluated the functionality, stability, cytotoxicity, toxicity, immunogenicity and in vivo biodistribution of nanolipoprotein particles (NLPs), which are mimetics of naturally occurring high-density lipoproteins (HDLs). We found that a wide range of molecules could be reliably conjugated to the NLP, including proteins, single-stranded DNA, and small molecules. The NLP was also found to be relatively stable in complex biological fluids and displayed no cytotoxicity in vitro at doses as high as 320 µg/ml. In addition, we observed that in vivo administration of the NLP daily for 14 consecutive days did not induce significant weight loss or result in lesions on excised organs. Furthermore, the NLPs did not display overt immunogenicity with respect to antibody generation. Finally, the biodistribution of the NLP in vivo was found to be highly dependent on the route of administration, where intranasal administration resulted in prolonged retention in the lung tissue. Although only a select number of NLP compositions were evaluated, the findings of this study suggest that the NLP platform holds promise for use as both a targeted and non-targeted in vivo delivery vehicle for a range of therapeutics.

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.

Abstract Therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms. As such, we have employed a nanolipoprotein (NLP) platform to enhance the efficacy of synthetic innate immune agonists. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. We demonstrate that the synthetic Toll-like receptor (TLR) ligands monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. Further, conjugation of both MPLA and CpG ODNs to NLPs significantly enhances their immunostimulatory profiles in vivo compared to administration of agonists alone. Specifically, conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, enhances and prolongs stimulation of innate immune cells, significantly increases cytokine production, and enhances upregulation of myriad immunoregulatory genes in vivo. Importantly, enhancement of cytokine production by agonists conjugated to NLPs was also observed in primary human dendritic cells. Strikingly, BALB/c mice pretreated with NLP-CpG constructs prior to a lethal influenza challenge were protected from weight-loss induced death, whereas pretreatment with CpG alone had no effect on survival. Taken together, these data suggest that NLP:agonist conjugates potentially represent a novel therapeutic measure against emerging pathogens.

Subunit antigen-based vaccines can provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, because of the inherently low immunogenicity of these antigens, methods for colocalized delivery of antigen and immunostimulatory molecules (i.e., adjuvants) are needed. Here we report a robust nanolipoprotein particle (NLP)-based vaccine delivery platform that facilitates the codelivery of both subunit antigens and adjuvants. Ni-chelating NLPs (NiNLPs) were assembled to incorporate the amphipathic adjuvants monophosphoryl lipid A and cholesterol-modified CpG oligodeoxynucleotides, which can bind His-tagged protein antigens. Colocalization of antigen and adjuvant delivery using the NiNLP platform resulted in elevated antibody production against His-tagged influenza hemagglutinin 5 and Yersinia pestis LcrV antigens. Antibody titers in mice immunized with the adjuvanted NLPs were 5-10 times higher than those observed with coadministration formulations and nonadjuvanted NiNLPs. Colocalized delivery of adjuvant and antigen provides significantly greater immune stimulation in mice than coadministered formulations.

Modulation of the innate immune system has the potential to provide transient, non-specific protection from a variety of infectious organisms. Activation of innate immunity is rapid and short-lived, therefore methods aimed at enhancing and prolonging the response will increase the effectiveness of innate immune targeting as a therapeutic measure. In order to enhance the stimulatory properties of innate immune agonists, we have employed a nanolipoprotein (NLP) platform. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins, analogous to reconstituted HDLs. NLPs have been successfully used in numerous biotechnology applications, including drug delivery and diagnostic imaging. Here, we show that two synthetic TLR ligands: monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. In addition, the delivery of agonist-conjugated NLPs significantly enhances immunostimulatory effects relative to agonists alone, at low doses, both in vitro and in vivo. Our data demonstrate that conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, increases cytokine production, and induces upregulation of immunoregulatory genes in peripheral organs. These data suggest that treatment with NLP:agonist conjugates may provide transient protection against lethal pathogen challenge, and experiments to determine their effectiveness at countering infection are ongoing.

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as "hypothetical" were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.

Characterizing proteins recovered from natural microbial communities affords the opportunity to correlate protein expression and modification with environmental factors, including species composition and successional stage. Proteogenomic and biochemical studies of pellicle biofilms from subsurface acid mine drainage streams have shown abundant cytochromes from the dominant organism, Leptospirillum Group II. These cytochromes are proposed to be key proteins in aerobic Fe(II) oxidation, the dominant mode of cellular energy generation by the biofilms. In this study, we determined that posttranslational modification and expression of amino-acid sequence variants change as a function of biofilm maturation. For Cytochrome 579 (Cyt 579 ), the most abundant cytochrome in the biofilms, late developmental-stage biofilms differed from early-stage biofilms in N-terminal truncations and decreased redox potentials. Expression of sequence variants of two monoheme c -type cytochromes also depended on biofilm development. For Cyt 572 , an abundant membrane-bound cytochrome, the expression of multiple sequence variants was observed in both early and late developmental-stage biofilms; however, redox potentials of Cyt 572 from these different sources did not vary significantly. These cytochrome analyses show a complex response of the Leptospirillum Group II electron transport chain to growth within a microbial community and illustrate the power of multiple proteomics techniques to define biochemistry in natural systems.

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.

The biochemistry of unusual cytochromes isolated from acidophilic Fe(II)-oxidizing biofilms STEVEN W. SINGER*, BRIAN ERICKSON, NATHAN C. VERBERKMOES, MONA HWANG, CLARA S. CHAN, CHRISTOPHER JEANS, JILLIAN F. BANFIELD, ROBERT L. HETTICH AND MICHAEL P. THELEN Lawrence Livermore National Laboratory, Livermore, CA 94551, USA (*correspondence: singer2@llnl.gov) Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA University of California, Berkeley, CA 94720, USA