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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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At least 793 records · Page 44

Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.

60 APPLIED LIFE SCIENCES↗

What are mycorrhizal traits?

Traits are inherent properties of organisms, but how are they defined for organismal networks such as mycorrhizal symbioses? Mycorrhizal symbioses are complex and diverse belowground symbioses between plants and fungi that have proved challenging to fit into a unified and coherent trait framework. Here we propose an inclusive mycorrhizal trait framework that classifies traits as morphological, physiological, and phenological features that have functional implications for the symbiosis. We further classify mycorrhizal traits by location – plant, fungus, or the symbiosis – which highlights new questions in trait-based mycorrhizal ecology designed to charge and challenge the scientific community. This new framework is an opportunity for researchers to interrogate their data to identify novel insights and gaps in our understanding of mycorrhizal symbioses.

59 BASIC BIOLOGICAL SCIENCES↗

Predicting battery capacity from impedance at varying temperature and state of charge using machine learning

Prediction of battery health from electrochemical impedance spectroscopy (EIS) data can enable rapid measurement of battery state in real-world applications without using additional sensors or time-consuming performance measurements. However, deconvoluting the effect of capacity, state of charge, and temperature on EIS response is complicated analytically. Here, various machine-learning models, such as linear, Gaussian process, random forest, and artificial neural network regression, are utilized to predict capacity from EIS using hundreds of capacity, direct current (DC) resistance, and EIS measurements recorded under varying conditions of health, temperature, and state of charge (SOC). Several feature extraction and selection methods from traditional electrochemical analysis and statistical modeling are explored using machine-learning pipelines. EIS data from just two frequencies can accurately predict capacity, and interrogation shows that the optimal set of frequencies is not usually intuitive. Best results are achieved with an ensemble model, which predicts battery capacity with a mean absolute error of 1.9% on data from unobserved cells.

25 ENERGY STORAGE↗

Instruction of hematopoietic lineage choice by cytokine signaling

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support the production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response. - Highlights: • Recent studies provided definite proof for lineage-instructive action of cytokines. • Signaling pathways involved in hematopoietic lineage instruction remain elusive. • New tools are emerging to quantitatively study dynamic signaling networks over time.

60 APPLIED LIFE SCIENCES↗

The effects of chemical fixation on the cellular nanostructure

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods. - Highlights: • PWS was employed to monitor fixation process for the same cells in vitro. • Dramatic changes in cellular nanostructure were observed after fixation. • However, the population difference of chromatin structure remains after fixation.

60 APPLIED LIFE SCIENCES↗

13 C-metabolic flux analysis of Clostridium ljungdahlii illuminates its core metabolism under mixotrophic culture conditions

Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO 2 fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using 13 C-metabolic flux analysis ( 13 C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our 13 C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. 13 C-MFA during mixotrophic growth using the parallel tracers [1– 13 C]fructose, [1,2– 13 C]fructose, [1,2,3– 13 C]fructose, and [U– 13 C]asparagine found that externally supplied CO 2 contributed the majority of carbon consumed. All internally-produced CO 2 from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.

13C metabolic flux analysis↗

IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomic biomarker discovery

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up to 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.

59 BASIC BIOLOGICAL SCIENCES↗

Stream lamination and rapid mixing in a microfluidic jet for X-ray spectroscopy studies

Microfluidic mixers offer new possibilities for the study of fast reaction kinetics down to the microsecond time scale, and methods such as soft X-ray absorption spectroscopy are powerful analysis techniques. These systems impose challenging constraints on mixing time scales, sample volume, detection region size and component materials. The current work presents a novel micromixer and jet device which aims to address these limitations. The system uses a so-called ‘theta’ mixer consisting of two sintered and fused glass capillaries. Sample and carrier fluids are injected separately into the inlets of the adjacent capillaries. At the downstream end, the two streams exit two micron-scale adjoining nozzles and form a single free-standing jet. The flow-rate difference between the two streams results in the rapid acceleration and lamination of the sample stream. This creates a small transverse dimension and induces diffusive mixing of the sample and carrier stream solutions within a time scale of 0.9 microseconds. The reaction occurs at or very near a free surface so that reactants and products are more directly accessible to interrogation using soft X-ray. We use a simple diffusion model and quantitative measurements of fluorescence quenching (of fluorescein with potassium iodide) to characterize the mixing dynamics across flow-rate ratios.

Huyke, Diego A. (ORCID:0000000283356613)↗

MeV bremsstrahlung X rays from intense laser interaction with solid foils

Laser-based compact MeV X-ray sources are useful for a variety of applications such as radiography and active interrogation of nuclear materials. MeV X rays are typically generated by impinging the intense laser onto ~mm-thick high-Z foil. Here, we have characterized such a MeV X-ray source from 120 TW (80 J, 650 fs) laser interaction with a 1 mm-thick tantalum foil. Our measurements show X-ray temperature of 2.5 MeV, flux of 3 × 10 12 photons/sr/shot, beam divergence of ~0.1 sr, conversion efficiency of ~1%, that is, ~1 J of MeV X rays out of 80 J incident laser, and source size of 80 m. Our measurement also shows that MeV X-ray yield and temperature is largely insensitive to nanosecond laser contrasts up to 10 -5 . Also, preliminary measurements of similar MeV X-ray source using a double-foil scheme, where the laser-driven hot electrons from a thin foil undergoing relativistic transparency impinging onto a second high-Z converter foil separated by 50–400 m, show MeV X-ray yield more than an order of magnitude lower compared with the single-foil results.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Less is More: Bigger Data from Compressive Measurements

Compressive sensing approaches are beginning to take hold in (scanning) transmission electron microscopy (S/TEM) [1,2,3]. Compressive sensing is a mathematical theory about acquiring signals in a compressed form (measurements) and the probability of recovering the original signal by solving an inverse problem [4]. The inverse problem is underdetermined (more unknowns than measurements), so it is not obvious that recovery is possible. Compression is achieved by taking inner products of the signal with measurement weight vectors. Both Gaussian random weights and Bernoulli (0,1) random weights form a large class of measurement vectors for which recovery is possible. The measurements can also be designed through an optimization process. The key insight for electron microscopists is that compressive sensing can be used to increase acquisition speed and reduce dose. Building on work initially developed for optical cameras, this new paradigm will allow electron microscopists to solve more problems in the engineering and life sciences. We will be collecting orders of magnitude more data than previously possible. The reason that we will have more data is because we will have increased temporal/spatial/spectral sampling rates, and we will be able ability to interrogate larger classes of samples that were previously too beam sensitive to survive the experiment. For example consider an in-situ experiment that takes 1 minute. With traditional sensing, we might collect 5 images per second for a total of 300 images. With compressive sensing, each of those 300 images can be expanded into 10 more images, making the collection rate 50 images per second, and the decompressed data a total of 3000 images [3]. But, what are the implications, in terms of data, for this new methodology? Acquisition of compressed data will require downstream reconstruction to be useful. The reconstructed data will be much larger than traditional data, we will need space to store the reconstructions during analysis, and the computational demands for analysis will be higher. Moreover, there will be time costs associated with reconstruction. Deep learning [5] is an approach to address these problems. Deep learning is a hierarchical approach to find useful (for a particular task) representations of data. Each layer of the hierarchy is intended to represent higher levels of abstraction. For example, a deep model of faces might have sinusoids, edges and gradients in the first layer; eyes, noses, and mouths in the second layer, and faces in the third layer. There has been significant effort recently in deep learning algorithms for tasks beyond image classification such as compressive reconstruction [6] and image segmentation [7]. A drawback of deep learning, however, is that training the model requires large datasets and dedicated computational resources (to reduce training time to a few days). A second issue is that deep learning is not user-friendly and the meaning behind the results is usually not interpretable. We have shown it is possible to reduce the data set size while maintaining model quality [8] and developed interpretable models for image classification [9], but the demands are still significant. The key to addressing these problems is to NOT reconstruct the data. Instead, we should design computational sensors that give answers to specific problems. A simple version of this idea is compressive classification [10], where the goal is to classify signal type from a small number of compressed measurements. Classification is a much simpler problem than reconstruction, so 1) much fewer measurements will be necessary, and 2) these measurements will probably not be useful for reconstruction. Other simple examples of computational sensing include determining object volume or the number of objects present in the field of view [11].

Stevens, Andrew↗

Improving precision in resonance ionization mass spectrometry : influence of laser bandwidth in uranium isotope ratio measurements.

The use of broad bandwidth lasers with automated feedback control of wavelength was applied to the measurement of {sup 235}U/{sup 238}U ratios by resonance ionization mass spectrometry (RIMS) to decrease laser-induced isotopic fractionation. By broadening the bandwidth of the first laser in a three-color, three-photon ionization process from a bandwidth of 1.8 GHz to about 10 GHz, the variation in sequential relative isotope abundance measurements decreased from 10% to less than 0.5%. This procedure was demonstrated for the direct interrogation of uranium oxide targets with essentially no sample preparation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Data Science Meets Physical Organic Chemistry

At the heart of synthetic chemistry is the holy grail of predictable catalyst design. In particular, researchers involved in reaction development in asymmetric catalysis have pursued a variety of strategies toward this goal. This is driven by both the pragmatic need to achieve high selectivities and the inability to readily identify why a certain catalyst is effective for a given reaction. While empiricism and intuition have dominated the field of asymmetric catalysis since its inception, enantioselectivity offers a mechanistically rich platform to interrogate catalyst-structure response patterns that explain the performance of a particular catalyst or substrate. In the early stages of an asymmetric reaction development campaign, the overarching mechanism of the reaction, catalyst speciation, the turnover limiting step, and many other details are unknown or posited based on related reactions. Considering the unclear details leading to a successful reaction, initial enantioselectivity data are often used to intuitively guide the ultimate direction of optimization. However, if the conditions of the Curtin-Hammett principle are satisfied, then measured enantioselectivity can be directly connected to the ensemble of diastereomeric transition states (TSs) that lead to the enantiomeric products, and the associated free energy difference between competing TSs (ΔΔ G ‡ = - RT ln[( S )/( R )], where ( S ) and ( R ) represent the concentrations of the enantiomeric products). We, and others, speculated that this important piece of information can be leveraged to guide reaction optimization in a quantitative way. Although traditional linear free energy relationships (LFERs), such as Hammett plots, have been used to illuminate important mechanistic features, we sought to develop data science derived tools to expand the power of LFERs in order to describe complex reactions frequently encountered in modern asymmetric catalysis. Specifically, we investigated whether enantioselectivity data from a reaction can be quantitatively connected to the attributes of reaction components, such as catalyst and substrate structural features, to harness data for asymmetric catalyst design. In this context, we developed a workflow to relate computationally derived features of reaction components to enantioselectivity using data science tools. The mathematical representation of molecules can incorporate many aspects of a transformation, such as molecular features from substrate, product, catalyst, and proposed transition states. Statistical models relating these features to reaction outputs can be used for various tasks, such as performance prediction of untested molecules. Perhaps most importantly, statistical models can guide the generation of mechanistic hypotheses that are embedded within complex patterns of reaction responses. Overall, merging traditional physical organic experiments with statistical modeling techniques creates a feedback loop that enables both evaluation of multiple mechanistic hypotheses and future catalyst design. In this Account, we highlight the evolution and application of this approach in the context of a collaborative program based on chiral phosphoric acid catalysts (CPAs) in asymmetric catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Emerging Electrochemical Techniques for Probing Site Behavior in Single-Atom Electrocatalysts

Single-atom catalysts (SACs) have aroused tremendous interest over the past decade, particularly in the community of energy and environment-related electrocatalysis. A rapidly growing number of recent publications have recognized it as a promising candidate with maximum atomic utilization, distinct activity, and selectivity in comparison to bulk catalysts and nanocatalysts. However, the complexity of localized coordination environments and the dispersion of isolated sites lead to significant difficulties when it comes to gaining insight into the intrinsic behavior of electrocatalytic reactions. Furthermore, the low metal loadings of most SACs make conventional ensemble measurements less likely to be accurate on the subnanoscale. Thus, it remains challenging to probe the activity and properties of individual atomic sites by available commercial instruments and analytical methods. In spite of this, continuing efforts have lately focused on the development of advanced measurement methodologies, which are very useful to the fundamental understanding of SACs. There have recently been a number of in situ/operando techniques applied to SACs, such as electron microscopy, spectroscopy, and other analysis methods, which support relevant functions to identify the active sites and reaction intermediates and to investigate the dynamic behavior of localized structures of the catalytic sites. This Account aims to present recent electrochemical probing techniques which can be used to identify single-atomic catalytic sites within solid supports. First, we describe the basic principles of molecular probe methods for the study and analysis of electrocatalytic site behavior. In particular, the in situ probing technique enabled by surface interrogation scanning electrochemical microscopy (SISECM) can measure the active site density and kinetic rate with high resolution. An alternative electrochemical probing technique is further demonstrated on the basis of single-entity electrochemistry, which allows the unique electrochemical imaging of the size and catalytic rate of single atoms, molecules, and clusters. Next, the merits and limitations of different electrochemical techniques are then discussed, along with perspectives for future prospects. Apart from this, we further showcase the powerful capability of emerging electrochemical probing techniques for determining significant effects and properties of SACs for various electrocatalytic reactions, including oxygen reduction and evolution, hydrogen evolution, and nitrate reduction. Overall, electrochemical techniques with atomic resolution have greatly increased opportunities for observing, measuring, and understanding the surface and interface chemistry during energy conversion. In the future, it is anticipated that the development of electrochemical probing techniques will be advanced with innovative perspectives on the behavior and features of SACs. We hope that this Account can contribute in several ways to promoting the fundamental knowledge and technical progress of emerging electrochemical measurements for studying SACs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Modular Homogeneous Chromophore–Catalyst Assemblies

Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate that molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule–nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Hybrid-Lipid Bilayers Induce n -Alkyl-Chain Order in Reversed-Phase Chromatographic Surfaces, Impacting their Shape Selectivity for Aromatic Hydrocarbon Partitioning

Shape selectivity is important in reversed-phase liquid chromatographic separations, where stationary phases are capable of separating geometric isomers, thereby resolving solutes based on their three-dimensional structure or shape rather than other chemical differences. Numerous chromatographic studies have been carried out using n-alkyl-chain-modified columns to understand how molecular shape affects retention. For polycyclic aromatic hydrocarbons (PAHs), it was found that planar compounds were selectively retained over non-planar structures of comparable molecular weight on surfaces with longer n-alkyl chains, higher chain-density, or at lower temperatures, where selectivity likely arises with greater ordering of the n-alkyl chains. A limitation of these studies, however, is the small range of chain ordering that can be achieved and lack of a direct measure of the n-alkyl-chain order of the stationary phases. In this work, we employ a C 18 stationary phase modified with a monolayer of phospholipid as a means of significantly varying the n-alkyl chain order. These hybrid-supported lipid bilayers, which have previously been employed as membrane-like stationary phases for measuring lipophilicity, provide a unique approach to control n-alkyl chain ordering by varying the acyl chain length and degree of unsaturation of the phospholipid modifier. The degree of alkyl-chain order of the resulting modified surfaces is determined from the ratio of trans- versus gauche-conformers, measured in situ within individual porous particles by confocal Raman microscopy. This methodology was also used to assess the affinity of these surfaces for planar versus non-planar PAH molecules. The retention selectivity for the planar versus non-planar compounds, thus determined, was found to vary significantly and systematically with the degree of order of the acyl/alkyl chains in the hybrid-supported lipid bilayers. Furthermore, the investigation also demonstrates the utility of confocal Raman microscopy for interrogating the impact of solute partitioning on stationary-phase structure within porous chromatographic particles.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

13 C-Isotope-Assisted Assessment of Metabolic Quenching During Sample Collection from Suspension Cell Cultures

Metabolomics and fluxomics are core approaches to directly profile and interrogate cellular metabolism in response to various genetic or environmental perturbations. In order to accurately measure the abundance and isotope enrichment of intracellular metabolites, cell culture samples must be rapidly harvested and cold-quenched to preserve the in vivo metabolic state of the cells at the time of sample collection. When dealing with suspension cultures, this process is complicated by the need to separate the liquid culture media from cellular biomass prior to metabolite extraction. Here, we examine the efficacy of several commonly used metabolic quenching methods, using the model cyanobacterium Synechocystis sp. PCC 6803 as an example. Multiple 13 C-labeled compounds, including 13 C-bicarbonate, 13 C-glucose and 13 C-glutamine, were used as tracers during the sample collection and cold-quenching process to assess the extent of metabolic turnover after cells were harvested from culture flasks. We show that the combination of rapid filtration followed by 100% cold (–80°C) methanol quenching exhibits the highest quenching efficiency, while mixing cell samples with a partially frozen 30% methanol slurry (–24°C) followed by centrifugation is slightly less effective at quenching metabolism but enables less laborious sample processing. By contrast, rapidly mixing the cells with a saline ice slurry (~0°C) is less effective as indicated by high isotope-labeling rates after sample harvest, while mixing the cells with 60% cold methanol (–65°C) prior to centrifugation causes significant metabolite loss. Furthermore, this study demonstrates a rigorous, quantitative, and broadly applicable method for assessing the metabolic quenching efficacy of protocols used for sample collection in metabolomics and fluxomics studies.

59 BASIC BIOLOGICAL SCIENCES↗

Controlling the Fluctuating Tip-Enhanced Raman Spectra of Chloramben on Silver Nanocubes

Tip-enhanced Raman (TER) scattering from molecules residing at plasmonic junctions can be used to detect, identify, and image single molecules. This is most evident for flat molecules interrogated under extreme conditions of temperatures and pressure. It is also the case for (bio)molecular systems that feature preferred orientations/conformations under ambient laboratory conditions. More complex molecules that can adopt multiple conformations and/or that feature different protonation and/or charge states give rise to complex TER spectra. Here we illustrate how the latter can be controlled in the case of chloramben molecules coated onto plasmonic silver nanocubes. We show that characteristic molecular Raman spectra cannot be obtained when tunneling plasmons are operative, i.e., when the tip is in direct contact with the chemically functionalized plasmonic nanoparticles. We rationalize these observations and propose an approach to less invasive, and hence, more analytical TER spectral imaging.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage

Here, the need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW) received in the proton-exchanged (SO 3 H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA + : SO 3 - ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 cm -1 - 500 cm -1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA + modified Aquivion ionomers were able to reach maximum power densities (P max ) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA + modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW -1 ), where the greater density of SO 3 - moieties in the Aquivion materials produces an environment more favourable to mass transport and higher TBA + concentrations.

36 MATERIALS SCIENCE↗