Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Bridging the Gap in Cryopreservation Mechanism: Unraveling the Interplay between Structure, Dynamics, and Thermodynamics in Cryoprotectant Aqueous Solutions.

Cryoprotectants play a crucial role in preserving biological material, ensuring their viability during storage and facilitating crucial applications such as the conservation of medical compounds, tissues, and organs for transplantation. However, the precise mechanism by which cryoprotectants modulate the thermodynamic properties of water to impede the formation and growth of ice crystals, thus preventing long-term damage, remains elusive. This is evident in the use of empirically optimized recipes for mixtures that typically contain DMSO, glycerol, and various sugar constituents. Here, we use terahertz calorimetry, Overhauser nuclear polarization, and molecular dynamics simulations to show that DMSO exhibits a robust structuring effect on water around its methyl groups, reaching a maximum at a DMSO mole fraction of XDMSO = 0.33. In contrast, glycerol exerts a smaller water-structuring effect, even at higher concentrations (Scheme 1). These results potentially suggest that the wrapped water around DMSO's methyl group, which can be evicted upon ligand binding, may render DMSO a more surface-active cryoprotectant than glycerol, while glycerol may participate more as a viscogen that acts on the entire sample. These findings shed light on the molecular intricacies of cryoprotectant solvation behavior and have potentially significant implications for optimizing cryopreservation protocols.

Nanoscale water-polymer interactions tune macroscopic diffusivity of water in aqueous poly(ethylene oxide) solutions.

The separation and anti-fouling performance of water purification membranes is governed by both macroscopic and molecular-scale water properties near polymer surfaces. However, even for poly(ethylene oxide) (PEO) - ubiquitously used in membrane materials - there is little understanding of whether or how the molecular structure of water near PEO surfaces affects macroscopic water diffusion. Here, we probe both time-averaged bulk and local water dynamics in dilute and concentrated PEO solutions using a unique combination of experimental and simulation tools. Pulsed-Field Gradient NMR and Overhauser Dynamic Nuclear Polarization (ODNP) capture water dynamics across micrometer length scales in sub-seconds to sub-nanometers in tens of picoseconds, respectively. We find that classical models, such as the Stokes-Einstein and Mackie-Meares relations, cannot capture water diffusion across a wide range of PEO concentrations, but that free volume theory can. Our study shows that PEO concentration affects macroscopic water diffusion by enhancing the water structure and altering free volume. ODNP experiments reveal that water diffusivity near PEO is slower than in the bulk in dilute solutions, previously not recognized by macroscopic transport measurements, but the two populations converge above the polymer overlap concentration. Molecular dynamics simulations reveal that the reduction in water diffusivity occurs with enhanced tetrahedral structuring near PEO. Broadly, we find that PEO does not simply behave like a physical obstruction but directly modifies water's structural and dynamic properties. Thus, even in simple PEO solutions, molecular scale structuring and the impact of polymer interfaces is essential to capturing water diffusion, an observation with important implications for water transport through structurally complex membrane materials.

Mutations in Tau Protein Promote Aggregation by Favoring Extended Conformations.

Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Some tauopathies can be inherited due to mutations in the gene encoding tau, which might favor the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the disease-associated single-point mutations promote amyloid formation. We combined biochemical and biophysical characterization, notably, small-angle X-ray scattering (SAXS), to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions, and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radii of gyration. We show that mutations disfavor intramolecular protein interactions, which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate the aggregation propensity of IDPs.

Spectroscopically dark phosphate features revealed by chemical exchange saturation transfer.

Phosphate is an essential anion in the human body, comprising approximately 1% of the total body weight, and playing a vital role in metabolism, cell membranes, and bone formation. We have recently provided spectroscopic, microscopic, and computational evidence indicating that phosphates can aggregate much more readily in solution than previously thought. This prior work provided indirect evidence through the observation of unusual 31 P NMR relaxation and line-broadening effects with increasing temperature. Here, we show that, under conditions of slow exchange and selective RF saturation, additional features become visible in chemical exchange saturation transfer (CEST) experiments, which appear to be related to the previously reported phosphate clustering. In particular, CEST shows pronounced dips several ppm upfield of the main phosphate resonance at low temperatures, while direct 31 P spectroscopy does not produce any signals in that range. We study the pH dependence of these new spectroscopic features and present exchange and spectroscopic parameters based on fitting the CEST data. These findings could be of importance in the investigation of phosphate dynamics, especially in the biological milieu.

P1 Center Electron Spin Clusters Are Prevalent in Type Ib Diamonds.

Understanding the spatial distribution of the P1 centers is crucial for diamond-based sensors and quantum devices. P1 centers serve as polarization sources for dynamic nuclear polarization (DNP) quantum sensing and play a significant role in the relaxation of nitrogen vacancy (NV) centers. Additionally, the distribution of NV centers correlates with the distribution of P1 centers, as NV centers are formed through the conversion of P1 centers. We utilized DNP and pulsed electron paramagnetic resonance (EPR) techniques that revealed strong clustering of a significant population of P1 centers that exhibit exchange coupling and produce asymmetric line shapes. The 13C DNP frequency profile at a high magnetic field revealed a pattern that requires an asymmetric EPR line shape of the P1 clusters with electron-electron (e-e) coupling strengths exceeding the 13C nuclear Larmor frequency. EPR and DNP characterization at high magnetic fields was necessary to resolve energy contributions from different e-e couplings. We employed a two-frequency pump-probe pulsed electron double resonance technique to show cross-talk between the isolated and clustered P1 centers. This finding implies that the clustered P1 centers affect all of the P1 populations. Direct observation of clustered P1 centers and their asymmetric line shape offers a novel and crucial insight into understanding magnetic noise sources for quantum information applications of diamonds and for designing diamond-based polarizing agents with optimized DNP efficiency for 13C and other nuclear spins of analytes. We propose that room temperature 13C DNP at a high field, achievable through straightforward modifications to existing solution-state NMR systems, is a potent tool for evaluating and controlling diamond defects.

Spontaneous Cortical Intracerebral Hemorrhage Causing Ipsilateral Hand and Oral Sensory Change Without Motor Deficit: A Case Report.

Intracerebral hemorrhage (ICH) is the second most common stroke subtype associated with high morbidity and mortality rates. Although various brain regions are susceptible to ICH, putaminal hemorrhage is the most common, whereas cortical ICH is less common. Here, we report the case of a 69-year-old man who developed a parietal cortical ICH. The patient developed hypoesthesia and paresthesia in the right upper lip and hand; however, the weakness was not severe. Twenty-five days after the ICH onset, the manual muscle test results were normal, but he had difficulty eating and shaving because of decreased hand dexterity. The rehabilitation focused on improving fine hand motor function and endurance. On the 94th day after ICH onset, paresthesia remained only in the fingertips, and the upper lip sensory change disappeared. Patients with sensory symptoms in the perioral area, hands, and brain lesions were previously referred to as having cheiro-oral syndrome (COS). With the advancement of neuroimaging, the use of this term has decreased, as cerebrovascular events can explain patient symptoms in correlation with neuroanatomy, etiology, and pathogenesis. We report a patient with cortical ICH, also known as COS, which is a stroke syndrome with a good prognosis.

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement.

Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross ß-sheets. We solve a 3 Å cryo-EM structure of jR2R3-P301L fibrils with a pseudo 2 1 screw symmetry in which each half of the fibril's cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion. Significance Statement: This study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L-among the widest used mutations in cell and animal models of Alzheimer's Disease-destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimer's Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

Multi Electron Spin Cluster Enabled Dynamic Nuclear Polarization with Sulfonated BDPA.

Dynamic nuclear polarization (DNP) can amplify the solid-state nuclear magnetic resonance (NMR) signal by several orders of magnitude. The mechanism of DNP utilizing α,γ-bisdiphenylene-ß-phenylallyl (BDPA) variants as Polarizing Agents (PA) has been the subject of lively discussions on account of their remarkable DNP efficiency with low demand for microwave power. We propose that electron spin clustering of sulfonated BDPA is responsible for its DNP performance, as revealed by the temperature-dependent shape of the central DNP profile and strong electron-electron (e-e) crosstalk seen by Electron Double Resonance. We demonstrate that a multielectron spin cluster can be modeled with three coupled spins, where electron J (exchange) coupling between one of the e-e pairs matching the NMR Larmor frequency induces the experimentally observed absorptive central DNP profile, and the electron T1e modulated by temperature and magic-angle spinning alters the shape between an absorptive and dispersive feature. Understanding the microscopic origin is key to designing new PAs to harness the microwave-power-efficient DNP effect observed with BDPA variants.

Gadolinium Spin Decoherence Mechanisms at High Magnetic Fields.

Favorable relaxation processes, high-field spectral properties, and biological compatibility have made spin-7/2 Gd3+-based spin labels an increasingly popular choice for protein structure studies using high-field electron paramagnetic resonance. However, high-field relaxation and decoherence in ensembles of half-integer high-spin systems, such as Gd3+, remain poorly understood. We report spin-lattice (T1) and phase memory (TM) relaxation times at 8.6 T (240 GHz), and we present the first comprehensive model of high-field, high-spin decoherence accounting for both the electron spin concentration and temperature. The model includes four principal mechanisms driving decoherence: energy-conserving electron spin flip-flops, direct "T1" spin-lattice relaxation-driven electron spin flip processes, indirect T1-driven flips of nearby electron spins, and nuclear spin flip-flops. Mechanistic insight into decoherence can inform the design of experiments making use of Gd3+ as spin probes or relaxivity agents and can be used to measure local average interspin distances as long as 17 nm.

Precision Proteoform Design for 4R Tau Isoform Selective Templated Aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau, folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naïve 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Ethanol-Extracted Acorn Induces Anti-Inflammatory Effects in Human Keratinocyte and Production of Hyaluronic Acid in Human Fibroblasts.

Acorn (Quercus acutissima CARR.) has been used in traditional food and medicinal ethnopharmacology in Asia, and it has shown multifarious functions such as antidementia, antiobesity, and antiasthma functions. However, there is limited scientific evidence about the efficacy of acorn for ameliorating skin problems. Treatment with ethanol-extracted acorns (EeA's) ablated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), monocyte chemoattractant protein-1 (MCP-1), and interleukin (IL)-8 stimulated by tumor necrosis factor (TNF)-α in human adult low calcium high temperature (HaCaT) cells under sublethal dosages. In addition, treatment with EeA dose dependently inhibited the ex vivo hyper keratin formation induced by TNF-α in HaCaT cells in conjunction with the blockade of cytokeratin-1 (CK-1) and cytokeratin-5 (CK-5) expression. Moreover, EeA treatment stimulated the expression of hyaluronic acid (HA) expression in human fibroblasts in a dose-dependent manner. Linoleamide was identified as the functional component of EeA using preparative high-performance liquid chromatography and ultra high performance liquid chromatography-mass spectrometry-mass spectrometry analysis, and the anti-inflammatory features and enhanced HA expression were verified. Collectively, these results suggest the efficacy of EeA supplementation in improving skin problems via anti-inflammation and upregulating HA production.

CBD and PSP cell-passaged Tau Seeds Generate Heterogeneous Fibrils with A sub-population Adopting Disease Folds.

The recent discovery by cryo-electron microscopy that the neuropatho-logical hallmarks of different tauopathies, including Alzheimer's disease, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP), are caused by unique misfolded conformations of the protein tau is among the most profound developments in neurodegenerative disease research. To capitalize on these discoveries for therapeutic development, one must achieve in vitro replication of tau fibrils that adopt the rep-resentative tauopathy disease folds - a grand challenge. To understand whether the commonly used, but imperfect, fragment of the tau pro-tein, K18, is capable of inducing specific protein folds, fibril seeds derived from CBD- and PSP-infected biosensor cells expressing K18, were used to achieve cell-free assembly of naïve, recombinant 4R tau into fibrils without the addition of any cofactors. Using Double Electron Electron Resonance (DEER) spectroscopy, we discovered that cell-passaged patho-logical seeds generate heterogeneous fibrils that are distinct between the CBD and PSP lysate-seeded fibrils, and are also unique from heparin-induced tau fibril populations. Moreover, the lysate-seeded fibrils contain a characteristic sub-population that resembles either the CBD or PSP disease fold, corresponding with the respective starting patient sam-ple. These findings indicate that CBD and PSP patient-derived fibrils retain strain properties after passaging through K18 reporter cells.

Local solvation structures govern the mixing thermodynamics of glycerol-water solutions.

Glycerol is a major cryoprotective agent and is widely used to promote protein stabilization. By a combined experimental and theoretical study, we show that global thermodynamic mixing properties of glycerol and water are dictated by local solvation motifs. We identify three hydration water populations, i.e., bulk water, bound water (water hydrogen bonded to the hydrophilic groups of glycerol) and cavity wrap water (water hydrating the hydrophobic moieties). Here, we show that for glycerol experimental observables in the THz regime allow quantification of the abundance of bound water and its partial contribution to the mixing thermodynamics. Specifically, we uncover a 1 : 1 connection between the population of bound waters and the mixing enthalpy, which is further corroborated by the simulation results. Therefore, the changes in global thermodynamic quantity - mixing enthalpy - are rationalized at the molecular level in terms of changes in the local hydrophilic hydration population as a function of glycerol mole fraction in the full miscibility range. This offers opportunities to rationally design polyol water, as well as other aqueous mixtures to optimize technological applications by tuning mixing enthalpy and entropy based on spectroscopic screening.

Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures.

Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions.

Relationships between Molecular Structural Order Parameters and Equilibrium Water Dynamics in Aqueous Mixtures.

Water's unique thermophysical properties and how it mediates aqueous interactions between solutes have long been interpreted in terms of its collective molecular structure. The seminal work of Errington and Debenedetti [Nature 2001, 409, 318-321] revealed a striking hierarchy of relationships among the thermodynamic, dynamic, and structural properties of water, motivating many efforts to understand (1) what measures of water structure are connected to different experimentally accessible macroscopic responses and (2) how many such structural metrics are adequate to describe the collective structural behavior of water. Diffusivity constitutes a particularly interesting experimentally accessible equilibrium property to investigate such relationships because advanced NMR techniques allow the measurement of bulk and local water dynamics in nanometer proximity to molecules and interfaces, suggesting the enticing possibility of measuring local diffusivities that report on water structure. Here, we apply statistical learning methods to discover persistent structure-dynamic correlations across a variety of simulated aqueous mixtures, from alcohol-water to polypeptoid-water systems. We investigate a variety of molecular water structure metrics and find that an unsupervised statistical learning algorithm (namely, sequential feature selection) identifies only two or three independent structural metrics that are sufficient to predict water self-diffusivity accurately. Surprisingly, the translational diffusivity of water across all mixed systems studied here is strongly correlated with a measure of tetrahedral order given by water's triplet angle distribution. We also identify a separate small number of structural metrics that well predict an important thermodynamic property, the excess chemical potential of an idealized methane-sized hydrophobe in water. Ultimately, we offer a Bayesian method of inferring water structure by using only structure-dynamics linear regression models with experimental Overhauser dynamic nuclear polarization (ODNP) measurements of water self-diffusivity. This study thus quantifies the relationships among several distinct structural order parameters in water and, through statistical learning, reveals the potential to leverage molecular structure to predict fundamental thermophysical properties. In turn, these findings suggest a framework for solving the inverse problem of inferring water's molecular structure using experimental measurements such as ODNP studies that probe local water properties.

Design of Soft Material Surfaces with Rationally Tuned Water Diffusivity.

Water structure and dynamics can be key modulators of adsorption, separations, and reactions at soft material interfaces, but systematically tuning water environments in an aqueous, accessible, and functionalizable material platform has been elusive. This work leverages variations in excluded volume to control and measure water diffusivity as a function of position within polymeric micelles using Overhauser dynamic nuclear polarization spectroscopy. Specifically, a versatile materials platform consisting of sequence-defined polypeptoids simultaneously offers a route to controlling the functional group position and a unique opportunity to generate a water diffusivity gradient extending away from the polymer micelle core. These results demonstrate an avenue not only to rationally design the chemical and structural properties of polymer surfaces but also to design and tune the local water dynamics that, in turn, can adjust the local activity for solutes.

Tutorial on Lateral Dielectrophoretic Manipulations in Microfluidic Systems.

Microfluidic lab-on-a-chip systems can offer cost- and time-efficient biological assays by providing high-throughput analysis at very small volume scale. Among these extremely broad ranges of assays, accurate and specific cell and reagent control is considered one of the most important functions. Dielectrophoretic (DEP)-based manipulation technologies have been extensively developed for these purposes due to their label-free and high selectivity natures as well as due to their simple microstructures. Here, we provide a tutorial on how to develop DEP-based microfluidic systems, including a detailed walkthrough of dielectrophoresis theory, instruction on how to conduct simulation and calculation of electric field and generated DEP force, followed with guidance on microfabricating two forms of DEP microfluidic systems, namely lateral DEP and droplet DEP, and how best to conduct experiments in such systems. Finally, we summarize most recent DEP-based microfluidic technologies and applications, including systems for blood diagnoses, pathogenicity studies, in-droplet content manipulations, droplet manipulations and merging, to name a few. We conclude by suggesting possible future directions on how DEP-based technologies can be utilized to overcome current challenges and improve the current status in microfluidic lab-on-a-chip systems.

Triangulating Dopant-Level Mn(II) Insertion in a Cs2NaBiCl6 Double Perovskite Using Magnetic Resonance Spectroscopy.

Lead-free metal halide double perovskites are gaining increasing attention for optoelectronic applications. Specifically, doping metal halide double perovskites using transition metals enables broadband tailorability of the optical bandgap for these emerging semiconducting materials. One candidate material is Mn(II)-doped Cs2NaBiCl6, but the nature of Mn(II) insertion on chemical structure is poorly understood due to low Mn loading. It is critical to determine the atomic-level structure at the site of Mn(II) incorporation in doped perovskites to better understand the structure-property relationships in these materials and thus to advance their applicability to optoelectronic applications. Magnetic resonance spectroscopy is uniquely qualified to address this, and thus a comprehensive three-pronged strategy, involving solid-state nuclear magnetic resonance (NMR), high-field dynamic nuclear polarization (DNP), and electron paramagnetic resonance (EPR) spectroscopies, is used to identify the location of Mn(II) insertion in Cs2NaBiCl6. Multinuclear (23Na, 35Cl, 133Cs, and 209Bi) one-dimensional (1D) magnetic resonance spectra reveal a low level of Mn(II) incorporation, with select spins affected by paramagnetic relaxation enhancement (PRE) induced by Mn(II) neighbors. EPR measurements confirm the oxidation state, octahedral symmetry, and low doping levels of the Mn(II) centers. Complementary EPR and NMR measurements confirm that the cubic structure is maintained with Mn(II) incorporation at room temperature, but the structure deviates slightly from cubic symmetry at low temperatures (<30 K). HYperfine Sublevel CORrelation (HYSCORE) EPR spectroscopy explores the electron-nuclear correlations of Mn(II) with 23Na, 133Cs, and 35Cl. The absence of 209Bi correlations suggests that Bi centers are replaced by Mn(II). Endogenous DNP NMR measurements from Mn(II) → 133Cs (<30 K) reveal that the solid effect is the dominant mechanism for DNP transfer and supports that Mn(II) is homogeneously distributed within the double-perovskite structure.

Identification of Microorganisms that Bind Specifically to Target Materials of Interest Using a Magnetophoretic Microfluidic Platform.

Discovery of microorganisms and their relevant surface peptides that specifically bind to target materials of interest can be achieved through iterative biopanning-based screening of cellular libraries having high diversity. Recently, microfluidics-based biopanning methods have been developed and exploited to overcome the limitations of conventional methods where controlling the shear stress applied to remove cells that do not bind or only weakly bind to target surfaces is difficult and the overall experimental procedure is labor-intensive. Despite the advantages of such microfluidic methods and successful demonstration of their utility, these methods still require several rounds of iterative biopanning. In this work, a magnetophoretic microfluidic biopanning platform was developed to isolate microorganisms that bind to target materials of interest, which is gold in this case. To achieve this, gold-coated magnetic nanobeads, which only attached to microorganisms that exhibit high affinity to gold, were used. The platform was first utilized to screen a bacterial peptide display library, where only the cells with surface peptides that specifically bind to gold could be isolated by the high-gradient magnetic field generated within the microchannel, resulting in enrichment and isolation of many isolates with high affinity and high specificity toward gold even after only a single round of separation. The amino acid profile of the resulting isolates was analyzed to provide a better understanding of the distinctive attributes of peptides that contribute to their specific material-binding capabilities. Next, the microfluidic system was utilized to screen soil microbes, a rich source of extremely diverse microorganisms, successfully isolating many naturally occurring microorganisms that show strong and specific binding to gold. The results show that the developed microfluidic platform is a powerful screening tool for identifying microorganisms that specifically bind to a target material surface of interest, which can greatly accelerate the development of new peptide-driven biological materials and hybrid organic-inorganic materials.