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At least 1,477 records · Page 82

An Integral Activity-Based Protein Profiling Method for Higher Throughput Determination of Protein Target Sensitivity to Small Molecules

Activity-based protein profiling (ABPP) is a chemoproteomic technique that uses small molecule probes to label active enzymes selectively and covalently in complex proteomes. Competitive ABPP, which involves treatment of the active proteome with an analyte of interest, is especially powerful for profiling how small molecules impact specific protein activities. Advances in higher throughput workflows have made it possible to generate extensive competitive ABPP data across diverse biological samples, making this approach highly appealing for characterizing shared and unique proteins affected by perturbations such as drug or chemical exposures. To use the competitive ABPP approach effectively to understand potential adverse effects of chemicals of concern (CoC), a wide range of concentrations may be needed, particularly for chemicals that lack potency or toxicity data. In this work, we present an integral competitive ABPP method that enables target sensitivity determination for different organophosphate (OP) pesticides as model toxicants. Using previously developed OP-ABPs, we optimized conditions for tandem mass tag (TMT) multiplexing of ABPP samples and compared conventional competitive ABPP involving samples at discrete paraoxon concentrations to pooled samples across that same concentration range. We then expanded our approach to compare protein target sensitivities toward two additional OP pesticides, chlorpyrifos oxon and malaoxon. The results showed that differences in integral intensities for the pooled competition sample can be used to evaluate the relative sensitivity of specific proteins without increasing the overall number of samples. For 8 CoC concentrations of interest, this strategy reduced the number of TMT plexes and the corresponding number of LC–MS/MS analyses 3-fold. In conclusion, we envision the integral ABPP (IABPP) method will provide a means to screen diverse chemicals more rapidly to identify both high and low sensitivity protein targets.

activity-based probes↗

Photoelectrochemical Nitrogen Reduction to Ammonia on Cupric and Cuprous Oxide Photocathodes

Photoelectrochemical N 2 reduction enables the production of NH 3 under ambient conditions using water as the hydrogen source. Furthermore, by utilizing solar energy, photoelectrochemical N 2 reduction can significantly reduce the energy input required for N 2 reduction. In this study, photoelectrochemical N 2 production was investigated using CuO and Cu 2 O photocathodes that are known to be poorly catalytic for water reduction, the major reaction competing with N 2 reduction. When tested under simulated solar illumination with isotopically labeled 15 N 2 in 0.1 M KOH solution, the CuO and Cu 2 O photocathodes produced 15 NH 3 with a Faradaic efficiency of 17% and 20% at 0.6 V and 0.4 V vs. RHE, respectively. These potentials are significantly more positive than the thermodynamic reduction potential of N 2 , which demonstrates how the use of photoexcited electrons in the CuO and Cu 2 O photocathodes can reduce the energy input required for NH 3 production. Here, the use of photoexcited electrons in these photocathodes for N 2 reduction, water reduction, and photocorrosion was carefully examined.

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Highly Bright and Photostable Two-Dimensional Nanomaterials Assembled from Sequence-Defined Peptoids

Free-standing two-dimensional (2D) organic nanomaterials are highly attractive for biological applications because of their unique structural properties and high biocompatibility. Herein, we designed and synthesized a new class of highly bright and photostable membrane-mimetic 2D nanosheets from sequence-defined peptoids. These nanosheets exhibited the high quantum yield and photostability as a result of the precise placement and ordering of dansyl dye molecules within crystalline nanosheets. We further showed the use of these nanosheets as biocompatible and programmable probes for live cell imaging and cell labeling. Furthermore, by programing these nanosheets with different surface charges, we achieved the enhanced lysosome escape of these nanosheets, showing their great potential as nanocarriers for the efficient intracellular delivery of macromolecular drugs.

2D nanomaterials↗

Insights into Nucleation and Growth of Colloidal Quaternary Nanocrystals by Multimodal X-ray Analysis

Copper chalcogenide nanocrystals find applications in photovoltaic inks, bio labels, and thermoelectric materials. We reveal insights in the nucleation and growth during synthesis of anisotropic Cu 2 ZnSnS 4 nanocrystals by simultaneously performing in situ X-ray absorption spectroscopy (XAS) and small-angle X-ray scattering (SAXS). Real-time XAFS reveals that upon thiol injection into the reaction flask, a key copper thiolate intermediate species is formed within fractions of seconds, which decomposes further within a narrow temperature and time window to form copper sulfide nanocrystals. These nanocrystals convert into Cu 2 ZnSnS 4 nanorods by sequentially incorporating Sn and Zn. Real-time SAXS and ex situ TEM of aliquots corroborate these findings. Our work demonstrates how combined in situ X-ray absorption and small-angle X-ray scattering enables the understanding of mechanistic pathways in colloidal nanocrystal formation.

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Self-Sorting vs Coassembly in Peptide Amphiphile Supramolecular Nanostructures

The functionality of supramolecular nanostructures can be expanded if systems containing multiple components are designed to either self-sort or mix into coassemblies. This is critical to gain the ability to craft self-assembling materials that integrate functions, and our understanding of this process is in its early stages. Here, in this work, we have utilized three different peptide amphiphiles with the capacity to form β-sheets within supramolecular nanostructures and found binary systems that self-sort and others that form coassemblies. This was measured using atomic force microscopy to reveal the nanoscale morphology of assemblies and confocal laser scanning microscopy to determine the distribution of fluorescently labeled monomers. We discovered that PA assemblies with opposite supramolecular chirality self-sorted into chemically distinct nanostructures. In contrast, the PA molecules that formed a mixture of right-handed, left-handed, and flat nanostructures on their own were able to coassemble with the other PA molecules. We attribute this phenomenon to the energy barrier associated with changing the handedness of a β-sheet twist in a coassembly of two different PA molecules. This observation could be useful for designing biomolecular nanostructures with dual bioactivity or interpenetrating networks of PA supramolecular assemblies.

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High-Purity Quantum Emission from an Au 24 (S-CH 2 Ph- t Bu) 20 Nanocluster at Room Temperature

Atomically precise gold nanoclusters have garnered significant attention for their diverse applications, ranging from biological labeling to optoelectronics. Their potential in optical quantum computing, which calls for ideal single-photon sources, has recently become a key area of interest. In the current work, we use photon antibunching experiments to explore the single-photon emission efficiency of atomically precise Au 24 nanoclusters protected by 4-tert-butylbenzyl mercaptan ligands (Au 24 (TBBM) 20 ). This cluster exhibits quantum emission with good photostability and without any observable blinking or spectral drift at room temperature under an inert gas atmosphere, with antibunching dips (g 2 (0)) as low as 0.07 in the solid state or, equivalently, a single-photon purity of 93% under time-gated conditions. Transient absorption and time-gated antibunching studies reveal that the short emission lifetime of this cluster and its high photoluminescence quantum yield in the solid state play critical roles in enhancing the emitted single-photon purity. This research advances the understanding of single-emitter behavior in atomically precise gold nanoclusters, contributing to the development of stable quantum emitters that are essential for quantum computing and cryptography.

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Etching-Chemistry-Driven Ruthenium Doping on Ti 3 C 2 T x MXene for Optimizing Electrochemical Performance

We demonstrate that the etching chemistry used during MXene synthesis from Ti 3 AlC 2 MAX phase significantly influences surface functionalization and structural vacancies, which in turn affect ruthenium (Ru) ion interactions. Using hydrofluoric acid (HF) and ammonium bifluoride (NH 4 HF 2 ) as etchants, we obtained MXene surfaces with distinct functional groups and Ti vacancies that impact Ru ion interactions and electrochemical performance. Both MXene variants (labeled MX(H) and MX(N), respectively) exhibited negative zeta potentials in their pristine state, but upon the addition of Ru the zeta potential for MX(H) reached 12.9 mV while that for MX(N) remained negative at −6.4 mV. This adsorption resulted in a 14.4-fold increase in the specific capacitance of MX(H)/Ru compared to pristine MX(H), whereas MX(N)/Ru exhibited only a 4.4-fold increase over its pristine counterpart. X-ray diffraction analysis identified the formation of ammonium titanium oxide fluoride, (NH 4 ) 3 TiOF 5 , on MX(N), which likely contributed to its reduced Ru adsorption. X-ray photoelectron spectroscopy suggested the presence of Ti vacancies in both MXene variants; however, their behavior toward Ru accommodation differed markedly, with MX(H) showing the most obvious shift in the Ti 2p peak in the XPS survey spectrum, while MX(N) showed the most obvious shift in the C 1s peak. Electron paramagnetic resonance spectroscopy further demonstrated a distinct alteration in the spectral signatures of MX(H) upon Ru addition, in contrast to the negligible changes in MX(N), indicating effective passivation of the Ti defect sites in MX(H) via vacancy-assisted Ru doping. Cyclic voltammetry showed that Ru-incorporated MX(H) nanocomposites exhibit more efficient redox-active sites, as reflected in their higher capacitance values. These findings highlight the pivotal role of MXene surface chemistry in controlling cation adsorption, providing valuable insights for the rational design of high-performance electrodes.

2D surface engineering↗

Scattering-type Scanning Near-Field Optical Microscopy of Polymer-Coated Gold Nanoparticles

Scattering-type scanning near-field optical microscopy (s-SNOM) has emerged over the past years as a powerful characterization tool that can probe important properties of advanced materials and biological samples in a label-free manner, with spatial resolutions lying in the nanoscale realm. In this work, we explore such usefulness in relationship with an interesting class of materials: polymer-coated gold nanoparticles (NPs). As thoroughly discussed in recent works, the interplay between the Au core and the polymeric shell has been found to be important in many applications devoted to biomedicine. We investigate bare Au NPs next to polystyrenesulfonate (PSS) and poly(diallyldimethylammonium chloride) (PDDA) coated ones under 532 nm laser excitation, an wavelength matching the surface plasmon band of the custom-synthesized nanoparticles. We observe consistent s-SNOM phase signals in the case of bare and shallow-coated Au NPs, whereas for thicker shell instances, these signals fade. For all investigated samples, the s-SNOM amplitude signals were found to be very weak, which may be related to reduced scattering efficiency due to absorption of the incident beam. We consider these observations important, as they may facilitate studies and applications in nanomedicine and nanotechnology where the precise positioning of polymer-coated Au NPs with nanoscale resolution is needed besides their dielectric function and related intrinsic optical properties, which are also quantitatively available with s-SNOM.

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Improved 140 Nd Production for the 140 Nd/ 140 Pr In Vivo Generator through Target Recycling and Radiochemical Optimization

Theranostic strategies that utilize f-block therapeutic radionuclides, including 161 Tb, 177 Lu, 225 Ac, and 227 Th, suffer from a shortage of positron emission tomography (PET) imaging counterparts in the same chemical space and often rely on 68 Ga as a surrogate. The 140 Nd/ 140 Pr in vivo PET generator, which belongs to the f-block, may address this issue and can be produced via the 141 Pr(p,2n) 140 Nd production route by using medium-energy cyclotrons. However, impurities in the target material, including stable Nd, and the inherent difficulty of adjacent lanthanide separations limit the achievable radionuclidic and chemical purity of 140 Nd. In this work, we address these challenges through the purification and recycling of praseodymium target material and optimization of Nd/Pr separation. The resulting purified 140Nd was evaluated using DOTA and Macropa chelators via radiolabeling and in vitro stability studies. A target material purification and recycling method was developed for the monoisotopic 141 Pr starting material to remove stable Nd impurities, yielding 90.3 ± 4.7% (n = 3) recovery. The purified 141 Pr was isolated as Pr 6 O 11 and irradiated with 24 MeV protons (20.07 MeV at the target surface) at 20 μA for 4 h, which produced 1417.0 ± 83.4 MBq (38.3 ± 2.2 mCi) of 140 Nd at the end of bombardment (EOB). The produced 140 Nd was purified through an optimized DGA normal method to recover 71.6 ± 6.3% pure 140 Nd. The amount of stable Nd reduced progressively in each target purification cycle from >340 ppm without purification to <250 ppb after three cycles, while other measured metallic impurities were below 30 ppb. This improvement in target purity was reflected in the direct increase of apparent molar activity (AMA), when purified 140 Nd was evaluated with DOTA and Macropa chelators. AMA of [ 140 Nd]Nd-DOTA and [ 140 Nd]Nd-Macropa increased from 70.3 MBq/μmol (1.9 mCi/μmol) and 74 MBq/μmol (2.0 mCi/μmol) to 8025.3 MBq/μmol (216.9 mCi/μmol) and 8473.0 MBq/μmol (229.0 mCi/μmol), respectively, after the third target purification cycle. Further evaluation of chelator-labeled 140 Nd showed that [ 140 Nd]Nd-DOTA was stable in phosphate-buffered saline (PBS), saline, human serum, and mouse serum, whereas [140Nd]Nd-Macropa was stable in all except human serum. This work established a practical methodological advance for the production of 140 Nd/ 140 Pr in vivo PET generators, combining optimized target recycling and radiochemical separation to enable scaled-up and high-molar activity 140 Nd suitable for preclinical imaging. These advances support broader development of 140 Nd/ 140 Pr as a robust PET analogue, especially for f-block therapeutics.

Irradiation↗

Hyperspectral and Nanosecond Temporal Resolution Widefield Infrared Photothermal Heterodyne Imaging

Label-free, bond-selective imaging offers new opportunities for fundamental and applied studies in chemistry, biology, and materials science. Preventing its broader application to investigating spectrally congested specimens are issues related to low sensitivity as well as low spatial and temporal resolution. Here, we demonstrate a widefield, mid-infrared (MIR) photothermal imaging technique, called widefield Infrared Photothermal Heterodyne imaging (wIR-PHI), that massively parallelizes acquisition of MIR absorption data through use of a high-speed complementary metal-oxide-semiconductor camera. wIR-PHI possesses notable features that include: spatial resolution significantly below the MIR diffraction limit, hyperspectral imaging capabilities, high sensitivity, and ~100 ns temporal resolution. Here, the first two features are highlighted by hyperspectral imaging of proximally close poly(methyl methacrylate) (PMMA) and polystyrene (PS) nanoparticles where clear, bond-specific imaging of nanoparticles, separated by less than the MIR diffraction limit, is demonstrated. Sensitivity is highlighted by imaging individual PMMA and PS nanoparticles with radii between r = 97-556 nm. This leads to a current, peak absorption cross-section limit-of-detection of σ abs = 1.9× 10 –16 cm 2 . wIR-PHI's 100 ns temporal resolution is simultaneously demonstrated by observing the decay of photothermal contrast on individual nanoparticles on a ~200-6000 ns timescale. In whole, wIR-PHI’s dramatic increase in acquisition speed opens opportunities for future MIR kinetic imaging and spectroscopic studies of important chemical, biological, and material processes.

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A Quantum Dot Nanobiosensor for Rapid Detection of Botulinum Neurotoxin Serotype E

Botulinum neurotoxins (BoNTs) are potent toxins produced by Clostridium bacteria that are responsible for the illness botulism and are listed as bioterrorism agents. BoNT serotype E (BoNT/E) is one of four BoNT serotypes that cause human botulism and is the second most frequent cause of foodborne botulism. Rapid detection and discrimination of BoNT serotypes implicated in human disease are critical for ensuring timely treatment of patients and identifying sources of toxins, but there have been few reported detection methods for BoNT/E and even fewer methods usable for BoNT serotyping. We report a nanobiosensor based on Forster resonance energy transfer (FRET) between semiconductor nanocrystals (quantum dots, QDs) and dark quencher-labeled peptide probes to detect biologically active BoNT/E in aqueous media. The peptide probes contain a specific cleavage site for active BoNT/E. QD photoluminescence, which changes intensity due to FRET when the peptide probe is cleaved, was used to indicate toxin presence and quantity. The detection of a BoNT/E light chain (LcE) and holotoxin was observed within 3 h. The limits of detection were 0.02 and 2 ng/mL for LcE and holotoxin, respectively. The nanobiosensor shows good specificity toward the target in tests with nontarget BoNT serotypes. The high sensitivity, simple operation, short detection time, and ability to be used in parallel with probes developed for other BoNT serotypes indicate that the nanobiosensor will be useful for rapid BoNT/E detection and serotype discrimination in food analysis.

botulinum neurotoxin E↗

DNA-PAINT Imaging with Hydrogel Imprinting and Clearing

Hydrogel-embedding is a versatile technique in fluorescence microscopy, offering stabilization, optical clearing, and the physical expansion of biological specimens. DNA-PAINT is a super-resolution microscopy approach based on the diffusion and transient binding of fluorescently labeled oligos, but its feasibility in hydrogels has not yet been explored. In this study, we demonstrate that polyacrylamide hydrogels support sufficient diffusion for effective DNA-PAINT imaging. Using acrydite-anchored oligonucleotides imprinted from patterned DNA origami nanostructures and microtubule filaments in fixed cells, we find that hydrogel embedding preserves docking strand positioning at the nanoscale. Sample clearing via protease treatment had minor structural effects on the microtubule structure and enhanced diffusion and accessibility to hydrogel-imprinted docking strands. Our work demonstrates promising potential for diffusion and binding-based fluorescence imaging applications in hydrogel-embedded samples.

DNA origami↗

Sequential Ammonia and Carbon Dioxide Adsorption on Pyrolyzed Biomass to Recover Waste Stream Nutrients

Untreated fecal waste in unsewered communities and reactive nitrogen (N) loading from wastewater and urine disposal are important environmental challenges. Through pyrolysis, untreated fecal waste can be converted into amine-rich carbon dioxide (CO 2 ) sorbents, the chemisorption of which tunes the surface chemistry toward ammonia (NH 3 ) uptake. We compared the N-loading potential within amine-rich pyrolyzed human solid waste through exposure to CO 2 followed by NH 3 with the N-loading potential on carboxyl-rich, pyrolyzed oxidized wood using sequential adsorption of isotopically labeled CO 2 and NH 3 . The total N content of pyrolyzed oxidized wood exposed once to NH3 was similar to native, pyrolyzed human solid waste (3.7% compared to 4.6%). However, sorption kinetics and spectroscopic evidence revealed distinct N forms between the two materials affecting subsequent CO 2 and NH 3 uptake. Tertiary amines comprised 70% of N in pyrolyzed waste, compared to only 53% in pyrolyzed oxidized wood. The high heat of adsorption of CO 2 on pyrolyzed human solid waste (50 kJ mol-1) at low concentrations (0.05 mmol CO 2 g -1 ) was indicative of CO 2 reactivity with tertiary amines, consistent with the formation of carbamic acid (Figure S1). Conversely, after exposure to NH 3 , pyrolyzed oxidized wood chemisorbed CO 2 at much higher concentrations (0.7 mmol CO 2 g-1), pointing to primary amines as the active functional moieties. Adsorption kinetics of pyrolyzed oxidized wood revealed continued, albeit diminishing NH 3 uptake with each consecutive CO 2 treatment, averaging 5.9 mmol NH 3 g -1 for the first NH 3 exposure event, and still 3.5 and 2.9 mmol NH 3 g -1 for the second and third. Penetration of 15 NH 3 and 13 CO 2 measured by NanoSIMS reached over 7 µm deep into both materials, explaining the large NH 3 capture. The significantly lower NH 3 adsorption compared to pyrolyzed oxidized wood may be a result of potassium (K) carbonate precipitation precluding a Lewis base interaction between NH 3 and chemisorbed CO 2 , suggested by a 2-fold increase in surface K concentrations after exposure of pyrolyzed human solid waste to CO 2 +NH 3 detected by XPS.

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Lignin Removal in Subcellular Location of Poplar Cell Wall During Pretreatment Significantly Impacts Cellulose Digestibility

The γ-valerolactone (GVL) pretreatment is one of the leading solvent-based methods for producing high-quality lignin under mild conditions. However, the glucan conversion yield from GVL pretreated biomass remains unsatisfactory. To explore the discrepancies between the relatively low glucan conversion and high lignin extraction, we conducted GVL−HCl and NaOH pretreatments on poplar and investigated their effects on lignin content and location, as well as on enzymatic hydrolysis of poplar cell walls at the subcellular level. Under designated pretreatment conditions of GVL−HCl (90% GVL, 0.1 M HCl, 100 °C, 1 h) and NaOH (1 M, 121 °C, 2 h), the glucan conversion yields were 69.4% and 95.8%, with lignin removal rates of 67.8% and 47.7%, respectively. Four types of GFP-labeled carbohydrate binding modules were used to identify different forms of cellulose in the pretreated cell walls. The overall binding intensities to pretreated poplar were stronger for NaOH compared to GVL−HCl pretreatment. Stimulated Raman scattering microscopy imaging revealed that GVL−HCl preferentially extracted lignin from the compound middle lamella and cell corner areas, while NaOH effectively dissolved lignin in the secondary cell walls. Real-time imaging of cellulase degradation of pretreated cell walls further indicated that digestion started from both the cell lumen and the compound middle lamella areas for GVL, whereas it occurred uniformly across the secondary cell walls for NaOH. Our findings suggest that the location of lignin removal during pretreatment is crucial for enzymatic cellulose degradation, in addition to the total amount of lignin extraction.

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Computational Framework for Machine-Learning-Enabled 13 C Fluxomics

13 C metabolic flux analysis (MFA) has emerged as a powerful tool for synthetic biology. This optimization-based approach suffers long computation time and unstable solutions depending on the initial guess. Here, we develop a machine-learning-based framework for 13 C fluxomics. Specifically, training and test data sets are generated by metabolic network decomposition and flux sampling, in which flux ratios at metabolic nodes and simulated labeling patterns of metabolites are used as training targets and features, respectively. To improve prediction accuracy and simplify the model, automated processes are developed for flux ratio selection based on solvability and feature screening based on importance. We found that predictive performance can be significantly improved using both amino acids and central carbon metabolites in comparison with amino acids alone. Together with measured external fluxes, the predicted flux ratios determine the mass balance system, yielding global flux distributions. This approach is validated by flux estimation using both simulated and experimental data in comparison with canonical 13 C MFA. The approach represents a reliable fluxomics method readily applicable to high-throughput metabolic phenotyping, which highlights the advances of intelligent learning algorithms in synthetic biology, specifically in the Test and Learn stage of the Design-Build-Test-Learn cycle.

13C metabolic flux analysis↗

Robust Synthetic Biology Toolkit to Advance Carboxysome Study and Redesign

Carboxysomes are polyhedral protein organelles that microorganisms use to facilitate carbon dioxide assimilation. They are composed of a modular protein shell that envelops an enzymatic core mainly composed of physically coupled Rubisco and carbonic anhydrase. While the modular construction principles of carboxysomes make them attractive targets as customizable metabolic platforms, their size and complexity can be a hindrance. In this work, we design and validate a plasmid set, the pXpressome toolkit, in which α-carboxysomes are robustly expressed and remain intact and functional after purification. We tested this toolkit by introducing mutations that influence carboxysome structure and performance. We find that deletion of vertex-capping genes results in formation of larger carboxysomes, while deletion of facet forming genes produces smaller particles, suggesting that adjusting the ratio of these proteins can rationally affect morphology. Through a series of fluorescently labeled constructs, we observe that this toolkit leads to more uniform expression and better cell health than previously published carboxysome expression systems. Overall, the pXpressome toolkit facilitates the study and redesign of carboxysomes with robust performance and improved phenotype uniformity. The pXpressome toolkit will support efforts to remodel carboxysomes for enhanced carbon fixation or serve as a platform for other nanoencapsulation goals.

59 BASIC BIOLOGICAL SCIENCES↗

Imaging from Macro to Nanoscale: Multimodal Advances in Chemical and Biomedical Imaging

Imaging increasingly serves as a multiscale framework for linking molecular mechanisms to cellular behavior, tissue architecture, and organ phenotypes in biology and unraveling fundamental processes in chemistry, physics and materials science. This Perspective highlights recent advances in chemical and biomedical imaging across macro-, micro-, and nanoscales, using representative examples published in Chemical and Biomedical Imaging (CBMI). At the macroscale, we discuss chemically selective MRI, including endogenous and exogenous CEST strategies, together with photoacoustic imaging as a hybrid modality with functional and chemical contrast. At the microscale, we consider fluorescence, label-free optical and vibrational imaging, and selected X-ray approaches that expand sensitivity, specificity, and temporal resolution in biological and materials systems. At the nanoscale, we highlight super-resolution fluorescence microscopy, single-molecule methods, tip-enhanced Raman spectroscopy, and correlative imaging strategies that resolve local heterogeneity and molecular organization. Across scales, a common theme emerges that advances in probes, contrast mechanisms, instrumentation, and sample handling are enabling chemically informed imaging that connects molecular specificity with biological context.

multiscale imaging↗