搜索结果：Biomolecules

Paramagnetic relaxation enhancement (PRE) provides valuable distance constraints with respect to unpaired electron for the structural and dynamic characterization of biomolecules. While transverse PRE (Γ 2) has been widely used, its quantitative interpretation is often confounded by chemical exchange and other factors. In contrast, longitudinal PRE (Γ 1), though much smaller in magnitude than Γ 2, is a more faithful reporter of electron-nuclei distances. In this work, we introduce the division after subtraction (DAS) approach, which utilizes a synchronized sampling scheme to analytically cancel out common-mode artifacts. We validate this approach using site-directed covalent labeling and solvent PRE systems, and demonstrate improved accuracy compared to standard separate fitting routine. DAS is particularly effective for labile residues, where traditional nonlinear regression breaks down due to solvent-mediated effects. The enhanced precision with DAS in Γ 1 measurement allows for more rigorous investigation into biomolecular conformational landscape, thus providing a powerful complement to Γ 2-based PRE methodology.

Spatial multi-omics analyzes biomolecules such as the proteome, metabolome, and lipidome within their native spatial context in tissues or cells. Mass spectrometry imaging (MSI) has emerged as a powerful technique for mapping the region-specific molecular distribution in regions of interest (ROIs). Laser capture microdissection coupled with mass spectrometry (LCM-MS) is another well-established workflow, enabling the accurate characterization of biomolecules in ROIs. To advance the current analytical application, we expanded a matrix-assisted laser desorption/ionization (MALDI)-MSI-guided LCM-MS workflow for integrated multi-omics analysis and applied it to mouse brain tissue as a proof-of-principle validation. MALDI-MSI annotated 387 putative metabolites and lipids, revealing distinct molecular distributions between the cortex and hippocampus. Both regions were subsequently isolated as ROIs using LCM and analyzed by LC-MS/MS metabolomics, lipidomics, and proteomics to achieve accurate biomolecular profiling. LC-MS/MS metabolomics and lipidomics annotated 249 compounds, several of which exhibited distinct abundance patterns between the two regions. LC-MS/MS proteomics matched to over 3500 protein groups across the two regions. Biological network analysis revealed strong associations between molecular pathways and known region-specific phenotypes. Overall, this MALDI-MSI-guided LCM-MS workflow enables comprehensive spatial multi-omics profiling and quantitative biomolecular analysis, providing valuable insights into complex biological systems and spatial molecular organization.

Coherent Raman imaging (CRI) enables label-free chemical imaging based on intrinsic molecular vibrations, but its applicability is often limited by low sensitivity, hindering the detection of low-abundance biomolecules. Although electronic resonance enhances sensitivity, most resonance CRI implementations rely on narrowband excitation and/or detection, which limits spectral coverage and complicates the differentiation of target molecules from complex backgrounds. Here, we address these challenges by developing broadband electronic resonance coherent anti-Stokes/Stokes Raman scattering (BER-CARS/CSRS) microscopy. We show that BER-CARS/CSRS enables highly sensitive, label-free imaging of endogenous chromophores with broad spectral coverage of the entire fingerprint region. Specifically, we captured time-lapse images, visualizing low-abundance cytochromes alongside abundant biomolecules (lipids, proteins, and nucleic acids) in living human embryonic kidney (HEK) 293 cells. Furthermore, we applied the method to mouse tissues, highlighting characteristic localizations of cytochromes in the brain cortex, the cerebral ventricle wall, and the liver. Our results demonstrate that BER-CARS/CSRS provides highly sensitive, label-free chemical imaging from organelle dynamics to tissue mapping, enabling quantitative phenotyping and slide-scale histopathology.

Solid-state nanopores can be combined with optical measurements for the detection of biomolecules. With a view to a reliable optical biosensing library and avoiding computationally demanding simulations or expensive experiments, we develop a learning approach based on deep neural networks that are being trained on electronic, conformational, and optical characteristics from density functional theory (DFT) simulations of single amino acids placed in a graphene nanopore. Principal component analysis is used to compress the high-dimensional data into a latent representation, improving training efficiency while preserving the most important physical features. For the five amino acids considered in this work, the learning model predicts the full orientation-dependent absorption spectra with consistently high accuracy in previously unseen molecular orientations within each amino acid dataset, with test R 2 values exceeding 0.9. These results indicate robust interpolation across rich conformational variations of the exact amino acids studied here. The developed workflow enables the construction of reliable optical biosensing libraries with near-DFT accuracy at a fraction of the computational cost. It thus provides a scalable and efficient pathway towards real-time, high-precision optical identification of biomolecules.

Vitamins A and C (VitA+C) are pivotal biomolecules, with synergistic effects, especially in conjoint presence, on cellular pluripotency and regenerative capabilities. This randomized trial aimed to evaluate (VitA+C)-augmented injectable platelet-rich fibrin (i-PRF) versus i-PRF alone with a modified minimally invasive surgical technique (M-MIST) in periodontal intraosseous defects in patients with stage III periodontitis. This parallel group, two-arm, triple-blinded, randomized controlled trial included patients with stage III grade B periodontitis (n&#xa0;=&#xa0;28) randomly allocated to the test (VitA+C/i-PRF+M-MIST; n&#xa0;=&#xa0;14) or the control (i-PRF+M-MIST; n&#xa0;=&#xa0;14) group. The changes in radiographic linear defect depth (RLDD) (primary outcome), radiographic bone fill (RBF), and radiographic bone density (RBD) were recorded at baseline, 6, and 9 months. Clinical attachment level (CAL), probing depth (PD), gingival margin level (GML), plaque index (PI), and gingival index (GI; secondary outcomes) were recorded at baseline, 3 6, and 9 months. The in vitro release kinetics of VitA and VitC from the i-PRF were characterized over 7 days using high-performance liquid chromatography (HLPC). VitA+C/i-PRF+M-MIST demonstrated a significantly higher RLDD-reduction at 6 months and a higher RBF percentage at 6 and 9 months compared with i-PRF+M-MIST (p&#xa0;<&#xa0;0.05), with no intergroup differences regarding RBD. Both groups independently demonstrated significant improvements in CAL, PD, GML, RLDD, and BDA over time (p&#xa0;<&#xa0;0.05). Significantly higher CAL-gain and PD-reduction were evident in the VitA+C/i-PRF+M-MIST group at 9 months (p&#xa0;<&#xa0;0.05). I-PRF with M-MIST significantly enhance periodontal clinical and radiographic parameters over time. Conjoint vitamin A/C augmentation of i-PRF at the described concentration results in significantly greater improvement in radiographic and clinical parameters of intraosseous defects compared with i-PRF alone. This study was registered in the US National Institutes of Health Clinical Trials Registry (NCT05499598; https://www. gov/). Platelet&#x2010;rich fibrins (PRF) could serve as sustained&#x2010;release carriers for biomolecules. The current trial assessed the clinical benefits of an injectable&#x2010;PRF (i&#x2010;PRF) as a sustained&#x2010;release vehicle for vitamins A/C, at specifically defined pluripotency&#x2010;inducing concentrations, in combination with a minimally invasive surgical technique for stage III periodontitis intraosseous defects. The current findings demonstrate that vitamins A/C provide additional significant benefits on periodontal clinical and radiographic outcomes when used with a minimally invasive surgical technique for the management of intraosseous defects compared with i&#x2010;PRF alone.

Mucosa can be found in the eyes, oral cavity, bladder, vagina, airways, and gastrointestinal tract. It is an attractive route of administration for systemic or topical delivery of therapeutics. However, the mucus layer acts as a protective barrier, limiting the amount of biomolecules that reach the underlying epithelium. Mucoadhesion, mucodiffusion, and mucolysis are well-established mucointeractive strategies that can improve therapeutic outcomes, but due to their individual limitations, the resulting delivery is often still unsatisfactory. In recent years, drug delivery systems have emerged that combine multiple mucointeractive strategies, which we define here as hybrid mucointeractive delivery systems. This work aims to provide a general overview of such drug delivery systems, which include particle-releasing macrostructures such as gels, foams, films, and fibers, as well as systems such as zeta potential-changing particles and self-emulsifying drug delivery systems. Their potential, possible future, and limitations are discussed as well.

Marine macroalgae are increasingly recognized as sustainable and chemically diverse sources of valuable biomolecules. However, the short-chain peptide fraction (2-4 amino acids) of edible and invasive seaweeds remains poorly explored, mainly because conventional proteomics workflows are generally optimized for longer peptide sequences. In this study, an untargeted LC-HRMS suspect screening workflow was developed to characterize short-chain peptides generated by hydrolysis with Alcalase&#xae;, an alkaline protease from Bacillus licheniformis&#xa0;hydrolysis in 14 marine macroalgae species belonging to green, brown, and red phyla collected along the Atlantic coast of southern Spain. Overall, 333 short-chain peptides, predominantly dipeptides, were putatively annotated through accurate-mass filtering and manual MS/MS interpretation. Multivariate and univariate analyses revealed distinct peptide distribution patterns among algal groups, with brown algae showing a higher abundance of hydrophobic dipeptides associated with predicted bioactive properties according to in silico screening tools. Notably, invasive brown algae displayed peptide profiles largely comparable to edible species, supporting their potential valorization as sustainable sources of short-chain peptides. Exploratory in silico analyses further suggested the occurrence of peptide sequences potentially associated with antioxidant, antihypertensive, antidiabetic, sensory, and antifungal properties. Several peptides were also predicted to contribute to umami-related flavor attributes, highlighting the relevance of these matrices for future investigation of food-related peptide functionality. Although the proposed annotations and predicted bioactivities require orthogonal confirmation and experimental validation, the results provide a comprehensive exploratory overview of marine macroalgal short peptidomes. Overall, this study expands current knowledge on algae-derived short-chain peptides and demonstrates the applicability of untargeted LC-HRMS workflows for the characterization of underexplored peptide fractions in complex marine matrices.

DNA origami has emerged as a groundbreaking approach in nanotechnology, offering unparalleled precision, programmability, and structural versatility at the molecular scale. Originally conceived as a method to fold DNA into arbitrary 2D and 3D shapes, DNA origami has rapidly evolved into a multifunctional platform, enabling the construction of dynamic, responsive, and addressable nanostructures. As we stand at the intersection of biology, physics, and engineering, this perspective explores how far we can truly "fold" DNA origami, not just structurally but functionally, toward the realization of advanced nanoenabled technologies. We examine the foundational design principles that have propelled DNA origami from static nanoshapes to reconfigurable architectures capable of precise molecular actuation. By integrating functional elements such as quantum dots, metallic nanoparticles, and biomolecules, DNA origami has unlocked novel possibilities in optoelectronics, ranging from plasmonic nanodevices to photonic nanostructures, and in biomedicine, where it serves as a vehicle for targeted drug delivery, biosensing, and immunomodulation. Despite these achievements, several grand challenges remain, including issues of scalability, structural stability under operational conditions, and integration with other nanomaterials and systems. This perspective reflects on the current state of the field and identifies opportunities for future innovation, particularly through convergence with artificial intelligence, machine-learning-guided design, and hybrid materials science. Ultimately, we posit that DNA origami is no longer just a tool for nanoscale construction but a foundational technology poised to redefine the frontiers of optoelectronics, diagnostics, and therapeutics. As we continue to push the boundaries of what can be folded, this article invites the scientific community to rethink the potential of DNA origami from blueprint to breakthrough in shaping the future of nanoenabled applications.

Metal-organic frameworks (MOFs) are at the forefront of materials research due to their high area-to-volume ratio, tunable porosity, host-guest adsorption capabilities, and improved optical, fluorescent, conductive, and electromagnetic properties. Van der Waals heterostructures (VWHs) are 2D materials, including MOFs and graphene oxide (GO), among others, stacked like building blocks defined with one-atomic-plane precision by weak, reversible interactions that synergistically integrate their features to create new structures with enhanced properties. For example, MOF-based architectures enable the adsorption of numerous small molecules, such as electroactive probes and biomolecules, which can be linked to GO to form VWHs and stably coupled to an electrode surface, thereby enhancing the electrode's response and making them promising for the development of highly sensitive biosensors. In this work, VWHs were assembled by stacking Ni3(HITP2) MOF and GO, which were deposited on screen-printed carbon electrodes (SPCE) by drop-casting and characterized by electrochemistry, spectroscopy, and microscopy. They showed better conductive properties than bare and modified electrodes with the corresponding materials separately, thus offering a highly conductive surface area that permits the adsorption of toluidine blue O (TB), an in-situ electrochemical probe, and multiple anchoring points of procalcitonin capture antibodies. As a result, a label- and reagent-free nanoimmunosensor platform was developed for the detection of procalcitonin in one step, showing high sensitivity (0.099&#x202f;&#x3bc;A*mL/pg) and high specificity over a linear range from 4.25 to 125.00&#x202f;pg/mL, with a limit of detection (LOD) of 4.25&#x202f;pg/mL by square wave voltammetry (SWV). The nanoimmunosensor performance was tested in spiked human serum, with recovery greater than 90%, and in real samples, demonstrating high discrimination between positive and negative samples and strong correlation with a gold standard method. Procalcitonin levels, in the context of a panel of infection biomarkers, provide information not only about diagnosis but also prognosis, infection severity, and sepsis risk.

Micro- and nanoplastics (MNPs) are emerging global pollutants that pose a significant threat to living organisms due to their widespread presence, ingestion by aquatic species, and ability to cross biological barriers, including the blood-brain barrier. Zebrafish is a well-established and convenient model for ecotoxicological research because of its small size, optical transparency, fully sequenced genome, high genetic homology to humans, ease of breeding, and short life cycle. Exposure to MNPs affects multiple organ systems in zebrafish, including the brain, eyes, liver, intestine, gills, and reproductive system. These particles can induce oxidative stress, inflammation, and interference with diverse biomolecules, leading to adverse biological effects. An analysis of transcriptomic alterations induced by MNPs exposure can contribute to understanding the mechanisms of these adverse effects. In this narrative review, we classify existing studies on MNPs exposure in zebrafish by affected organ system and summarize the gene expression-based evidence of MNP-induced toxicity with a particular focus on high-throughput approaches such as RNA sequencing and single-cell RNA sequencing.

Self-assembled monolayers of biomolecules, particularly peptides, are important for a wide range of applications, including surface coatings, biosensing, and bioelectronics. The performance of peptide two-dimensional crystal patterns on solid substrates as molecular scaffolds strongly depends on the stability of the assemblies, which is closely related to their desorption behavior. However, investigation on peptide desorption remains poorly understood, especially across distinct sizes and time scales. Here, we investigated the desorption characteristics and underlying mechanisms of peptide assemblies on graphite/MoS2 driven by water incubation mediated soft-interfacial concentration gradient (CG). We captured, recorded and quantified morphological and structural changes in peptide nanowires (pNWs) using three key parameters: surface coverage, width and length of pNWs. We further examined the effects of peptide sequence, substrate, humidity, and temperature on the desorption behaviors and characteristics of peptide assemblies by modulating interpeptide and peptide-substrate interactions. Quantitative analysis of the time-dependent changes in surface coverage revealed exponential desorption kinetics with an exponent index of 0.51&#xa0;&#xb1;&#xa0;0.05. In addition, we observed a water-assisted phase transition of pNWs, performed quantitative analysis, and mapped the corresponding energy landscape. Finally, we propose a multistep desorption mechanism involving desorption/adsorption, attachment/detachment, and phase transitions accompanied by peptide diffusion. This work provides fundamental insight into CG-induced peptide desorption on graphite surfaces and helps bridge the knowledge gap between nanoscale desorption phenomena and behavior at micro--/macroscales. It also highlights the potential applications of peptide-based systems in self-cleaning surfaces, controllable release, and related technologies.

Aging is associated with the deterioration of various biological processes including disrupted proteostasis and impaired macroautophagy/autophagy. Biomolecules can undergo liquid-liquid phase separation (LLPS) to form biomolecular condensates that exert specific biological functions. Trr1 (thioredoxin reductase 1) is a pivotal enzyme in the thioredoxin antioxidant system. Deletion of TRR1 results in impaired autophagy; however, the underlying mechanism is largely unexplored. In this study, we explored whether LLPS of Trr1 affected autophagy. Trr1 formed dynamic LLPS condensates during replicative aging in yeast. Phase separation of Trr1 occurred in response to endoplasmic reticulum (ER) stress generated by cellular aging, rather than to oxidative stress. Furthermore, Trr1 condensates participated at the phagophore assembly site during endoplasmic reticulophagy and promoted autophagosome development by affecting lipidation of the Atg8 protein. Additionally, maintaining the liquid-like dynamic nature of Trr1 condensates was essential for cellular fitness. Our findings revealed an unconventional role of Trr1 through LLPS in aging. The function of phase-separated condensates of Trr1 in mitigating aging-associated ER stress offers insights into the mechanisms underlying healthy cellular aging. These findings highlight a potential target for developing interventions to combat aging and associated diseases.Abbreviations: Atg: autophagy related; DTT: dithiothreitol; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; ERphagy: endoplasmic reticulophagy; FRAP: fluorescence recovery after photobleaching; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; PAS: phagophore assembly site; PLDs: prion-like domains; RFP: red fluorescent protein; RLS: replicative lifespan; Trr1: thioredoxin reductase 1; Trx: thioredoxin; UPR: unfolded protein response; IDRs: intrinsically disordered regions.

Poly(ADP-ribose) or PAR regulates multiple aspects of cell biology, both as an independent signaling molecule and as a modification on biomolecules. As a posttranslational modification, PAR can modulate the biochemical properties of target proteins. Isolated free PAR molecules function in cellular signaling. This chapter describes two methods to isolate and purify free PAR and protein-linked PAR from biochemical reactions, one using chemical fractionation and another using physical separation. A method to isolate free PAR and protein-linked PAR from human cells is also presented. These methods allow monitoring of free PAR and protein-linked PAR levels under different biochemical conditions or in response to different cellular stimuli.

Bioorthogonal reactions offer a powerful tool for site-specific labeling of biomolecules in living systems. Among them, the recently reported hydrazonyl sultone (HS)-bicyclo[6.1.0]non-4-yne (BCN) ligation reaction stands out for its fast reaction kinetics and tunable aqueous stability. Herein, we describe the experimental protocols of using HS-BCN ligation for site-specific modification of a recombinant nanobody in vitro and a G protein-coupled receptor (GPCR) on a live mammalian cell surface. These protocols include the genetic encoding of BCN-lysine (BCNK) into the target protein, bioorthogonal modification of the BCNK-encoded proteins, and characterization of the reaction rate and selectivity. Together with the robust genetic encoding of the strained alkyne BCN in any protein structure, the HS-BCN ligation reaction promises to expand the capabilities of bioorthogonal chemistry to enable facile modifications of domain antibodies in vitro for diagnostic applications and selective fluorescent labeling of GPCRs for biophysical studies of receptor dynamics in live cells.