Search Results (437)

Extracellular vesicles (EVs) mediate intercellular communication by delivering proteins and RNAs, with their molecular cargo often reflecting the biological context of their source. Perinatal tissues are promising sources of EV-related biomaterials with potential dermatologic applications. In this study, we compared EV-related molecular cargo from two umbilical cord-associated sources, umbilical cord mesenchymal stem cell (UCMSC)-derived EVs and cord blood plasma (CBP), to investigate whether these materials exhibit distinct functional effects on melanogenesis. UCMSC-derived EVs were isolated from conditioned culture medium and characterized using nanoparticle tracking analysis (NTA), cryo-electron microscopy (cryo-EM), and canonical EV marker detection, while cord blood samples were processed to obtain plasma following centrifugation and filtration, containing EVs together with soluble plasma components. Functional assays in the murine melanocyte cell line B16F10 demonstrated that UCMSC-derived EVs suppressed melanin production, whereas CBP treatment enhanced melanogenesis. Integrative omics analyses combining microRNAs (miRNAs) microarray profiling and proteomic characterization revealed distinct molecular signatures between UCMSC-derived EVs and CBP samples. Functional validation using miRNA mimic assays showed that selected miRNAs, including miR-6862-5p, miR-3622b-5p, miR-7847-3p, miR-6774-5p, and miR-4685-5p, reduced melanin production, whereas others, including miR-203a-3p, miR-126-3p, miR-139-5p, and miR-15b-5p, increased melanin levels. Pathway analysis using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc.) associated these miRNA subsets with signaling pathways involved in melanogenesis. Together, these findings indicate that UCMSC-derived EVs and CBP exhibit opposite functional effects on melanogenesis and possess distinct miRNA and protein cargo profiles, providing potential molecular targets for modulating pigmentation and supporting the development of EV-related therapeutic strategies for pigmentation disorders. Full article

Renal organic anion transporters 1 (OAT1) and 3 (OAT3) mediate the excretion of endogenous metabolites and xenobiotics. Flavonoids interact significantly with these transporters, but the structural determinants—especially regarding in vivo phase II metabolism—remain unclear. This review integrates recent cryogenic electron microscopy (cryo-EM) structural biology and transporter kinetics to delineate the molecular basis of flavonoid–OAT interactions. We highlight phase II metabolites as key in vivo effectors. Structurally, OAT1 strictly favors compact, planar anionic scaffolds, whereas OAT3 accommodates bulkier, conjugated forms. Crucially, flavonoids exert a “double-edged” toxicological effect: high-affinity OAT inhibition risks herb–drug interactions, yet competitively limits the tubular uptake of nephrotoxins. Furthermore, disease states and post-translational regulation reshape these interactions. By bridging structural insights with biomarker-guided pharmacokinetics, we propose a mechanistic framework to improve the precise safety assessment of flavonoid-containing therapeutics. Full article

Exosomes, a major subpopulation of small extracellular vesicles (sEV), are conserved mediators of intercellular communication, yet the properties of their endosomal precursors, intraluminal vesicles (ILV), have not been systematically quantified across species or imaging modalities. This study systematically evaluates ILV sizes across diverse eukaryotic species and modalities while assessing their relationship to secreted sEV sizes. We carried out two complementary meta-analyses of ILV sizes based on transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM) data across species. This was followed by in situ assessment of sEVs secreted by HEK293T cells with TEM, nanoparticle tracking analysis and super-resolution microscopy characterization. Across species, imaging modalities, and cellular contexts, ILV sizes were under approximately 200 nm, with a mean diameter of 100.5 nm, overlapping with the size range of sEVs. This study addresses an existing knowledge gap by systematically evaluating ILV size across species and revealing an upper size limit of approximately 200 nm. Full article

Protein condensates and filaments are both intracellular structures characterized by their ability to facilitate specific biological functions. Their formation is primarily driven by phase separation, which can be elucidated by fluorescence microscopy or electron microscopy. Here we summarize the main studies on protein condensates and filaments organized according to the techniques used, including fluorescence methods like localization screening, fluorescence co-localization spectroscopy, methods based on photobleaching, super-resolution imaging, and electron methods including negative-stain electron microscopy and cryo-EM. We also discuss correlative light/electron microscopy (CLEM), which integrates fluorescence microscopy and electron microscopy to provide complementary insights. Collectively, these methods offer temporal and spatial insights into investigating the phase separation of protein condensates and filaments, and promote the discovery of unexplored structures and their yet-to-be-characterized biological roles. Full article

RNA origami enables genetically encoded, single-stranded RNA nanostructures that can self-assemble through co-transcriptional folding and are increasingly deployed as scaffolds for biosensing, synthetic biology, and nanomedicine. A recurring practical bottleneck is scalable, solution-phase readout of whether a designed scaffold has reached its intended base-paired architecture, whether it undergoes slow maturation or kinetic trapping, and how its stability is distributed across motifs. Here, we adapt microfluidic modulation spectroscopy (MMS) as a label-free structural biosensor for RNA folding by exploiting the rich 1760–1600 cm⁻¹ vibrational fingerprints of RNA bases and base pairs. MMS alternates between sample and composition-matched buffer measurements in a microfluidic transmission cell to automatically subtract the solvent background, enabling high-quality spectral measurement from microliter volumes under native solution conditions. Using a six-helix-bundle-with-clasp (6HBC) RNA origami as a model, we established an analysis workflow (baselined second derivative and constrained deconvolution) to quantify paired versus unpaired populations. Thermal ramping resolves multiple unfolding events and yields an unfolding barcode that differs between young and mature ensembles. Importantly, MMS tracks post-transcriptional maturation from a kinetically trapped young conformer toward a more compact, base-paired mature state, consistent with prior cryo-EM/SAXS observations for 6HBC RNA origami. Together, these results position MMS as a rapid, automated, and scalable complement to high-resolution structure determination for engineering dynamic RNA origami biosensors. Full article

The human mitochondrial Lon protease (LonP1) is a central regulator of mitochondrial DNA copy number and metabolic reprogramming. However, the structural basis for how LonP1 recognizes native physiological substrates remains elusive. Here, we present the high-resolution cryo-EM structure of the human LonP1 hexamer actively engaging its native substrate, TFAM. The reconstruction reveals a distinct bipartite search-and-shred mechanism. Unlike its bacterial homologs, the human N-terminal domain (NTD) adopts a compact architecture acting as a selective vestibule to recruit and initially unfold the substrate tertiary structure. Subsequently, the polypeptide is threaded through the central channel via a hand-over-hand mechanism driven by a spiral array of aromatic pore-loops. This structural framework provides a mechanistic rationale for the spatial segregation of LonP1 and offers a template for targeting mitochondrial proteostasis in human diseases. Full article

Cardiac Ca²⁺ handling is critical for excitation–contraction coupling (ECC), with the ryanodine receptor type 2 (RyR2) serving as the key sarcoplasmic reticulum (SR) Ca²⁺ release channel in cardiomyocytes. The dysfunction of RyR2 is linked to fatal cardiac arrhythmias, including heart failure (HF) and catecholaminergic polymorphic ventricular tachycardia (CPVT). This review aims to elucidate the structural basis of RyR2, its core role in cardiac ECC and Ca²⁺ homeostasis, and the regulatory mechanisms of key modulators on its activity. By integrating recent high-resolution cryo-EM structural analyses with molecular and cellular studies on RyR2 regulation, as well as clinical evidence of RyR2 mutations in arrhythmogenic heart diseases, we provide a comprehensive overview of the field. Cryo-EM has unraveled RyR2’s gating mechanisms, ligand-binding sites, and structural features. Functionally, RyR2 mediates calcium-induced calcium release (CICR) and maintains Ca²⁺ homeostasis through coordination with SERCA2a and NCX. Key modulators (CaM, FKBP12.6, and PKA/CaMKII) and disease-linked mutations regulate RyR2 activity through distinct pathways, with defective RyR2 leading to store-overload-induced Ca²⁺ release (SOICR) and arrhythmias. Furthermore, reactive oxygen species (ROS) can induce RyR2 oxidation, establishing a pathological Ca²⁺ leak-ROS cycle in heart disease. In conclusion, RyR2 is a pivotal sensor of myocardial function, with its structural and regulatory mechanisms now well-characterized by recent studies. However, the effects of numerous RyR2 mutations remain unclear, and deeper mechanistic insights will lay a key foundation for developing novel therapies against RyR2-related cardiac diseases. Full article

Short-chain acyl-CoA dehydrogenase (SCAD) is a critical enzyme in mitochondrial fatty acid β-oxidation, catalyzing the initial dehydrogenation of short-chain acyl-CoAs. Mutations in the ACADS gene cause SCAD deficiency (SCADD), a disorder with remarkably heterogeneous clinical presentation. However, the molecular mechanisms underlying substrate specificity and the pathogenicity of most ACADS variants remain poorly understood. Here, we present high-resolution cryo-EM structures of human SCAD in complex with its physiological substrate butyryl-CoA (C₄) and the longer substrate hexanoyl-CoA (C₆). The butyryl-CoA-bound structure at 2.1 Å resolution details a pre-catalytic geometry ideal for hydride transfer, with Glu392 positioned as the catalytic base. We systematically characterized nineteen disease-associated mutations, which we classify into three functional categories: those disrupting FAD binding, those impairing substrate binding, and those compromising protein folding and stability. In addition, using the W177R mutant as a representative model, we demonstrate that folding-defective mutations provoke protein aggregation, leading to proteotoxicity, oxidative stress, and apoptosis, revealing a pathogenic mechanism beyond mere catalytic loss. In brief, our integrated findings elucidate the structural determinants of substrate specificity and catalytic mechanism in SCAD, and provide mechanistic insights into the functional impairments caused by mutations linked to SCADD. Full article

The use of microalgae as a food source is limited by consumers’ dislike of their organoleptic traits, primarily the intense green color and bitter taste associated with high chlorophyll content. The eukaryotic microalgae Chlorella vulgaris can grow under heterotrophic conditions, providing the opportunity to cultivate chlorophyll-less strains. In this work we applied random mutagenesis for breeding chlorophyll-deficient C. vulgaris strains. Wild-type strain was UVC-radiated, and 12 colonies with changed pigmentation were selected. Based on phenotypic stability two mutants, M6 and M11, were selected for characterization of growth, pigment and biomass accumulation. Cultivation under photo-, mixo- and heterotrophic conditions revealed distinct phenotypes for the two mutants. M6 remained chlorophyll-deficient in all cultivation conditions tested, while chlorophyll was observed in M11 when grown under light. Under heterotrophic and mixotrophic growth conditions, both mutants were chlorophyll-deficient while biomass productivity, protein content, and amino acid composition remained similar to wild type. Characterization of the cellular ultrastructure of the wild type and mutants using cryo Focused Ion-Beam Scanning Electron Microscopy revealed that functional chloroplasts and thylakoid membranes were absent in the mutants. Our work demonstrates how a simple approach using UV mutagenesis and visual screening can provide novel strains of C. vulgaris with traits for improved consumer acceptance, without compromising the use of the algae biomass as a protein-rich food source. Full article

Extracellular vesicles (EVs) are membrane-bound particles secreted by most cell types that play a pivotal role in intercellular communication via transporting protein, nucleic acid, lipid, and metabolite cargos. Among EVs, exosomes are a well-characterized subtype, typically ranging from 10–150 nm in diameter and originating from the endosomal pathway via the formation of multivesicular bodies that fuse with the plasma membrane. EVs/exosomes can be isolated from various biological fluids and cultured cells, with production and yield influenced by the cell type and culture conditions. Isolation methods, including ultracentrifugation or density-based ultracentrifugation, tangential flow filtration, size-exclusion chromatography, immunoaffinity and membrane-affinity capture, and recently developed commercial equipment, offer distinct advantages and limitations in terms of purity, scalability, and exosome integrity. Characterization techniques, such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), cryogenic electron microscopy (cryo-EM), atomic force microscopy (AFM), Western blotting, flow cytometry, and dynamic light scattering (DLS), assess exosome size, morphology, and biomarker expression. Given their biocompatibility and inherent targeting capabilities across a diverse range of diseases, EVs/exosomes hold clinical promise as diagnostic biomarkers, cell-free therapeutics, drug delivery vehicles, immune modulators, and in regenerative medicine. However, these emerging fields in exosome medicine continue to face challenges in standardizing EV sourcing, production, purification, yield, bio-targeting, drug loading, and drug delivery. While EVs/exosomes represent a rapidly advancing frontier in biomedical science, robust protocols for standardization and scalable production will be essential for their successful translation into clinical applications. This article provides a comprehensive overview of EV/exosome origins, their biological functions, the approaches for their isolation and characterization, and their therapeutic potential. Full article

Background/Objectives: Antisense oligonucleotides (ASOs) hold great therapeutic potential due to their precise ability to modulate gene expression, particularly for treating genetic and neurological disorders. However, effective delivery of ASOs remains a major challenge. While most recent research focused on lipid nanoparticles (LNPs) as ASO carriers, alternative formulations, preparation methods and lipid compositions on delivery optimization are not fully explored. In this study, we investigated two types of formulations, lipoplexes (LPXes) and LNPs, prepared using lysine-type cationic lipids, K3C14 or K3C16, selected from a lysine-type lipid mini-library for their superior formulation stability and distinct cellular entry mechanisms. Methods: The physicochemical properties of the formulations were characterized using dynamic light scattering. Cytotoxicity was evaluated in spleen and liver cell lines. LPXes and LNPs were assessed for ASO delivery efficiency using an engineered HEK293 split-luciferase cell line, while immune response was evaluated in human peripheral blood mononuclear cells. Cryogenic electron microscopy (Cryo-EM) images were captured for structural analysis. Results: Lysine-type lipid mini-library screening identified lipids with either a hydrocarbon spacer K3 or C14 fatty acid tail exhibiting great stability and safety. Among the tested LPX and LNP formulations, the K3C16 lipoplex demonstrated ASO delivery efficiency and immune responses comparable to the benchmark SpikeVax LNP formulation. Notably, Cryo-EM imaging revealed novel structures that have not been reported previously; the K3C14 lipoplex formed a rouleaux-like structure, whereas the K3C16 lipoplex exhibited a lipid nanosheet-like structure, distinct from the conventional LNP structure. Conclusions: These results highlight the potential of an unconventional type of lipid assembly for efficient ASO delivery. Full article

With the advancements in instrumentation, image processing algorithms, and computational capabilities, single-particle cryo-electron microscopy (cryo-EM) has achieved atomic resolution in determining the 3D structures of viruses. The virus structures play a crucial role in studying their biological function and advancing the development of antiviral vaccines and treatments. Despite the effectiveness of artificial intelligence (AI) in general image processing, its development for identifying and extracting virus particles from cryo-EM micrographs has been hindered by the lack of manually labeled high-quality datasets. To fill the gap, we introduce CryoVirusDB, a labeled dataset containing the coordinates of expert-picked virus particles in cryo-EM micrographs. CryoVirusDB comprises 9941 micrographs from nine datasets representing seven distinct non-enveloped viruses exhibiting icosahedral or pseudo-icosahedral symmetry, along with coordinates of 339,398 labeled virus particles. It can be used to train and test AI and machine learning (e.g., deep learning) methods to accurately identify virus particles in cryo-EM micrographs for building atomic 3D structural models for viruses. Full article

Modern advances in artificial intelligence have accelerated the development of computational tools for protein–ligand structure prediction, yet their real-world performance remains uneven across receptor classes and ligand chemotypes. Recently published cryo-EM structures of several different psychedelics bound to the serotonin 5HT_2A receptor provide a unique opportunity to explore how modern AI-based modeling performs in a pharmacologically important GPCR system. Here, we compare three major approaches: AI-based protein–ligand cofolding (Boltz-2), a leading AI-driven docking module (Uni-Mol Docking v2), and a widely used classical physics-based docking pipeline (AutoDock Vina) across a series of tryptamine and phenethylamine psychedelics. Predicted binding poses were comparatively assessed through structural alignment with these newly available cryo-EM complexes. Additionally, calcium-mobilization assays were performed to provide a coarse functional readout for comparison with computationally predicted binding affinities. This study integrates methodological review with exploratory benchmarking to illustrate how different modeling paradigms behave on a shared receptor–ligand test set. Our results highlight substantial variation between modeling strategies, with AI-based cofolding often producing global binding orientations more closely resembling experimental structures, and classical docking showing greater variability across ligands, while still outperforming AI-driven docking on average. These observations underscore both the growing utility and current limitations of AI-assisted structure prediction in serotonergic drug discovery, and emphasize the importance of careful, experimentally anchored evaluation as such tools continue to advance. Full article

Hyperuricemia affects approximately 20% of the global adult population and serves as the primary etiological factor for gout. Glucose transporter 9 (GLUT9) plays a critical role in renal urate reabsorption, representing a promising therapeutic target for hyperuricemia treatment. This study employed an integrated computational and experimental approach to identify novel flavonoid-based putative GLUT9 binders, combining molecular docking, molecular dynamics (MD) simulations, ADMET prediction, antioxidant evaluation, and density functional theory (DFT) calculations. Eight structurally diverse flavonoids were docked against the human GLUT9 cryo-EM structure, and antioxidant activities were assessed using DPPH, ABTS, and FRAP assays. All tested flavonoids exhibited favorable binding affinities ranging from −7.67 to −9.10 kcal/mol. Epigallocatechin gallate (EGCG) demonstrated the highest binding affinity (−9.10 kcal/mol) with an extensive hydrogen bonding network, while chrysin exhibited the second-highest affinity (−8.35 kcal/mol) with favorable drug-like properties. MD simulations over 100 ns confirmed the structural stability of the complexes. EGCG displayed exceptional antioxidant capacity (DPPH IC₅₀ = 3.28 μM) superior to ascorbic acid, whereas chrysin showed lower radical scavenging activity despite favorable GLUT9 binding. DFT calculations revealed that higher HOMO energies correlated with enhanced antioxidant activity. These findings suggest that EGCG and chrysin exhibit favorable GLUT9 binding profiles, warranting further functional and pharmacokinetic optimization. Full article

Cryo-electron microscopy (cryo-EM) has revolutionized structural virology, enabling routine structure determination at 2–4 Å resolution, with exceptional cases reaching 1.56 Å. The structural diversity of viruses across vertebrate, plant, and insect hosts provides fundamental insights into infection mechanisms, host–pathogen coevolution, and therapeutic target identification. However, analysis of Electron Microscopy Data Bank entries reveals notable disparities in structural coverage: among 11,717 eukaryotic virus structures (excluding bacteriophages), vertebrate viruses constitute 97.6% (n = 11,432) of deposited entries, while plant viruses (1.0%; n = 117) and insect viruses (1.4%; n = 168) remain significantly underrepresented. This bias stems from distinct technical barriers including size limitations for giant viruses exceeding 200 nm, the loss of asymmetric information during symmetry-imposed processing, and the morphological complexity of filamentous and pleomorphic viruses. Each barrier has driven the development of specialized methodological solutions: block-based local refinement overcomes through-focus variations in giant viruses, cryo-electron tomography (cryo-ET) validates and reveals asymmetric features lost in symmetrized reconstructions, and subtomogram averaging enables structural analysis of pleomorphic assemblies. This review synthesizes recent methodological advances, critically evaluates their capacity to address specific technical barriers, and proposes strategies for expanding structural investigations across underrepresented host systems to achieve comprehensive understanding of viral structural biology. Full article