Search Results (579)

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

HIV-1 entry into host cells culminates in integration of the reverse transcribed double-stranded viral DNA into host genes. Several lines of evidence suggest that intact, or nearly intact, HIV-1 cores—large, ~60 nm-wide structures—pass through the nuclear pore complex (NPC), and that this passage is associated with pore remodeling. Cryo-electron tomography studies support the dynamic nature of NPCs and their regulation by cytoskeleton and ATP-dependent processes. To explore NPC remodeling, we used super-resolution Stochastic Optical Reconstruction Microscopy (STORM) of U2OS cells endogenously expressing nucleoporin 96 tagged with SNAP. Single-molecule localization imaging and computational averaging resolved 8-fold symmetric nuclear pores with an average radius of ~51 nm. Depletion of cellular ATP using sodium azide or antimycin A, previously reported to reduce the size of yeast NPCs, did not significantly alter the nuclear pore radius in U2OS cells. Similarly, stressing the nuclear envelope by hypotonic or hypertonic conditions failed to induce detectable expansion or contraction of NPCs. These results indicate that the NPCs in U2OS cells do not respond to ATP depletion nor mechanical stresses on changes in pore morphology that can be resolved by STORM. Since these cells are infectable by HIV-1, we surmise that direct multivalent interactions between HIV-1 capsid and phenylalanine-glycine nucleoporins lining the pore’s interior drive the core penetration into the nucleus and the associated changes in the pore structure. Full article

Alcohol-associated hepatitis (AH) is the most severe clinical manifestation of alcohol-associated liver disease. Corticosteroids are the only disease-specific therapy shown to improve short-term survival. Currently, no non-invasive markers are available to predict patient response to corticosteroids or long-term survival in AH. This study investigates whether surface antigens on plasma extracellular vesicles (EVs), key mediators of intercellular communication, can reflect the underlying immune dysregulation in AH and serve as prognostic markers. Patients with AH were prospectively enrolled between 2020 and 2024. Blood samples were collected before corticosteroid initiation during the first 24 h of hospitalization. EVs were characterized using nanoparticle tracking analysis, cryo-electron microscopy, and flow cytometry. Interleukin-6 (IL-6), soluble (s)CD62p, Circulating Vascular Cell Adhesion Molecule-1 (sVCAM), tumor necrosis factor receptor superfamily member 1 (TNRFS1a), and Intercellular Adhesion Molecule 1 (ICAM-1) were quantified by ELISA. Key outcome variables included response to corticosteroids and mortality. A total of 46 patients with AH and 28 healthy donors (HD) were included. EV concentration was significantly higher in AH patients than in HD (9.3 × 10¹¹ [IQR 4–24] versus 2.4 × 10¹¹ [IQR 2–4], p = 0.03). Specific EV antigens were associated with key clinical outcomes: CD20 and CD2 levels differed between patients with or without infections (bacterial, viral, and fungal) developed during hospitalization; CD40 and CD146 were elevated in patients who developed acute kidney injury. EVs enriched in monocyte (CD14) and T-reg (CD25) markers were associated with plasma IL-6 levels, while endothelial markers CD105 and CD146 correlated with sVCAM and sCD62p. EVs enriched in platelet (CD49e) and endothelial (CD31) markers were associated with corticosteroid response, whereas EVs enriched with endothelial (CD105 and CD146) and B lymphocyte (CD19) markers were associated with mortality. Overall, EVs enriched in endothelial and monocyte markers may represent a candidate non-invasive tool for predicting corticosteroid response and mortality in AH, aiding risk stratification and early identification of non-responders for timely transplant evaluation. Full article

It has been known for decades that eukaryotic cellular mRNAs are frequently translated by multiple ribosomes organized into polysomes of diverse topology, including circular arrangements. The closed-loop model, in which the 5′ cap and 3′ poly(A) tail are bridged by initiation factors, provided a mechanistic basis for mRNA circularization and suggested that the spatial proximity of termini facilitates ribosome recycling. Various biochemical, structural, and imaging approaches—including electron microscopy, atomic force microscopy, cryo-electron tomography, and single-molecule fluorescence—have since demonstrated that polysomes indeed adopt compact and heterogeneous conformations, with circular assemblies representing a significant fraction. Although direct visualization of ribosome recycling remains technically challenging, ribosome turnover experiments, kinetic analyses and modeling support the concept of closed-loop-assisted reinitiation (CLAR), whereby terminating ribosomes are re-utilized to sustain translation efficiency. Together, the findings suggest that mRNA circularization is a dynamic and regulated state that enhances protein synthesis under specific conditions, while linear or modular polysome architectures may dominate in others. Understanding the balance between these modes of translation remains central to elucidating the interplay between mRNA topology, ribosome dynamics, and translational control. Full article

Determining RNA secondary structures is a fundamental challenge in computational biology and molecular sensing. Experimental techniques such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy can reveal RNA structures with atomic precision, but their high cost and time consuming nature limit large-scale applications. To address this issue, we introduce the Structure-Sensing Nucleotide Attention Learning framework (NTFold), a virtual sensing framework based on deep learning for accurate RNA secondary structure prediction. NTFold integrates a Nucleotide Attention Module (NAM) to explicitly model dependencies among nucleotides, thereby capturing fine-grained sequence correlations. The resulting correlation map is subsequently refined by a Structural Refinement Module (SRM), which preserves hierarchical spatial information and enforces structural consistency. Through this two stage learning paradigm, NTFold produces high-precision contact maps that enable reliable RNA secondary structure reconstruction. Extensive experiments demonstrate that NTFold outperforms existing deep learning-based predictors, highlighting its capability to learn both local and global nucleotide interactions in an sensor inspired manner. This study provides a new direction for integrating attention driven correlation modeling with structure-sensing refinement toward efficient and scalable RNA structural sensing. Full article

Lipid raft-associated gangliosides facilitate the early stages of SARS-CoV-2 entry by triggering the exposure of the receptor-binding domain (RBD) within the trimeric spike protein, which is initially sequestered. A broad range of in silico, cryoelectron microscopy and physicochemical approaches indicate that the RBD becomes accessible after a ganglioside-induced conformational rearrangement originating in the N-terminal domain (NTD) of one protomer and propagating to the neighboring RBD. We previously identified a triad of amino acids, Q134-F135-N137, as a strictly conserved element on the NTD. In the present review, we integrate a series of structural and experimental data revealing that this triad may act as a conformational transducer connected to a chain of residues that are capable of transmitting an internal conformational wave within the NTD. This wave is generated at the triad level after physical interactions with lipid raft gangliosides of the host cell membrane. It propagates inside the NTD and collides with the RBD of a neighboring protomer, triggering its unmasking. We also identify a chain of aromatic residues that are capable of controlling electron transfer through the NTD, leading us to hypothesize the existence of a dual conformational/quantum wave. In conclusion, the complete conservation of the Q134-F135-N137 triad despite six years of extensive NTD remodeling underscores its critical role in the viral life cycle. This triad represents a potential Achilles’ heel within the hyper-variable NTD, offering a stable target for therapeutic or vaccinal interventions to disrupt the conformational wave and prevent infection. These possibilities are discussed. Full article

The function of the highly conserved GTPase LepA, a homolog of elongation factor EF-G, remains unknown in translation. However, there is biochemical data that it implicates in the 30S ribosomal subunit biogenesis. Here, using cryo-electron microscopy, we characterized 30S subunits isolated from an Escherichia coli strain with a deleted lepA gene. The cryo-EM maps for ∆lepA 30S particles were divided into classes corresponding to consecutive assembly intermediates: from particles characterized by unformed helices h44/h45 of the central decoding center (CDR) and highly flexible head, through intermediates with a distorted CDR and a partial stabilization of the head, to near-mature 30S subunits with correctly docked h44 in the CDR, accessible 3′ end of 16S rRNA for translation but significant flexibility in head domain. Cryo-EM analysis of ΔlepA 30S intermediates revealed that they predominantly proceed to nearly mature functional state and exhibit suboptimal flexibility in the head domain. This finding suggests that LepA likely contributes to the final proper stabilization of the 3′ domain of the 30S subunit during ribosome assembly. Full article

The National Institute of Standards and Technology (NIST) is developing analytical methods to characterize extracellular vesicles (EVs) to support the urgent need for standardized EV reference materials (RMs). This study used orthogonal techniques, cryogenic electron microscopy (Cryo-EM), particle tracking analysis (PTA), asymmetrical flow field-flow fractionation (AF⁴), and microfluidic resistive pulse sensing (MRPS), to evaluate particle size distributions (PSDs) and particle number concentrations (PNCs) of human mesenchymal stem cells (MSCs) and LNCaP prostate cancer cell EVs. Proteomic profiles were assessed by mass spectrometry (MS), and microRNA (miRNA) content of LNCaP EVs was evaluated by small RNA-seq at two independent laboratories. A commercial green fluorescent protein exosome served as a control, except in Cryo-EM, proteomic, and miRNA analyses. Cryo-EM, regarded as the gold standard for morphological resolution, served as PSD reference. PSDs from all methods skewed larger than Cryo-EM, with MRPS closest, AF⁴ most divergent, and PTA intermediate with broader distributions. All techniques reported broad PSDs (30 nm to >350 nm) with PNCs decreasing with increasing particle size, except for AF⁴. Quantitative discrepancies in PNCs reached up to two orders of magnitude across methods and cell sources. MS identified global and EV-specific proteins, including syntenin-1 and tetraspanins CD9, CD63, and CD81. RNA-seq revealed notable inter-laboratory variation. These findings highlight the variability across measurement platforms and emphasize the need for reproducible methods to support NIST’s mission of developing reliable EV reference materials. Full article

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen associated with healthcare-related infections and is of particular concern due to its high level of antibiotic resistance and its ability to form biofilms. The global emergence of carbapenem-resistant A. baumannii highlights the urgent need for alternative therapeutic strategies. This study investigated the antibacterial and antibiofilm activities of two scorpion venom-derived peptides, pantinin-1 and pantinin-2, against a reference strain and a clinical isolate of A. baumannii. We found that both peptides, in the non-cytotoxic concentration range, have strong bactericidal activity, showing a minimum inhibitory concentration (MIC) of 6.25 μM and 12.5 μM for pantinin 1 and 2, respectively. Scanning electron microscopy (SEM) analysis showed that the peptides cause extensive damage to the bacterial membrane. Furthermore, both peptides showed potent antibiofilm activity, inhibiting adhesion and maturation, arresting biofilm expansion, and reducing the expression of key biofilm-associated genes (bap, pgaA, and smpA). Altogether, these findings indicate that pantinin-1 and pantinin-2 act through a dual mechanism, combining bactericidal and antivirulence activities. Their strong efficacy at low micromolar concentrations, together with low cytotoxicity, underscores their potential as innovative therapeutic candidates against infections caused by carbapenem-resistant, biofilm-forming A. baumannii. Full article

Volume electron microscopy (Volume-EM) has transformed structural cell biology by enabling nanometre-resolution imaging across cellular and tissue scales. Serial-section TEM, Serial Block-Face Scanning Electron Microscopy (SBF-SEM), Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and multi-beam SEM now routinely generate terabyte-scale volumes that capture organelles, synapses and neural circuits in three dimensions, while cryogenic Volume-EM extends this landscape by preserving vitrified, fully hydrated specimens in a near-native state. Together, these room-temperature and cryogenic modalities define a continuum of approaches that trade off volume, resolution, throughput and structural fidelity, and increasingly interface with correlative light microscopy and cryo-electron tomography. In parallel, advances in computation have turned Volume-EM into a data-intensive discipline. Multistage preprocessing pipelines for alignment, denoising, stitching and intensity normalisation feed into automated segmentation frameworks that combine convolutional neural networks, affinity-based supervoxel agglomeration, flood-filling networks and, more recently, diffusion-based generative restoration. Weakly supervised and self-supervised learning, multi-task objectives and human-AI co-training mitigate the scarcity of dense ground truth, while distributed storage and streaming inference architectures support segmentation and proofreading at the terascale and beyond. Open resources such as COSEM, MICRONS, OpenOrganelle and EMPIAR provide benchmark datasets, interoperable file formats and reference workflows that anchor method development and cross-laboratory comparison. In this review, we first outline the physical principles and imaging modes of conventional and cryogenic Volume-EM, then describe current best practices in data acquisition and preprocessing, and finally survey the emerging ecosystem of AI-driven segmentation and analysis. We highlight how cryo-Volume-EM expands the field towards native-state structural biology, and how multimodal integration with light microscopy, cryo-electron tomography (cryo-ET) and spatial omics is pushing Volume-EM from descriptive imaging towards predictive, mechanistic, cross-scale models of cell physiology, disease ultrastructure and neural circuit function. Full article

Modern olive breeding points to a plant model characterized by low vigour, high productivity, and resistance to biotic and abiotic stresses, all traits required by the intensive and superhigh-density (SHD) systems of olive tree growing. The Italian Don Carlo and FS-17 Favolosa stand out among the new cultivars that are being tested. They were obtained not by breeding but by mass selection from two seedling populations of the Frantoio cultivar (maternal parent). Here, a multidisciplinary approach was used to determine the paternal parent of Don Carlo and FS-17, and then to investigate the inheritance of interesting traits such as fruit cell dimensions and oil content in these cultivars. Microsatellites were applied in phylogeny and kinship analyses, along with two functional markers previously developed on OeACP1 and OeACP2 genes. Ascolana Tenera cultivar was identified as the paternal parent of both new cultivars. This result was also supported by the analysis of the self-incompatibility group of the new cultivars and their most likely paternal parents. Light and electron microscopy [Cryo Scanning Electronic Microscopy (CRYO-SEM), Electronic Scanning Microscopy (E-SEM), and Transmission Electron Microscope (TEM)] techniques were used to analyze the fruit development concerning oil accumulation. Significant differences in cuticle thickness, size and shape of mesocarp and exocarp cells, and oil content were detected among cultivars. Our results suggested that the rearrangement of the traits studied led to an improved progeny compared to the parents. FS-17 exhibited an oil storage efficiency higher than Frantoio. Don Carlo showed fruit traits and oil content almost intermediate between the parents, making it a dual-purpose cultivar. Full article

Evidence increasingly indicates that synaptic vesicle dysfunction emerges early in Parkinson’s disease (PD), preceding overt dopaminergic neuron loss rather than arising solely as a downstream consequence of neurodegeneration. α-Synuclein (αSyn), a presynaptic protein that regulates vesicle clustering, trafficking, and neurotransmitter release under physiological conditions, exhibits dose-, conformation-, and context-dependent actions that distinguish its normal regulatory roles from pathological effects observed in disease models. This narrative review synthesizes findings from a structured search of PubMed and Scopus, with emphasis on α-syn-knockout (αSynKO) and BAC transgenic (αSynBAC) mouse models, which do not recapitulate the full human PD trajectory but provide complementary insights into αSyn physiological function and dosage-sensitive vulnerability. Priority was given to studies integrating ultrastructural approaches—such as cryo-electron tomography, high-pressure freezing/freeze-substitution TEM, and super-resolution microscopy—with proteomic and lipidomic analyses. Across these methodologies, several convergent presynaptic alterations are consistently observed. In vivo and ex vivo studies associate αSyn perturbation with impaired vesicle acidification, consistent with altered expression or composition of vacuolar-type H⁺-ATPase subunits. Lipidomic analyses reveal age- and genotype-dependent remodeling of vesicle membrane lipids, particularly curvature- and charge-sensitive phospholipids, which may destabilize αSyn–membrane interactions. Complementary biochemical and cell-based studies support disruption of SNARE complex assembly and nanoscale release-site organization, while ultrastructural analyses demonstrate reduced vesicle docking, altered active zone geometry, and vesicle pool disorganization, collectively indicating compromised presynaptic efficiency. These findings support a synapse-centered framework in which presynaptic dysfunction represents an early and mechanistically relevant feature of PD. Rather than advocating αSyn elimination, emerging therapeutic concepts emphasize preservation of physiological vesicle function—through modulation of vesicle acidification, SNARE interactions, or membrane lipid homeostasis. Although such strategies remain exploratory, they identify the presynaptic terminal as a potential window for early intervention aimed at maintaining synaptic resilience and delaying functional decline in PD. Full article

A quaternized and phenyl-functionalized hyperbranched PEI-based sponge (S_HPEI-QP) was successfully prepared, and its adsorption performance was investigated to evaluate its potential for removing the anionic non-steroidal anti-inflammatory drug (ibuprofen (IBU)). We reported that the synthesis of polyethyleneimine-based sponges was achieved through cryo-polymerization using 1,4-butanediol diglycidyl ether (BDDE) as the crosslinking agent. Subsequent functionalization with resorcinol diglycidyl ether (RDGE) and trimethylamine introduced quaternary ammonium cations, imparting strong basicity and hydrophilicity, as well as phenyl groups, conferring hydrophobic characteristics, respectively. The aforementioned sponge material, S_HPE-QPI, primarily facilitates the efficient adsorption of IBU in aqueous solutions through the anion exchange properties of quaternary ammonium groups and the π-π interactions associated with oxygen-activated benzene rings. Various characterizations, such as scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and specific surface area determination method (BET), confirmed the successful synthesis of the bifunctionalized S_HPEI-QP adsorbent. This adsorbent features a porous structure (specific surface area of 77.2 m² g⁻¹ and pore size distribution of 25–100 nm) and an isoelectric point (pH_pzc) of 9.38. The adsorption kinetics of the adsorbent for IBU were extremely rapid and conformed to a pseudo-second-order kinetic model, and the adsorption isotherm aligned with the Langmuir isotherm model. Noteworthily, S_HPEI-QP demonstrated an exceptionally high adsorption capacity for IBU, achieving a maximum uptake of 905.73 mg g⁻¹ at pH 7.0, which surpassed that of most of the previous reported adsorbents. Moreover, the sponge material can be chemically regenerated. After eight cycles of use, the adsorption efficiency decreased by only 4%. These findings suggest that the synthesized dendritic anion exchange adsorbent represents a promising candidate for the removal of IBU from contaminated water sources. Full article

One of the most important events in the pathogenesis of Parkinson’s disease and related disorders is the formation of abnormal fibrils via the aggregation of α-synuclein (α-syn) with β-sheet-rich organization. The use of Cryo-EM has uncovered different polymorphs of the fibrils, each having unique structural interfaces, which has made the design of inhibitors even more challenging. Here, a structure-guided framework incorporating AI-assisted peptide generation was set up with the objective of targeting the conserved β-sheet motifs that are present in various forms of α-syn fibrils. The ProteinMPNN, then, AlphaFold-Multimer, and PepMLM were employed to create short peptides that would interfere with the growth of the fibrils. The two selected candidates, T1 and S1, showed a significant inhibition of α-syn fibrillation, as measured by a decrease in the ThT fluorescence and the generation of either amorphous or fragmented aggregates. The inhibitory potency of the peptides was in line with the predicted interface energies. This research work illustrates that the integration of cryo-EM structural knowledge with the computational design method leads to the quick discovery of the wide-spectrum peptide inhibitors, which is a good strategy for the precision treatment of neurodegenerative diseases. Full article

The application of cryo-electron microscopy (cryo-EM) in membrane protein structural biology has catalyzed unprecedented advances in our understanding of fundamental biological processes and transformed drug discovery paradigms. This review briefly describes the biological achievements enabled using cryo-EM techniques, including single particle analysis (SPA), micro-electron diffraction (microED), and subtomogram averaging (STA), in elucidating the structures and functions of membrane proteins, ion channels, transporters, and viral glycoproteins. We highlight how these structural insights have revealed druggable sites, enabled structure-based drug design, and provided mechanistic understanding of disease processes. Key biological targets include G protein-coupled receptors (GPCRs), ion channels implicated in neurological disorders, respiratory chain complexes, viral entry machinery, and membrane transporters. The integration of cryo-EM with computational drug design has already yielded clinical candidates and approved therapeutics, marking a new era in membrane protein pharmacology. Full article