Search Results (532)

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing. Full article

Amyloids are protein-based biomaterials composed of fibrils with cross-β cores. Previously only associated with degenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and diabetes, amyloids remain active and functional both in vivo and in vitro conditions, enabling a variety of applications in medicine, nanotechnology, and biotechnology. This review aims to review the most advanced methods for amyloid fibril structural studies, with special attention on amyloid thin films. Selected advances of biomedical and biotechnological relevance will be outlined, and perspectives for future studies in the context of ongoing methodological progress will be discussed. Full article

ABCG2 is a crucial ATP-binding cassette (ABC) transporter involved in multidrug resistance and essential physiological and pharmacological processes. In recent years, multiple ABCG2 structures have been resolved using cryo-electron microscopy (cryo-EM), providing significant insights into its conformational states during its transport cycle. However, even more than 25 years after its description, a high-resolution X-ray crystallographic structure is still unavailable, limiting the understanding of its dynamic transitions, as well as leaving aspects of the transport cycle unresolved and open to discussion. Given the complexity of ABCG2, a multidisciplinary approach is essential in order to fully elucidate its mechanism. This review compiles recent advances in ABCG2 structural biology, highlights unresolved controversies, and explores future directions to bridge the gap between structure and function. Moving forward, integrating multiple structural and functional approaches will be key to uncovering the intricate workings of this enigmatic transporter. In particular, detailed structural insights will be crucial to identifying new ABCG2 substrates and designing selective inhibitors, with important implications for therapeutic development. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

Extracellular vesicles (EVs) are key mediators of intercellular communication and play a vital role in cancer progression. While EVs in the blood are well-studied, those in local body fluids, such as gastric juice (GJ), remain underinvestigated. Previously, we first characterized GJ-derived EVs and demonstrated their potential for gastric cancer (GC) screening. Here, we conducted a detailed morphological analysis of GJ-EVs using cryo-electron microscopy, identifying both typical and atypical EV subtypes, and categorized their relative abundances. A subsequent comparison of the size distribution of GJ-derived EVs by nanoparticle tracking analysis revealed significant differences between samples obtained from GC patients (n = 40) and healthy subjects (n = 25). Additionally, the mean EV sizes differed significantly according to the presence of the tetraspanin protein CD9. Furthermore, the ratio of CD9-positive to CD9-negative EV samples differed between cancer patients and healthy donors. These data suggest that GJ contains distinct subpopulations of EVs that vary in size and CD9 expression, as well as EVs with certain types of atypical morphology. The identification of discrepancies in EV size and the presence of CD9 between GJ from cancer patients and healthy individuals offers potential avenues for the identification of new GC markers. Full article

Anesthesia is a cornerstone of modern medicine, enabling surgery, pain management, and critical care. Despite its widespread use, the precise molecular mechanisms of anesthetic action remain incompletely understood. Recent advancements in structural biology, including cryo-electron microscopy (Cryo-EM), X-ray crystallography, and computational modeling, have provided high-resolution insights into anesthetic–target interactions. This review examines key molecular targets, including GABA_A receptors, NMDA receptors, two-pore-domain potassium (K2P) channels (e.g., TREK-1), and voltage-gated sodium (Nav) channels. General anesthetics modulate GABA_A and NMDA receptors, affecting inhibitory and excitatory neurotransmission, while local anesthetics primarily block Nav channels, preventing action potential propagation. Structural studies have elucidated anesthetic binding sites and gating mechanisms, providing a foundation for drug optimization. Advances in computational drug design and AI-assisted modeling have accelerated the development of safer, more selective anesthetics, paving the way for precision anesthesia. Future research aims to develop receptor-subtype-specific anesthetics, Nav1.7-selective local anesthetics, and investigate the neural mechanisms of anesthesia-induced unconsciousness and postoperative cognitive dysfunction (POCD). By integrating structural biology, AI-driven drug discovery, and neuroscience, anesthesia research is evolving toward safer, more effective, and personalized strategies, enhancing clinical outcomes and patient safety. Full article

The solubility of soybean isolate protein (SPI) undergoes significant degradation during storage and transportation. This study investigates the formulation and assessment of SPI-stabilized medium internal phase emulsions (MIPEs) with different solubilities, namely SPI80, SPI70, SPI60, and SPI50, corresponding to solubility levels of about 80%, 70%, 60%, and 50%, respectively. The contact angles of these SPI variants ranged from 79.35 to 86.55 degrees, with SPI60 and SPI50 exhibiting significantly higher values compared to SPI80 and SPI70. All SPI samples were successfully utilized for the preparation of MIPEs. However, as SPI solubility decreases, emulsion stability progressively declines, accompanied by a reduction in the absolute value of zeta potential. Additionally, interfacial protein adsorption in emulsions decreases with decreasing SPI solubility, a trend that is similarly observed in viscosity characteristics, storage modulus (G′), and loss modulus (G″). Confocal laser scanning microscopy (CLSM) and cryo-scanning electron microscopy (Cryo-SEM) analyses revealed that emulsions exhibit reduced uniformity and a less interconnected microstructural network as SPI solubility decreases. These findings provide a theoretical foundation for utilizing low-solubility SPI in MIPEs applications. Full article

Biological control of plant diseases is important for crop production. Botrytis cinerea and Fusarium graminearum are two common pathogenic fungi which result in great harm to crop production, processing, and storage of foodstuffs. Yeasts have unique advantages to be the focus of biological control of plant diseases through multiple mechanisms, including producing volatile organic compounds (VOCs) with inhibitory effect. However, the discontinuous display of inhibitory effect by yeast VOCs on pathogenic fungi is restricted by the conventional confrontation method, and the inhibitory mechanisms are unclear. We developed a new method to detect the inhibitory effect of Saccharomyces cerevisiae (yeast) VOCs on B. cinerea and F. graminearum. Our results showed that the yeast VOCs inhibited the growth and development of B. cinerea and F. graminearum and the strength of the inhibitory effect is positively related to the yeast inoculation amount. We confirmed the inhibition effect of ethyl acetic, one of the main yeast VOCs, on both pathogenic fungi. We further found that the deletion or overexpression of the ethyl acetic synthesis-related genes (ATF1 and/or ATF2) did not change the inhibitory effect much. The overexpression of ATF1 changed the main composition of VOCs. One of the changed VOCs, phenethyl acetic, even had stronger inhibitory effect than ethyl acetic on F. graminearum when they were added alone. These results suggest that the inhibitory effect of yeast VOCs on pathogenic fungi is a complex module. The lonely added individual component of VOCs may inhibit the growth and development of pathogenic fungi, while the partial alternation of VOC composition through gene modification may not be enough to change the total inhibitory effect. Full article

In this study, novel biodegradable polycaprolactone (PCL)-based composites for sustainable packaging applications were developed by adding surface-treated wood fibers (WFs). Specifically, the WFs were treated with citric acid (CA) to improve the fiber/matrix adhesion and then melt compounded with a PCL matrix. The presence of an absorption peak at 1720 cm⁻¹ in the Fourier transform infrared (FTIR) spectra of CA-treated WFs, coupled with the increase in the storage modulus and complex viscosity in the rheological analysis, confirmed the occurrence of an esterification reaction between CA and WFs, with a consequent increase in interfacial interactions with the PCL matrix. Scanning electron microscopy (SEM) of the cryo-fractured surface of the composites highlighted that PCL was able to efficiently wet the fibers after the CA treatment, with limited fiber pull-out. Quasi-static tensile tests showed that the composites reinforced with CA-treated wood fibers exhibited a significant increase in yield strength (about 30% with a WF amount of 10% at 0 °C) and also a slight improvement in the VICAT softening temperature (about 6 °C with respect to neat PCL). Water absorption tests showed reduced water uptake in CA-treated composites, consistent with the reduced hydrophilicity confirmed by higher water contact angle values. Therefore, the results obtained in this work highlighted the potential of CA-treated WFs as reinforcement for PCL composites, contributing to the development of eco-sustainable and high-performance packaging materials. Full article

Voltage-gated potassium (Kv) channels are e ssential for shaping action potentials and rely on anionic lipids for proper gating, yet the mechanistic basis of lipid–channel interactions remains unclear. Cryo-electron microscopy studies suggest that, in the down state, arginine residues of the voltage sensor draw lipid phosphates upward, leading to a local membrane thinning of ~5 Å—an effect absent in the open state. To test whether membrane thickness directly affects voltage sensor function, we reconstituted Kv channels from Aeropyrum pernix (KvAP) into planar lipid bilayers containing photoswitchable lipids. Upon blue light illumination, the membrane thickened, and KvAP activity increased; UV light reversed both effects. Our findings indicate that membrane thickening weakens the interaction between lipid phosphates and voltage-sensing arginines in the down state, lowering the energy barrier for the transition to the up state and thereby promoting channel opening. This non-genetic, membrane-mediated approach provides a new strategy to control ion channel activity using light and establishes a direct, reversible link between membrane mechanics and voltage sensing, with potential applications in the remote control of neuronal excitability. Full article

Health benefits associated with hyaluronic acid, along with its properties such as water-binding capacity and antimicrobial activity, suggest that incorporating it into meat systems could provide a basis for formulating functional meat products. This study aimed to evaluate the properties of myofibrillar protein gels and emulsions with varying concentrations of hyaluronic acid. The results indicate that increasing the hyaluronic acid concentration (0.008% to 0.04%) does not significantly affect the cooking loss, while a concentration of 0.08% enhances cooking loss. This, in turn, increased gel hardness, while the water-holding capacity remains unaffected. Cryo-scanning electron microscopy (Cryo-SEM) images revealed a partial disruption of the gel structure, with rising hyaluronic concentrations. In pork myofibrillar protein emulsions, smaller droplets and higher stability were observed after HA incorporation. Samples containing hyaluronic acid were more viscous and exhibited shear-thinning properties. Overall, the hyaluronic acid used in this study improved emulsion properties, whereas the gel structure was compromised. Full article

Type IV pili (T4Ps) are multifunctional surface fibers essential for bacterial motility, adhesion, and virulence, found across Gram-negative and Gram-positive bacteria and archaea. Detailed descriptions of T4P structural biology are allowing progress in understanding T4P biogenesis. Secretins, large outer membrane channels, are crucial for T4P extrusion in Gram-negative bacteria. Using cryo-EM and AlphaFold, we modeled the structure of BfpB, the secretin of the Bundle-Forming Pilus (BFP) of enteropathogenic Escherichia coli. BfpB exhibits a unique 17-fold symmetry, correlating with the thicker BFP filaments, and diverging from the 12–15 subunits typical of T4P, type 2 secretion (T2S), and type 3 secretion (T3S) systems. Additionally, we identified an extended β-hairpin loop in the N3 domain, resembling features of distantly related T3SS secretins, and an N-terminal helix where a C-terminal S-domain is seen in some T2S and T3S secretins. These findings reveal evolutionary parallels and structural adaptations in secretins, highlighting the link between oligomerization and pilus structure. This work advances our understanding of T4P biogenesis, secretin evolution, and bacterial secretion systems, offering insights into pathogenic diversity and future research directions. Full article

Understanding the formation, structural evolution, and response of water ice at the nanoscale is essential for advancing research in fields such as cryo-electron microscopy and atmospheric science. In this work, we used environmental transmission electron microscopy (ETEM) to investigate the formation of water ice nanostructures and the etching and charging behaviors of ice under fast electron irradiation. These nanostructures were observed to be suspended along the edges of copper grids and supported on few-layer graphene. We varied growth parameters (temperature and time) to produce water ice nanostructures characterized by uniform thickness and enhanced crystallinity. Moreover, we examined the lithographic patterning of water ice at the copper grid edges and its localized etching effects on graphene substrates. Off-axis electron holography experiments further revealed charging phenomena induced by electron beam irradiation, enabling a quantitative assessment of charge accumulation on the ice nanostructures. Our findings demonstrate the controlled growth of ice thin films under high vacuum conditions at cryogenic temperatures, elucidate the etching behavior and charging phenomena of water ice under rapid electron beam irradiation. Full article

The rapid regulatory mechanism of light-induced state transitions (STs) in oxygenic photosynthesis is particularly appealing for membrane-based applications. This interest stems from the unique ability of the thylakoid membrane protein cytochrome b₆f (cytb₆f) to increase or decrease its hydrophobic thickness (d_P) in parallel with the reduction or oxidation of the PQ pool induced by changes in light quality. This property appears to be the long-sought biophysical driver behind the reorganizations of membrane proteins during STs. This study decisively advances the hydrophobic mismatch (HMM) model for cytb₆f-driven STs by thoroughly analyzing thirteen X-ray crystal and eight cryo-electron microscopy cytb₆f structures. It uncovers the lipid nanoenvironments that cytb₆f, with different hydrophobic thicknesses, selectively attracts. Under optimal, stationary conditions for photosynthesis in low light, when there is hydrophobic matching between the hydrophobic thicknesses of cytb₆f d_P and that of the bulk thylakoid lipid phase d_L, d_P = d_L, cytb₆f predominantly binds to anionic lipids—several phosphatidylglycerol (PG) molecules and one sulfoquinovosyldiacylglycerol (SQDG) molecule. Upon the induction of the transition to State 2, when d_P increases and induces a positive HMM (d_P > d_L), the neutral, non-bilayer-forming lipid monogalactosyldiacylglycerol (MGDG) replaces some of the bound PGs. Upon the induction of the transition to State 1, when d_P decreases and induces a negative HMM (d_P < d_L), PGs and SQDG detach from their binding sites, and two neutral, bilayer-forming lipids such as digalactosyldiacylglycerol (DGDG) occupy two sites. Additionally, this research uncovers two lipid-mediated signaling pathways from Chla to the center of flexibility, the Phe/Tyr124^fg-loop-suIV residue—one of which involves β-carotene. This study identifies two novel types of lipid raft-like nanodomains that are devoid of typical components, such as sphingomyelin and cholesterol. These findings firmly validate the HMM model and underscore the STs as the first recognized functional process that fully utilizes the unique and evolutionarily conserved composition of just four thylakoid lipid classes. This research contributes to our understanding of membrane dynamics in general and STs in particular. It introduces a novel and simple approach for reversible protein reorganization driven purely by biophysical mechanisms, with promising implications for various membrane-based applications. Full article