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Vesna Spasovski,1,2,* Anna Romolo,1,3,* Urška Zagorc,4 Vesna Arrigler,4 Matic Kisovec,5 Apolonija Bedina Zavec,5 Matevž Arko,1 Adrienn Molnár,6,7 Gitta Schlosser,7 Aleš Iglič,3,8 Ksenija Kogej,4 Veronika Kralj-Iglič1 1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; 2Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; 3University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia; 4University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia; 5National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia; 6Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 7MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Faculty of Science, Institute of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 8University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia*These authors contributed equally to this workCorrespondence: Veronika Kralj-Iglič, Email veronika.kralj-iglic@zf.uni-lj.siIntroduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered.Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy, interferometric light microscopy and liquid chromatography–mass spectrometry.Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes.Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.Plain Language Summary: Cells shed into their exterior nanoparticles (here referred to as extracellular vesicles – EVs) that are free to move, reach distant cells and are taken up by them. As they carry bioactive constituents, EVs may have important impact on the recipient cells. The mechanisms of EV formation and mediation can be employed in designing therapeutic, prophylactic and diagnostic methods for various medical issues. EVs can be harvested from biological samples; however, their yield is small,12 and there are potential side effects. Artificial vesicles – liposomes – have high yield; however, in vivo, they can be degraded before reaching the target and their reproducibility is yet insufficient. In order to combine advantages of both types of nanoparticles, we have composed nanohybridosomes (NSHs) from soya lecithin, water and supernatant of isolation of EVs from spruce needle homogenate, visualized them by cryogenic electron microscopy and characterized them with respect to their size, concentration and protein/nucleic acid content. We have applied a recently developed interferometric light microscopy to determine the hydrodynamic radius and the concentration of EVs. We found that the majority of composed particles are nano-sized and that they enclose more than 25% of the incoming volume of liquid, which is considerably more than about 1% that can be harvested by isolation of EVs from spruce needle homogenate by (ultra)centrifugation.Keywords: hybridosomes, liposomes, nanovesicles, extracellular particles, small cellular particles, drug delivery

Extracellular nanoparticles (EPs) are a subject of increasing interest for their biological role as mediators in cell-cell communication; however, their harvesting and assessment from bodily fluids are challenging, as processing can significantly affect samples. With the aim of minimizing processing artifacts, we assessed the number density ( n ) and hydrodynamic diameter ( D h ) of EPs directly in diluted plasma and blood using the following recently developed technique: interferometric light microscopy (ILM). We analyzed 613 blood and plasma samples from human patients with inflammatory bowel disease (IBD), collected in trisodium citrate and ethylenediaminetetraacetic acid (EDTA) anticoagulants, and 163 blood and plasma samples from canine patients with brachycephalic obstructive airway syndrome (BOAS). We found a highly statistically significant correlation between n in the plasma and n in the blood only in the human (i.e., but not canine) blood samples, between the samples with trisodium citrate and EDTA, and between the respective D h for both species (all p < 10 -3 ). In the human plasma, the average < D h > was 139 ± 31 nm; in the human blood, < D h > was 158 ± 11 nm; in the canine plasma, < D h > was 155 ± 32 nm; and in the canine blood, < D h > was 171 ± 33 nm. The differences within species were statistically significant ( p < 10 -2 ), with sufficient statistical power (P > 0.8). For < n >, we found no statistically significant differences between the human plasma and blood samples or between the samples with trisodium citrate and EDTA. Our results prove that measuring n and D h of EPs in minimally processed fresh blood and in diluted fresh plasma by means of ILM is feasible for large populations of samples.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 μm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g . The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (R h ) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In R h , ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by R h of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger R h obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and R h of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum ( P. tricornutum ) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum , mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Unprotected dipeptides are attractive building blocks for environmentally friendly hydrogel biomaterials by virtue of their low-cost and ease of preparation. This work investigates the self-assembling behaviour of the distinct stereoisomers of Ile-Phe and Phe-Ile in phosphate buffered saline (PBS) to form hydrogels, using transmission electron microscopy (TEM), attenuated total reflectance infrared spectroscopy (ATR-IR), circular dichroism (CD), and oscillatory rheometry. Each peptide purity and identity was also confirmed by 1 H- and 13 C-NMR spectroscopy and HPLC-MS. Finally, single-crystal XRD data allowed the key interactions responsible for the supramolecular packing into amphipathic layers or water-channels to be revealed. The presence of the latter in the crystal structure is a distinctive feature of the only gelator of this work that self-organizes into stable hydrogels, with fast kinetics and the highest elastic modulus amongst its structural isomers and stereoisomers., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius ( R h ), intensity of scattered light ( I ), and the shape parameter ( ρ ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the R h (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato ( Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC-MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry-based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.

A major drawback of the IgG capture step is the high cost of the protein A resin. For a better utilization of the resin, a continuous multi-column operation was recently proposed. In this method, accurate detection of leaking IgG is crucial to divert the breakthrough fluid from the waste to the next column and prolong the loading step without product loss. The detection of a breakthrough point as a change in UV absorption is based on a relatively small signal addition of IgGs to the bulk signal of host cell proteins. To achieve specificity, we used a quartz crystal microbalance and immobilized protein A as specific ligand on the sensor surface. We integrated the quartz crystal microbalance sensor in-line after the protein A column for real-time detection of IgGs in the breakthrough fluid. We show that this specific IgG detection in the breakthrough fluid can be more sensitive than with the UV detector. The use of the same product-specific ligand in the affinity column and in the sensor allows simultaneous in-line regeneration of column and sensor in a single step. Such a sensor could support cost-efficient load control during the entire continuous multi-column capture step in downstream processing., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein-membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein-membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery.

Background: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell., Results: As a "proof of concept", we present here the use of purified His-LLO H311A mutant protein and its conjugation on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparticles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins., Conclusions: Endosomal acidification leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.

Aerolysin-like pore-forming proteins are an important family of proteins able to efficiently damage membranes of target cells by forming transmembrane pores. They are characterized by a unique domain organization and mechanism of action that involves extensive conformational rearrangements. Although structures of soluble forms of many different members of this family are well understood, the structures of pores and their mechanism of assembly have been described only recently. The pores are characterized by well-defined β-barrels, which are devoid of any vestibular regions commonly found in other protein pores. Many members of this family are bacterial toxins; therefore, structural details of their transmembrane pores, as well as the mechanism of pore formation, are an important base for future drug design. Stability of pores and other properties, such as specificity for some cell surface molecules, make this family of proteins a useful set of molecular tools for molecular recognition and sensing in cell biology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'., (© 2017 The Author(s).)

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH., Competing Interests: The authors declare no competing financial interests.

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.

Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.

Therapeutic nucleic acids (TNAs) including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) have emerged as promising treatment strategies for a wide variety of diseases, offering the potential to modulate gene expression with a high degree of specificity. These small, synthetic nucleic acid-like molecules provide unique advantages over traditional pharmacological agents, including the ability to target previously "undruggable" genes. Despite this promise, several biological barriers severely limit their clinical efficacy. Upon administration, TNAs primarily enter cells through endocytosis, becoming trapped inside membrane-bound vesicles known as endosomes. Studies estimate that only 1–2% of TNAs successfully escape endosomal compartments to reach the cytosol, and in some cases the nucleus, where they bind target mRNA and exert their therapeutic effect. Endosomal entrapment and inefficient nuclear localization are therefore critical bottlenecks in the therapeutic application of TNAs. This review explores the current understanding of TNA endosomal escape and nuclear transport along with strategies aimed at overcoming these challenges, including the use of endosomal escape agents, peptide-TNA conjugates, non-viral delivery vehicles, and nuclear localization signals. By improving both endosomal escape and nuclear localization, significant advances in TNA-based therapeutics can be realized, ultimately expanding their clinical utility. [ABSTRACT FROM AUTHOR]

Membrane Attack Complex/Perforin (MACPF) and Cholesterol-Dependent Cytolysins (CDC) form the MACPF/CDC superfamily of important effector proteins widespread in nature. MACPFs and CDCs were discovered separately with no sequence similarity at that stage being apparent between the two protein families such that they were not, until recently, considered evolutionary related. The breakthrough showing they are came with recent structural work that also shed light on the molecular mechanism of action of various MACPF proteins. Similarity in structural properties and conserved functional features indicate that both protein families have the same evolutionary origin. We will describe the distribution of MACPF/CDC proteins in nature and discuss briefly their similarity and functional role in different biological processes.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans, the severity of which depends on multiple factors, including intrinsic characteristics of the affected individuals and the pathogen itself. Additionally, emerging evidence suggests that epigenetic modifications may also modulate host susceptibility to infection. Therefore, different clinical outcomes can be expected, ranging from self-limiting gastroenteritis to severe central nervous system and maternal-neonatal infections, and bacteremia. Furthermore, L. monocytogenes is a genetically and phenotypically diverse species, resulting in a large variation in virulence potential between strains. Multilocus sequence typing (MLST) has been widely used to categorize the clonal structure of bacterial species and to define clonal complexes (CCs) of genetically related isolates. The combination of MLST and epidemiological data allows to distinguish hypervirulent CCs, which are notably more prevalent in clinical cases and typically associated with severe forms of the disease. Conversely, other CCs, termed hypovirulent, are predominantly isolated from food and food processing environments and are associated with the occurrence of listeriosis in immunosuppressed individuals. Reports of genetic traits associated with this diversity have been described. The Food and Agriculture Organization (FAO) is encouraging the search for virulence biomarkers to rapidly identify the main strains of concern to reduce food waste and economical losses. The aim of this review is to comprehensively collect, describe and discuss the methodologies used to discriminate the virulence potential of L. monocytogenes CCs. From the exploration of in vitro and in vivo models to the study of expression of virulence genes, each approach is critically explored to better understand its applicability and efficiency in distinguishing the virulence potential of the pathogen. [ABSTRACT FROM AUTHOR]

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore‐forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re‐examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin‐luciferase reaction. Activation of calcium‐activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two‐electrode voltage‐clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx‐sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut‐like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles. [ABSTRACT FROM AUTHOR]

Cholesterol-dependent cytolysins (CDCs) are the distinct class of β-barrel pore-forming toxins (β-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane β-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the poreforming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membranebinding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far. [ABSTRACT FROM AUTHOR]

Nano- and micro-sized vesicular and colloidal structures mediate cell–cell communication. They are important players in the physiology of plants, animals, and humans, and are a subject of increasing interest. We investigated the effect of three surfactants, N-cetylpyridinium chloride (CPC), sodium dodecyl sulfate (SDS), and Triton X-100 (TX100), and two anionic polyelectrolytes, sodium polystyrene sulfonate (NaPSS) and sodium polymethacrylate (NaPMA), on nanoliposomes. In addition, the effect of SDS and TX100 on selected biological membranes (erythrocytes and microalgae) was investigated. The liposomes were produced by extrusion and evaluated by microcalorimetry and light scattering, based on the total intensity of the scattered light (Itot), hydrodynamic radius (Rh), radius of gyration (Rg), shape parameter p (=Rh/Rg,0), and polydispersity index. The EPs shed from erythrocytes and microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum were visualized by scanning electron microscopy (SEM) and analyzed by flow cytometry (FCM). The Rh and Itot values in POPC liposome suspensions with added CPC, SDS, and TX100 were roughly constant up to the respective critical micelle concentrations (CMCs) of the surfactants. At higher compound concentrations, Itot dropped towards zero, whereas Rh increased to values higher than in pure POPC suspensions (Rh ≈ 60–70 nm), indicating the disintegration of liposomes and formation of larger particles, i.e., various POPC–S aggregates. Nanoliposomes were stable upon the addition of NaPSS and NaPMA, as indicated by the constant Rh and Itot values. The interaction of CPC, SDS, or TX100 with liposomes was exothermic, while there were no measurable heat effects with NaPSS or NaPMA. The SDS and TX100 increased the number density of EPs several-fold in erythrocyte suspensions and up to 30-fold in the conditioned media of Dunaliella tertiolecta at the expense of the number density of cells, which decreased to less than 5% in erythrocytes and several-fold in Dunaliella tertiolecta. The SDS and TX100 did not affect the number density of the microalgae Phaeodactylum tricornutum, while the number density of EPs was lower in the conditioned media than in the control, but increased several-fold in a concentration-dependent manner. Our results indicate that amphiphilic molecules need to be organized in nanosized particles to match the local curvature of the membrane for facilitated uptake. To pursue this hypothesis, other surfactants and biological membranes should be studied in the future for more general conclusions. [ABSTRACT FROM AUTHOR]

Cancer cell dormancy is a reversible process whereby cancer cells enter a quiescent state characterized by cell cycle arrest, inhibition of cell migration and invasion, and increased chemoresistance. Because of its reversibility and resistance to treatment, dormancy is a key process to study, monitor, and interfere with, in order to prevent tumor recurrence and metastasis and improve the prognosis of cancer patients. However, to achieve this goal, further studies are needed to elucidate the mechanisms underlying this complex and dynamic dual process. Here, we review the contribution of extracellular vesicles (EVs) to the regulation of cancer cell dormancy/awakening, focusing on the cross-talk between tumor and non-tumor cells in both the primary tumor and the (pre-)metastatic niche. Although EVs are recognized as key players in tumor progression and metastasis, as well as in tumor diagnostics and therapeutics, their role specifically in dormancy induction/escape is still largely elusive. We report on the most recent and promising results on this topic, focusing on the EV-associated nucleic acids involved. We highlight how EV studies could greatly contribute to the identification of dormancy signaling pathways and a dormancy/early awakening signature for the development of successful diagnostic/prognostic and therapeutic approaches. [ABSTRACT FROM AUTHOR]

This study investigated the adaptability of Dunaliella salina to different salinity levels, with an emphasis on growth, pigment concentration, and desalination potential. It was found that among the 21 salinity levels, Salinity 75 produced consistently favorable results in cell count (13.08 × 103 ± 1.41 × 103 cells/mL), dry biomass (2.46 ± 0.06 g/L), pigment content (chlorophyll a = 97,500,000 ± 100,000 pg/L, chlorophyll b = 123,600,000 ± 300,000 pg/L), and desalination (9.32 ± 0.47 reduction). Therefore, Salinity 75 was selected for the final trial (scale-up), which revealed unanticipatedly high cell counts (58.96 × 103 ± 535.22 cells/mL), with the dry biomass weight being statistically different (higher) than expected (4.21 ± 0.02 g/L) (p < 0.0001), most likely due to the high cell count and energy reserve storage for high-salinity adaption in the form of bio-compounds. Pigment growth continued (chlorophyll a = 95,400,000 ± 2,200,000 pg/L, chlorophyll b = 128,100,000 ± 5,100,000 pg/L), indicating pigment production under salt stress. Notably, desalination did not occur in this stage, possibly due to the necessity for a bigger initial inoculate, prolonged exposure or bioaccumulation becoming the prevailing mechanism over desalination. Nevertheless, the trial highlights D. salina's strong adaptation to various salinity levels. This suggests a promising future in halophyte research, particularly in understanding the mechanisms that prevent salt accumulation in cells and how to overcome this barrier. Additionally, these results suggest that microalgae could be a viable resource in saline-rich environments unsuitable for conventional agriculture, promoting industrial adaptation to adverse conditions. [ABSTRACT FROM AUTHOR]

This study described the efficiency of pineapple peel extract (PPE) in increasing the immunity of Nile tilapia (Oreochromis niloticus) and in inhibiting Aeromonas hydrophila infections. The PPE concentrations used in this study were: 62.5, 125, and 250 mg/mL. 0.1 mL of PPE was administered via intraperitoneal injection into the fish. On day 7, fish was intraperitoneally injected with 0.1 mL A. hydrophila (density 108 CFU/mL). Hematology and biochemical serum parameters were observed on day 1 and 7 after injected with PPE and day 7 and 14 post-challenge with A. hydrophila. The results indicated that, red blood cells, white blood cells, and hematocrit of PPE-injected fish were significantly higher than those in the control group (p<0.05). Alkaline phosphatase, aspartate transaminase, and alanine transaminase in the fish groups receiving PPE were lower than those of the control group. After challenge test, fish injected with 125 mg/mL PPE concentration was able to significantly reduce the mortality rate compare to control (from 80.50% to 47.22%). It can be concluded that PPE was effective in preventing A. hydrophila infections, and had a positive impact on the immunity of fish. So that pineapple is an alternative plant that can be used to improve fish health. [ABSTRACT FROM AUTHOR]

Background: Salmonid species have followed markedly divergent evolutionary trajectories in their interactions with sea lice. While sea lice parasitism poses significant economic, environmental, and animal welfare challenges for Atlantic salmon (Salmo salar) aquaculture, coho salmon (Oncorhynchus kisutch) exhibit near-complete resistance to sea lice, achieved through a potent epithelial hyperplasia response leading to rapid louse detachment. The molecular mechanisms underlying these divergent responses to sea lice are unknown. Results: We characterized the cellular and molecular responses of Atlantic salmon and coho salmon to sea lice using single-nuclei RNA sequencing. Juvenile fish were exposed to copepodid sea lice (Lepeophtheirus salmonis), and lice-attached pelvic fin and skin samples were collected 12 h, 24 h, 36 h, 48 h, and 60 h after exposure, along with control samples. Comparative analysis of control and treatment samples revealed an immune and wound-healing response that was common to both species, but attenuated in Atlantic salmon, potentially reflecting greater sea louse immunomodulation. Our results revealed unique but complementary roles of three layers of keratinocytes in the epithelial hyperplasia response leading to rapid sea lice rejection in coho salmon. Our results suggest that basal keratinocytes direct the expansion and mobility of intermediate and, especially, superficial keratinocytes, which eventually encapsulate the parasite. Conclusions: Our results highlight the key role of keratinocytes in coho salmon's sea lice resistance and the diverged biological response of the two salmonid host species when interacting with this parasite. This study has identified key pathways and candidate genes that could be manipulated using various biotechnological solutions to improve Atlantic salmon sea lice resistance. [ABSTRACT FROM AUTHOR]

The delivery of therapeutic agents faces significant hurdles posed by the endo-lysosomal pathway, a bottleneck that hampers clinical effectiveness. This comprehensive review addresses the urgent need to enhance cellular delivery mechanisms to overcome these obstacles. It focuses on the potential of smart nanomaterials, delving into their unique characteristics and mechanisms in detail. Special attention is given to their ability to strategically evade endosomal entrapment, thereby enhancing therapeutic efficacy. The manuscript thoroughly examines assays crucial for understanding endosomal escape and cellular uptake dynamics. By analyzing various assessment methods, we offer nuanced insights into these investigative approaches’ multifaceted aspects. We meticulously analyze the use of smart nanocarriers, exploring diverse mechanisms such as pore formation, proton sponge effects, membrane destabilization, photochemical disruption, and the strategic use of endosomal escape agents. Each mechanism’s effectiveness and potential application in mitigating endosomal entrapment are scrutinized. This paper provides a critical overview of the current landscape, emphasizing the need for advanced delivery systems to navigate the complexities of cellular uptake. Importantly, it underscores the transformative role of smart nanomaterials in revolutionizing cellular delivery strategies, leading to a paradigm shift towards improved therapeutic outcomes. [ABSTRACT FROM AUTHOR]

Animal venoms are a complex mixture of highly specialized toxic molecules. Among them, pore-forming proteins (PFPs) or toxins (PFTs) are one of the major disease-causing toxic elements. The ability of the PFPs in defense and toxicity through pore formation on the host cell surface makes them unique among the toxin proteins. These features made them attractive for academic and research purposes for years in the areas of microbiology as well as structural biology. All the PFPs share a common mechanism of action for the attack of host cells and pore formation in which the selected pore-forming motifs of the host cell membrane-bound protein molecules drive to the lipid bilayer of the cell membrane and eventually produces water-filled pores. But surprisingly their sequence similarity is very poor. Their existence can be seen both in a soluble state and also in transmembrane complexes in the cell membrane. PFPs are prevalent toxic factors that are predominately produced by all kingdoms of life such as virulence bacteria, nematodes, fungi, protozoan parasites, frogs, plants, and also from higher organisms. Nowadays, multiple approaches to applications of PFPs have been conducted by researchers both in basic as well as applied biological research. Although PFPs are very devastating for human health nowadays researchers have been successful in making these toxic proteins into therapeutics through the preparation of immunotoxins. We have discussed the structural, and functional mechanism of action, evolutionary significance through dendrogram, domain organization, and practical applications for various approaches. This review aims to emphasize the PFTs to summarize toxic proteins together for basic knowledge as well as to highlight the current challenges, and literature gap along with the perspective of promising biotechnological applications for their future research. [ABSTRACT FROM AUTHOR]

Plants are attacked by various pathogens that secrete a variety of effectors to damage host cells and facilitate infection. One of the largest and so far understudied microbial protein families of effectors is necrosis- and ethylene-inducing peptide-1-like proteins (NLPs), which are involved in important plant diseases. Many NLPs act as cytolytic toxins that cause cell death and tissue necrosis by disrupting the plant's plasma membrane. Their mechanism of action is unique and leads to the formation of small, transient membrane ruptures. Here, we capture the interaction of the cytotoxic model NLP from the oomycete Pythium aphanidermatum, NLP Pya , with plant cell-mimicking membranes of giant unilamellar vesicles (GUVs) and tobacco protoplasts using confocal fluorescence microscopy. We show that the permeabilization of GUVs by NLP Pya is concentration- and time-dependent, confirm the small size of the pores by observing the inability of NLP Pya monomers to pass through them, image the morphological changes of GUVs at higher concentrations of NLP Pya and confirm its oligomerization on the membrane of GUVs. In addition, NLP Pya bound to plasma membranes of protoplasts, which showed varying responses. Our results provide new insights into the interaction of NLP Pya with model lipid membranes containing plant-derived sphingolipids., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Measurement of antibody and antibody fusion protein concentration is vital for process development and manufacturing. Continuous, in-line monitoring of antibody concentration could be useful in a variety of applications, such as controlling the loading of protein A columns to prevent breakthrough, monitoring bioreactor titer, and detecting leaks past ultrafiltration/diafiltration membranes. Molecule-specific monitoring techniques are advantageous for antibody detection in cell culture fluid in the presence of complex process impurities. Here we report a continuous in-line, real-time IgG monitoring platform using a fiber-optic biosensor with a replaceable sensor tip covalently functionalized with a fluor-labeled protein consisting of a pentamer of the Z domain (a more stable form of the B domain) of protein A. The sensor demonstrates concentration-dependent fluorescence enhancement in the presence of human IgG (0.01-0.75 g/L), with consistent signals during five runs each with 1 and 0.1 g/L IgG, and maintains its specificity in the presence of Chinese hamster ovary (CHO) cell culture fluid. A 5% breakthrough of a typical 10 g/L load would be detected in less than 20 s in a flowing stream emerging from a protein A column without prior sample preparation. This sensor platform may be suitable for monitoring IgG and fragment, crystallizable (Fc) fusion proteins in diverse upstream and downstream bioprocess applications., (© 2024 Wiley Periodicals LLC.)

Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella -specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella , we provide the first proof for reverse recruitment from venom to organismal development. [ABSTRACT FROM AUTHOR]

Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated. [ABSTRACT FROM AUTHOR]

Earthworms are used to cure wounds in Chinese villages for thousands of years. Recently, scientists realized their extracts could promote wound healing and they have anti-inflammatory, antioxidant, anti-apoptosis, and anti-microbial properties, but its mechanism of promoting wound healing remains unclear. In the presented study, electronic literature databases and LC-MS/MS were used to determine earthworms' ingredients and differential metabolites. Swiss Target Prediction database was used for ingredients' target prediction and wound disease-relevant genes were found from GeneCards, OMIM, and DrugBank databases. Network pharmacology was conducted to demonstrate filtering hub targets, biological functions, and the signaling pathways of earthworms extract against wounds. Molecular docking and metabolism analysis were used to look for core target genes and key bioactive molecules from earthworms. Finally, the investigation shows 5 most important signal pathways, 5 core genes, and 6 bioactive ingredients-related cell–cell adhesion, cell proliferation, and cell migration processes could be affected by earthworms' extract. On 3rd day, the extract could regulate HIF1A and EGFR targets to make the differences of quantities of 4-pyridoxate, tetradecanoic acid, and L-kynurenine. While on 7th day, the regulation refers 6 earthworms' bioactive ingredients, 4 core genes (CTNNB1, EGFR, SRC, and CASP3), and 4 differential metabolites (4-hydoxy-2-quinolinecarboxylic acid, urocanate, deoxyinosine, creatine, and sn-glycerol-3-phosphocholine). on 14th day, 2 core genes (EGFR, SRC) are influenced in the biological processes. Briefly, we found that 6 ingredients from earthworms have most bioactive and 5 core genes play an important role in promoting wound-healing processes. These discovers indicates earthworms could against wound via AGE-RAGE, PI3K-Akt, HIF1A, MAPK, and Axon guidance pathways. [ABSTRACT FROM AUTHOR]

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6–pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid. [ABSTRACT FROM AUTHOR]

Glioblastoma (GB) is a rare but extremely aggressive brain tumor that significantly impacts patient outcomes, affecting both duration and quality of life. The protocol established by Stupp and colleagues in 2005, based on radiotherapy and chemotherapy with Temozolomide, following maximum safe surgical resection remains the gold standard for GB treatment; however, it is evident nowadays that the extreme intratumoral and intertumoral heterogeneity, as well as the invasiveness and tendency to recur, of GB are not compatible with a routine and unfortunately ineffective treatment. This review article summarizes the main challenges in the search for new valuable therapies for GB and focuses on the impact that extracellular vesicle (EV) research and exploitation may have in the field. EVs are natural particles delimited by a lipidic bilayer and filled with functional cellular content that are released and uptaken by cells as key means of cell communication. Furthermore, EVs are stable in body fluids and well tolerated by the immune system, and are able to cross physiological, interspecies, and interkingdom barriers and to target specific cells, releasing inherent or externally loaded functionally active molecules. Therefore, EVs have the potential to be ideal allies in the fight against GB and to improve the prognosis for GB patients. The present work describes the main preclinical results obtained so far on the use of EVs for GB treatment, focusing on both the EV sources and molecular cargo used in the various functional studies, primarily in vivo. Finally, a SWOT analysis is performed, highlighting the main advantages and pitfalls of developing EV-based GB therapeutic strategies. The analysis also suggests the main directions to explore to realize the possibility of exploiting EVs for the treatment of GB. [ABSTRACT FROM AUTHOR]

Exosomes distributed by extracellular vesicles carry various information highly consistent with cells, becoming a new type of biomarker for tumor screening. However, although conventional characterization technologies can quantify size and morphology for exosomes, they are limited in related fields such as function tracing, protein quantification at unit point, and microstructural information. In this paper, firstly, different exosome characterization methods are systematically reviewed, such as dynamic light scattering, nanoparticle tracking analysis, flow cytometry, electron microscope, and emerging super-resolution imaging technologies. Then, advances in applications are described one by one. Last but not least, we compare the features of different technologies for exosomes and propose that super-resolution imaging technology can not only take into account the advantages of conventional characterization techniques but also provide accurate, real-time, and super-resolution quantitative analysis for exosomes. It provides a fine guide for exosome-related biomedical research, as well as application in liquid biopsy and analysis techniques. [ABSTRACT FROM AUTHOR]

The toxicological impact of airborne polluting ultrafine particles (UFPs, also classified as nanoparticles with average sizes of less than 100 nm) is an emerging area of research pursuing a better understanding of the health hazards they pose to humans and other organisms. Hemolytic activity is a toxicity parameter that can be assessed quickly and easily to establish part of a nanoparticle's behavior once it reaches our circulatory system. However, it is exceedingly difficult to determine to what extent each of the nanoparticles present in the air is responsible for the detrimental effects exhibited. At the same time, current hemolytic assessment methodologies pose a series of limitations for the interpretation of results. An alternative is to synthesize nanoparticles that model selected typical types of UFPs in air pollution and evaluate their individual contributions to adverse health effects under a clinical assay of osmotic fragility. Here, we discuss evidence pointing out that the absence of hemolysis is not always a synonym for safety; exposure to model nanopollutants, even at low concentrations, is enough to increase erythrocyte susceptibility and dysfunction. A modified osmotic fragility assay in combination with a morphological inspection of the nanopollutant–erythrocyte interaction allows a richer interpretation of the exposure outcomes. Membrane–nanoparticle interplay has a leading role in the vulnerability observed. Therefore, future research in this line of work should pay special attention to the evaluation of the mechanisms that cause membrane damage. [ABSTRACT FROM AUTHOR]

The remarkable diversity of lymphocytes, essential components of the immune system, serves as an ingenious mechanism for maximizing the efficient utilization of limited host defense resources. While cell adhesion molecules, notably in gut-tropic T cells, play a central role in this mechanism, the counterbalancing molecular details have remained elusive. Conversely, we've uncovered the molecular pathways enabling extracellular vesicles secreted by lymphocytes to reach the gut's mucosal tissues, facilitating immunological regulation. This discovery sheds light on immune fine-tuning, offering insights into immune regulation mechanisms. [ABSTRACT FROM AUTHOR]

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell–cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant–pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management. [ABSTRACT FROM AUTHOR]

Aegerolysin proteins are involved in various interactions by recognising a molecular receptor in the target organism. The formation of pores in combination with larger, non-aegerolysin-like protein partners (such as membrane attack complex/perforin proteins [MACPFs]) is one of the possible responses in the presumed competitive exclusion of other organisms from the ecological niche. Bicomponent pairs are already observed at the gene level. Fungi growing under extreme conditions can be divided into ubiquitous and extremotolerant generalists which can compete with mesophilic species and rare, isolated extremophilic and extremotolerant specialists with narrow ecological amplitude that cannot compete. Under extreme conditions, there are fewer competitors, so fungal specialists generally produce less diverse and complicated profiles of specialised molecules. Since extremotolerant and extremophilic fungi have evolved in numerous branches of the fungal tree of life and aegerolysins are unevenly distributed across fungal genomes, we investigated whether aegerolysins, together with their partner proteins, contribute to the extreme survival ecology of generalists and specialists. We compiled a list of 109 thermo-, psihro-, acido-, alkali-, halo-, metallo- and polyextremo-tolerant/-philic fungal species. Several challenges were identified that affected the outcome: renaming fungal species, defining extremotolerant/extremophilic traits, identifying extremotolerant/extremophilic traits as metadata in databases and linking fungal isolates to fungal genomes. The yield of genomes coding aegerolysins or MACPFs appears to be lower in extremotolerant/extremophilic fungi compared to all fungal genomes. No candidates for pore-forming gene pairs were identified in the genomes of extremophilic fungi. Aegerolysin and MACPFs partner pairs were identified in only two of 69 species with sequenced genomes, namely in the ubiquitous metallotolerant generalists Aspergillus niger and A. foetidus. These results support the hypothesised role of these pore-forming proteins in competitive exclusion., (© 2024 The Author(s). IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

pH regulation is a serious concern in the industrial fermentation process as pH adjustment heavily utilizes acid/base and pollutes the environment. Under pH-stress conditions, microbial growth and production of valuable target products may be severely affected. Furthermore, some strains generating acidic or alkaline products require self pH regulation and increased tolerance against pH-stress. For pH control, synthetic biology has provided advanced engineering approaches to construct robust and more intelligent microbial strains, exhibiting tolerance to pH-stress to cope with limitations of pH regulation. This study reviewed the current progress of advanced strain evolution strategies to engineer pH-stress tolerant strains via synthetic biology. In addition, a large number of pH-responsive elements, including promoters, riboswitches, and some proteins have been investigated and applied for construction of pH-responsive genetic circuits and intelligent pH-responsive microbial strains., (© 2021 Wiley-VCH GmbH.)

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Urinary bladder cancer can be treated by intravesical application of therapeutic agents, but the specific targeting of cancer urothelial cells and the endocytotic pathways of the agents are not known. During carcinogenesis, the superficial urothelial cells exhibit changes in sugar residues on the apical plasma membranes. This can be exploited for selective targeting from the luminal side of the bladder. Here we show that the plant lectins Jacalin (from Artocarpus integrifolia), ACA (from Amaranthus caudatus) and DSA (from Datura stramonium) selectively bind to the apical plasma membrane of low- (RT4) and high-grade (T24) cancer urothelial cells in vitro and urothelial tumours ex vivo. The amount of lectin binding was significantly different between RT4 and T24 cells. Endocytosis of lectins was observed only in cancer urothelial cells and not in normal urothelial cells. Transmission electron microscopy analysis showed macropinosomes, endosome-like vesicles and multivesicular bodies filled with lectins in RT4 and T24 cells and also in cells of urothelial tumours ex vivo. Endocytosis of Jacalin and ACA in cancer cells was decreased in vitro after addition of inhibitor of macropinocytosis 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and increased after stimulation of macropinocytosis with epidermal growth factor (EGF). Clathrin, caveolin and flotillin did not colocalise with lectins. These results confirm that the predominant mechanism of lectin endocytosis in cancer urothelial cells is macropinocytosis. Therefore, we propose that lectins in combination with conjugated therapeutic agents are promising tools for improved intravesical therapy by targeting cancer cells. [ABSTRACT FROM AUTHOR]