Molecular Recognition Mechanism Research Articles

Preparation and characterization of aptamer-based sorbent for the selective extraction of zearalenone and its derivatives from human urine.

The aim of this work is the development of a biomimetic strategy involving a molecular recognition mechanism using aptamers immobilized on a solid support for the analysis of the mycotoxin zearalenone (ZEA) and two of its derivatives in human urine: alpha-zearelenol (α-ZEL) and beta-zearelenol (β-ZEL). Three oligonucleotide sequences reported in theliterature as beingspecific to ZEA were thus covalently grafted onto activated sepharose, and a thorough study of the percolation and washing conditions was performed to promote the selective retention of the three targeted compounds. With the optimized extraction procedure, a strong and selective retention was obtained for ZEA and to a lesser extent α-ZEL and β-ZEL, with extraction recoveries of 88±9%, 77±15%, and 45±12% respectively, in standard solutions. Application of this procedure to spiked human urine strongly highlighted the efficiency of the clean-up effect resulting from the use of this selective sorbent. Limits of quantification of the whole analytical procedure including extraction on oligosorbent andLC-MS analysis were 0.18 and 0.24ngmL-1, for ZEA and α-ZEL, respectively, thus demonstrating clearly the potential of the developed method for monitoring human dietary exposure to these compounds.

Establishment of Broad-Specificity Monoclonal Antibody-Based Immunoassay for Rapid Detection of Indole-Type and Indazole-Type Synthetic Cannabinoids and Metabolites.

Synthetic cannabinoids (SCs) have emerged as one of the most severely abused categories of new psychoactive substances (NPS), exacerbating the global drug problem and posing significant threats to public health. Presently, a class of new amide-type SCs featuring an indazole or indole core has been identified in numerous cases of illegal drug use, but there is still a lack of comprehensive analysis methods of SC detection. Herein, monoclonal antibodies (mAbs) 2E4 and AE6 targeting 36 indole-type and indazole-type SCs and their metabolites with IC50 ranging from 0.14 to 85.28 ng/mL were prepared and the molecular mechanism of antibody recognition was elaborated. We established the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and gold immunochromatography assay (GICA) based on mAbs 2E4 and AE6 to detect indazole-type and indole-type SCs in urine and hair samples. Under optimal conditions, the proposed method detected ADB-BUTINACA (an indazole-type SC) with limits of detection (LODs) of 0.11 ng/mL for urine and 0.024 ng/mg for hair by ic-ELISA, and 1.02 ng/mL for urine and 0.046 ng/mg for hair by GICA; the LODs of 4F-MDMB-BUTICA (an indole-type SC) detection was 0.036 ng/mL for urine and 0.012 ng/mg for hair by ic-ELISA, and 0.54 ng/mL for urine and 0.03 ng/mg for hair by GICA. Collectively, our study provides a comprehensive foundation for the rapid screening and quantitation of SC derivatives in biological samples.

Unveil the molecular recognition mechanisms of triazine haptens and monoclonal antibodies: Establishing ic‐ELISA methods for triazine herbicide analysis in tea

AbstractThis study introduced two triazine haptens (with 0 or 1 carbon connecting arms at nonisopropyl positions) and developed two heterologous indirect competitive enzyme‐linked immunosorbent assay (ic‐ELISA) methods. The IC50 values for the 17 triazine pesticides ranged from 0.84 ng/mL to 225.63 ng/mL, and the established method was applied to determine their concentrations in tea samples. This study aimed to address the current debate on the recognition mechanism of triazine haptens with shorter connecting arms. This study produced two new findings by investigating the recognition mechanism of triazine haptens and antibodies. On the one hand, the specific amino acid isoleucine (ILE‐219) is crucial for isopropyl recognition. On the other hand, the selection of the connecting site of the triazine hapten has a significant effect on the specificity of the antibody, with the influence of the length of the carbon connecting arm being secondary. Our newly designed haptens show promise for diverse triazine pesticide immunoassay applications. This discovery established a foundational framework for constructing a tailored antibody library for pollutants.

Generation of a Nonbilayer Lipid Nanoenvironment after Epitope Binding Potentiates Neutralizing HIV-1 MPER Antibody.

Establishment of interactions with the envelope lipids is a cardinal feature of broadly neutralizing antibodies (bnAbs) that recognize the Env membrane-proximal external region (MPER) of HIV. The lipid envelope constitutes a relevant component of the full "quinary" MPER epitope, and thus antibodies may be optimized through engineering their capacity to interact with lipids. However, the role of the chemically complex lipid nanoenvironment in the mechanism of MPER molecular recognition and viral neutralization remains poorly understood. To approach this issue, we computationally and experimentally investigated lipid interactions of broadly neutralizing antibody 10E8 and optimized versions engineered to enhance their epitope and membrane affinity by grafting bulky aromatic compounds. Our data revealed a correlation between neutralization potency and the establishment of favorable interactions with small headgroup lipids cholesterol and phosphatidylethanolamine, evolving after specific engagement with MPER. Molecular dynamics simulations of chemically modified Fabs in complex with an MPER-Transmembrane Domain helix supported the generation of a nanoenvironment causing localized deformation of the thick, rigid viral membrane and identified sphingomyelin preferentially occupying a phospholipid-binding site of 10E8. Together, these interactions appear to facilitate insertion of the Fabs through their engagement with the MPER epitope. These findings implicate individual lipid molecules in the neutralization function of MPER bnAbs, validate targeted chemical modification as a method to optimize MPER antibodies, and suggest pathways for MPER peptide-liposome vaccine development.

A highly sensitive electrochemical aptasensor for detecting broad-spectrum estrogen molecules in clinical samples

The lack of sensitive and accurate monitoring methods for in vivo estrogen levels presents challenges for better prevention of estrogen-induced diseases. We have developed a label-free electrochemical aptasensor that demonstrates high sensitivity and selectivity for the broad-spectrum detection of estrogen molecules. This biosensor uses gold nanoparticles for electrochemical signal amplification and aptamers for broad-spectrum target recognition, enabling precise detection of trace amounts of estrogen in serum samples. The aptasensor demonstrates high sensitivity in estrogen detection, with a linear detection range of 0.01–1 nM and a minimum detection limit of 3 pM. It also exhibits excellent selectivity and interference resistance, with a detection error of less than 19 %, even in the presence of high concentrations of other biological substances. Additionally, molecular dynamics simulations were performed to provide insights into the molecular mechanism of aptamer broad-spectrum recognition and construction principles underlying the sensor. We anticipate that this aptasensor will serve as a robust, convenient, and cost-effective detection method, offering a valuable solution for the prevention of estrogen-induced diseases.

End Group Effects on Anion Binding in Tetraglycine Peptide: A Computational Study.

The importance of anions in various processes has led to a search for molecules that can effectively recognize and interact with these anions. This study explores how the tetraglycine (Gly)4 peptide in its zwitterionic, neutral, and terminally capped forms acts as a receptor for H2PO4- and HSO4- anions within the framework of supramolecular host-guest chemistry. Using molecular dynamics (MD) simulations, we obtained the conformations of the receptor-anion complexes. Density functional theory (DFT), quantifies the complexes' interaction energies in both gas and solvent phases. Proton transfer within the zwitterionic complex with H2PO4- anion alters peptide charge distribution, affecting its conformation and binding site arrangement, as analysed by quantum mechanics/molecular mechanics (QM/MM) methods. Symmetry-adapted perturbation theory (SAPT) and noncovalent interactions analysis highlight the role of electrostatic interactions in these receptor-anion complexes. It emphasizes the key interactions such as N-H···O and O-H···O=C between the peptide backbone and anions and elucidates the molecular recognition mechanism driven by crucial noncovalent interactions. The termination of the peptide's end groups modulates anion binding sites from the backbone to the charged N-terminal, resulting in distinct binding sites. Our findings provide insights for designing peptides tailored to function as anion receptors in diverse supramolecular chemistry applications.

Broad-spectrum detection of benzimidazoles with lateral flow immunoassay: A computational chemistry-assisted hapten design strategy and explore of molecular recognition mechanism

Benzimidazoles (BMZs) are a class of veterinary drugs with a benzimidazole ring, the abuse of which poses a serious threat to ecological balance and human health. Consequently, the development of broad-spectrum antibodies and rapid assays are crucial for detecting BMZs in food samples. Herein, we scientifically designed three hapten structures, predicted the availability of the hapten with computational chemistry, and subsequently verified the broad-spectrum with immunological experiments. A broad-spectrum monoclonal antibody (6F10) was prepared based on the predicted hapten-2. Molecular recognition studies illustrated intricate interactions between mAb 6F10 binding to BMZs attributed to halogen bonds and π-π/π-alkyl interactions, revealing key amino acid sites and demonstrating the reliability of the hapten prediction strategies. Finally, a broad-spectrum, rapid, and sensitive lateral flow immunoassay based on aggregation-induced emission microspheres with high fluorescence intensity was established. The LOD values of the proposed method for eight kinds of BMZs were 0.027, 0.032, 0.058, 0.091, 0.087, 0.246, 0.369, and 0.311 ng mL−1, respectively. In this work, a hapten prediction strategy based on a computational chemistry method effectively guided the preparation of antibodies for broad-spectrum recognition of BMZs, and the molecular recognition studies verified the interaction of mAb 6F10 with BMZs, enabling broad-spectrum and sensitive detection of BMZs in milk.

Exploring phosphorene-protein interactions: An integrated computational and spectroscopic investigation

Among 2D materials, exfoliated black phosphorus (or phosphorene) shows great promise for applications in biological domains. However, despite its performances, little is known about the intricate and dynamic interactions that this material can form with proteins. This increases the risk of off-target effects and adds complexity in designing phosphorene-based devices with tailored properties. In this study, we present a straightforward and easily implementable pipeline that integrates spectroscopies with Molecular Dynamics simulations to explore the dynamic interplay between phosphorene and a protein system. Using lysozyme as a deeply investigated reference protein, we employed two theoretical protein models with unique secondary structure folds to increase the descriptive power of the approach and disentangle the complexity and variability of experimental data into a few primary drivers of protein-phosphorene interactions. Our results show that the 2D material does not significantly alter the protein structure, but the observed conformational changes are influenced by the secondary fold. Indeed, while the beta structure interacts mainly through unfolded regions, the alpha fold favours phosphorene binding through structured clusters of residues, leading to more significant structural and dynamic perturbations. By utilizing this pipeline, we have gained valuable insights into the molecular recognition mechanism of phosphorene, enhancing the development of improved phosphorene-based devices. In addition, our methodology offers potential for further applications in biomedicine to characterise interfaces between other 2D (nano)materials and biological entities.

Virtual screening, molecular docking, and molecular dynamics simulation studies on the hypoglycemic function of oat peptides

Oat contains a large amount of polysaccharides and peptides, among which oat peptides have the function of reducing blood sugar levels in the body. This paper reviewed the peptides obtained from oat extraction and identification from literature and constructed the corresponding oat peptide database. Based on the DPP4 protein, a virtual screening of the peptide database was performed. One hundred nanosecond molecular dynamics simulations were performed for the six peptides obtained from the screening. The interaction information between different peptide molecules and DPP4 was analyzed from the stable binding conformations after the simulation, and the binding free energy between different peptide molecules and DPP4 was calculated. The results show that the peptide molecules obtained from the virtual screening can all stably bind to the DPP4 protein, among which two peptide molecules have relatively strong affinity with DPP4 and can be used as lead molecules for the subsequent design and modification of DPP4 inhibitors. The simulation results are informative for a deeper understanding of the structural characteristics of DPP4 and the molecular recognition mechanism between DPP4 and oat peptides.

Biocatalytic Desymmetrization for the Atroposelective Synthesis of Axially Chiral Biaryls Using an Engineered Imine Reductase.

The enzymatic atroposelective synthesis of biaryl compounds is relatively rare, despite considerable attention received by biocatalysis in academic and industrial sectors. Imine reductases (IREDs) are an important class of enzymes that have been applied in the asymmetric synthesis of chiral amine building blocks. In this work, two IREDs (IR140 and IR189) were identified to catalyze the efficient desymmetrization of biaryls utilizing various amine donors. Further protein engineering enabled the identification of variants (IR189 M8-M9 and IR189 M13-M14) that are able to catalyze the formation of both (R) and (S) atropisomers in excellent yields and atroposelectivities for up to 24 examples (up to 99% ee and yield). The absolute configuration and rotational barriers were confirmed, and the reactions were readily enlarged to allow isolation of the atropisomeric products in 99% ee and 82% isolated yields. The optically pure biaryl amines were further derivatized into various synthetically useful atropisomers. To shed light on the molecular recognition mechanisms, molecular dynamics (MD) simulations were performed, offering plausible explanations for the improved atroposelectivities and enzymatic activities. The current strategy expands the scope of IRED-catalyzed synthesis of axially chiral biaryl amines, contributing significantly to the field of atroposelective biocatalysis.

Decomposition of Forces in Protein: Methodology and General Properties.

In contrast to the central role played by the structure of biomolecules, the complementary force-based view has received little attention in past studies. Here, we proposed a simple method for the force decomposition of multibody interactions and provided some techniques to analyze and visualize the general behavior of forces in proteins. It was shown that atomic forces fluctuate at a magnitude of about 3000 pN, which is huge in the context of cell biology. Remarkably, the average scalar product between atomic force and displacement universally approximates -3kBT. This is smaller by an order of magnitude than the simple product of their fluctuation magnitudes due to the unexpectedly weak correlation between the directions of force and displacement. The pairwise forces are highly anisotropic, with elongated fluctuation ellipsoids. Residue-residue forces can be attractive or repulsive (despite being more likely to be attractive), forming some kind of tensegrity structure stabilized by a complicated network of forces. Being able to understand and predict the interaction network provides a basis for rational drug design and uncovering molecular recognition mechanisms.

Stereoselective Synthesis of Oxetanes Catalyzed by an Engineered Halohydrin Dehalogenase.

Although biocatalysis has garnered widespread attention in both industrial and academic realms, the enzymatic synthesis of chiral oxetanes remains an underdeveloped field. Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that have been engineered to accomplish the reversible transformation of epoxides. In our work, a biocatalytic platform was constructed for the stereoselective kinetic resolution of chiral oxetanes and formation of 1,3-disubstituted alcohols. HheC from Agrobacterium radiobacter AD1 was engineered to identify key variants capable of catalyzing the dehalogenation of γ-haloalcohols (via HheC M1-M3) and ring opening of oxetanes (via HheC M4-M5) to access both (R)- and (S)-configured products with high stereoselectivity and remarkable catalytic activity, yielding up to 49% with enantioselectivities exceeding 99% ee and E>200. The current strategy is broadly applicable as demonstrated by expansion of substrate scope to include up to 18 examples for dehalogenations and 16 examples for ring opening. Additionally, the functionalized products are versatile building blocks for pharmaceutical applications. To shed light on the molecular recognition mechanisms for the relevant variants, molecular dynamic (MD) simulations were performed. The current strategy expands the scope of HHDH-catalyzed chiral oxetane ring constructions, offering efficient access to both enantiomers of chiral oxetanes and 1,3-disubstituted alcohols.

Stereoselective Synthesis of Oxetanes Catalyzed by an Engineered Halohydrin Dehalogenase

Although biocatalysis has garnered widespread attention in both industrial and academic realms, the enzymatic synthesis of chiral oxetanes remains an underdeveloped field. Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that have been engineered to accomplish the reversible transformation of epoxides. In our work, a biocatalytic platform was constructed for the stereoselective kinetic resolution of chiral oxetanes and formation of 1,3‐disubstituted alcohols. HheC from Agrobacterium radiobacter AD1 was engineered to identify key variants capable of catalyzing the dehalogenation of γ‐haloalcohols (via HheC M1‐M3) and ring opening of oxetanes (via HheC M4‐M5) to access both (R)‐ and (S)‐configured products with high stereoselectivity and remarkable catalytic activity, yielding up to 49% with enantioselectivities exceeding 99% ee and E>200. The current strategy is broadly applicable as demonstrated by expansion of substrate scope to include up to 18 examples for dehalogenations and 16 examples for ring opening. Additionally, the functionalized products are versatile building blocks for pharmaceutical applications. To shed light on the molecular recognition mechanisms for the relevant variants, molecular dynamic (MD) simulations were performed. The current strategy expands the scope of HHDH‐catalyzed chiral oxetane ring constructions, offering efficient access to both enantiomers of chiral oxetanes and 1,3‐disubstituted alcohols.

Developing and Benchmarking Sulfate and Sulfamate Force Field Parameters via Ab Initio Molecular Dynamics Simulations To Accurately Model Glycosaminoglycan Electrostatic Interactions.

Glycosaminoglycans (GAGs) are negatively charged polysaccharides found on cell surfaces, where they regulate transport pathways of foreign molecules toward the cell. The structural and functional diversity of GAGs is largely attributed to varied sulfation patterns along the polymer chains, which makes understanding their molecular recognition mechanisms crucial. Molecular dynamics (MD) simulations, thanks to their unmatched microscopic resolution, have the potential to be a reference tool for exploring the patterns responsible for biologically relevant interactions. However, the capability of molecular dynamics force fields used in biosimulations to accurately capture sulfation-specific interactions is not well established, partly due to the intrinsic properties of GAGs that pose challenges for most experimental techniques. In this work, we evaluate the performance of molecular dynamics force fields for sulfated GAGs by studying ion pairing of Ca2+ to sulfated moieties─N-methylsulfamate and methylsulfate─that resemble N- and O-sulfation found in GAGs, respectively. We tested available nonpolarizable (CHARMM36 and GLYCAM06) and explicitly polarizable (Drude and AMOEBA) force fields, and derived new implicitly polarizable models through charge scaling (prosECCo75 and GLYCAM-ECC75) that are consistent with our developed "charge-scaling" framework. The calcium-sulfamate/sulfate interaction free energy profiles obtained with the tested force fields were compared against reference ab initio molecular dynamics (AIMD) simulations, which serve as a robust alternative to experiments. AIMD simulations indicate that the preferential Ca2+ binding mode to sulfated GAG groups is solvent-shared pairing. Only our scaled-charge models agree satisfactorily with the AIMD data, while all other force fields exhibit poorer agreement, sometimes even qualitatively. Surprisingly, even explicitly polarizable force fields display a notable disagreement with the AIMD data, likely attributed to difficulties in their optimization and possible inherent limitations in depicting high-charge-density ion interactions accurately. Finally, the underperforming force fields lead to unrealistic aggregation of sulfated saccharides, which qualitatively disagrees with our understanding of the soft glycocalyx environment. Our results highlight the importance of accurately treating electronic polarization in MD simulations of sulfated GAGs and caution against over-reliance on currently available models without thorough validation and optimization.

Molecular mechanism of prolactin-releasing peptide recognition and signaling via its G protein-coupled receptor

Prolactin-releasing peptide (PrRP) is an RF-amide neuropeptide that binds and activates its cognate G protein-coupled receptor, prolactin-releasing peptide receptor (PrRPR), also known as GPR10. PrRP and PrRPR are highly conserved across mammals and involved in regulating a range of physiological processes, including stress response, appetite regulation, pain modulation, cardiovascular function, and potentially reproductive functions. Here we present cryo-electron microscopy structures of PrRP-bound PrRPR coupled to Gq or Gi heterotrimer, unveiling distinct molecular determinants underlying the specific recognition of the ligand’s C-terminal RF-amide motif. We identify a conserved polar pocket that accommodates the C-terminal amide shared by RF-amide peptides. Structural comparison with neuropeptide Y receptors reveals both similarities and differences in engaging the essential RF/RY-amide motifs. Our findings demonstrate the general mechanism governing RF-amide motif recognition by PrRPR and RF-amide peptide receptors, and provide a foundation for elucidating activation mechanisms and developing selective drugs targeting this important peptide–receptor system.

Insights into lysophosphatidylserine recognition and Gα12/13-coupling specificity of P2Y10.

The lysophosphatidylserine (LysoPS) receptor P2Y10, also known as LPS2, plays crucial roles in the regulation of immune responses and holds promise for the treatment of autoimmune diseases. Here, we report the cryoelectron microscopy (cryo-EM) structure of LysoPS-bound P2Y10 in complex with an engineered G13 heterotrimeric protein. The structure and a mutagenesis study highlight the predominant role of a comprehensive polar network in facilitating the binding and activation of the receptor by LysoPS. This interaction pattern is preserved in GPR174, but not in GPR34. Moreover, our structural study unveils the essential interactions that underlie the Gα13 engagement of P2Y10 and identifies key determinants for Gα12-vs.-Gα13-coupling selectivity, whose mutations selectively disrupt Gα12 engagement while preserving the intact coupling of Gα13. The combined structural and functional studies provide insights into the molecular mechanisms of LysoPS recognition and Gα12/13 coupling specificity.

Interglycosidic C5–C6 rotamer distributions of alkyl O-rutinosides

The conformational study of carbohydrates is critical to understand the molecular recognition mechanisms underlying their biological functions. Moreover, the systematic study of their conformational patterns can unlock useful tools to design optimized glycomimetics and drug candidates. Using nuclear magnetic resonance, we studied the interglycosidic rotamer equilibria of ester-protected and deprotected alkyl O-rutinosides (α-L-Rha(1,6)β-D-GlcOR). In the protected series, the equilibrium about the C5–C6 bond distributes among the three possible rotamers gg, gt, and tg, being gt the predominant conformer. In these series, the flexibility about C5–C6 shows a marked dependency on the aglycone's structure, where the increase on the aglycone's volume leads to a progressive increment on the tg contributions at the expense of gt, with gg remaining practically constant along the series. The removal of the protective groups results in rutinosides displaying an equilibrium equally distributed between gg and gt with no tg contributions regardless of the aglycone's structure.

Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

Structural basis for DNA recognition by a viral genome-packaging machine

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

Extracellular vesicles (EVs)' journey in recipient cells: from recognition to cargo release.

Extracellular vesicles (EVs) are nano-sized bilayer vesicles that are shed or secreted by virtually every cell type. A variety of biomolecules, including proteins, lipids, coding and non-coding RNAs, and mitochondrial DNA, can be selectively encapsulated into EVs and delivered to nearby and distant recipient cells, leading to alterations in the recipient cells, suggesting that EVs play an important role in intercellular communication. EVs play effective roles in physiology and pathology and could be used as diagnostic and therapeutic tools. At present, although the mechanisms of exosome biogenesis and secretion in donor cells are well understood, the molecular mechanism of EV recognition and uptake by recipient cells is still unclear. This review summarizes the current understanding of the molecular mechanisms of EVs' biological journey in recipient cells, from recognition to uptake and cargo release. Furthermore, we highlight how EVs escape endolysosomal degradation after uptake and thus release cargo, which is crucial for studies applying EVs as drug-targeted delivery vehicles. Knowledge of the cellular processes that govern EV uptake is important to shed light on the functions of EVs as well as on related clinical applications.