Search Results (42)

Membrane tubes, a class of soft biological confinement for ubiquitous transport intermediates, are essential for cell trafficking and intercellular communication. However, the confinement interaction and directional migration of diffusive nanoparticles (NPs) are widely dismissed as improbable due to the surrounding environment compressive force. Here, combined with the mechanics analysis of nanoparticles (such as extracellular vesicles, EVs) to study their interaction in confinement, we perform dissipative particle dynamics (DPD) simulations to construct a model that is as large as possible to clarify the submissive behavior of NPs. Both molecular simulations and mechanical analysis revealed that the interactions between NPs are controlled by confinement deformation and the centroid distance of the NPs. When the centroid distance exceeds a threshold value, the degree of crowding variation becomes invalid for NPs motion. The above conclusions are further supported by the observed dynamics of multiple NPs under confinement. These findings provide new insights into the physical mechanism, revealing that the confinement squeeze generated by asymmetric deformation serves as the key factor governing the directional movement of the NPs. Therefore, the constraints acting on NPs differ between rigid confinement and soft confinement environments, with NPs maintaining relative stillness in rigid confinement. Full article

Rhodopsin, a G-protein-coupled receptor (GPCR) comprising the protein opsin covalently linked to the chromophore 11-cis retinal, is pivotal in visual phototransduction. Mutations in the gene encoding rhodopsin (RHO) can cause opsin misfolding or reduce its stability, resulting in retinal degenerative disorders such as retinitis pigmentosa (RP). Current therapeutic strategies employing retinoid-based chaperones partially rescue the folding and trafficking of mutant rhodopsin, but are limited by inherent toxicity and instability due to photoinduced isomerization. In the present work, a pharmacophore-based virtual screening protocol combined with molecular docking and molecular dynamics simulations was employed, leading to the identification of a novel non-retinoid opsin ligand that can potentially act as a pharmacological chaperone. Biological validation confirmed that the compound VS1 binds opsin effectively, representing a valuable starting point for structure-based optimization studies aimed at identifying new opsin stabilizers. Full article

Fluorescent cholesterol probes are indispensable tools for studying membrane structure, dynamics, and trafficking. To better understand the structure–function relationship of fluorescent cholesteryl probes, we developed a series of five new modular naphthalimide-containing cholesteryl probes (CND15–CND19). These probes incorporate an L-lysine linker between the cholesterol moiety and the fluorophore, along with a series of distinct head groups. We conducted extensive biophysical characterizations of these probes, including the determination of their solvatochromic properties and lipid partitioning behavior using giant unilamellar vesicles. Molecular dynamics simulations were employed to identify key molecular interactions of these probes within model lipid membranes. Furthermore, live-cell imaging in 3T3 fibroblasts demonstrated the potential applications of these analogs in live-cell imaging, measuring cellular membrane dynamics and studying cholesterol-related processes. The results of this study underscore the critical role of the linker and head group in designing fluorescent cholesterol-mimicking probes. These findings provide valuable insights into optimizing probe designs for future cholesterol and membrane biology research. Full article

In the human ABCG2 (ATP Binding Casette transporter G2/BCRP/MXR) multidrug transporter, a so-called “leucin plug/valve” (a.a. L554/L555) has been suggested to facilitate substrate exit and the coupling of drug transport to ATPase activity. In this work, we analyzed the effects of selected variants in this region by expressing these variants, both in mammalian and Sf9 insect cells. We found that, in mammalian cells, the L554A, L554F, L555F, and a combination of L554F/L555F variants of ABCG2 were functional, were processed to the plasma membrane, and exhibited substrate transport activity similar to the wild-type ABCG2, while the L555A and L554A/L555A mutants were poorly expressed and processed in mammalian cells. In Sf9 cells, all the variants were expressed at similar levels; still, the L555A and L554A/L555A variants lost all transport-related functions, while the L554F and L555F variants had reduced dye transport and altered substrate-stimulated ATPase activity. In molecular dynamics simulations, the mutant variants exhibited highly rearranged contacts in the central transmembrane helices; thus, alterations in folding, trafficking, and function can be expected to occur. Our current studies reinforce the importance of L554/L555 in ABCG2 folding and function, while they do not support the specific role of this region in selective substrate handling and show a general reduction in the coupling of drug transport to ATPase activity in the mutant versions. Full article

Background: Detecting intracellular diffusion dynamics with high spatiotemporal resolution is critical for understanding the complex molecular mechanisms that govern viral infection, drug delivery, and sustained receptor signaling within cellular compartments. Although considerable progress has been made, accurately distinguishing between different types of diffusion in three dimensions remains a significant challenge. Methods: This study extends a previously established two-dimensional, machine learning-based diffusional fingerprinting approach into a three-dimensional framework to overcome this limitation. It presents an algorithm that predicts intracellular motion types based on a comprehensive feature set, including custom statistical descriptors and standard Imaris-derived trajectory features, which capture subtle variations in individual trajectories. The approach employs an extended gradient-boosted decision trees classifier trained on an array of synthetic trajectories designed to simulate diffusion behaviors typical of intracellular environments. Results: The machine learning classifier demonstrated a classification accuracy of over 90% on synthetic datasets, effectively capturing and distinguishing complex diffusion patterns. Subsequent validation using an experimental dataset confirmed the robustness of the approach. The incorporation of the Imaris track features streamlined diffusion classification and enhanced adaptability across diverse volumetric imaging modalities. Conclusions: This work advances our ability to classify intracellular diffusion dynamics in three dimensions and provides a method that is well-suited for high-resolution analysis of intracellular receptor trafficking, intracellular transport of pathogenic agents, and drug delivery mechanisms. Full article

Cosmic ray muon tomography is a promising method for the non-invasive inspection of shipping containers and trucks. It leverages the highly penetrating cosmic muons and their interactions with various materials to generate three-dimensional images of large and dense objects, such as inter-modal shipping containers, which are typically opaque to conventional X-ray radiography techniques. One of the key tasks of customs and border security is verifying shipping container declarations to prevent illegal trafficking, and muon tomography offers a viable solution for this purpose. Common imaging methods using muons rely on data analysis of either muon scattering or absorption–transmission. We design a compact muon tomography system with dimensions of 3 × 3 × 3 m³, consisting of 2D position-sensitive detectors. These detectors include plastic scintillators, wavelength-shifting (WLS) fibers, and SiPMs. Through light transport modeling with GEANT4, we demonstrate that the proposed detector design—featuring 1 m × 1 m scintillator plates with 2 mm² square-shaped WLS fibers—can achieve a spatial resolution of approximately 0.7–1.0 mm. Through Monte Carlo simulations, we demonstrate that combining muon scattering and absorption data enables the rapid and accurate identification of cargo materials. In a smuggling scenario where tobacco is falsely declared as paper towel rolls, this combined analysis distinguishes the two with 3 $\sigma$ confidence at a spatial resolution of 1 mm (FWHM) for the muon detector, achieving results within a scanning time of 40 s for a 20-foot shipping container. Full article

Microgravity is one of the main stressors that astronauts are exposed to during space missions. This condition has been linked to many disorders, including those that feature dysfunctional immune homeostasis and inflammatory damage. Over the past 30 years, a significant body of work has been gathered connecting weightlessness—either authentic or simulated—to an inefficient reaction to pathogens, dysfunctional production of cytokines and impaired survival of immune cells. These processes are also orchestrated by a plethora of bioactive lipids, produced by virtually all cells involved in immune events, which control the induction, magnitude, outcome, compartmentalization and trafficking of immunocytes during the response to injury. Despite their crucial importance in inflammation and its modulation, however, data concerning the role of bioactive lipids in microgravity-induced immune dysfunctions are surprisingly scarce, both in quantity and in variety, and the vast majority of it focuses on two lipid classes, namely eicosanoids and endocannabinoids. The present review aims to outline the accumulated knowledge addressing the effects elicited by microgravity—both simulated and authentic—on the metabolism and signaling of these two prominent lipid groups in the context of immune and inflammatory homeostasis. Full article

Iron overload in many brain regions is a common feature of aging and most neurodegenerative diseases. In this review, the causes, mechanisms, mathematical models, and possible therapies are summarized. Indeed, physiological and pathological conditions can be investigated using compartmental models mimicking iron trafficking across the blood–brain barrier and the Cerebrospinal Fluid-Brain exchange membranes located in the choroid plexus. In silico models can investigate the alteration of iron homeostasis and simulate iron concentration in the brain environment, as well as the effects of intracerebral iron chelation, determining potential doses and timing to recover the physiological state. Novel formulations of non-toxic nanovectors with chelating capacity are already tested in organotypic brain models and could be available to move from in silico to in vivo experiments. Full article

We extend network analysis to directed criminal networks in the context of asymmetric links. We computed selected centralities, centralizations and the assortativity of a drug trafficking network with 110 nodes and 295 edges. We also monitored the centralizations of eleven temporal networks corresponding to successive stages of investigation during the period 1994–1996. All indices reach local extrema at the stage of highest activity, extending previous results to directed networks. The sharpest changes ($90\%$) are observed for betweenness and in-degree centralization. A notable difference between entropies is observed: the in-degree entropy reaches a global minimum at month 12, while the out-degree entropy reaches a global maximum. This confirms that at the stage of highest activity, incoming instructions are precise and focused, while outgoing instructions are diversified. These findings are expected to be useful for alerting the authorities to increasing criminal activity. The disruption simulations on the time-averaged network extend previous results on undirected networks to directed networks. Full article

Integrins are heterodimeric cell-surface receptors that regulate cell–cell adhesion and cellular functions through bidirectional signaling. On the other hand, anomalous trafficking of integrins is also implicated in severe pathologies as cancer, thrombosis, inflammation, allergies, and multiple sclerosis. For this reason, they are attractive candidates as drug targets. However, despite promising preclinical data, several anti-integrin drugs failed in late-stage clinical trials for chronic indications, with paradoxical side effects. One possible reason is that, at low concentration, ligands proposed as antagonists may also act as partial agonists. Hence, the comprehension of the specific structural features for ligands’ agonism or antagonism is currently of the utmost interest. For α4β1 integrin, the situation is particularly obscure because neither the crystallographic nor the cryo-EM structures are known. In addition, very few potent and selective agonists are available for investigating the mechanism at the basis of the receptor activation. In this account, we discuss the physiological role of α4β1 integrin and the related pathologies, and review the few agonists. Finally, we speculate on plausible models to explain agonism vs. antagonism by comparison with RGD-binding integrins and by analysis of computational simulations performed with homology or hybrid receptor structures. Full article

This study mainly investigates the maximum length of CAV (Connected and Autonomous Vehicle) platoons in a heterogeneous traffic flow environment. By employing MATLAB to simulate the heterogeneous traffic flow on expressways, this study focuses on the maximum platoon length of CAV platoons and explores their impact on the traffic flow characteristics on expressways. Firstly, based on four different car-following modes of heterogeneous traffic flow, F-STCA (Flexible–Symmetric Two-Lane Cellular Automata Model) and the symmetric lane-changing strategy, the study refines and improves the construction of the NaSch (Nagel and Schreckenberg) model introduced into the Gipps safety distance formula. The whole improvement process is based on the acceleration decay characteristics of vehicles on expressway. Secondly, the congestion situations under different maximum platoon lengths are compared using fundamental heat maps of the simulation data. The evolution of the fundamental diagram with changes in maximum platoon length is studied to investigate the impact and magnitude of maximum platoon lengths on the road capacity under different CAV permeabilities. Finally, the study explores the stability and safety of heterogeneous traffic flow involving CAV platoons using SD (Standard Deviation) and TERCRI (Time-Exposed Rear-End Collision Risk Index). The results show that when the CAV’s permeability does not reach a high level on heavily trafficked sections of the expressway, considering the overall average speed, the maximum platoon length should not be set too long and should be around five vehicles. This restriction does not apply when the CAV permeability approaches 100%. Full article

Methylmalonic aciduria and homocystinuria type C protein (MMACHC) is required by the body to metabolize cobalamin (Cbl). Due to its complex structure and cofactor forms, Cbl passes through an extensive series of absorptive and processing steps before being delivered to mitochondrial methyl malonyl-CoA mutase and cytosolic methionine synthase. Depending on the cofactor attached, MMACHC performs either flavin-dependent reductive decyanation or glutathione (GSH)-dependent dealkylation. The alkyl groups of Cbl have to be removed in the presence of GSH to produce intermediates that can later be converted into active cofactor forms. Pathogenic mutations in the GSH binding site, such as R161Q, R161G, R206P, R206W, and R206Q, have been reported to cause Cbl diseases. The impact of these variations on MMACHC’s structure and how it affects GSH and Cbl binding at the molecular level is poorly understood. To better understand the molecular basis of this interaction, mutant structures involving the MMACHC-MeCbl-GSH complex were generated using in silico site-directed point mutations and explored using molecular dynamics (MD) simulations. The results revealed that mutations in the key arginine residues disrupt GSH binding by breaking the interactions and reducing the free energy of binding of GSH. Specifically, variations at position 206 appeared to produce weaker GSH binding. The lowered binding affinity for GSH in the variant structures could impact metabolic pathways involving Cbl and its trafficking. Full article

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354–367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357–360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter. Full article

Receptor-mediated transcytosis is an elegant and promising strategy for drug delivery across biological barriers. Here, we describe a novel ligand–receptor pair based on a dimeric, engineered derivative of the Pseudomonas aeruginosa lectin LecA, here termed Di-LecA, and the host cell glycosphingolipid Gb3. We characterized the trafficking kinetics and transcytosis efficiencies in polarized Gb3-positive and -negative MDCK cells using mainly immunofluorescence in combination with confocal microscopy. To evaluate the delivery capacity of dimeric LecA chimeras, EGFP was chosen as a fluorescent model protein representing macromolecules, such as antibody fragments, and fused to either the N- or C-terminus of monomeric LecA using recombinant DNA technology. Both LecA/EGFP fusion proteins crossed cellular monolayers in vitro. Of note, the conjugate with EGFP at the N-terminus of LecA (EGFP-LecA) showed a higher release rate than the conjugate with EGFP at the C-terminus (LecA-EGFP). Based on molecular dynamics simulations and cross-linking studies of giant unilamellar vesicles, we speculate that EGFP-LecA tends to be a dimer while LecA-EGFP forms a tetramer. Overall, we confidently propose the dimeric LecA chimeras as transcytotic drug delivery tools through Gb3-positive cellular barriers for future in vivo tests. Full article

Uranyl–protein interactions participate in uranyl trafficking or toxicity to cells. In addition to their qualitative identification, thermodynamic data are needed to predict predominant mechanisms that they mediate in vivo. We previously showed that uranyl can substitute calcium at the canonical EF-hand binding motif of calmodulin (CaM) site I. Here, we investigate thermodynamic properties of uranyl interaction with site II and with the whole CaM N-terminal domain by spectrofluorimetry and ITC. Site II has an affinity for uranyl about 10 times lower than site I. Uranyl binding at site I is exothermic with a large enthalpic contribution, while for site II, the enthalpic contribution to the Gibbs free energy of binding is about 10 times lower than the entropic term. For the N–terminal domain, macroscopic binding constants for uranyl are two to three orders of magnitude higher than for calcium. A positive cooperative process driven by entropy increases the second uranyl-binding event as compared with the first one, with ΔΔG = −2.0 ± 0.4 kJ mol⁻¹, vs. ΔΔG = −6.1 ± 0.1 kJ mol⁻¹ for calcium. Site I phosphorylation largely increases both site I and site II affinity for uranyl and uranyl-binding cooperativity. Combining site I phosphorylation and site II Thr7Trp mutation leads to picomolar dissociation constants Kd₁ = 1.7 ± 0.3 pM and Kd₂ = 196 ± 21 pM at pH 7. A structural model obtained by MD simulations suggests a structural role of site I phosphorylation in the affinity modulation. Full article